Patent Application: US-77893504-A

Abstract:
the present invention concerns the isolation of plasmids from extremely thermophilic anaerobic microorganisms and their use in genetic transformation of thermophilic and mesophilic microorganisms . more particular the invention concerns the use of thermostable plasmid vectors as tools for creating shuttle vectors for genetic transformation of extremely thermophilic anaerobic microorganisms .

Description:
anaerocellum thermophilum dsm6725 is a strict anaerobic microorganism with a temperature optimum at 72 - 75 ° c . which is freely available from a public culture collection at dsm — deutsche sammiung von mikroorganismen und zelikulturen gmbh , mascheroder weg 1b , d - 3300 braunschweig , germany , under the accession number dsm6725 . anaerocellum thermophilum dsm6725 was found to harbour three plasmids , pbas2 , pbas and pbal , noner of which have previously been reported . pbas2 is 3653 bp ( fig1 ) with seq . id no : 15 , pbas is 1863 bp ( fig2 ) with seq id no : 22 and pbal is 8294 bp ( fig3 ) with seq . id no : 1 , and the gc content of the plasmids is 43 %, 43 % and 39 %, respectively . sequencing of pbas2 revealed 8 open reading frames , where orfs 11 and 61 showed homology to genes encoding a recombinase protein and a replicase protein , respectively . the following orfs of pbas2 have been designated the sequence id numbers : seq id no : 16 = recombinase , seq id no : 17 = replicase ( orf 11 ), seq id no : 18 = orf41 , seq id no : 19 = orf42 , seq id no : 20 = orf43 , seq id no : 21 = orf44 . furthermore , a detailed analysis of the dna structure of pbas2 revealed a putative replication initiation site and replication terminus . sequencing of pbas revealed that it was a sub - species plasmid ( fig2 ), showing complete sequence identity to a portion of pbas2 , and hence containing only some of the orfs predicted in pbas2 . nucleotides 1 - 1863 in pbas correspond to nucleotides 638 - 2501 in pbas2 . plasmid pbal contains 14 open reading frames , of which three showed similarity to known proteins . one orf showed homology to replication proteins found on various staphylococcus plasmids . these plasmids , pnvh97 , psk1 , pi9789 and pip680 , all replicate via the theta mechanism , indicating that pbal also employs this type of replication mechanism . significant homology was also found to sigma factor k from bacillus thuringiensis and to dna repair protein from campylobacter jejuni . the orfs of pbal have been designated the following sequence id numbers : seq . id no : 2 = orf 31 ( dna polymerase ); seq . id no : 3 = orf32 ; seq . id no : 4 = orf 33 ( sigma factor ); seq . id no : 5 = orf 34 ( replicase ); seq . id no : 6 = orf41 ; seq . id no : 7 = orf42 ; seq . id no : 8 = orf43 ; seq id no : 9 = orf44 ; seq . id no : 10 = orf51 ; seq . id no : 11 = orf52 ; seq . id no : 12 = orf53 ; seq . id no : 13 = orf61 ; seq . id no : 14 = orf62 . the pbas2 and pbal plasmids found in anaerocellum thermophilum dsm6725 , showed no nucleotide sequence similarity to each other . similarly the proteins encoded by the orf &# 39 ; s in the pbas2 and pbal plasmids did not reveal any similarity . the described plasmids , pbas2 , pbas and pbal , are derived from an extremely thermophilic anaerobic microorganism with a temperature optimum at 72 - 75 ° c . this makes them the most thermostable plasmids isolated from extremely thermophilic anaerobic microorganisms . pbas2 , pbas and pbal are therefore expected to be considerably more thermostable than the plasmids reported by others , since most of the latter plasmids are isolated from cultures with temperature optimum at 60 ° c . as opposed to all other plasmids reported , with the exception of pnb2 , the full dna sequence of pbas and pbal has been determined . knowledge of the dna sequence allows for identification of replication regions and proteins with potential importance for plasmid copy number control , thus providing a far more solid basis for the construction of vectors . in addition both plasmids are small and easy to handle , making them especially useful as the basis for genetic systems for extremely thermophilic anaerobic microorganisms . the thermostable expression plasmid ( peaks ) is provided according to the invention as an example of several possible expression plasmids . it is to be understood that peaks is intended only to be illustrative and in no way limitative . a person skilled in the art will readily understand that once the extremely thermophilic plasmids pbas2 , pbas and pbal have been isolated and sequenced , one may derive extremely thermophilic expression plasmids based on these genetic elements . marker genes are included in plasmid and shuttle vectors to facilitate the identification and selection of host cells transformed with the vector / plasmid . marker genes may encode proteins conferring resistance to antibiotics , such as an ampicillin resistance gene , a kanamycin resistance gene , a tetracycline resistance gene , or an erythromycin resistance gene . alternatively they may encode visual markers , such as those based on beta - galactosidase marker gene expression . particularly useful marker genes are those which encode proteins which can be actively expressed in the host cell of interest . in the case of an extreme thermophilic anaerobic host cell , it is important to select a marker gene encoding a heat - stable protein . the kanamycin resistance gene is particularly useful in this context , since both the kanamycin resistance protein and the kanamycin antibiotic are rather heat - stable . this is particular important for plasmid stability , where the maintenance of the plasmid in the extreme thermophilic anaerobic host may depend on antibiotic selection pressure . the inclusion of more than one selection marker is particularly useful in shuttle vectors , which should be maintained in two host cell types . multiple cloning sites are included in the shuttle vector provided by the invention to facilitate the introduction of additional expression cassettes , which may comprise marker genes or heterogenous protein expression cassettes . expression vectors of the invention are used for transformation of host cells , thus constructing microorganisms suitable for industrial production processes at temperatures of 60 - 75 ° c . for the manufacture of recombinant proteins which are subsequently recovered . said proteins can be an alcohol dehydrogenase , a carbohydrase , an amylase , a cellulase , a beta - glucanase , a beta - glucosidase , an oxidoreductase , a protease , an oxidoreductase , or a lipase and they may be applied in a wide variety of industrial processes characterised by high temperatures . examples of recombinant proteins used in industrial processes are cellulases , amylases , xylanases , β - galactosidases , β - glucosidases , α - glucosidases , and glucanases . expression vectors of the invention are also used for transformation of host cells to modify the catalytic properties of the cells , thus improving their performance as biocatalysts in industrial processes at temperatures of 60 - 75 ° c . this is known as metabolic engineering and the modified cells may be used for e . g . degradation of biopolymers such as lignocellulosic materials . of course the expression vectors of the invention may also be effective at lower temperatures between room temperature and 60 ° c . screening for plasmids was done on thermophilic anaerobic isolates from our strain collection and on dsm6725 , which was purchased from dsmz — deutsche sammiung von mikroorganismen und zelikulturen gmbh , germany many . anaerobic cultures were grown in a ba medium as previously described ( angelidaki et al ., 1990 ), but the medium was amended with 1 g / l yeast extract ( difco ) and cysteine was not added . the medium was reduced with 0 . 25 g / l sodium sulfide . appropriate carbon sources , cellulose , glucose , xylose , mannose or galactose was added at 5 g / l and incubation was at 70 ° c . and ph 6 . 8 . 60 ml overnight cultures were used for extraction of plasmids . cells were harvested by centrifugation and washed once in 10 ml te ph 8 . 0 , pelleted and resuspended in te containing 10 mg / ml lysozyme . the cells were lysed according to the method described by ( o &# 39 ; sullivan et al ., 1993 ), starting from step 2 , and the plasmid dna was subsequently purifed according to standard molecular methods ( ausubel , 1987 ). the plasmids pbas2 , pbas and pbal were detected and isolated from anaerocellum thermophilum dsm6725 as described in example 1 . the presence of the plasmids &# 39 ;, designated pbas2 , pbas and pbal , in anaerocellum thermophilum dsm6725 was verified by agarose gel electrophoresis using 1 . 2 % agarose , stained with ethidium bromide and illuminated by uv light . for cloning purposes the cloning vector pbluescript sk + and cloning host e . coli dh 5 - α a was used . initially all three plasmids were characterized by digestion with 23 different restriction endonucleases . restriction endonuclease digests were carried out according to the suppliers recommendation ( new england biolabs ). plasmids pbas and pbal showed a unique ecori site and were cloned into ecori / sap ( shrimp alkaline phosphatase , boeringer mannheim ) treated pbluescript sk + vector . pbas was sequenced by “ plasmid walking ”. in the first round of sequencing the pbluescript ks + primers t3 and t7 were used and the sequence information obtained was then used to design sequencing primers for the next round of sequencing . pbal revealed an additional unique sali site , which was used for subcloning into sali / ecori treated pbluescript ks + , eventually yielding two plasmids pbmn 4 . 2 kb and pbab 4 . 0 kb . these to plasmids were then sequenced by “ plasmid walking ”. restriction mapping and sequencing of pbas and pbas2 revealed that pbas was a plamsid sub - species of pbas2 . since part of the nucleotide sequence of pba2 was homologous to pbas , the sequence of the unknown part of the plasmid was determined according to the following methods . the unknown part of pbas2 was amplified using the following primers : this fragment was then sequenced by plasmid walking starting with primers : the known part of the plasmid , common to pbas , was verified by restriction enzyme digestion . the nucleotide residues 1 - 1863 of pbas correspond to nucleotide residues 638 to 2501 of pbas2 . fig1 and 3 show the three plasmids and the complete sequences of pbas2 , pbas and pbal are given in seq id nos 15 , 22 and 1 , respectively . pbas was used as the thermostable backbone for construction of a vector replicating in thermophilic anaerobic microorganisms . a 1841 bp fragment of plasmid pbas , including potential replication region and replication protein was amplified by pcr using the following primer sequences : pbas upper1 - 20 : gcgggatccgtcgacgaaftcgaaaagcagataag ( seq . id no : 27 ) pbas lower184 : gggctgcagtctcgtttaacaactaca ( seq . id no : 28 ) thereby introducing unique bamh1 and pst1 restriction sites in the pcr pruduct . subsequently the bamh1 , pst1 digested pbas pcr product was cloned into pbluescript sk + / kan r which was restriction enzyme digested with bamh1 and pst1 . pbluescript sk + / kan r was constructed by introducing the kanamycin resistance gene from pub110 into the pbluescript vector . the kan r gene in pub110 was excised by digestion with the restriction enzymes , mun1 and nci1 , treated with t4 dna polymerase , and then ligated into ecorv digested pbluescript . the resulting thermophilic expression plasmid peaks is shown in fig3 . the kanamycin - sensitive , thermophilic anaerobic ( clostridium - like ) isolate ( sonne et al . 1993 ), x7b and the kanamycin sensitive strain thermoanaerobacter mathranli a3m4 were transformed to kanamycin resistance by the thermostable shuttle vector peaks ( including the kanamycin resistance gene ), by using electroporation and peg mediated transformation . initially transformations were conducted at 55 c and kanamycin resistant colonies were selected . the selected colonies were cultivated at both 55 ° c . and at 70 ° c . in selective liquid medium ( kanamycin added ), without any apparent differences in cell growth rates . following growth of the transformants in selective liquid medium for 20 generations , they were still stable . since both plasmids pbas2 , pbas and pbal are small and easy to handle , they are ideal as thermophilic backbone for constructing plasmid vectors for genetic engineering and metabolic engineering of thermophilic microorganisms . for example for improving ethanol yield from thermophilic anaerobic microorganisms , by introduction of additional copies of genes responsible for ethanol formation ( e . g . adh , alcohol dehydrogenase ). in addition the plasmids pbas2 , pbas and pbal can be used as basis for constructing expression vectors for over - expressing industrially important thermostable enzymes e . g . cellulases , amylases , xylanases , β - galactosidases β - glucosidases etc . the shuttle vector , peaks , which incorporates pbas , can be used for the expression of thermostable enzyme . the inducible promoter of the xyla gene from thermoanaerobacter ethanolicus ( erbeznik et al . 1998 ), is cloned into the pst1 or ecor1site of peaks , together with a downstream mcr sequence , and xyl a terminator . a gene of interest , encoding for example an ethanol dehydrogenase , can be cloned into the mcs of the plasmid , and subsequently transformed into the extreme thermophilic anaerobic microbial host . expression of the heterogenous protein from the gene inserted into peaks can be regulated by the level of xylose or glucose added to the medium . sequence analysis revealed that pbal encodes at least three proteins : 1 ) a dna polymerase / dna repair protein 2 ) a sigma k factor and 3 ) a replication protein . their gene products , especially dna polymerase / dna repair protein encoded by pbal can be used in dna manipulating processes used in molecular biology techniques . for example in the pcr reaction . this example disclosed the construction of a high copy number shuttle vector based on pbas2 comprising a kanamycin resistance gene . the entire pbas2 plasmid is amplified using pbascw1 and pbasccw1 primers . the resulting molecule is digested with pst i restriction enzyme and inserted into the pst i digested e . coli vector pbluescript sk +. the resulting plasmid is called pbluebas2 . pub110 is digested with mun i and nci i and the fragment containing the kanamycin resistance gene is treated with t4 dna polymerase . the kanamycin resistance casette is then inserted into the ecorv site of pbluebas2 . this example disclosed the construction of a low copy number shuttle vector based on pbas2 comprising a kanamycin resistance gene . the entire pbas2 plasmid is amplified using pbascw3 and pbasccw3 primers . the product is digested with bamh i and inserted into the low copy number plasmid pou71 digested with bamh i . the resulting plasmid is called p71bas2 . pub110 is digested with mun i and nci i and the fragment containing the kanamycin resistance gene is treated with t4 dna polymerase . the kanamycin resistance casette is then inserted into the ecor i and t4 dna polymerase treated p71bas2 . the resulting plasmid is called p7ibas2k . japanese unexamined published patent application no . jp 60 - 186287 a , agency of industrial science and technology ( agen ). new plasmid derived from anaerobic thermophilic bacteria — is used as vector in breeding anaerobic thermophilic bacterial . sep . 21 , 1985a . japanese unexamined published patent application no . jp 60 - 186277 a , agency of industrial science and technology ( agen ). new microorganism thermoanaerobacter no . 62 — contains new plasmid useful as vector in recombinant dna experiment . sep . 21 , 1985b . japanese unexamined published patent application no . jp 60 - 186278 a , agency of industrial science and technology ( agen ). new microorganism thermoanaerobacter no . 29 — containing new plasmid characterised by specified restriction end nuclease cleavage map . sep . 21 , 1985c . japanese unexamined published patent application no . jp 60 - 186286 a , agency of industrial science and technology ( agen ). new plasmid - derived from anaerobic thermophilic bacteria . sep . 21 , 1985d . japanese unexamined published patent application no . jp 60 - 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