WO2009147673A1 - Bacterie issue de paracoccus marcusii exprimant une voie de sequestration et de secretion et utilisations associees - Google Patents

Bacterie issue de paracoccus marcusii exprimant une voie de sequestration et de secretion et utilisations associees Download PDF

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WO2009147673A1
WO2009147673A1 PCT/IL2009/000555 IL2009000555W WO2009147673A1 WO 2009147673 A1 WO2009147673 A1 WO 2009147673A1 IL 2009000555 W IL2009000555 W IL 2009000555W WO 2009147673 A1 WO2009147673 A1 WO 2009147673A1
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marcusii
bacterium
particles
isolated
seq
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Joseph Hirschberg
Varda Mann
Mark Harker
Oori Weisshaus
Eszter Hoffmann
Ilya Pankratov
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Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
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Publication of WO2009147673A1 publication Critical patent/WO2009147673A1/fr

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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • Paracoccus marcusii for use in producing biomaterials, including lipophilic materials and proteins that are synthesized and accumulated in the bacterial cells, and/or secreted to the growth medium.
  • Vectors and nucleic acid constructs for genetic manipulation of bacteria in general, and P. marcusii in particular are disclosed.
  • Bacterial cell transport and secretion is disclosed.
  • Transport of organic compounds, including DNA, proteins and smaller molecules, across the bacterial cell envelope is a basic function found in all groups of bacteria.
  • Secreted compounds have various functions in processes such as the biogenesis of the cell envelope, the acquisition of nutrients, motility, intercellular communication and many more.
  • Aggressive bacterial virulence factors that enable a progressive colonization of host organisms are commonly secreted proteins, with toxins and translocated effector compounds as well-studied examples.
  • GSP General Secretory Pathway
  • organelles chloroplasts and endoplasmic reticulum
  • OM outer membrane
  • Type I secretion systems TlSS
  • Type II secretion systems T2SS
  • Type III secretion system T3SS
  • Type IV secretion systems T4SS
  • Type V secretion systems T5SS
  • Chaperone usher (CU) pathway Type VI secretion systems (T6SS).
  • OM vesicles may contribute to bacterial survival by reducing levels of toxic compounds (such as toluene) and by aiding in the release of attacking phage.
  • toxic compounds such as toluene
  • OM vesicle production has been known for more than 50 years, the mechanisms responsible for vesicle secretion, while maintaining bacterial viability, remain elusive. Components such as porins have been isolated and analyzed (see, for example, Nikaido, Microb. MoI. Biol. Rev, 2003; 67:593-656).
  • Certain Gram-negative bacteria release outer membrane vesicles that contain secreted proteins, apparently without the intervention of any known 'secretion' factor but also independently of membrane perturbation.
  • Paracoccus marcusii :
  • US Patent No. 5,935,808 to Hirschberg et al. discloses a coccoid bacterial species belonging to the genus Paracoccus, Paracoccus marcusii MHl, which produces and actively secretes to the growth medium small particles that contain various lipophilic materials, including carotenoids, lipids and proteins, enabling the production of biomaterials in high concentration and relatively easy separation from the medium by harvesting the secreted particles.
  • an isolated genetically modified bacterium comprising a sequestration and secretion pathway capable of secreting particles, which particles, when secreted, comprise a 39 kDa P. marcusii polypeptide having an amino acid sequence as set forth in SEQ ID NO: 38 or a 39 kDa protein homologue.
  • a process for production of at least one desired recombinant gene product or metabolite thereof comprising the steps of: (a) providing a genetically modified bacterial species comprising a sequestration and secretion pathway capable of secreting particles, which particles, when secreted, comprise a 39 kDa P. marcusii polypeptide having an amino acid sequence as set forth in SEQ ID NO: 38, a 39 kDa homologue or portions thereof and wherein the bacterial species is modified to express said at least one recombinant gene; (b) providing the bacterial species with growing conditions for production of the at least one gene product or metabolite thereof; and
  • the isolated bacterium bacterium is a Gram-negative bacterium. According to some embodiments of the invention the isolated bacterium is of genus Paracoccus.
  • the isolated bacterium is Paracoccus marcusii.
  • the isolated bacterium is Paracoccus marcusii strain MHl, which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen as strain DSM 11574 T .
  • the secreted particles comprise at least one lipophilic compound.
  • the lipophilic compound is a carotenoid.
  • the particles are devoid of carotenoid.
  • the secreted particles are 30 to 150 nm in size. According to some embodiments of the invention the secreted particle comprises a genetically modified gene product or a metabolite thereof.
  • the isolated bacterium exogenously expresses at least one P. marcusii gene of the pathway.
  • the at least one P. marcusii gene is located within the P. marcusii sequestration and secretion pathway gene cluster as set forth in SEQ ID NO: 39.
  • the at least one P. marcusii gene comprises an open reading frame as set forth in any of SEQ ID NOs: 40 to 54.
  • the secreted particle can be isolated from said bacterium by ultracentrifigation.
  • a growth medium of the bacterium comprising said secreted particles.
  • the recovering the at least one desired recombinant gene product or metabolite thereof comprises extracting the desired recombinant gene product or metabolite thereof from cells of the bacterial species.
  • the recovering is from the medium.
  • the recovering is from the secreted particles.
  • the particle which comprises at least one lipophilic compound.
  • the lipophilic compound is a carotenoid.
  • the particle being of dimensions 30 to 150 nm in size.
  • the particle further comprises a genetically modified gene product or a metabolite thereof.
  • the particle is isolated from said bacterium by ultracentrifigation.
  • the nucleic acid construct is capable of replicating in both E.coli and P. marcusii.
  • the nucleic acid construct comprising an E. coli transposon sequence linked to elements required for plasmid maintenance and replication (Rep3) in P. marcusii.
  • the nucleic acid construct further comprising a nucleic acid sequence encoding a polypeptide of interest.
  • the nucleic acid construct wherein the polypeptide of interest is selected from the group consisting of a carotenoid synthase.
  • nucleic acid construct further comprising a selectable marker.
  • the nucleic acid construct is designed capable of expressing an exogenous gene in a bacterium of the genus Paracoccus.
  • the nucleic acid construct is designed capable of expressing an exogenous gene in Paracoccus marcusii.
  • nucleic acid construct is selected from the group consisting of SEQ ID NOs: 3 to 5.
  • nucleic acid construct is as set forth in SEQ ID NO: 3 (PMSH9). According to some embodiments of the invention the nucleic acid construct is as set forth in SEQ ID NO: 65 (Hybrid ⁇ ).
  • a bacterial cell transformed with the nucleic acid constructs.
  • FIGs. 2A-2C are TEM microscope images showing the maturation of particle accumulation and secretion of P. marcusii cells in culture.
  • 2A shows a mid-logarithmic growth stage when several small particles are visible;
  • 2B shows a late-logarithmic growth stage when larger particles can be observed;
  • 2C shows two exemplary cells after four days at stationary growth stage, when particles have traversed the cell envelope and are now attached to the outer surface of the cell;
  • FIG. 3 is a TEM microscope image showing secreted particle accumulation around the cell surface in P. marcusii cells at stationary growth phase of culture;
  • FIGs. 4A-4C are TEM microscope images showing particle secretion across the cell envelope in P. marcusii cells.
  • FIG. 4A shows a close-up of a particle (arrow) in the periplasmic compartment between the inner cytoplasmic membrane (IM) and the outer membrane (OM);
  • FIG. 4B shows the movement of particles from the cytoplasm (circled) across the inner membrane into the periplasmic compartment (arrows);
  • FIG. 4C is a close-up of particles protruding from the outer envelope;
  • FIGs. 5A and 5B are a photograph showing pellets of secreted particles following centrifugation of medium from exemplary P. marcusii cultures grown 4 days at stationary phase;
  • FIG. 6 is a photograph of SDS-PAGE analysis of the proteins extracted from P. marcusii cells and the secretory particles. Proteins were extracted from whole P. marcusii cells (lane 1), the bottom of the pellet (lane 2) or the side of the pellet (lane 3), pellet as depicted in FIGs. 5A and 5B. The arrow indicates the relative size (in kDa) of the major polypeptide component of the secretory particle;
  • FIG. 7 is a photograph of SDS-PAGE analysis of the proteins extracted from P. marcusii secretory particles, following detergent treatment. Isolated particles secreted from P. marcusii cells were treated with Triton X-IOO. Lane 1- protein from total untreated particle pellet (control); Lane 2- supernatant from detergent treatment of pellets, after ultracentrifugation; Lane 3- protein from untreated particles from the bottom of the pellet (see, FIGs. 5A and 5B); Lane 4- protein from untreated particles from the side of pellet (see, FIGs. 5A and 5B); Lane 5- supernatant after ultracentrifugation of particles treated with 1% Triton x-100.
  • FIG. 8 is a photograph of SDS-PAGE analysis of the proteins extracted from P. marcusii secretory particles, following detergent treatment.
  • Isolated particles excreted from P. marcusii cells were treated with Triton X-100: Lane 1-Proteins from supernatant after ultracentrifugation of particles treated with 1% Triton X-100; Lane 2- Proteins from pellet after ultracentrifugation of particles treated with 1% Triton X-100; Lane 3- Proteins from supernatant after ultracentrifugation of particles treated with 0.1% Triton X-100; Lane 4- Proteins from pellet after ultracentrifugation of particles treated with 0.1% Triton x-100.
  • FIG. 9 is a schematic representation of the open reading frames (ORFs) and annotation of the genes of the 39kDa protein (SEQ ID NO: 39) cluster.
  • Genes 1-8 SEQ ID NOs: 40-47;
  • FIG. 10 is a photograph showing an example of phenotypic variation in Tn-5- tagged (Kanamycin+Rifampicin resistant) mutants producing caroteniods but impaired in particle secretion;
  • FIG. 11 is a schematic representation of the P. marcusii shuttle vector constructs pMSH9 and Hybrid ⁇ . Open reading frames (ORF) incorporated into the vectors are:
  • FIG. 12 is a sequence alignment of the CrtE sequence (encoding geranylgeranyl phosphatase synthase), from wild type and genetically modified mutant 140 P. marcusii, showing the frameshift mutation in the crtE gene;
  • FIGs. 13A and 13B are SEM micrographs of wild-type (FIG. 13A) and mutant 140 (FIG. 13B) P. marcusii cells from a suspension culture at late logarithmic phase. Note the presence of secreted particles, even in the absence of significant carotenoid synthesis in strain 140 (FIG. 13B); FIGs. 14A and 14B are photographs showing carotenoid synthesis complementation in E. coli.
  • E. coli strain XL IB (harboring pIPI+pEIB ⁇ crtB), which does not produce carotenoids, was transformed with either pABWl(PM-Wt crtB) (FIG.
  • FIG. 14A Note restoration of the carotenoid synthesis and accumulation of lycopene (and phytoene) with complementation in correct orientation (FIG. 14A), but not in inverted orientation (FIG. 14B);
  • FIGs. 15A- 15D are photographs showing functional complementation of mutant P. marcusii with plasmid carrying the crtB (phytoene synthase) gene.
  • P. marcusii mutant #125 harbors a frameshift mutation in crtB and Rifampicin resistance.
  • Transgenic expression of wild type crtB from plasmid ABWl restored carotenoid synthesis (FIGs. 15B-15D), while transgenic expression of a control, inverted crtB failed to restore carotenoid synthesis (FIG. 15A);
  • FIGs. 16A- 16C are photographs showing functional complementation of mutant P. marcusii with plasmid carrying the crtB (phytoene synthase) gene.
  • Conjugation of the mutant P. marcusii #125 with E. coli S17(pABW-crtB) after 8 days shows indication of orange carotenoid-producing colonies.
  • Growth of mutant IL #125 on increased Kanamycin resistance (IRK) plates show no growth, ruling out contamination.
  • the present invention in some embodiments thereof, relates to the genetic modification of the bacterial species Paracoccus marcusii for the production of biomaterials, such as lipophilic materials and proteins that are synthesized and accumulated in the bacterial cells, and/or secreted into the growth medium and, more particularly, but not exclusively, to vectors and nucleic acid constructs which can be used in genetic manipulation of bacteria in general, and P. marcusii in particular.
  • P. marcusii has a unique sequestration and accumulation mechanism that can 'sequester' organic materials, lipophilic metabolites, hydrophilic peptides and proteins having hydrophobic regions within membrane-less secretory particles, thus eliminating cytotoxicity and enabling the cells to accumulate organic materials as secondary metabolites at extremely high concentrations. Compartmentalization and concentration of the products of genetically modified P. marcusii into secretory particles can effectively sequester such lipophilic and hydrophobic molecules within the cell, avoiding potentially damaging interaction with lipid bilayer membranes. These materials can be extracted from cells grown in cultures (aerobic or unaerobic fermentation) at high cell concentration.
  • P. marcusii excretes particles containing high concentrations of organic materials, mainly lipophilic compounds and proteins, from the cells to the medium, unique to P. marcusii.
  • P. ma?-cusii can be grown in continuous cultures and the desired compounds can be collected without harvesting the biomass, at high concentrations, by relatively simple physical means.
  • P. marcusii can grow at room temperatures without a requirement for illumination. 4. Being a prokaryote, P. marcusii is amenable to genetic manipulations aimed at altering metabolism, improving yield and/or altering growth properties. Moreover, component genes of this secretion mechanism can be incorporated into nucleic acid constructs and vectors for heterologous expression in other bacterial species.
  • nucleic acid vectors effective in transforming P. marcusii While reducing the present invention to practice, the inventors have constructed nucleic acid vectors effective in transforming P. marcusii, and have succeeded in genetically modifying the bacterial host cells to produce altered phenotypes including selectable antibiotic resistance and disruption of carotenoid synthesis and secretion (see Examples II and III, below, and FIG. 10). Further, the inventors have isolated, sequenced and cloned a 39 kDa polypeptide (SEQ ID NO: 38) which is a major protein component of P. marcusii secretory particles (see Example I, below, FIGs. 6, 7 and 8). Identifying this major component of secretory particles allows better understanding of the structure of the secretory particle, and of the characteristics of biomolecules that can be introduced therein, e.g. hydrophobic molecules.
  • SEQ ID NO: 38 39 kDa polypeptide
  • an isolated genetically modified bacterium comprising a sequestration and secretion pathway capable of secreting particles, which particles, when secreted, comprise a 39 kDa P. marcusii polypeptide having an amino acid seq ⁇ ence as set forth in SEQ ID NO: 38, a 39 kDa homologue or portions thereof.
  • the 39 kDa polypeptide was found to be closely associated with lipid contents of the secretory particles under non-denaturing conditions, but could be solubilized by detergent treatment.
  • the genetically modified bacterium can be modified to express or indirectly produce lipophilic and hydrophobic molecules. Organic compounds can be produced in P. marcusii or other bacteria expressing the P.
  • marcusii sequestration, accumulation and/or secretion pathway by expressing genes from other organisms that encode biosynthetic enzymes of these compounds.
  • these can include polypeptides, polynucleotides, carbohydrates, lipids, minerals, small molecules (smaller than 1 kDa) or combinations thereof.
  • bio-materials suitable for expression or production in the genetically modified bacterium according to some embodiments of the invention as claimed are detailed below.
  • proteins and biomolecules synthesized in a bacterial cell can be modified to include regions enhancing their sequestration, accumulation and/or secretion in the secreted particles.
  • the bacterium of the present invention can be any bacterium having the sequestration and secretion pathway. According to some embodiments of the present invention, the bacterium is Paracoccus marcusii.
  • P. marcusii is defined as Paracoccus marcusii, classified as a member of the genus Paracoccus (see Harker et al, Intl J Syst Biol, 1998;48:543-48, and US Patent No. 5,935,808).
  • the genus Paracoccus consists of gram-negative cocci or short rods, showing a substantial metabolic versatility. Representatives are able to grow aerobically on a wide range of organic compounds. A number of species can grow anaerobically as well, using nitrate as electron acceptor, and some representatives are able to use hydrogen as electron donor for chemoautotrophic growth.
  • the genus belongs to the ⁇ -3 subclass of the Proteobacteria.
  • P. denitrificans the type species of the genus
  • P. aestuarii the type species of the genus
  • P. alcaliphilus P. alkenifer
  • P. aminovorans P. bengalensis
  • P. carotenifaciens P. haeundaensis
  • P. halodenitrificans P. halophilus
  • P. homiensis P. kocurii
  • p. kondratievae P. koreensis
  • P. marinus P. marcusii
  • P. methylutens P. pantrophus
  • P. seriniphilus P.
  • P. marcusii produces and secretes significant amounts of carotenoids into the medium.
  • P. marcusii is Paracoccus marcusii strain MHl, which has been deposited with the Deutsche Sammlung von Mikroorganisman und Zellkulturen as strain DSM 11574 T .
  • bacterial species other than P. marcusii which possess the sequestration and secretion pathway can be used in the present invention.
  • Bacterial species lacking sequestration and secretion pathways, and those possessing only portions of the pathway can be genetically modified to express polypeptides having component portions of the P. marcusii sequestration and secretion pathway.
  • a gene cluster in proximity to the 39 kDa secretory particle polypeptide gene in P. marcusii has been identified (SEQ ID NO: 39), and sequences encoding putative sequestration and secretion pathway components have been identified and isolated (see SEQ ID NOs. 40- 54). Modification of bacteria to express these sequences (for example, SEQ ID NOs.
  • P. marcusii bacteria can be genetically modified to reduce, enhance or otherwise modify the components of the endogenous sequestration and secretion pathway, for example, to better accommodate concentration and inclusion of specific molecules.
  • the bacterium is a Gram negative bacterium, defined as a bacterium that does not retain the Gram stain used in Gram's method.
  • Gram negative bacteria include proteobacteria such as E. coli and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, acetic acid bacteria, Legionella, alpha-proteobacteria, cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria, Neisseria sp., H. influenzae, K. pneumoniae, Salmonella, and others.
  • the Gram negative bacterium is of the Paracoccus genus.
  • genetic material refers generally to nucleotides and polynucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded.
  • Genetic material refers also to sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.
  • polynucleotide is understood as a polyribonucleotide or a polydeoxyribonucleotide that can be a modified or non-modified DNA or an RNA.
  • the term polynucleotide includes, for example, a single strand or double strand DNA, a DNA composed of a mixture of one or several single strand region(s) and of one or several double strand region(s), a single strand or double strand RNA and an RNA composed of a mixture of one or several single strand region(s) and of one or several double strand region(s).
  • the term polynucleotide can also include an RNA and/or a DNA including one or several triple strand regions.
  • polynucleotide is equally understood the DNAs and RNAs containing one or several bases modified in such a fashion as to have a skeleton modified for reasons of stability or for other reasons.
  • nucleic acid constructs capable of replicating in P. marcusii.
  • Such nucleic acid constructs can contain DNA sequence elements from P.
  • the nucleic acid construct includes sequences homologous to P. marcusii plasmid DNA.
  • the nucleic acid construct is a "shuttle" plasmid vector capable of replication in both E.coli and P. marcusii.
  • Shuttle vectors suitable for use with the present invention include, but are not limited to
  • the nucleic acid construct is a vector carrying a moveable element such as a transposon (for example, Tn- 5). Transformation of host bacteria with transposon-bearing constructs results in disruption of endogenous bacterial DNA, sequences of which are ultimately retrievable by virtue of antibiotic resistance markers incorporated into the vector and selective DNA amplification.
  • An exemplary transposon-bearing nucleic acid construct suitable for use with the present invention is plasmid pRL27. A number of procedures exist for the preparation of competent bacteria and the introduction of DNA into those bacteria. A very simple, moderately efficient transformation procedure for use with E.
  • coli involves re-suspending log-phase bacteria in ice-cold 50 mM calcium chloride at about lO.sup.lO bacteria/ml and keeping them ice-cold for about 30 min. Plasmid DNA (0.1 mg) is then added to a small aliquot (0.2 ml) of these now competent bacteria, and the incubation on ice continued for a further 30 minutes, followed by a heat shock of 2 minutes at 42.degree. C. The bacteria are then usually transferred to nutrient medium and incubated for some time (30 minutes to 1 hour) to allow phenotypic properties conferred by the plasmid to be expressed, e.g., antibiotic resistance commonly used as a selectable marker for plasmid-containing cells.
  • secretory particles are commonly 30-150 nm in diameter, and are characterized, in native P. marcusii, by a 39 kDa major polypeptide protein component (see Example I, FIGs. 6-8) having an amino acid * sequence as set forth in SEQ ID NO: 38.
  • a process for production of at least one desired recombinant gene product or metabolite thereof in the genetically modified bacterium is cultured in an aqueous or solid nutrient medium including sources of carbon, nitrogen and inorganic substances to support its growth, production of the recombinant gene product and, optionally, their secretion to the growth medium.
  • the recombinant gene product can then be recovered either from the medium and/or the bacterial cells.
  • One ordinarily skilled in the art would know how to design the exact growth and recovering procedures to maximize yields.
  • Exemplary medium for bacterial growth and production of recombinant gene products suitable for culturing the genetically modified bacteria contains a carbon source, a nitrogen source and inorganic salts necessary for the growth, as well as if necessary special required substances (for example, vitamins, amino acids, nucleic acids etc.).
  • sugars such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, maltose, etc.
  • organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, pyruvic acid, malonic acid
  • alcohols such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol, glycerol
  • oil or fat such as soybean oil, rice bran oil, olive oil, corn oil, sesame oil, linseed oil, and the like are mentioned.
  • Amount of the carbon source added varies according to the kind of the carbon source, and usually 1 to 100 g, preferably 2 to 50 g per 1 liter medium.
  • nitrogen sources for example, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, urea etc., are used alone or in combination.
  • Amount of the nitrogen source added varies according to the kind of the nitrogen source, and usually 0.1 to 30 g, and preferably 1 to 10 g per 1 liter medium.
  • potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric sulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganous sulfate, manganous chloride, zinc sulfate, zinc, chloride, cupric sulfate, calcium chloride, calcium carbonate, sodium carbonate, etc. may be used alone or in combination.
  • Amount of inorganic acid varies according to the kind of the inorganic salt, and usually 0.001 to 10 g per 1 liter medium.
  • vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract, corn steep liquor, soybean meal, dried yeast etc. may be used alone or in combination.
  • Amount of the special required substance used varies according to the kind of the substance, and usually ranges between 0.2 g to 200 g, and preferably 3 to 100 g per 1 liter medium.
  • a pH value 5 of a medium is adjusted to pH 2 to 12, preferably 6 to 9.
  • Culturing is carried out at temperature of 15 to 40 0 C, and preferably 20 to 35 0 C, usually for 1 to 20 days, and preferably 1 to 4 days, under aerobic condition provided by shaking or aeration/agitation.
  • the genetically modified bacteria can be cultured in conventional fermentation bioreactor using methods well known in the art such as described, for example, in U.S. Pat. Nos. 6,214,221, 6,100,061, 5,998,184 and 5,571,720.
  • the recombinant gene product, or metabolite thereof may be isolated and purified from the culture. Namely, microbial cells are separated from the culture by a conventional means such as centrifugation or filtration, and the cells or the medium are subjected to an extraction with a suitable solvent.
  • secretory particles may be recovered from the medium, by for example, ultracentrifugation, density separation (salt flotation, for example), size filtration and the like. Particles can be recovered from the medium, or recovered from disrupted bacterium.
  • any gene product or metabolite thereof of interest can be purified therefrom.
  • immunosuppressants such as the polyene metabolites aureothin, iV-acetyl- aureothamine and spectinabilin- nitrophenyl pyrones SNF4435C and D, isolated from Streptomyces species such as strains of Streptomyces spectabilis and Streptomyces thioluteu; higher-chain alcohols including isobutanol, 1-butanol, 2-methyl-l-butanol, 3- methyl-1-butanol and 2-phenylethanol from Clostridium acetobutylicum (see Inui, M.
  • non-fermentative enzymes such as pyruvate decarboxylases from Zymomonas mobilis and alcohol dehydrogenase and acyltransferase from Acinetobacter baylyi to produce higher-chain alcohols; triacylglycerols (TAGs), waxes and wax esters (WE) produced using genes from Acinetobacter sp.
  • TAGs triacylglycerols
  • WE wax esters
  • fatty acid ethyl ester FAEE
  • fatty acid methyl ester FAME
  • triacylglycerol TAG
  • wax ester synthase/acyl-coenzyme A WS/DGAT: diacylglycerol acyltransferase; modified lipids from renewable resources using acyltransferases; surface-active compounds such as sophorolipids and surfactants; fatty acids, or biodiesel fuels, produced in P.
  • marcusii from carbon sources glycerol and glycerol derivatives such as acyl-l-(acyl-6'- mannobiosyl)-3-glycerol; carotenoids, polyhydroxyalkanoates (PHAs); polyhydroxybutyrate (PHB); isoprenoid precursors -isopentol; isoprenoids- monoterpenes; isoprenoids and derived compounds (such as sterols, dolichols etc.); coenzyme-Q (or ubiquinone, UQ); essential oils and sterols using 3-hydroxy-3- methylglutaryl CoA reductase (Munoz-Bertomeu, J. et al.
  • bio-fuel molecules other than ethanol artemisinin; resveratrol (3,5,4'-trihydroxy-tr ⁇ n,s-stilbene); organic acids such as citric, lactic and succinic acid; natural rubber polymer; aromatic compounds such as propenylbenzenes; long-chain n-3 polyunsaturated fatty acids (LC- PUFA); starches and branched oligosaccharides (LPS ,glycans, sulfated glycans (GAG's) branched polymers); ectoine - a compatible solute accumulated in halophilic bacteria in response to high salt -derived from aspartate metabolism; terpenoids (mono/di/tri/sesqui/poly),qinones,gibberellins, geranylgeranylated proteins; mono/ di- glycosylated isoprenoids derivatives; ladderane lipids (these lipids are comprised of three to five linearly
  • genes products and metabolites can be purified by conventional means known in the art. They can be used, in unpurified or purified form, for example, in agricultural, pharmaceutical, cosmetic compositions, can be used for fuel, in detergent compositions, or other applications.
  • the recombinant gene products or metabolites of the present invention can also be included in agricultural compositions, which also preferably include an agricultural acceptable carrier.
  • An agriculturally acceptable carrier can be a solid or a liquid, preferably a liquid, more preferably water.
  • the agricultural composition of the invention may also contain other additives such as fertilizers, inert formulation aids, i.e. surfactants, emulsifiers, defoamers, dyes, extenders and the like. Reviews describing methods of preparation and application of agricultural compositions are available. See, for example, Couch and Ignoffo (1981) in Microbial Control of Pests and Plant Disease 1970-1980, Burges (ed.), chapter 34, pp. 621-634; Corke and Rishbeth, ibid, chapter 39, pp.
  • compositions of the present invention are leaf application, seed coating and soil application, as disclosed in U.S. Pat. NO: 5,039,523, which is fully incorporated herein.
  • Gene products or metabolites of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a pharmaceutical composition refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the polypeptide accountable for the intended biological effect.
  • pharmaceutically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal, or parenteral delivery, including intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries as desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, and sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, or carbon dioxide.
  • the dosage may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with, optionally, an added preservative.
  • the compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.
  • a suitable vehicle e.g., a sterile, pyrogen-free, water-based solution
  • the pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a "therapeutically effective amount” means an amount of active ingredients (e.g., a nucleic acid construct) effective to prevent, alleviate, or ameliorate symptoms of a disorder (e.g., ischemia) or prolong the survival of the subject being treated.
  • a therapeutically effective amount means an amount of active ingredients (e.g., a nucleic acid construct) effective to prevent, alleviate, or ameliorate symptoms of a disorder (e.g., ischemia) or prolong the survival of the subject being treated.
  • the dosage or the therapeutically effective amount can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl, E. et al. (1975), "The Pharmacological Basis of Therapeutics," Ch. 1, p.l.)
  • Dosage amount and administration intervals may be adjusted individually to provide sufficient plasma or brain levels of the active ingredient to induce or suppress the biological effect (i.e., minimally effective concentration, MEC).
  • MEC minimally effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.
  • Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks, or until cure is effected or diminution of the disease state is achieved.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser device may also be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration.
  • Such notice may include labeling approved by the U.S. Food and Drug
  • compositions comprising a preparation of the invention formulated in a pharmaceutically acceptable carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as further detailed above.
  • food compositions comprise one or more gene product or metabolite thereof of the present invention as food additives.
  • the phrase "food additive” [defined by the FDA in 21 C.F.R. 170.3(e)(l)] includes any liquid or solid material intended to be added to a food product. This material can, for example, include an agent having a distinct taste and/or flavor or physiological effect (e.g., vitamins).
  • the food additive composition may comprise the polypeptide of the present invention.
  • the food additive composition of the present invention can include the polypeptide per se, or an encapsulated form of the polypeptide (described hereinabove with respect to pharmaceutical compositions) .
  • the food additive composition of the present invention can be added to a variety of food products.
  • the phrase "food product” describes a material consisting essentially of protein, carbohydrate and/or fat, which is used in the body of an organism to sustain growth, repair and vital processes and to furnish energy. Food products may also contain supplementary substances such as minerals, vitamins and condiments. See Merriani- Webster's Collegiate Dictionary, 10th Edition, 1993.
  • the phrase "food product” as used herein further includes a beverage adapted for human or animal consumption.
  • Representative examples of food products in which the food additive of the present invention can be incorporated include, without limitation, baked goods, soft drinks, cereals, candy, jams, jellies, tofu, cheese and ice cream.
  • a food product containing the food additive of the present invention can also include additional additives such as, for example, antioxidants, sweeteners, flavorings, colors, preservatives, enzymes, nutritive additives such as vitamins and minerals, emulsifiers, pH control agents such as acidulants, hydrocolloids, antifoams and release agents, flour improving or strengthening agents, raising or leavening agents, gases and chelating agents, the utility and effects of which are well-known in the art.
  • Cosmetical Compositions such as, for example, antioxidants, sweeteners, flavorings, colors, preservatives, enzymes, nutritive additives such as vitamins and minerals, emulsifiers, pH control agents such as acidulants, hydrocolloids, antifoams and release agents, flour improving or strengthening agents,
  • compositions are usually prepared for aesthetic use and may comprise the gene product or metabolite thereof of the present invention as either the active ingredient or as a carrier.
  • cosmetically or cosmeceutically acceptable carrier describes a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the applied active ingredient(s).
  • acceptable carriers examples include carrier materials that are well-known for use in the cosmetic and medical arts as bases for e.g., emulsions, creams, aqueous solutions, oils, ointments, pastes, gels, lotions, milks, foams, suspensions, aerosols and the like, depending on the final form of the composition.
  • suitable carriers therefore include, without limitation, water, liquid alcohols, liquid glycols, liquid polyalkylene glycols, liquid esters, liquid amides, liquid protein hydrolysates, liquid alkylated protein hydrolysates, liquid lanolin and lanolin derivatives, and like materials commonly employed in cosmetic and medicinal compositions.
  • suitable carriers include, without limitation, alcohols, such as, for example, monohydric and polyhydric alcohols, e.g., ethanol, isopropanol, glycerol, sorbitol, 2-methoxyethanol, diethyleneglycol, ethylene glycol, hexyleneglycol, mannitol, and propylene glycol; ethers such as diethyl or dipropyl ether; polyethylene glycols and methoxypolyoxyethylenes (carbowaxes having molecular weight ranging from 200 to 20,000); polyoxyethylene glycerols, polyoxyethylene sorbitols, stearoyl diacetin, and the like.
  • alcohols such as, for example, monohydric and polyhydric alcohols, e.g., ethanol, isopropanol, glycerol, sorbitol, 2-methoxyethanol, diethyleneglycol, ethylene glycol, hexyleneglycol, mannito
  • compositions of the present invention may be formulated into any pharmaceutical, cosmetic or cosmeceutical form normally employed for topical application.
  • the compositions of the present invention can be, for example, in a form of a cream, an ointment, a paste, a gel, a lotion, a milk, a suspension, an aerosol, a spray, a foam, a shampoo, a hair conditioner, a serum, a swab, a pledget, a pad and a soap.
  • compositions of the present invention can optionally further comprise a variety of components that are suitable for rendering the compositions more cosmetically or aesthetically acceptable or to provide the compositions with additional usage benefits.
  • Such conventional optional components are well known to those skilled in the art and are referred to herein as "ingredients". These include any cosmetically acceptable ingredients such as those found in the CTFA International Cosmetic Ingredient Dictionary and Handbook, 8th edition, edited by Wenninger and Canterbery, (The Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, D.C., 2000).
  • ingredients include humectants, deodorants, antiperspirants, sun screening agents, sunless tanning agents, hair conditioning agents, pH adjusting agents, chelating agents, preservatives, emulsifiers, occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti- irritants, colorants, propellants (as described above) and surfactants.
  • compositions of the present invention can comprise, in combination with ammonium lactate and urea, one or more additional humectants or moisturizing agents.
  • additional humectants or moisturizing agents include, without limitation, guanidine, glycolic acid and glycolate salts (e.g.
  • aloe vera in any of its variety of forms (e.g., aloe vera gel), allantoin, urazole, polyhydroxy alcohols such as sorbitol, glycerol, hexanetriol, propylene glycol, butylene glycol, hexylene glycol and the like, polyethylene glycols, sugars and starches, sugar and starch derivatives (e.g., alkoxylated glucose), hyaluronic acid, lactamide monoethanolamine, acetamide monoethanolamine and any combination thereof.
  • aloe vera gel e.g., aloe vera gel
  • allantoin urazole
  • polyhydroxy alcohols such as sorbitol, glycerol, hexanetriol, propylene glycol, butylene glycol, hexylene glycol and the like
  • polyethylene glycols sugars and starches
  • sugar and starch derivatives e.g., alkoxylated glucose
  • compositions of the present invention can further comprise a pH adjusting agent.
  • a pH adjusting agent As is discussed hereinabove, although the ammonium lactate or any corresponding ammonium salt may serve as a pH adjusting agent, it is preferable for the compositions of the invention to have a pH value of between about 4 and about 7, preferably between about 5 and about 6, most preferably about 5.5 or substantially 5.5 and hence the presence of a pH adjusting agent is preferred.
  • Suitable pH adjusting agents include, for example, one or more of adipic acids, glycines, citric acids, calcium hydroxides, magnesium aluminometasilicates, buffers or any combinations thereof.
  • deodorant agents that are usable in the context of the present invention include, without limitation, quaternary ammonium compounds such as cetyl-trimethylammonium bromide, cetyl pyridinium chloride, benzethonium chloride, diisobutyl phenoxy ethoxy ethyl dimethyl benzyl ammonium chloride, sodium N-lauryl sarcosine, sodium N-palmlthyl sarcosine, lauroyl sarcosine, N-myristoyl glycine, potassium N-lauryl sarcosine, stearyl, trimethyl ammonium chloride, sodium aluminum chlorohydroxy lactate, tricetylmethyl ammonium chloride, 2,4,4'-trichloro-2'-hydroxy diphenyl ether, diaminoalkyl amides such as L-lysine hexadecyl amide, heavy metal salts of citrate, salicylate, and
  • deodorant agents include, without limitation, odor absorbing materials such as carbonate and bicarbonate salts, e.g. as the alkali metal carbonates and bicarbonates, ammonium and tetraalkylammonium carbonates and bicarbonates, especially the sodium and potassium salts, or any combination of the above.
  • odor absorbing materials such as carbonate and bicarbonate salts, e.g. as the alkali metal carbonates and bicarbonates, ammonium and tetraalkylammonium carbonates and bicarbonates, especially the sodium and potassium salts, or any combination of the above.
  • Antiperspirant agents can be incorporated in the compositions of the present invention either in a solubilized or a particulate form and include, for example, aluminum or zirconium astringent salts or complexes.
  • sun screening agents include, without limitation, p-aminobenzoic acid, salts and derivatives thereof (ethyl, isobutyl, glyceryl esters; p-dimethylaminobenzoic acid); anthranilates (i.e., o- amino-benzoates; methyl, menthyl, phenyl, benzyl, phenylethyl, linalyl, terpinyl, and cyclohexenyl esters); salicylates (amyl, phenyl, octyl, benzyl, menthyl, glyceryl, and di- pro-pyleneglycol esters); cinnamic acid derivatives (menthyl and benzyl esters, a-phenyl cinnamonitrile; butyl cinnamoyl pyruvate); dihydroxycinnamic acid derivatives (umbelliferone,
  • sunless tanning agents usable in context of the present invention include, without limitation, dihydroxyacetone, glyceraldehyde, indoles and their derivatives.
  • the sunless tanning agents can be used in combination with the sunscreen agents.
  • Suitable hair conditioning agents that can be used in the context of the present invention include, for example, one or more collagens, cationic surfactants, modified silicones, proteins, keratins, dimethicone polyols, quaternary ammonium compounds, halogenated quaternary ammonium compounds, alkoxylated carboxylic acids, alkoxylated alcohols, alkoxylated amides, sorbitan derivatives, esters, polymeric ethers, glyceryl esters, or any combinations thereof.
  • the chelating agents are optionally added to the compositions of the present invention so as to enhance the preservative or preservative system.
  • Preferred chelating agents are mild agents, such as, for example, ethylenediaminetetraacetic acid (EDTA), EDTA derivatives, or any combination thereof.
  • Suitable preservatives that can be used in the context of the present composition include, without limitation, one or more alkanols, disodium EDTA (ethylenediamine tetraacetate), EDTA salts, EDTA fatty acid conjugates, isothiazolinone, parabens such as methylparaben and propylparaben, propylene glycols, sorbates, urea derivatives such as diazolindinyl urea, or any combinations thereof.
  • Suitable emulsifiers that can be used in the context of the present invention include, for example, one or more sorbitans, alkoxylated fatty alcohols, alkylpolyglycosides, soaps, alkyl sulfates, monoalkyl and dialkyl phosphates, alkyl sulphonates, acyl isothionates, or any combinations thereof.
  • Suitable occlusive agents that can be used in the context of the present invention include, for example, petrolatum, mineral oil, beeswax, silicone oil, lanolin and oil- soluble lanolin derivatives, saturated and unsaturated fatty alcohols such as behenyl alcohol, hydrocarbons such as squalane, and various animal and vegetable oils such as almond oil, peanut oil, wheat germ oil, linseed oil, jojoba oil, oil of apricot pits, walnuts, palm nuts, pistachio nuts, sesame seeds, rapeseed, cade oil, corn oil, peach pit oil, poppyseed oil, pine oil, castor oil, soybean oil, avocado oil, safflower oil, coconut oil, hazelnut oil, olive oil, grape seed oil and sunflower seed oil.
  • saturated and unsaturated fatty alcohols such as behenyl alcohol
  • hydrocarbons such as squalane
  • various animal and vegetable oils such as almond oil, peanut oil, wheat germ oil, lins
  • Suitable emollients other than ammonium lactate, that can be used in the context of the present invention include, for example, dodecane, squalane, cholesterol, isohexadecane, isononyl isononanoate, PPG Ethers, petrolatum, lanolin, safflower oil, castor oil, coconut oil, cottonseed oil, palm kernel oil, palm oil, peanut oil, soybean oil, polyol carboxylic acid esters, derivatives thereof and mixtures thereof.
  • Suitable thickeners that can be used in the context of the present invention include, for example, non-ionic water-soluble polymers such as hydroxyethylcellulose (commercially available under the Trademark Natrosol.RTM. 250 or 350), cationic water-soluble polymers such as Polyquat 37 (commercially available under the Trademark Synthalen.RTM. CN), fatty alcohols, fatty acids and their alkali salts and mixtures thereof.
  • solubilizing agents that are usable in this context of the present invention include, without limitation, complex-forming solubilizers such as citric acid, ethylenediamine-tetraacetate, sodium meta-phosphate, succinic acid, urea, cyclodextrin, polyvinylpyrrolidone, diethylammonium-ortho-benzoate, and micelle- forming solubilizers such as TWEENS and spans, e.g., TWEEN 80.
  • complex-forming solubilizers such as citric acid, ethylenediamine-tetraacetate, sodium meta-phosphate, succinic acid, urea, cyclodextrin, polyvinylpyrrolidone, diethylammonium-ortho-benzoate, and micelle- forming solubilizers such as TWEENS and spans, e.g., TWEEN 80.
  • solubilizers that are usable for the compositions of the present invention are, for example, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene n-alkyl ethers, n-alkyl amine n-oxides, poloxamers, organic solvents, phospholipids and cyclodextrines.
  • Suitable penetration enhancers usable in context of the present invention include, but are not limited to, dimethylsulfoxide (DMSO), dimethyl formamide (DMF), allantoin, urazole, N,N-dimethylacetamide (DMA), decylmethylsulf oxide (Cio MSO), polyethylene glycol monolaurate (PEGML), propylene glycol (PG), propylene glycol monolaurate (PGML), glycerol monolaurate (GML), lecithin, the 1-substituted azacycloheptan-2-ones, particularly l-n-dodecylcyclazacycloheptan-2-one (available under the trademark Azone R TM from Whitby Research Incorporated, Richmond, Va.), alcohols, and the like.
  • the permeation enhancer may also be a vegetable oil. Such oils include, for example, safflower oil, cottonseed oil and corn oil.
  • Suitable anti-irritants that can be used in the context of the present invention include, for example, steroidal and non steroidal anti-inflammatory agents or other materials such as aloe vera, chamomile, alpha-bisabolol, cola nitida extract, green tea extract, tea tree oil, licoric extract, allantoin, caffeine or other xanthines, glycyrrhizic acid and its derivatives.
  • compositions of the present invention are preferably devoid of an enduring perfume composition.
  • the incorporation of such a perfume composition in pharmaceutical compositions is considered in the art disadvantageous for skin and scalp medical treatment, as it oftentimes cause undesirable irritation of a sensitive skin.
  • an enduring perfume composition describes a composition that comprises one or more perfumes that provide a long lasting aesthetic benefit with a minimum amount of material. Enduring perfume compositions are substantially deposited and remain on the body throughout any rinse and/or drying steps. Representative examples of such compositions are described, for example, in U.S. Patent No. 6,086,903.
  • fragrances other than enduring perfume compositions, perfumes or perfume compositions, which are fast removable from the surface they are deposited on, can be included in the compositions of the present invention.
  • the recombinant biomolecules of the present invention are preferably retrieved in "substantially pure” form to be used in pharmaceutical compositions and/or agricultural compositions, described below.
  • substantially pure refers to a purity that allows for the effective use of the protein in the diverse applications, described hereinabove optionally, 50%, 60%, 70%, 80%, 90 %, 95%, 99% or effectively 100% pure (or lesser or intermediate levels of purity).
  • composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases "ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Paracoccus marcusii strain MHl was routinely grown at 25. degree. C. in medium containing (per liter): 10 g of Bacto-tryptone, 5 g of Bacto-yeast extract, and 5 g of NaCl, pH 7.0. Liquid cultures were grown on a shaking water bath. For solid media 15 g of Bacto-agar was added per liter. The composition of this medium was modified by the addition of NaNC> 3 , starch, increased NaCl concentrations, and other components, as specified in the following Examples.
  • Microscopy Cultures were examined and photographed using a Zeiss Standard microscope, equipped with phase contrast optics.
  • Carotenoids analysis Aliquots of P. marcusii cells were harvested by centrifugation at 13,000Xg for 10 min and washed once in water. Aliquots of secretory particles of secreted by P. marcusii were harvested by ultracentrifugation at 40,000 rpm in an ultracentrifuge (Beckman LS, rotor SW50.1, 192,000Xg). The cells, secretory particles were resuspended in 200 ⁇ l acetone and incubated at 65 0 C for 10 min in the dark. For cells-depleted medium extraction, 10 ⁇ l of medium was mixed with 200 ⁇ l acetone and incubated at 65 0 C. for 10 min in the dark. The samples were centrifuged again at 13,000Xg for 10 min and the acetone supernatant containing the pigments was placed in a clean tube. The pigment extract was dried under a stream of N 2 and stored at
  • UV/Visible electronic absorption spectra were recorded in redistilled or HPLC grade acetone and diethyl ether. The spectra were recorded using a Cecil CE 5501 computing double beam UV/Visible spectrophotometer. The degree of fine structure is expressed as the ratio of the peak heights of %III/II where the zero value is taken as the minimum between the two absorption peaks, the peak height of the longest wavelength absorption wavelength is designated III, and that of the middle absorption wavelength as II. In the case of conjugated ketocarotenoids, such as astaxanthin, which exhibits a single absorption peak with no fine structure, the %III value is zero.
  • Electron microscopy Samples for electron microscopy were fixed overnight in 3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4, washed in the same buffer and post-fixed with 1% OsO 4 in 0.1 M cacodylate buffer for 2 hours. Dehydration was done in a series of ethyl alcohol and propylene oxide concentrations and embedded in Epon- 812. The ultra-thin sections were stained with uranyl acetate and lead citrate prior to viewing in a Jeol-1000 CX electron microscope.
  • Table I Carotenoid concentration and composition in cells and secreted particles off. marcusii suspension culture after 48 hours at stationary phase.
  • the kinetics of particle accumulation in P. marcusii cells was determined at different stages of cultivation (FIG. 1), indicating that by 48 hours in stationary phase culture, most cells have accumulated large secretory particles, or have already secreted them into the medium.
  • the amino acid sequence of the 39 kDa major secretory particle polypeptide component was determined using routine protein sequencing protocol. Based on the amino acid sequence of the 39 kDa protein (SEQ ID NO: 38), a gene comprising an open reading frame (ORF) encoding the 39 kDa polypeptide was detected and cloned (SEQ ID NO: 37). By analyzing the neighboring nucleotide sequences on the P. marcusii DNA, a 12 kb DNA region (SEQ ID NO: 39) with a cluster of additional ORFs surrounding the "39 kDa" gene was identified.
  • ORF open reading frame
  • Table II details the nucleic acid sequence coordinates and homology data of the ORFs of the 39 kDa protein cluster.
  • ORF #3 (6702-7241): riboflavin synthase subunit beta ribH, riboflavin synthase subunit beta
  • Rhodobacter sphaeroides ATCC 17025, gb
  • KpsS Capsule polysaccharide export protein [Cell envelope biogenesis, outer membrane] a family of periplasmic proteins involved in polysaccharide biosynthesis and/or export.
  • L-U-I Capsule polysaccharide biosynthesis [Paracoccus denitrificans PD1222] Length 440 GENE ID: 4580860 Pden_1558
  • Paracoccus denitrificans PD1222 gb
  • Specimen preparation for the scanning electron microscopy 1. Grow a single colony over night in 2 ml of IM growth medium.
  • Bacteria were grown for 48 to 72 hours on growth medium plates.
  • the DNA sequence was determined with the ABI Prism 377 DNA Sequencer (Perkin Elmer) and processed with ABI sequence analysis software. Vector NTI Suit software InforMax, North Bethesda MD USA) was used for sequence analysis. Sequencing of every fragment was performed twice and for every analysis separate genomic DNA extraction and PCR reaction were conducted.
  • a Rifampicin resistant strain of P. marciusii was developed for gene transfer.
  • Cells of P. marcusii MHl were plated on LB medium with serial dilutions of Rifampicin (Sigma ca#R3501), a Rifamycin derivative.
  • Rifampicin Sigma ca#R3501
  • An antibiotic resistant strain was selected following spontaneous mutation. This strain was resistant to 50 ⁇ g/ml Rifampicin in LB medium. Expression of foreign genes in transformed P. marcusii has been demonstrated in this strain.
  • “Shuttle” vectors The “shuttle” vectors capable of replicating in both E. coli and P. marcusii were constructed using a conjugating strain of E. coli, harboring a suicide vector with no need to replicate in P. marcusii and a strong selective pressure (Kanamycin resistance).
  • Three recombinant plasmid vectors were constructed by combining sequences from P. marcusih natural plasmids with the transposon sequence from EZ -Tn5TM (Epicentre Biotechnologies, Madison Wisconson). Gel electrophoresis analysis of non- chromosomal DNA reveals at least six plasmids ranging 5 to 60 kb in size and the presence of > 100 kb mega-plasmid is presumed. On the basis of these sequences, "shuttle" plasmid vectors [PMSH3 (SEQ ID NO: 5), 8 (SEQ ID NO: 4), and 9(SEQ ID NO: 3, Fig. H)] capable of replication in both E.coli and P. marcusii were developed.
  • Vector PMSH9 (FIG. 11) also contains a homologue of the E.coli
  • Mob gene facilitating its conjugative transfer from E.coli to P. marcusii, further enhancing it's usefulness as a shuttle vector.
  • P. marcusii transformation protocol A protocol to introduce recombinant DNA into P. marcusii was developed using
  • Escherichia coli S 17-1 strain as a donor strain.
  • E. coli S 17-1 genotype [Tpr, Smr, recA thi pro hsdR-M+ RP4:2-Tc::Mu:Km Tn7, ⁇ pir ] (de, Lorenzo, V and Timmis, K. N. 1994).
  • E.coli S17-1 cells carrying the desired plasmid were grown from single colony in 3 ml LB medium with 50 ⁇ g/ml Streptomycin and 50 ⁇ g/ml Kanamycin* at 37 0 C , rolling at 200 rpm, to a cell density corresponding OD 6 oonm of 0.8 ⁇ 1.0.
  • heterologous, foreign genes in P. marcusii was the functional expression of the aminoglycoside 3 '-phosphotransferase gene from Tn5, conferring kanamycin resistance in the genetically modified P. marcusii. P. marcusii cells were transformed via conjugation with E.coli strain S 17-1 carrying PMSH9 plasmid, according to the transformation protocol. Orange-colored P. marcusii colonies were obtained on LB agar plates containing Rifampicin (50 ⁇ g/ml) and Kanamycin (50 ⁇ g/ml). Plasmid DNA was extracted from these clones and was electroporated to E.coli MC1061 cells.
  • Hybrid 6 A stable genetic element capable of both replicating in P .marcusii and of directing expression of foreign genes in P. marcusii was constructed, based on PMSH9 vector. By inserting a DNA fragment from pBluscript SK+ into PMSH9 the 11,200 bp vector "Hybrid ⁇ " (FIG. 11) was developed, having the advantages of Ampicillin resistance, CoIEl on (high copy number in E.coli) and a multiple cloning site.
  • Suicide plasmid pRL27 carries a Tn5 transposon with Kan r gene and a conditional Ori R6K, and a transposase gene, which enables random transposon insertion into bacterial genomes (Larsen, R. A. et al. 2002).
  • pRL27 was transferred via conjugation from E. coli to P. marcusii and Kanamycin + Rifampicin resistant colonies were selected.
  • a single insertion of Tn5 in the P. marcusii genome provided a stable kanamycin resistant phenotype.
  • Retrieval of the Tn5 -interrupted loci was achieved by genomic DNA purification from P. marcusii mutant clones, followed by restriction enzyme digestion (for example, BamHI) and self ligation (circularization) of the digested genomic DNA.
  • the self-ligated circular sequences (artificial plasmids) were transfected into cells of E. coli strain MC1061 [Genotype: hsdR2 hsdM+ hsdS ⁇ araD139 ⁇ (ara-leu)7697 ⁇ (lac)X74 gal ⁇ 15 galK16 rpsL (StrR) mcrA mcrBl , ⁇ ir] (Casadaban, M. J. and Cohen, S. N. 1980) via electroporation. Transformed E. coli cells were then selected on Kanamycin LB agar plates for Kanamicin resistance.
  • Plasmid DNA was extracted from the Kanamycin-resistant E. coli cells. Sequence analysis was carried out by the ABI prism 377 DNA sequencer (PerkinElmer, Waltham, MASS) using the following primers:
  • TpnRL13-2 5'-CAGCAACACCTTCTTCACGA (SEQ ID NO: 1)
  • TpnRL 17-1 5'-AACAAGCCAGGGATGTAACG (SEQ ID NO: 2)
  • genes and sequences identified include: (plasmid identifier-sequence length bp) p6m2-1132 789 bp(SEQ ID NO: 7), p6m2-1171 801 bp(SEQ ID NO: 8), p27m5-1132 815 bp(SEQ ID NO: 9), p27m5-1171 779 bp(SEQ ID NO: 10), p27m6-L132 824 bp(SEQ ID NO: 11), p27m6-1171 527 bp(SEQ ID NO: 12), p4ml-1132 809 bp(SEQ ID NO: 13), p4ml-1171 755 bp(SEQ ID NO: 14), p5ml-L132 828 bp(SEQ ID NO: 15), p5ml-L171 536 bp(SEQ ID NO: 16), p27m2-L132 806 bp(SEQ ID NO: 17), p27m2- 11
  • Table III below lists the P. marcusii sequences recovered by "transposon tagging" with the transposon vectors developed for random mutagenesis and tagging.
  • the genes are identified as putative P. marcusii secretory genes, originating from the 39 kDa secretory particle protein gene cluster, and having homology (similarity) with specific genes from other, closely related bacterial species.
  • Mutant 140 was isolated as a white colony. Carotenoid analysis indicated that it did not contain any pigments (Table IV). The mutation that abolished carotenoid biosynthesis in mutant 140 had occurred in the gene crtE that encodes geranylgeranyl pyrophosphate synthase, an enzyme in an early step of the carotenoid biosynthesis enzyme (FIG. 12).
  • mutant 140 does not produce carotenoids it is evident by S ⁇ M that it is capable to form forms and secrete particles that emerge on the surface of the cells (FIG. 13A and 13B). This result indicates that particle formation and secretion may occur without sequestration and accumulation of carotenoids.
  • Table IV Accumulation of carotenoids in overproducing and non-producing mutant strains of P. marcusii. Cultures were harvested at stationary phase.
  • Bacterial strains P. marcusii Wt Rif- carotenoid producing, Rifampicin resistant strain by selected as described above.
  • Plasmids and constructs Plasmids and constructs: pABWl (RPK)- Plasmid pABWl(Re ⁇ -Par KiI) was felicitly provided by Dariusz Bartosik. pABWl (Dziewit et al. 2007, J Bacteriol;
  • pABW is an effective vector for a range of bacteria in the Paracoccus group (Dziewit et al. 2007).
  • the most important element in pABWl is a novel stabilizing element (tad-atd) encoded by plasmid pAMI2 of Paracoccus aminophilus JCM 7686.
  • the nucleotide sequence of the pAMI2 replicon has been deposited in the GenBank database under accession number DQ855273.
  • pABWl(RPK)-crtB- pABWl(RPK) with P. marcusii phytoene synthase.
  • pABWl(RPK)-crtB inverted- ⁇ ABWl(RPK) with inverted orientation P. marcusii phytoene synthase, non-functional.
  • pEIB ⁇ crtB- carries deletion of the crtB sequence from plasmid pEIB, for color complementation in E. coli. Protocols
  • E. coli color complementation crtB gene was amplified by PCR from native P. marcusii and cloned into plasmid pABWl(RPK) using standard cloning techniques. The resulting construct (pABWl(RPK)-crtB) was electroporated into E. coli strain
  • P. marcusii pABWl-crtB construct was electroporated into E. coli strain S 17-1 and selected for Kanamycin resistance. Conjugation was carried out as described above between donor (E. coli S17I(pABWl-crtB) and acceptor P. marcusii strain IL#125 "White mutant", and carotenoid synthesis (color complementation) was assessed. Results
  • Electroporation of the P. marcusii crtB- bearing construct pABWl(RPK)-crtB into E.coli strain XLlBlue containing crtB null plasmids resulted in lycopene synthesis and color complementation (FIG. 14A).
  • E. coli electroporated with a control construct with crtB in inverse orientation did not display any complementation, indicating the capability of functional complementation of carotenoid synthesis by the P. marcusii crtB gene.

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Abstract

L’invention concerne des espèces bactériennes Paracoccus marcusii génétiquement modifiées destinées à être utilisées dans la production de biomatériaux, notamment des matériaux lipophiles et des protéines qui sont synthétisés et accumulés dans les cellules bactériennes, et/ou sécrétées dans le milieu de croissance. L’invention concerne également des vecteurs et des structures d’acides nucléiques destinés à la manipulation génétique de bactéries en général et de P. marcusii en particulier.
PCT/IL2009/000555 2008-06-03 2009-06-03 Bacterie issue de paracoccus marcusii exprimant une voie de sequestration et de secretion et utilisations associees WO2009147673A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103966138A (zh) * 2014-05-14 2014-08-06 北京农业生物技术研究中心 孟加拉副球菌n74-1及其在水产养殖水质净化中的应用
WO2015136467A1 (fr) * 2014-03-12 2015-09-17 Uniwersytet Warszawski Plasmide pcrt01 et sa construction, nouvelles souches bactériennes, utilisations de ces dernières et procédés de production de caroténoïdes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0635576A1 (fr) * 1993-07-22 1995-01-25 NIPPON OIL Co. Ltd. Bactéries de genre nouveau et procédé de production de caroténoides par utilisation de ces bactéries
WO1999006586A1 (fr) * 1997-07-29 1999-02-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Nouvelles especes bacteriennes produisant des carotenoides et procede de production de carotenoides au moyen de ces nouvelles especes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0635576A1 (fr) * 1993-07-22 1995-01-25 NIPPON OIL Co. Ltd. Bactéries de genre nouveau et procédé de production de caroténoides par utilisation de ces bactéries
WO1999006586A1 (fr) * 1997-07-29 1999-02-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Nouvelles especes bacteriennes produisant des carotenoides et procede de production de carotenoides au moyen de ces nouvelles especes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BARTOSIK DARIUSZ ET AL: "Identification and characterization of transposable elements of Paracoccus pantotrophus.", JOURNAL OF BACTERIOLOGY, vol. 185, no. 13, July 2003 (2003-07-01), pages 3753 - 3763, XP002543304, ISSN: 0021-9193 *
DONOVAN P KELLY ET AL: "The genus paracoccus", THE PROKARYOTES: VOLUME 5: PROTEOBACTERIA: ALPHA AND BETA SUBCLASSES, SPRINGER-VERLAG, NEW YORK, NY, vol. 5, 1 January 2006 (2006-01-01), pages 232 - 249, XP008107202, ISBN: 978-0-387-30745-9, [retrieved on 20061127] *
DZIEWIT LUKASZ ET AL: "The SXT conjugative element and linear prophage N15 encode toxin-antitoxin-stabilizing systems homologous to the tad-ata module of the Paracoccus aminophilus plasmid pAM12", JOURNAL OF BACTERIOLOGY, vol. 189, no. 5, March 2007 (2007-03-01), pages 1983 - 1997, XP002543306, ISSN: 0021-9193 *
MIKOSA MALGORZATA ET AL: "Identification of a transposable genomic island of Paracoccus pantotrophus DSM 11072 by its transposition to a novel entrapment vector pMMB2", MICROBIOLOGY (READING), vol. 152, no. Part 4, April 2006 (2006-04-01), pages 1063 - 1073, XP002543305, ISSN: 1350-0872 *
SZUPLEWSKA MAGDALENA ET AL: "Identification of a mosaic transposable element of Paracoccus marcusii composed of insertion sequence ISPmar4 (ISAs1 family) and an IS1247a-driven transposable module (TMo)", FEMS MICROBIOLOGY LETTERS, vol. 292, no. 2, March 2009 (2009-03-01), pages 216 - 221, XP002543307, ISSN: 0378-1097 *
XU X Y ET AL: "Cloning and sequencing of a gene encoding a novel salt stress-induced membrane protein from Rhodobacter sphaeroides f. sp. denitrificans", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 56, no. 3-4, August 2001 (2001-08-01), pages 442 - 447, XP002543308, ISSN: 0175-7598 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015136467A1 (fr) * 2014-03-12 2015-09-17 Uniwersytet Warszawski Plasmide pcrt01 et sa construction, nouvelles souches bactériennes, utilisations de ces dernières et procédés de production de caroténoïdes
CN103966138A (zh) * 2014-05-14 2014-08-06 北京农业生物技术研究中心 孟加拉副球菌n74-1及其在水产养殖水质净化中的应用
CN103966138B (zh) * 2014-05-14 2016-03-30 北京农业生物技术研究中心 孟加拉副球菌n74-1及其在水产养殖水质净化中的应用

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