EP2145005A1 - Système de vecteur plasmidique archéen - Google Patents

Système de vecteur plasmidique archéen

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Publication number
EP2145005A1
EP2145005A1 EP08758462A EP08758462A EP2145005A1 EP 2145005 A1 EP2145005 A1 EP 2145005A1 EP 08758462 A EP08758462 A EP 08758462A EP 08758462 A EP08758462 A EP 08758462A EP 2145005 A1 EP2145005 A1 EP 2145005A1
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EP
European Patent Office
Prior art keywords
archaeal
sulfolobus
plasmid vector
vector system
gene
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EP08758462A
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German (de)
English (en)
Inventor
Georg Lipps
Silvia Berkner
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Universitaet Bayreuth
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Universitaet Bayreuth
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Priority to EP08758462A priority Critical patent/EP2145005A1/fr
Publication of EP2145005A1 publication Critical patent/EP2145005A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Definitions

  • the present invention relates to an archaeal plasmid vector system comprising: I) one or more selectable marker gene(s) for selection in archaea, and II) at least one component selected from the group consisting of a) an archaeal origin of replication, b) a homology region to an archaeal chromosome, c) a gene coding for a molecule conferring the ability to integrate the vector into an archaeal chromosome, and d) gene orf904; wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the present invention also relates to an archaeal plasmid vector system comprising: I) one or more selectable marker gene(s) for selection in archaea, and II) at least one component selected from the group consisting of a) an archaeal origin of replication, b) a homology region to an archaeal chromosome, and c) a gene coding for a molecule conferring the ability to integrate the vector into an archaeal chromosome; wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the present invention relates to a plasmid vector system comprising: I) gene orf904, and II) one or more selectable marker gene(s); wherein the vector is non-integrative.
  • the present invention relates to host cells transformed with the vector system as well as to kits comprising the vector system or a host cell of the present invention.
  • the present invention also relates to a method for producing a protein of interest.
  • Sulfolobus a genus of thermoacidophilic crenarchaeotes, has provided much of the information currently available on the physiology and molecular biology of archaea from geothermal environments.
  • Seminal studies of Sulfolobus spp. have addressed, for example, chromatin binding proteins (1 ;2), replication (3), cell cycle (4), repair (5), transcription (6), translation (7;8) as well as metabolism (9).
  • Sulfolobus spp. are manipulated in the laboratory.
  • Sulfolobus cells can be grown aerobically and heterotrophically on a variety of complex and defined carbon sources, either in liquid media or on plates, with doubling times as short as a few hours.
  • Sulfolobus spp. are hyperthermophiles with optimal growth temperatures around 80 0 C, their proteins are intrinsically stable and resistant to proteolysis.
  • Sulfolobus enzymes expressed in mesophilic hosts can often be purified with the aid of a heat step, which removes most proteins of the host.
  • the structural rigidity of thermophilic proteins also appears to be an advantage for crystallization, which is a prerequisite for X-ray analysis of three-dimensional structure.
  • Aucelli et ah An exceptional example of a plasmid-based vector is described by Aucelli et ah.
  • Aucelli et a/. (16) constructed a shuttle vector based on the sequence of the pSSVx genetic element from Sulfolobus islandicus.
  • pSSVx is a hybrid between a plasmid and a Fusellovirus and it is able to spread as a virus satellite in the presence of its helper virus SSV2.
  • the pSSVx chromosome was fused with an £ coli plasmid replicon and the ampicillin resistance gene and used as a vector.
  • a comparative approach is described in the international patent application WO 2004/106527.
  • This application relates to Sulfolobus expression vectors which comprise genes encoding structural proteins and the site-specific integrase of SSV1 , SSV2 or pSSVx.
  • Vectors based on viral sequences are packaged into virus particles - which are generated from structural proteins, e.g. of SSV1 or SSV2 - and spread easily in a host cell culture. Spreading throughout a host cell culture and thus circumventing a stringent selection of transformed cells is the most prominent of viral-based vectors.
  • working with viral particles in archaeal/bacterial systems is often connected with enormous difficulties in handling thereof. For example, there is a considerable risk of contamination of different cell culture samples with virus particles.
  • a further disadvantage is a negative influence of viral sequences on growth behavior of infected host cells.
  • the presence of viruses decreases the growth and reproduction rate of an infected host cell. Therefore, in view of a vector system, the replication cycle of virus-based vectors will be generally slower than replication of plasmid-based vectors.
  • vectors based on viruses are also disadvantageous due to their large molecular size (e.g. the genomes of SSV1 or SSV2 comprise 15.5 kb or 14.8 kb, respectively). Large molecular size vectors constrain the length of the sequences to be incorporated therein (i.e. inserts). Thus, inserts cloned into most viral vectors are limited to rather short nucleic acid sequences. Vectors having high molecular weight inserts cannot be packed into infectious virus particles.
  • plasmid-based vector is the shuttle vector pEXSs generated by Cannio eta/. (17).
  • the pEXSs vector is composed of an £ co/Zplasmid sequence and the putative SSV1 viral autonomously replicating sequence (ARS).
  • ARS viral autonomously replicating sequence
  • the technical problem underlying the present invention was to provide a stable plasmid vector system which is highly efficient in archaea and that may be utilizable for industrial application.
  • the solution to this technical problem is achieved by the subject-matter as characterized in the claims.
  • the present invention relates to an archaeal plasmid vector system comprising: I) one or more selectable marker gene(s) for selection in archaea, and II) at least one component selected from the group consisting of a) an archaeal origin of replication, b) a homology region to an archaeal chromosome, c) a gene coding for a molecule conferring the ability to integrate the vector into an archaeal chromosome, and d) gene orf904; wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the present invention further relates to an archaeal plasmid vector system comprising: I) one or more selectable marker gene(s) for selection in archaea, and II) at least one component selected from the group consisting of a) an archaeal origin of replication, b) a homology region to an archaeal chromosome, and c) a gene coding for a molecule conferring the ability to integrate the vector into an archaeal chromosome; wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the plasmid vector system of the present invention can be applied for archaea where it is replicated in high amounts.
  • the inventive vector system was already used for electroporation and cultivation in Sulfolobus under various conditions whereby reproducible and satisfying results were obtained. Therefore, an industrial application of that plasmid-based vector system according to the invention is possible.
  • sequences of several kb can be cloned into the vector for genetic studies and/or expression.
  • inventive vector does not carry any genes which might allow the assembly of a functional virus particle, there is no risk of contamination of cell cultures as with an alternative virus vector system when using the vector system of the invention. Therefore, the inventive vector system is easy and safe to handle and does not require the precaution that is needed for the handling of virus-based vector systems.
  • a characteristic feature of the archaeal plasmid vector system is that it comprises one or more selectable marker gene(s) for selection in archaea. Thus, the plasmid vector system is applicable for selection in archaea.
  • archaea or "archaeal” refers to a microorganism which represents (besides bacteria and eukaryota) one of the three kingdoms of live. Based on their rRNA, archaea can be sub-divided into two ma journey groups, crenarchaeota and euryarchaeota, and two minor groups, korarcheota and nanoarcheota.
  • euryarchaeota encompasses the classes Archaeglobi, Halobacteria, Methanobacteria, Methanococci, Methanomicrobia, Methanopyri, Thermococci, Thermoplasmata and further unclassified euryarchaeota. If “euryarchaeota” are used as host system according to the invention, thermophilic euryarchaeota such as Thermococci and Thermoplasmata are preferred euryarchaeota.
  • crenarchaeota encompasses the class "Thermoprotei” which comprises, e.g., the orders Desulfurococcales (such as the families Desulfurococcaceae and Pyrodictiaceae), Thermoproteales (such as the families Thermoproteaceae and Thermofilaceae), and Sulfolobales.
  • Desulfurococcales such as the families Desulfurococcaceae and Pyrodictiaceae
  • Thermoproteales such as the families Thermoproteaceae and Thermofilaceae
  • Sulfolobales Sulfolobales.
  • the aforementioned "crenarchaeota” are the most preferred host cell system for the inventive plasmid vector.
  • plasmid vector system which is suitable to be used for archaea, in particular for "crenarchaeota” or the more specific forms of “crenarchaeota” as disclosed herein, e.g. "Sulfolobales” or its genera.
  • Sulfolobales comprises the family Sulfolobaceae and other unclassified Sulfolobales.
  • Sulfolobaceae refers to the genera Acidianus, Metal losphaera, Stygiolobus, Sulfolobus, Sulfurisphaera and other unclassified Sulfolobaceae.
  • Sulfolobus refers to hyperthermophilic archaea genera Sulfolobus which comprises, in particular, the species Sulfolobus acidocaldarius, Sulfolobus brierleyi, Sulfolobus hakonensis, Sulfolobus islandicus, Sulfolobus metallicus, Sulfolobus neozealandicus, Sulfolobus shibatae, Sulfolobus solfataricus, Sulfolobus tengchongensis, Sulfolobus thuringiensis, Sulfolobus tokodaii, Sulfolobus yangmingensis and cultured Sulfolobus sp..
  • the term "vector” refers to a means for transferral of nucleic acid sequences.
  • the vector of the invention may contain an expression control sequence including, e.g. a promoter, enhancer, silencer, TATA-box, ribosome binding site, attachment sites for transcription factors, terminator, and/or one or more restriction sites into which an insert sequence can be cloned.
  • the vector may have a size of 1 kb to 50 kb, preferably 2 to 30 kb, more preferably 3 to 20 kb, and most preferably 3 to 10 kb. Specific preferred are 1 to 10kb, 2 to 7kb or 3 5o 5kb.
  • Plasmid refers to a circular, extrachromosomal nucleic acid molecule capable of replication in a cell.
  • Plasmid vector system refers to a vector system which is based on a plasmid or parts thereof.
  • vector system is synonymously used to the terms "plasmid vector” or "vector”.
  • archaeal plasmid vector system refers to a vector system as defined above which is functional in archaea, i.e. can be applied for archaea, for example, for cloning and/or expression of genes in archaea.
  • the vector system is preferably functional in crenarchaea, more preferably Sulfolobales, even more preferably Sulfolobaceae and most preferably Sulfolobus.
  • the vector system is functional in euryarchaeota, more preferably thermophilic euryarchaeota and most preferably Thermococci and/or Thermoplasmata.
  • selectable marker gene refers to a gene encoding a protein or an RNA that, when expressing said gene in a cell, confers a phenotype onto the cell which allows a selection of the cell expressing said selectable marker gene.
  • the selectable marker gene does not naturally occur on the plasmid on which the plasmid vector is based.
  • the selectable marker gene was introduced into the vector by recombinant biochemical methods, e.g. via cloning.
  • the selectable marker gene allows for selection of cells which were transformed with the inventive vector carrying the selectable marker gene.
  • the selectable marker gene encodes a protein that provides for synthesis of a compound necessary for the cell metabolism under natural or modified environment conditions or which, in general, allows the cell to survive under said conditions.
  • the selectable marker gene is an essential gene or a gene conferring a growth advantage for the cell.
  • the selectable marker gene can encode a protein that confers resistance to an antibiotic on said cell.
  • Antibiotics can be divided into several subclasses according to their structural elements or function on the cell treated therewith: aminoglycoside, arsenic-based, bacteriostatic, ⁇ -lactam, fluoroquinolone, glycopeptide, polyketide, and sulfonamide antibiotics.
  • the selectable marker gene preferably, confers resistance to all kinds of antibiotics. More preferably, the selectable marker gene confers resistance to the antibiotics selected from the group consisting of ampicillin, carbenicillin, penicillin
  • C cephalosporins, cefotaxim, cefalexin, vancomycin, cycloserine, chloramphenicol, erythromycin, lincomycin, tetracyclin, spectinomycin, clindamycin, chlortetracycline, gentamycin, hygromycin (B), kanamycin, neomycin, streptomycin, G418, tobramycin, rifampicin, mitomycin (C), nalidixic acid, doxorubicin, 5-flurouracil, 6-mercaptopurine, micanozole, trimethoprim, methotrexat, metronidazole, sulfmetoxazole, benylpenicillin, propicillin, azidocillin, flucloxacillin, dicloxacillin, novobiocin and oxacillin. Selection of cells which were transformed with a vector carrying the antibiotic selectable gene and are therefore resistant to that antibiotic is performed using an
  • the selectable marker gene may encode a protein which influences the growth behavior of a cell when expressed inside this cell under the presence of an inhibitor or activator/enhancer of said protein.
  • the influence can be in a positive or negative manner, whereby the positive manner is preferred.
  • the protein encoded by the selectable marker gene influences the cell, in the presence of an inhibitor, in a positive manner.
  • the selectable marker gene is the 3- hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (44), most preferably, the selectable marker gene is a thermostable HMG-CoA reductase.
  • HMG- CoA 3-hydroxy-3-methylglutaryl coenzyme A reductase reductase reductase to synthesize mevalonate, which eukaryotes employ in the production of sterols and archaea need for the isoprenoid side chains of their unique and characteristic lipids.
  • Preferred inhibitors for HMG-CoA are statins selected from the group consisting of simvastatin, mevinolin, lovastatin, atorvastatin and pravastatin and analogues thereof.
  • the selectable marker gene provides for the synthesis of a metabolic compound. More preferably, the selectable marker gene encodes substances which are necessary for formation of nucleotides (adenine (A), guanine (G), cytosine (C), uracil (U) or thymine (T)) or amino acids (alanine (A, Ala), arginine (R, Arg), asparagine (N, Asn), aspartic acid (D, Asp), cysteine (C, Cys), glutamate (G, GIu), glutamine (Q, GIn), glycine (G, GIy), histidine (H, His), isoleucine (I, lie), leucine (L, Leu), lysine (K, Lys), methionine (M, Met), phenylalanine (F, Phe), proline (P, Pro), serine (S, Ser), threonine (T, Thr), tryptophan (W, Trp),
  • genes encoding orotidine-5'-monophosphate pyrophosphorlyase and/or orotidine-5'-monophosphate decarboxylase are especially preferred.
  • Orotidine-5'- monophosphate pyrophosphorlyase (PyrE) and orotidine-5'-monophosphate decarboxylase (PyrF) are necessary for the synthesis of uracil.
  • Both genes can be used in vectors for transformation of uracil-auxotrophs, e.g. cells which do not carry functional pyrE and/or pyrF genes, neither on their chromosome nor on their plasmids (26). Selection of cells which were transformed with a vector carrying either or both genes of pyrE and pyrF genes is performed using uracil-free mediums for cultivating cells.
  • any of the above described selectable marker genes may be used for selection in archaea.
  • selection in archaea means that only cells carrying a plasmid encoding the selectable marker genes will survive, have a growth advantage or are specifically recognized under the culturing conditions.
  • An origin of replication may also be used as a selectable marker gene enabling propagation of a plasmid vector.
  • reporter gene may be used as a selectable marker gene.
  • Preferred reporter genes are genes encoding ⁇ -galactosidase, luciferase, green fluorescent protein or peptides and proteins which preferably bind or interact with reporter molecules such as fluorescent dyes, as well as variants thereof.
  • a further characteristic feature of the plasmid vector according to the present invention is that the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the term "functional virus particle” refers to a virus particle which is capable of encapsulating or integrating a nucleic acid molecule in any possible way.
  • the plasmid vector does not contain any sequences which allow for formation of virus or virus-like particles.
  • the vector sequence DNA or RNA
  • the plasmid vector does not contain any sequences which could lead to formation of virus or virus-like particles, solely or in combination with helper components.
  • the plasmid-virus hybrid pSSVx is not able to form virus or virus-like particles on its own. However, in combination with its helper plasmid SSV2, pSSVx generates virus-like particles. Even more preferably, the plasmid vector of the present invention does not contain any (functional) structural genes of viruses and/or any genes encoding components necessary for packaging of nucleic acids (e.g. packaging signals which are capable of directing nucleic acid molecules into the envelope of a virion). Accordingly, if the plasmid vector of the invention contains one or more of the above genes, these genes are provided in dysfunctional form (e.g. mutated). Most preferably, the plasmid vector neither comprises the above genes in their natural forms, i.e. without mutations, nor any mutated forms (including deletions, insertions or substitutions) of those genes.
  • the vector does not contain any structural genes of viruses and/or any genes encoding components necessary for packaging of nucleic acids, originally or after elimination of those genes. More preferably, the genes are eliminated when constructing the vector according to the invention.
  • the vector system has more capacity for cloning of genes of interest.
  • the plasmid vector of the present invention exclusively comprises, if any, viral genes/sequences selected from the group consisting of viral integrases and viral origins of replication.
  • the present invention relates to an archaeal plasmid vector system comprising one or more selectable marker gene(s) for selection in archaea and an archaeal origin of replication, wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the specific advantage of this vector system is based on the archaeal origin of replication which enables the vector to be replicated within an archaeal cell without being integrated into an archaeal chromosome.
  • This vector can easily be replicated in a cell and removed by standard biotechnological method.
  • the vector therefore, is especially usable for preparation of high (e.g. industrial) amounts of plasmids for further use in archaea.
  • oil of replication or "replication origin” refers to a DNA region that is essential for starting its replication.
  • archaeal origin of replication refers to a DNA region which is essential for starting its replication in archaea.
  • any replication origin which can start a replication in archaea may be used in connection with the present invention.
  • archaeal species are known to incorporate one to three origins of replication in their chromosomes (or/ ' Q. Further, archaea also contain a variety of plasmids which carry origins of replication. For example, the crenarchaea Sulfolobus incorporates several plasmids. In detail, these are the two types of plasmids: 1 ) rather small cryptic plasmids with genome size of 5-14 kb and 2) a family of conjugative plasmids with genomes larger than 25 kb (28;47).
  • crenarchaeal plasmids of type 1 are plasmids of the pRN family such as pRN1 , pRN2, pSSVx, pDL10, pHEN7, pXQ1 and pST1 ; plT3, pTAU4, pORAl and pTIK4 and variants thereof.
  • Plasmids of the plNG family e.g. plNG to plNG4 and plNG6
  • pTC pNOB ⁇
  • pKEF9, pHVE14 and pARN4 are examples of conjugative (type 2) plasmids.
  • archaea harbor various classes of viruses such as Siphoviridae, Myoviridae, Salterprovirus, SH1 , Rudiviridae, Lipothrixviridae, Guttaviridae, Globuloviridae, Ampullaviridae, Bicaudaviridae, Fuselloviridae (e.g. SSV1 , SSV2, SSV-K1 and SSV-RH) and STIV.
  • viruses such as Siphoviridae, Myoviridae, Salterprovirus, SH1 , Rudiviridae, Lipothrixviridae, Guttaviridae, Globuloviridae, Ampullaviridae, Bicaudaviridae, Fuselloviridae (e.g. SSV1 , SSV2, SSV-K1 and SSV-RH) and STIV.
  • the origin of replication may be an archaeal chromosomal, archaeal viral or archaeal plasmidal replication origin.
  • the origin of replication is an archaeal chromosomal or archaeal plasmidal replication origin.
  • the archaeal chromosomal origin of replication may be a chromosomal crenarchaeal origin of replication, preferably an origin of replication of a member of the class Thermoprotei, more preferably of a member of the order Sulfolobales, even more preferably of a member of the family Sulfolobaceae and most preferably of a member of the genus Sulfolobus.
  • a member of the genus Sulfolobus is selected from the group consisting of Sulfolobus acidocaldarius, Sulfolobus islandicus, Sulfolobus metallicus, Sulfolobus neozealandicus, Sulfolobus shibatae, Sulfolobus solfataricus, Sulfolobus tengchongensis, Sulfolobus thuringiensis, Sulfolobus tokodaii, Sulfolobus yangmingensis and other cultured Sulfolobus sp..
  • the archaeal chromosomal origin of replication may also be a chromosomal euryarchaeal origin of replication, preferably an origin of replication of a thermophilic euryarchaeota, more preferably of Thermococci or Thermoplasmata.
  • the archaeal plasmidal origin of replication may be selected from replication origins of any known archaeal plasmid, preferably from replication origins of the pRN plasmid family, or from replication origins of the plasmids of type 1 ) as defined above, more preferably from replication origins of the pRN plasmids, even more preferably from replication origins of the pRN1 plasmids and most preferably from replication origins of the pRN1 plasmid from Sulfolobus islandicus.
  • the present invention relates to an archaeal plasmid vector system comprising one or more selectable marker gene(s) for selection in archaea and a homology region to an archaeal chromosome, wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the specific characteristic of this vector system is that the homology region to an archaeal chromosome allows for stable integration of the plasmid vector into an archaeal chromosome which leads to replication of the vector in an integrated form. Integration of vectors into the chromosome ensures stable localization and replication of the vectors in the cell.
  • the inventive vector may encode a specific protein which has to be replicated over many cell division cycles. Integration of the vector into the chromosome ensures the coding sequence to be propagated to subsequent cell generations, even without selection for said vector. These cells can be grown in rich and optimized media allowing for rapid cellular growth.
  • the term "homology region to an archaeal chromosome” relates to a region on the piasmid vector which shows homology to a region on the chromosome of an archaeon.
  • the archaeon is preferably a crenarchaeon, preferably selected from a member of the class Thermoprotei, more preferably a member of the order Sulfolobales, even more preferably a member of the family Sulfolobaceae and most preferably a member of the genus Sulfolobus.
  • the member of the genus Sulfolobus is selected from the group consisting of Sulfolobus acidocaldarius, Sulfolobus islandicus, Sulfolobus metallicus, Sulfolobus neozealandicus, Sulfolobus shibatae, Sulfolobus solfataricus, Sulfolobus tengchongensis, Sulfolobus thuringiensis, Sulfolobus tokodaii, Sulfolobus yangmingensis and other cultured Sulfolobus sp..
  • the archaeon is an euryarchaeon, more preferably a thermophilic euryarchaeota, most preferably Thermococci or Thermoplasmata.
  • the homology region may comprise a length of 10-50,000 bp, preferably 30-10,000 bp, more preferably 50-5,000 bp and most preferably 100-1 ,000 bp.
  • the region on the plasmid vector may differ from the chromosomal sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted nucleic acid(s), and show a sequence homology of at least 60%, preferably at least 80%, more preferably at least 85%, equally more preferably at least 90%, even more preferably at least 95% and most preferably at least 97%, to the chromosomal sequence.
  • the sequences can be aligned in order to be subsequently compared to one another. Therefore, gaps can be inserted into one sequence at that positions where the sequence lacks a homolog at a sequence which is compared thereto. If a position in the first sequence is occupied by the same compound as is the case at a position in the second sequence, the two sequences are identical at this position.
  • the percentage to which two sequences are identical is a function of the number of identical positions divided by the total number of positions.
  • the percentage to which two sequences are identical can be determined using a mathematical algorithm.
  • a preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm as described by Karlin et a/. (45) and Altschul et a/. (46). Such an algorithm is integrated in the BLAST program. Sequences which are identical or similar to the sequences of the present invention to a certain extent can be identified by this program.
  • sequence homology encompasses sequence identity and sequence similarity.
  • sequences having a sequence homology are either identical or similar to each other to a certain extent, as defined above.
  • the homology region on the vector enables integration of the vector into the archaeal chromosome.
  • the vector may be integrated in a stable way and may or may not be removed again from the chromosome. If the vector is removed again, the excision sites may be the same or different compared to the sites for integration of the vector. Thus, the vector may be excised completely or only partially. Further, additional sections of the chromosome may be removed together with the (full length or partial) vector.
  • the integration process may occur via recombination of homologous sequences on the vector and chromosome. Preferably, the integration takes place via crossover, more preferably via single crossover.
  • the present invention relates to an archaeal plasmid vector system comprising one or more selectable marker gene(s) for selection in archaea and a gene coding for a molecule conferring the ability to integrate the vector into an archaeal chromosome, wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the vector system of the third embodiment also allows for replication of the vector, which is integrated within the archael chromosome sequence. Integration of plasmids into the chromosomes is advantageous because those plasmids are more difficult to be eliminated by the cell when cultivating the cell, e.g. in the absence of a compound for selection. Thus, cells carrying the plasmid within their chromosome can, for example, easily be cultivated without compounds for selection, usually leading to faster cell growth.
  • molecule conferring the ability to integrate the vector into an archaeal chromosome refers to a molecule which is capable of catalyzing the integration of
  • the molecule is an integrase or a transposase.
  • any integrase and transposase molecule is comprised by the present invention as long as it catalyses the integration of the vector into the chromosomal DNA of an archaeal cell
  • lntegrases are enzymatic proteins which are naturally present in retroviruses or retrotransposons and which catalyze integration of the nucleic acid thereof into the host chromosome.
  • the integrase molecule is an integrase from an archaeal virus selected from Fuselloviridae, even more preferably from the viruses SSV1 , SSV2, SSV-K1 and SSV-RH and related viruses.
  • the integrase molecule is an integrase from the conjugative plasmids, preferably of the plNG family (e.g. plNG1 to plNG4 and plNG6), pTC, pNOB8, pKEF9, pHVE14 and pARN4 plasmids. lntegrases from other archaeal viruses are also preferred.
  • the attachment site for integration may be located within the coding sequence of the integrase gene on the vector or outside in the remaining nucleotide sequences on the vector.
  • the attachment site is located inside the coding sequence, whereby a re-excision of the gene form the chromosome can be prevented leading to stable integration.
  • Transposases are enzymes which cut out DNA from one location and insert this DNA at another location.
  • the transposase molecule is an archaeal transposase.
  • the transposase molecule can be a transposase from a crenarchaeaota, preferably from a member of the class Thermoprotei, more preferably from a member of the order Sulfolobales, even more preferably from a member of the family Sulfolobaceae and most preferably from a member of the genus Sulfolobus.
  • the transposase molecule is a transposase from the same taxonomic order as the host cell.
  • the transposase molecule is sequence- specific for the chromosomal sequence enabling the construction of cells with defined integration sites.
  • the present invention relates to an archaeal plasmid vector system comprising one or more selectable marker gene(s) for selection in archaea and gene orf904, which is typically selected from one of the orf904 sequences occurring in the naturally available pRN plasmids, wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the inventive vector comprises the gene or/904 and the orf56 ox a functional derivative thereof of plasmid pRN, e.g. plasmid pRN1.
  • the multifunctional protein orf904 appears to be the essential open reading frame for plasmid replication of the pRN1 plasmid and is shown to form the replication initiation site and function. The inventors discovered the importance of orf904 for construction of self replicating vector systems due to the multifunctionality of that gene and the proteins encoded thereby. A detailed description of orf904 is given below.
  • gene orf904 or “functional derivatives” of “gene orf904" refers to the open reading frame sequence orf904 in the pRN plasmid family of Sulfolobus, preferably the pRN plasmid of Sulfolobus, more preferably the pRN1 plasmid of Sulfolobus and most preferably the pRN1 plasmid of Sulfolobus islandicus, e.g.
  • gene orf904 derivative or "orf56 derivative” also encompasses gene sequences which differ in one or more mutation(s), such as one or more substituted, inserted and/or deleted nucleic acid(s), and show, e.g. over a stretch of about 300 amino acids or over the entire sequence, a sequence homology of at least 60%, preferably at least 70%, more preferably at least 80%, equally more preferably at least 85%, even more preferably at least 90% and most preferably at least 97%, to the gene orf904 of the pRN1 plasmid of Sulfolobus, e.g. to SEQ ID No. 2.
  • the derivatives of "gene orf904" are functional.
  • inventive vectors comprising a native "gene ori904" , e.g. SEQ ID No. 2, may easily be compared with identical vectors comprising a derivative of SEQ ID No. 2. Functionality is given, if at least 50% of the biological activity of the vector containing the native sequence is realized by the vector containing the derivative orf904 sequence. Methods for determination of sequence homology are described above.
  • the vector may comprise the selectable marker gene(s) and any one of the components (a), (b), (c) and/or (d).
  • the vector may comprise the selectable marker gene(s) and component (a), the selectable marker gene(s) and component (b), the selectable marker gene(s) and component (c) or the selectable marker gene(s) and component (d), according to embodiments 1 to 4 disclosed above.
  • the vector may also comprise two, three or four of components (a), (b), (c) and (d) in combination with the selectable marker gene(s) as defined herein.
  • the vector may comprise the selectable marker gene(s) and components (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (C); (a), (b), and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c) and (d) together, in more specific embodiments according to the present invention.
  • the archaeal plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea, an archaeal origin of replication (a) and a homology region to an archaeal chromosome (b), wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the archaeal plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea, an archaeal origin of replication (a) and a gene coding for a molecule conferring the ability to integrate the vector into an archaeal chromosome (c), wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the archaeal plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea, an archaeal origin of replication (a), a homology region to an archaeal chromosome (b) and a gene coding for a molecule conferring the ability to integrate the vector into an archaeal chromosome (c), wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the plasmid vector systems according to the preferred embodiments described above are especially useful for construction of plasmid vector systems which can be replicated, both, as isolated plasmids and integrated into the archaeal chromosome. Thus, they combine advantages of both replication alternatives which are described above.
  • the vectors may, e.g., first be replicated in a fast manner in its integrated form in the absence of a substance for selection, and can be used subsequently in another cell as isolated plasmids in the presence of a substance for selection. Also the reverse order of usage is possible.
  • the vectors may initially be replicated as plasmids and then may be stably integrated into the archaeal chromosome.
  • the archaeal plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea, a homology region to an archaeal chromosome (b) and gene orf904 (d); wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the archaeal plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea, a gene coding for a molecule conferring the ability to integrate the vector into an archaeal chromosome (c) and gene orf904 (d); wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the archaeal plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea, a homology region to an archaeal chromosome (b), a gene coding for a molecule conferring the ability to integrate the vector into an archaeal chromosome (c) and gene orf904 (d); wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the above mentioned preferred embodiments of the present invention all relate to plasmid vector systems comprising open reading frame or/904 and, optionally, orf56 or functional derivatives thereof.
  • gene orf904 is especially advantageous for construction of vectors without complex interplay of various components, since this gene encodes a multifunctional protein which incorporates all functions required for replication. Combination of that gene with any of components (b) and/or (c) necessary for integration of the plasmid into the archaeal chromosome, leads to vector systems combining integrative and non- integrative functions, as described herein.
  • the plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea selected from essential gene(s) for a host organism and gene(s) conferring a growth advantage, and an archaeal origin of replication, wherein the archaeal origin of replication is an archaeal plasmidal origin of replication; wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea selected from the genes coding for orotidine-5'-monophosphate pyrophosphorlyase and/or orotidine-5'- monophosphate decarboxylase, or HMG-CoA reductase, and an archaeal origin of replication, wherein the archaeal origin of replication is an origin of replication of the pRN1 plasmid; wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea selected from essential gene(s) for a host organism and gene(s) conferring a growth advantage, and a homology region to an archaeal chromosome; wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea selected from the genes coding for orotidine-5'-monophosphate pyrophosphorlyase and/or orotidine-5'- monophosphate decarboxylase, or HMC-CoA reductase, and a homology region to an archaeal chromosome, wherein the homology region to an archaeal chromosome is 10 to 50,000 bp, preferably 30 to 10,000 bp, more preferably 50 to 5,000 bp and most preferably 100 to 1 ,000 bp in length; wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the plasmid vector system comprises one or more selectable marker gene(s) for selection in archaea selected from the genes coding for orotidine-5'-monophosphate pyrophosphorlyase and/or orotidine-5'- monophosphate decarboxylase, or HMG-CoA reductase, and a homology region to an archaeal chromosome, wherein the homology region to an archaeal chromosome is a homology region of a crenarchaeal chromosome, preferably a chromosome of a member of the class Thermoprotei, more preferably of the order Sulfolobales, even more preferably of the family Sulfolobaceae and most preferably of the genus Sulfolobus; wherein the genes of the plasmid vector do not allow an assembly of a functional virus particle.
  • the vector system of embodiments as described above comprise an £ coli origin of replication and one or more selectable marker gene(s) for selection in £ coli, as described below, as component(s) of further embodiments.
  • the present invention also relates to a plasmid vector system comprising: I) gene orf904 and, optionally, orf56, or functional derivatives thereof as defined above and II) one or more selectable marker gene(s); wherein the vector is non-integrative.
  • plasmid vector system comprising: I) gene orf904 and, optionally, orf56, or functional derivatives thereof as defined above and II) one or more selectable marker gene(s); wherein the vector is non-integrative.
  • the vector system comprises one or more selectable marker gene(s) which is/are essential gene(s) or gene(s) conferring a growth advantage for the cell.
  • the vector system comprises a selectable marker gene(s) selected from the genes coding for orotidine-5'-monophosphate pyrophosphorlyase and/or orotidine-5'-monophosphae decarboxylase, or HMG-CoA reductase
  • non-integrative refers to a feature of the plasm id vector wherein the vector is not integrated into a chromosomal DNA of a cell.
  • a non-integrative vector is characterized by its capability to replicate in a cell without being integrated into the cell's chromosome.
  • the vector preferably lacks any of the components (b) and/or (c), or any other component which would enable the vector to be integrated into the archaeal chromosome.
  • the inventive vector systems may be applied in a variety of biochemical and biotechnological fields.
  • the vector systems can be used in connection with genetic studies, especially on archaeal species. With respect to such studies, the vector is integrated into the archaeal chromosome or replicated episomally. The integration can be carried out either via integrases, transposases and/or homology regions as described above.
  • the vector system can be also applied for expression of genes, e.g., coding for proteins of interest (i.e. as expression vectors) or as shuttle vectors for transfer of genes between different organisms.
  • the vector of the invention may be used as a cloning vector.
  • a cloning vector is in general a vector which is used for insertion of foreign DNA fragments.
  • the cloning vector is further characterized by a multiple cloning site into which the DNA fragment is inserted after treating the fragment and the vector with the same restriction enzyme.
  • the vector serves for cloning of genes into archaea, preferably crenarchaeota, more preferably Sulfolobales, even more preferably Sulfolobaceae and most preferably Sulfolobus.
  • the vector serves for cloning into archaea, preferably crenarchaeota, more preferably Sulfolobales, even more preferably Sulfolobaceae and most preferably Sulfolobus.
  • the vector serves for cloning into
  • the vector serves for cloning into euryarchaeota, more preferably thermophilic euryarchaeota, most preferably Thermococci and/or Thermoplasmata.
  • the vector of the invention may be used as an expression vector.
  • An expression vector serves to express a gene of interest. It additionally includes an expression cassette comprising a promoter, a ribosome biding site, a terminator and the gene which has to be expressed.
  • An especially useful promoter is the mal promoter (SEQ ID NO:3), which is disclosed herein as component of the inventive vector systems or, alternatively, also as such, namely as SEQ ID No: 3.
  • any vector not necessarily a vector of the invention, comprising SEQ ID No: 3.
  • the gene which has to be expressed is a gene of interest, e.g., encoding a protein or polypeptide of interest.
  • the substance (e.g. gene, protein or peptide) of interest may be naturally occurring (in any organism), may be modified, e.g. having a mutated sequence as compared to a naturally occurring substance, or may be artificial.
  • the substance of interest may also be modified by comprising a fusion such as a purification tag.
  • the expression vector serves for expression in archaea. More preferably, the expression vector serves for expression in crenarchaea, still more preferably Sulfolobales, even more preferably Sulfolobaceae and most preferably Sulfolobus.
  • the expression vector serves for expression in Sulfolobus selected from the group consisting from Sulfolobus acidocaldarius, Sulfolobus brierleyi, Sulfolobus hakonensis, Sulfolobus islandicus, Sulfolobus metallicus, Sulfolobus neozealandicus, Sulfolobus shibatae, Sulfolobus solfataricus, Sulfolobus tengchongensis, Sulfolobus thuringiensis, Sulfolobus tokodaii, Sulfolobus yangmingensis and Sulfolobus sp.
  • the expression vector serves for expression in euryarchaeota, more preferably in thermophilic euryarchaeota, most preferably in Thermococci and/or Thermoplasmata.
  • the vector of the invention may be used as a shuttle vector.
  • the term "shuttle vector" refers to a vector able to replicate in two different organisms.
  • a shuttle vector contains sequences which allow for replication and usually also selection in two organisms.
  • Shuttle vectors are used to transfer DNA cloned therein between different organisms.
  • a shuttle vector may be used in one organism for efficient replication and can be transformed into another organism for expression of genes of interest.
  • the shuttle vector is able to replicate in an archaeal and a bacterial or eukaryotic (such as yeast) organism. More preferably, the shuttle vector is able to replicate in an archaeal and a bacterial organism. Most preferably, the shuttle vector is able to replicate in an archaeal organism and in £ col/. Among archaeal organisms, crenarchaea are preferred, Sulfolobales are more preferred,
  • the shuttle vector is able to replicate in £ col/ and Sulfolobus acidocaldarius, Sulfolobus brierleyi, Sulfolobus hakonensis, Sulfolobus islandicus,
  • the shuttle vector is able to replicate in £ col/ and in crenarchonta, e.g. Sulfolobus, or eventually in euryarchaeota, e.g. in thermophilic euryarchaeota, e.g. in Thermococci and/or Thermoplasmata.
  • vector replication occurs extrachromosomally as a stable plasmid.
  • vector replication can either take place extrachromosomally or integrated into the chromosome.
  • the shuttle vector further comprises an E coli origin of replication.
  • E coli origin of replication refers to a DNA region which is essential for starting its replication in £ coli.
  • any origin of replication which is functional in £ coli can be used in accordance with the present invention, i.e. origins of other bacterial or non-bacterial species are also encompassed by the present invention.
  • an archaeal plasmid vector system comprising a non-£ coli origin of replication, in particular bacterial non-£ coli origin of replication, can also be constructed using the instructions provided herein.
  • a vector system can be initially constructed which incorporates a further origin of replication (e.g. an E coli replication origin). Then, the origin of replication can be removed from the vector again using e.g. cloning methods known in the art.
  • a further origin of replication e.g. an E coli replication origin
  • the inventive shuttle vector comprises one or more selectable marker gene(s) for selection in £ coli.
  • any of the selectable marker gene(s) as defined previously can be used in accordance with the present invention.
  • any selectable marker gene for selection in £ coli known in the art can be applied for selection.
  • the selectable marker gene for selection in £ coli is a gene conferring antibiotic resistance.
  • a gene encoding a reporter molecule is used as a selectable marker gene.
  • a typical shuttle vector of the invention comprises an origin of replication of another microorganism, e.g. E. coli (e.g. CoI E1 Ori) and downstream thereof a gene of interest, which is operably linked to a promoter (e.g. a mal promoter). Downstream thereof is located either, optionally, the orf56 sequence of a pRN plasmid, or, rather an orf904 selected from one of the pRN plasmids (or functional derivatives thereof). Downstream thereof at least one selection marker is positioned. All these elements are preferably positioned in the same 5'-3' direction. Upstream of the selection marker (but downstream of orf 904) additional elements may be positioned depending on the specific requirements.
  • E. coli e.g. CoI E1 Ori
  • a gene of interest which is operably linked to a promoter (e.g. a mal promoter).
  • Downstream thereof is located either, optionally, the orf56 sequence of a pRN plasmid, or
  • selctable markers e.g. pyrF or pyrE
  • inventive vector which does not have shuttle properties, may be based on an essentially similar construction mode and comprises the components mentioned above, but does not contain a functional second origin of replication.
  • the vector system of any of the inventive embodiments described above comprises an additional expression cassette for a reporter molecule.
  • reporter molecule refers to a protein or peptide which visibly indicates (“reports") its presence in a cell or in a certain compartment of a cell. Reporter molecules are well known in the art. A reporter molecule can report its presence with or without interaction with other substances. For example, the reporter molecule can undergo a reaction with another substance which leads to a change of color. Further, the reporter molecule can interact with a colorful or fluorescent dye or a radioactive substance for indication. According to the invention, a reporter molecule can also be used for detection of successful transformation of a cell.
  • the reporter molecule is preferably selected from the group consisting of ⁇ - galactosidase, luciferase, green fluorescent protein and variants thereof.
  • genes of several kb in length can be cloned into the inventive vectors. When applied for expression of genes of interest, the use of the inventive vectors may lead to production of proteins of high molecular weights.
  • the present invention also relates to a host cell transformed with the vector of the invention.
  • the host cell is an archaeal cell as the vector of the invention is suitable to transform archaea.
  • An archaeal cell may be a crenarchaeal cell, preferably a cell of a member of the class Thermoprotei, more preferably of a member of the order Sulfolobales, even more preferably of a member of the family Sulfolobaceae and most preferably of a member of the genus Sulfolobus.
  • the member of the genus Sulfolobus is selected from the group consisting of Sulfolobus acidocaldarius, Sulfolobus islandicus, Sulfolobus metallicus, Sulfolobus neozealandicus, Sulfolobus shibatae, Sulfolobus solfataricus, Sulfolobus tengchongensis, Sulfolobus thuringiensis, Sulfolobus tokodaii, Sulfolobus yangmingensis and other cultured Sulfolobus s ⁇ .
  • the archaeal cell is an euryarchaeal cell, preferably a cell of a thermophilic euryarchaeota, more preferably of Thermococci and/or Thermoplasmata.
  • the host cell is a bacterial cell, preferably an £ col/ cell, or a eukaryotic cell, preferably a yeast cell.
  • Preferred £ col/ strains are BL21 , BL21 (DE3) or BL21 (DE3) pLysS and BL21 , HB101 , JM109, XL-I blue, DH10B, DH12S, DH5alpha, DB3.1 , Stb14, TOPl O and derivatives thereof.
  • the host cell transfected with the vector according to the invention produces a protein of interest.
  • the vectors of the present invention can be introduced into the host cell using the same techniques as those used for introduction of common plasmid vectors. Examples of such techniques are electroporation, calcium phosphate-DNA transfection, liposome, and immunogene technique and the like.
  • the present invention also relates to a method of producing a protein of interest comprising culturing the host cell of the present invention under suitable conditions and isolating said polypeptide from the cells or the cell culture supernatant.
  • the present invention is also directed to the use of the vector system for production of a protein of interest.
  • the present invention relates to a kit comprising a vector system or a host cell of the present invention in one or more container(s).
  • the invention is based on the finding that vectors based on the sequence of Sulfolobus plasmids are stable and effective, especially suitable for use in Sulfolobus, e.g. for transformation of Sulfolobus.
  • vectors based on the pRN1 plasmid, a plasmid from the pRN family of Sulfolobus islandicus were found to be advantageous for various applications in the field of industrial and non- industrial biotechnology.
  • plasmids of the Sulfolobus pRN family share three conserved open reading frames (ORFs).
  • ORFs conserved open reading frames
  • plasmid pRN1 those three ORFs are named orf56, orfSO, and orf904.
  • ORFs conserved open reading frames
  • no pRN plasmid has been discovered outside the crenarchaeal phylum.
  • plasmids of the pRN family are not related to any other known eubacterial or archaeal plasmid.
  • the first pRN plasmid which was completely sequenced was plasmid pRNl isolated from the Sulfolobus islandicus strain RENHl (41). A more detailed description of the pRN1 plasmid is given below.
  • inventive vector although derived from pRN, does typically not comprise other ORFs, which are typical for the pRN plasmids other than orf904 and, optionally orf56. Accordingly, the inventive vector does typically not comprise at least one of orf72, orf ⁇ Oa, orf ⁇ Ob and orf ⁇ O, more preferably does not comprise at least two and even more preferably at least three of the afore-mentioned ORFs.
  • the pRN1 plasmid is notable for its relatively small size (5.4 kb), copy numbers ranging from 2 to 20 in mid-log phase, and the three genes (coding for orf56, orf ⁇ O, and orf904) which are also are conserved in other Sulfolobus plasmids.
  • the inventors have analyzed the three conserved proteins (representing two DNA- binding proteins and the replication protein) and the transcriptional activity of the plasmid (23-25). Those studies revealed that orf56 binds upstream of its own gene and down-regulates the expression of the cotranscript orf56/orf904. It therefore appears that orf56 could be involved in regulating the copy number of the plasmid.
  • Orf904 encodes the third conserved protein, a multifunctional 1 10 kDa replication enzyme that appears to play a central role in replication.
  • the gene orf904 is by far the largest ORF of the pRN plasmid family.
  • the corresponding genes occupy about a third to a half of the plasmids and encode proteins of about 900-1 ,000 amino acids.
  • Within the C-terminal part of the proteins a helicase domain of superfamily 3 can be identified.
  • the best-studied protein containing this domain is the bacteriophage P4 ⁇ protein, which is a multifunctional replication protein harboring a helicase domain in its C-terminal part.
  • the N-terminal 40 amino acids are not found in corresponding proteins of other plasmids.
  • the inventors developed a variety of stable vectors based on the sequence of pRN1. Further, the inventors also found out that - for a specific embodiment - orf904 and the replication operon orf56/orf904 are essential for construction of stable plasmids. However, neither orf80 nor the remaining open reading frames, orf72, orf90a or orf ⁇ Ob, are essential for plasmid replication.
  • the inventors developed a series of vectors which are also based on the pRNl sequence. In contrast to the vectors described above, however, a different system was used as a selectable marker gene - HMG-CoA reductase instead of pyrEF - (Fig. 11 -13).
  • the inventors realized that establishing a suitable and reliable selection is very important for the generation of stably replicating plasmid vectors, in particular derived from the Sulfolobus plasmid pRN1. For example, problems related to reversion of the uracil- auxotrophy were avoided by the use of a pyrE deletion mutant of 5. acidocaldarius. These deletion mutants could be easily selected for transformation with a plasmid carrying genes coding for orotidine-5'-monophosphate pyrophosphorlyase. In addition to the PyrEF selection, selection using genes encoding HMG-CoA reductase turned out to be advantageous for selection in archaea.
  • the inventors developed vectors ("pD-suicide") comprising a homology region to an archaeal chromosome (Fig. 7). As shown in Fig. 8 and 9, the vectors can be stably integrated into an archaeal chromosome via single crossover. Further, the vectors may be removed from the chromosome via recombination (Fig. 10).
  • the Figures indicate:
  • Fig. 1 shows physical maps of vectors.
  • A positions of the insertion sites of the £ co// replicon and the pyrEF marker genes for shuttle vectors pA-pN are indicated.
  • B shows conserved features of pRNl : crosshatched arrows: conserved open reading frames, grey area: conserved on the nucleotide level within the pRN family plasmids, black arrows: transcripts.
  • C is a vector map of the shuttle construct pC.
  • Fig. 2 is directed to the analysis of S. acidocaldarius transformants.
  • A shows a Southern blot of Hin ⁇ W digested genomic DNA preparations (-c: untransformed MR31 , A+c: positive control: plasmid pA from £ coli, G+c: separate positive control (plasmid pG from £ coli) because of the additional Hin ⁇ W restriction site present in pG).
  • B indicates a restriction analysis (Sad) of retransformation experiments for all shuttle constructs (o: original plasmid prepared from £ coli, r: retransformed plasmid).
  • C indicates a restriction analysis (Sad) of shuttle vectors isolated directly from S.
  • D shows a Southern blot like in A for controls and cultures transformed with pC and pE after 200 generations of consecutive cultivation.
  • E shows colony hybridizations for cultures from D with pRN1 specific probes.
  • FIG. 3 depicts growth of transformants.
  • A shows growth curves for MR31 transformed with pA to pN.
  • B shows growth curves for the recipient strain MR31 without addition of uracil (U), with the addition of uracil and transformed with pC and pE.
  • Fig. 4 shows the plasmid copy number per cell (triangles) for MR31 transformed with pC (left panel) and pE (right panel) and corresponding growth curves (line).
  • Fig. 5 indicates the replication of pj lacS in 5. acidocaldarius.
  • A is a Southern blot (Sad) of plasmid p ⁇ /acS from £ coli, MR31 transformed with p ⁇ /acS and untransformed MR31 (-c).
  • B shows the retransformation of pj lacS (Sad), +c: p]/acS from £ coli, 1 ,2,3: retransformants.
  • C indicates an X-gal test with untransformed MR31 and MR31 transformed with p)lacS( ⁇ O min at 75°C).
  • D depicts growth curves for MR31 transformed with pj (control) and with p)lacS.
  • E illustrates a reporter gene experiment showing copy numbers of pjlacS per cell and the corresponding ⁇ - galactosidase activities.
  • Fig. 6 indicates the replication of pjlacS ln S. solfataricus.
  • A is a Southern blot (Sad) of untransformed PBL2025 (-c), PBL2025 transformed with pj/ ⁇ 3c5and £ coli (+c).
  • B depicts growth curves of untransformed PBL2025 in 0,4% lactose medium, PBL2025 transformed with p)lacS in 0.4% lactose medium and in 0.2% tryptone medium.
  • C indicates the copy numper per cell for p)lacS in PBL2025 in 0.4% lactose medium.
  • D shows the retransformation of pj/ ⁇ 3c5 back into £ coli.
  • E indicates the plating of PBL2025 transformed with p)lacS after 100 generations of consecutive cultivation on non-selective tryptone and selective lactose plates stained with X-gal.
  • Fig. 7 shows the structure of the integrative vectors pD-Suicide. Integration into the chromosome of 5. acidocaldarius MR31 could be demonstrated for two distinct target sequences.
  • Fig. 8 demonstrates the site-specific integration of the vector pD-Suicide-ori2 into the genome of S. acidocaldarius MR31.
  • A is a scheme for the location of integration sites.
  • B is a Southern blot showing site specific single crossover integration of vector pD-Suicide-ori2: bands of the linearized non-integrated vector (5424 kb) and the integrated form of the vector (7284 kb) after digestion with Pstl and hybridization to a pyrE specific probe.
  • Fig. 9 demonstrates a "pop-in" method for selective modification of the Sulfolobus genome.
  • A is a scheme of a single crossover integration ("pop-in").
  • Fig. 10 demonstrates a "pop-in" method for selective modification of the Sulfolobus genome.
  • a and B are schemes for two possible removals of the selection marker pyrE via a second recombination from the genome (A: original sequence are restored; B: target genes knocked out: (successful pop-out))
  • Fig. 11 illustrates the structure of the vector pK-HMGCoA-Reductase. After transformation of Sulfolobus acidocaldarius, transformants could be obtained by selection with the inhibitor simvastatin.
  • Fig. 12 demonstrates a positive selection using simvastatin.
  • A demonstrates the selection: plating of cells after 3 rounds of preselection. Cells were transformed with pK-HMGCoA-Reductase. A control without addition of vector was included. After cultivation in medium containing 10 ⁇ g/mL simvastatin 10 6 dilutions of the control and the transformed culture were plated on simvastatin containing plates. No growth was observed for the untransformed control, whereas similar colony numbers were obtained with the transformed culture for different simvastatin concentrations. These clones were tested for plasmid presence by PCR (B) and retransformation (C). Clone #9 is correct.
  • B shows the detection of successive transformation of MR31 by the Hmg expression vector pK-HMGCoA-Reductase.
  • C demonstrates the retransformation of successful vector pK-HMGCoA-Reductase into £ coli.
  • Fig. 13 shows reduced growth inhibition of cells transformed with pK-HMGCoA- Reductase (pKtfhmg). Growth curves for transformed and untransformed MR31 in the presence of 5 and 10 ⁇ g/mL simvastatin are shown.
  • Fig. 14 shows the gene sequence of the pRN1 plasm id of Sulfolobus islandicus.
  • Fig. 15 shows the orf904 gene sequence from the pRN1 plasmid of Sulfolobus islandicus.
  • Fig. 16 shows the vector map of the Sulfolobus-E.coli shuttle vector for overexpression pCmalX.
  • the gene of interest is cloned under control of the mal promoter (here the gene of interest is lacS).
  • Fig. 17 depicts the sequence of the mal promoter region starting with the restriction site Sacll and ending with the restriction site Ncol used for cloning and comprising 262 bp of the promoter sequence of the gene saci_1 165.
  • Fig. 18 shows the effects of different sugars on the mal promoter.
  • A, B and C demonstrate the induction of the mal promoter with maltose and dextrin (0.2% and 0.4%, respectively).
  • D demonstrates the influence of tryptone, glucose, dextrin, xylose, sucrose and maltose and combinations thereof on the induction of the mal promoter.
  • E shows the expression of the C-terminally his-tagged single stranded binding protein from S. sol/a ta ricus in 5. acidocaldarius MR31 and purification over a talon affinity column (M: marker, CE: crude extract, FT: flowthrough, W: wash, EI - E6: elution fractions 1 -6).
  • Fig. 19 depicts the map of the knockout plasmid d2updownXpyrEF.
  • Fig. 20 is a schematic representation of the tryptophane synthesis operon from 5. acidocaldari ⁇ s.
  • the targeted gene saci_1422 (trpA) is shown with the homologous regions used in the knockout vector d2kosaci1422.
  • Fig. 21 is a schematic overview of steps in the recombination process of the knockout vector d2kosaci1422 into the genome.
  • the knockout plasmid is integrated by one double crossover or two subsequent single crossover events site specifically into the chromosome replacing the region of interest (here: innermost part of the trpA gene).
  • Fig. 22 demonstrates the correct integration of the knockout plasmid into the genome.
  • A shows the correct integration of the knockout plasmid into the genome by PCR (Lane wt: wild type genomic DNA as template, lanes labelled 1 -4 genomic DNA from 4 clonally purified 5. acidocaldarius knockout strains).
  • B and C demonstrates the correct integration of the knockout plasmid into the genome by a phenotypic test on solid medium (Knockout strains cannot grow on medium without tryptophane (B) whereas they can grow well on the same medium supplemented with 20 ⁇ g/mL of tryptophane (C)).
  • D depicts the correct integration of the knockout plasmid into the genome by a phenotypic test in liquid medium (Knockout strains cannot grow in liquid medium without tryptophane (open rectangles) whereas they can grow well on the same medium supplemented with 20 ⁇ g/mL of tryptophane (triangles) comparable to the wildtype (filled rectangles)).
  • Fig. 23 depicts the nucleotide sequence of the vector pCmalX whereby the gene of interest X is the lacS gene.
  • Fig. 24 shows the nucleotide sequence of the vector d2updownXpyrEF whereby the homologous regions of interest X are the flanking regions of the trpA gene from 5. acidocaldarius.
  • the following examples illustrate the invention.
  • shuttle vectors can be constructed from two plasmids that replicate in different hosts simply by fusing them at two points that preserve all the important functions of each plasmid.
  • ORFs or intergenic regions may be important for successful replication in Sulfolobus hosts. Therefore, transposition to generate pRN1 constructs interrupted at a number of different sites without regard to the location or its sequence context were used. From the initial transposition mixture, 13 distinct insertion points were chosen for further development, which included addition of the pyrEF genes of S. solfataricus as selectable marker (Fig. 1 , Table 1 ).
  • Table 1 Insertion sites of the transposon in constructs pA-pN.
  • replication operon orf56/orf904 was expected to be essential, only one construct interrupted within this region was chosen for analysis. The other constructs were chosen to have the interruption sites distributed as evenly as possible over the remaining part of pRN1.
  • the open reading frames orf ⁇ O, orf ⁇ Oa, orf72 and orf ⁇ Ob are also interrupted in at least one construct. It was already shown that orf ⁇ Oa, orf72 and orf ⁇ Ob are very unlikely to play a role in plasmid replication or maintenance in view of their very low levels of expression (36).
  • Acid- hydolyzed casein i.e., NZAmine AS (Sigma)
  • enzymatically hydrolyzed casein i.e., tryptone (BD Biosciences)
  • D-(+)-xylose was added at 0.2%
  • D-(+)-lactose in "lactose-only" medium at 0.4%.
  • PHI -16, R20, Sl RI , R1 , H1 , and MR31 20 ⁇ g ml/ 1 of uracil was added to the medium. Plates were solidified by addition of 0.6 % Gelrite (Sigma) and 10 mM CaCI 2 . Plates and shake flask cultures were incubated at 75°C.
  • Tn5-derived transposon TnPA21 (32) was amplified by PCR using a primer (5'- CTGTCTCTTATACACATCT) complementary to the mosaic end sequence, the terminal inverted repeat sequence found on both ends of the transposon.
  • Native pRN1 accession number NC 001771
  • REN1 H1 containing only the pRN1 plasmid (33) using the Nucleo Spin plasmid extraction Kit (Macherey Nagel). Plasmid preparations from approximately 100 mL of culture were combined and ethanol-precipitated to obtain 0.1 ⁇ g of pRN1 for the transposition reaction.
  • the transposition reaction was carried out in vitro using the EZ-Tn5 transposase (Epicentre) according to the instructions of the manufacturer. Transposon and plasmid were mixed at a molar ratio of 1 :1. The transposition reaction products were transformed into £ co// EC100 piY (Epicentre). The resulting transformants were screened for correctly inserted transposons by restriction digestion with Sad and NoA. 20% of the screened colonies (a total of 80 plasmids) showed two restriction bands with a combined length of 7.2 kb and were kept for further analysis.
  • the plasmid deita2pyrEF is a derivative of pBluescript with the /acZand f1 origin regions deleted. Specifically, pBluescriptSKII(+) was cut with Ssp ⁇ and Kpn ⁇ , religated, cut with Sad and Sap ⁇ and religated.
  • the pyrEF genes from S. solfataricus P2 were cloned into the SaA and PsA sites.
  • the transitional region from the delta2pyrEF part to the pRN1 part was sequenced to determine the exact insertion site and the direction of the transposon insertion and the direction of the cloning into the NoA site of delta2pyrEF.
  • construct pG by restriction analysis using H/nc ⁇ U an additional HindW site was found to be present and confirmed by sequencing.
  • the pRN1 part was not PCR-amplified, but stems from the native plasmid, we conclude that pRN1 had this mutation already when isolated, or that this mutation was introduced in £ col/ during propagation of pG. Except for this point mutation, pG corresponds to the expected sequence.
  • the plasmids pA - pN were electroporated into Sulfolobus strains representing different species. As the 5. solfataricus pyrEF genes provide the selectable marker, stable uracil auxotrophs were needed as recipient strains. Table 2 gives an overview of the SuI 'folobus pyrEF mutants tested as recipients for the various pRN1 constructs.
  • Table 2 Sulfolobus species and strains tested as recipient strains for the shuttle constructs pA-pN.
  • plasmids pC and pE were chosen to evaluate long- term stability under selective conditions. Cultures of pC and pE transformants were cultivated continuously for approximately 200 generations without uracil supplementation. Then retransformation experiments and Southern blots were repeated (Fig. 2D), with the same results.
  • the data of Fig. 3 indicate that direction of the insertion in a given region does not influence performance of the vector.
  • the vectors pF, pi and pG for example, have insertion sites within 15 nt of each other.
  • the pyrEF genes are oriented clockwise, in pi and pF counter clockwise, without detectable effects on plasmid stability or growth (Fig. 3).
  • the growth phenotype of transformed cells is comparable to that of the untransformed recipient strain when supplemented with uracil.
  • Genomic DNA was prepared from 1 mL of culture using the Chemagenic DNA Bacteria Kit (Chemagen, Baesweiler, Germany) according to the instructions of the manufacturer. After digestion with either HindW for constructs pA-pN or Sad for p]/acS restriction fragments were resolved in 1 % agarose gels, transferred to a Hybond N membrane (Amersham) by capillary transfer, fixed by UV irradiation for 5 min on a UV transilluminator and hybridized to digoxigenin-labeled probes complementary to the pyrF gene (position 9-320 from the start of the pyrEgene from S.
  • solfataricus P2 solfataricus P2
  • pRN1 position 4892-5048 in pRN1
  • lacS position 1 124-1438 from the start of the / ⁇ ?c5gene from S. islandicus REN1 H1
  • pRN1 for p]/acS. Labeling and detection was done using the PCR DIG Probe Synthesis Kit and the Digoxigenin Labeling and Detection Kit (Roche).
  • Colony hybridization Colonies from plates were transferred to Hybond N membranes and subsequently incubated for 10 min on a filter paper soaked with 0.5 M NaOH, 1.5 M NaCI then for 10 min on a filter paper soaked with 1 M Tris-HCI (pH 7,5), 1.5 M NaCI, then for 5 min on a filter paper soaked with 10x SSC. Membranes were cross-linked for 5 min on the 10x SSC filter paper using a transilluminator. Hybridization and detection were done as described for Southern blots with pRN1 -specific probes. Plasmid copy number determination
  • Copy numbers of the different shuttle constructs were determined as already described (36) by qPCR and cell number determination through plating, respectively.
  • Fig. 3C shows the retention of 13 constructs over approximately 10 generations, corresponding to about 1000-fold numerical expansions of the host cell populations. Most constructs showed measurable loss under these conditions, resulting in about 10% Pyr + cells in the cultures. In a few cases, however, plasmid retention was much lower. The most severe instability was seen in construct pH, in which the orf ⁇ O gene is interrupted. This result provided evidence that the small DNA-binding protein encoded by orf ⁇ O has an important role in the stable replication of pRN1 and related plasmids. Intermediate instability was observed for construct p)/acS (described below). It was suspected that this construct with the very strong tfSS ⁇ promoter is a burden for the cell. Both p]lacS and pH yielded small or heterogeneous colonies when streaked on selective plates, consistent with the observed instability under non-selective conditions.
  • ⁇ -D-glycosidase (37;38) encoded by the S. solfataricus Ia cS gene was assayed as ⁇ -galactosidase activity.
  • Crude extracts were prepared by a freeze-thaw method (20) in which cells were resuspended in 50 mM Na-phosphate buffer, pH 7, and subjected to 5 freeze-thaw cycles (-196°C/+50°C). After centrifugation for 30 min at 10000 rpm the supernatant was stored at -20 0 C, or assayed directly. All ⁇ -galactosidase assays were conducted in triplicate in a 75°C bench top shaker.
  • the reaction mixture consisted of 1 ⁇ L of crude extract (or water for blanks), 92 ⁇ L of 50 mM Na-phosphate buffer, pH 7 and the assay was started by addition of 7 ⁇ L of 12 mg mL "1 ortho-nitrophenyl- ⁇ -D-galactopyranoside (ONPG) solution. Incubation was continued for 5 min before the tubes were rapidly cooled on ice and 100 ⁇ L of 1 M Na 2 CO 3 solution was added to stop the reaction. Concentration of ONP was subsequently determined in a 96-well plate in a plate reader at 410 nm using a standard curve generated with ONP. Protein concentration of the crude extracts was determined by the method of Ehresmann (39).
  • a qualitative ⁇ -galactosidase assay was based on hydrolysis of 5-bromo-4-chloro-3- indolyl- ⁇ -D-galactoside (X-gal). For liquid cultures 200 ⁇ L of culture were mixed with 20 ⁇ L of substrate solution (20 mg/mL in dimethylformamide) and incubated at 75°C until color development was observed. To score colonies, plates were sprayed with the same X-gal solution and incubated at 75°C.
  • the primary obstacle to establishing stably replicating shuttle vectors derived from Sulfolobus plasmid pRNl is not preservation of critical plasmid functions or identification of a required host species, but creation of a suitably reliable selection.
  • point and transposon mutants of S. islandicus or 5. solfataricus that showed low reversion frequencies in small scale fluctuation tests (15*10 9 - ⁇ 6*10 9 reversions per cell division, unpublished results) displayed for unknown reasons higher reversion frequencies after electroporation with a shuttle construct. These problems could be avoided by the use of a pyrE deletion mutant of 5. acidocaldarius. In contrast to 5.
  • orf80 The other conserved open reading frame, orf80, also called plrA (plasmid regulatory), that is present on almost all sequenced genetic elements of Sulfolobus (42) is interrupted in pH. Interestingly, pH shows growth comparable to the other constructs and yields the same transformation efficiencies. Therefore orf80 seems not to be essential for replication and maintenance of pRNl , at least not when selective pressure is applied. However, under non-selective conditions, construct pH was lost at a much faster rate than any other construct that could be successfully established in S. acidocaldarius. The relative instability of this construct provides the first experimental evidence that the DNA-binding protein ORF80 has an important role in stable replication of pRN1. The instability of this construct may also have practical uses. For example, it may facilitate transfer of pyrEF-marked genes to the host chromosome, by allowing such genes to be first established on an episome, and then stabilized in the population by recombinational integration at the homologous locus.
  • S. acidocaldarius does not contain any integrated copies of pRN1 or genes homologous to pRNl genes. This enables detailed experiments on essential regions and proteins for pRN1 replication and maintenance without interference from plasmid-gene homologues located on the host chromosome. Because of the low sequence similarity between 5. acidocaldarius and S. solfataricus there is also minimal risk of undesired homologous recombination when cloning genes of S. solfataricus into the shuttle vector, e.g. for protein expression. S.
  • Example 4 Inducible promoter for protein expression with the Sulfolobus-E.coli shuttle vector
  • Fig. 16 An inducible promoter (map promoter) was integrated into a vector and analyzed for the controlled overexpression of proteins or other biomacromolecules in Sulfolobus acidocaldarius.
  • the map of that constructed vector named as pCmalX carrying the mal promoter is depicted in Fig. 16.
  • the exemplary gene of interest is lacS.
  • the sequence of a region of the mal promoter starting with the restriction site Sacll and ending with the restriction site Ncol used for cloning and comprising 262 bp of the promoter sequence of the gene saci_1 165 is shown in Fig. 17.
  • Fig. 23 shows the sequence of the vector pCmalX (X being the lacS gene).
  • the vector was analyzed for its function. Different sugars were tested for their ability to induce or repress the expression of the reporter gene (Fig. 18 A- D).
  • the reporter gene activity was measured by a beta-galactosidase activity assay using the substrate ortho-nitrophenylgalactopyranoside.
  • the promoter has some basal activity and is inducible upon addition of maltose and dextrin up to 17fold (Fig. 18 A-C).
  • the influence of the sugars glucose, xylose and sucrose on the basal promoter activity were also studied. Sucrose was identified as the sugar that has no inducing activity.
  • the measured beta-galactosidase activity after addition of sucrose is in the same range as the basal activity measured in tryptone alone, whereas glucose, dextrin and xylose addition cause elevated beta- galactosidase activities (Fig. 18 D).
  • the C-terminally his-tagged single stranded binding protein from S. solfataricus was expressed in S. acidocaldarius MR31 and purified over a talon affinity column. The successful expression is shown in Fig. 18 E.
  • Example 5 Targeted gene knockout using double crossover recombination A knockout plasmid was constructed based on the vector d2pyrEF and an upstream and downstream homologous region of the 5. acidocaldarius genome for targeted gene deletion in 5. acidocaldarius.
  • Fig. 19 The map of that knockout plasmid (d2updownXpyrEF) is shown in Fig. 19. Nucleotide sequence of the vector d2updownXpyrEF whereby the homologous regions of interest X are the flanking regions of the trpA gene from 5. acidocaldarius is depicted in Fig. 24. The tryptophane synthesis operon from 5. acidocaldarius is illustrated in Fig. 20. Fig. 21 demonstrates schematically the steps in the recombination process of the plasmid into the chromosome. The integration was analyzed and confirmed using different detection methods such as PCR (Fig. 22 A) and phenotypic tests (Fig. 22 B-D).
  • Lipps,G., Stegert,M. and Krauss,G. (2001 ) Thermostable and site- specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1 , a putative archael plasmid copy control protein. Nucleic. Acids. Res., 29, 904-913.

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Abstract

L'invention concerne un système de vecteur plasmidique archéen comprenant : I) un ou plusieurs gènes marqueurs pouvant être sélectionnés dans des archées, et II) au moins un composant sélectionné dans le groupe constitué par a) une origine archéenne de réplication, b) une région d'homologie avec un chromosome archéen, c) un gène codant pour une molécule qui confère une susceptibilité d'intégrer le vecteur dans un chromosome archéen, et d) le gène orf904 ; les gènes du vecteur plasmidique ne permettant pas l'assemblage d'une particule virale fonctionnelle. L'invention concerne également un vecteur plasmidique comprenant : I) le gène orf904, et II) un ou plusieurs gènes marqueurs pouvant être sélectionnés; le vecteur étant non intégratif. L'invention concerne en outre des cellules hôtes transformées par le système de vecteur ainsi que des kits comprenant ledit système de vecteur ou une cellule hôte de l'invention. L'invention concerne enfin un procédé de production de protéine d'intérêt.
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