WO2014151723A1 - Utilisation de nucléases thermophiles pour la dégradation d'acides nucléiques - Google Patents

Utilisation de nucléases thermophiles pour la dégradation d'acides nucléiques Download PDF

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WO2014151723A1
WO2014151723A1 PCT/US2014/026317 US2014026317W WO2014151723A1 WO 2014151723 A1 WO2014151723 A1 WO 2014151723A1 US 2014026317 W US2014026317 W US 2014026317W WO 2014151723 A1 WO2014151723 A1 WO 2014151723A1
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nuclease
host cell
cell
thermophilic
dna
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Joshua Trueheart
Jessica MCGRATH
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DSM IP Assets BV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
    • C12Y301/21003—Type I site-specific deoxyribonuclease (3.1.21.3)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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

Definitions

  • This invention relates to a method for degrading the nucleic acids of a host cell in vivo and/or in situ, in particular when the host cell comprises a recombinant DNA, using a heterologous thermophilic nuclease.
  • the present invention is beneficial in inactivating the biological activity of recombinant DNA in a biomass.
  • the inactivation of the biological activity of recombinant DNA helps to prevent active recombinant DNA molecules from remaining in the end product isolated from the biomass or in the biomass itself.
  • the inactivation of the biological activity of recombinant DNA helps to prevent active recombinant DNA molecules from being released into the environment.
  • Biotechnological production processes are increasingly employed to obtain biological compounds and fine chemicals.
  • the progress of molecular biology techniques makes possible the mass production of a wide variety of biological compounds and fine chemicals such as proteins, antibodies, polysaccharides, antibiotics, amino acids, vitamins, alcohols, etc.
  • the gene producing the desired end product(s) is genetically modified and/or introduced into a heterologous organism.
  • the production of the desired end product(s) then takes place in fermenters controlled by modern control techniques.
  • the end products are either the cells themselves, extracted from the cells, or collected from the cell culture broth if the compounds are found therein (either by active or passive processes).
  • the biomass produced at the end of the fermentation process contains the desired end product(s) but also active DNA molecules.
  • the active DNA molecules are recombinant DNAs. If left untreated, the recombinant DNA molecules could remain in the isolated end product and could also be released into the environment.
  • concerns about the potential impact of the recombinant DNA on the environment has caused the authorities and institutions in most countries to issue statutory requirements and regulations calling for inactivation of the waste materials produced from the fermentation process before it being released into the environment.
  • nucleic acids can be inactivated physically, and most commonly, by heat.
  • U.S. Patent No. 5,417,862 reports a method of inactivating the biological activity of DNA by heating the DNA to 60 °C to 100 °C in the presence of an acid.
  • a similar method of degrading DNA by a combination of heat and acid is reported in U.S. Patent No. 5,118,603.
  • the heating methods require a large amount of energy. In a large scale fermentation process, this method can be especially cost-intensive.
  • the harsh heat (and/or acidic) conditions could be detrimental to the integrity and activity of said product.
  • the nucleic acids can also be inactivated by mechanical means.
  • 6,165,711 reports a method using laser beams for disintegrating nucleic acids in a biologically active proteinaceous material.
  • a method for inactivating microorganism using high-intensity pulsed polychromatic light is reported in U.S. Patent Application No.
  • the nucleic acids can also be inactivated chemically by acids or alkali.
  • acids or alkali for example, in U.S. Patent No. 7,435,567, a method using hypochlorous acids for induction of autodigestion of nucleic acids in a microorganism is reported.
  • U.S. Patent Nos. 5,417,862 and 5,118,603 described above use other types of acid for the degradation of nucleic acids. While these methods cause the disruption of nucleic acids, the acid and alkali conditions are severe. The severe condition may cause the unwanted denaturation of certain biological compounds such as proteins.
  • the amount of the acid and alkali used can be relatively large, which makes the method disadvantageous from the industrial production viewpoint as well.
  • U.S. Patent Application No. 13/127,825 reports a method for degrading host cell nucleic acids associated with vaccine production, where the method comprises a step of nucleic acid degradation by adding purified nuclease into the cell culture.
  • U.S. Patent Application No. 10/607,903 the construction of a transgenic bacterial strain expressing a heterologous nuclease gene in an amount effective to degrade nucleic acids is reported. While the method of adding nuclease in vitro such as the one reported in U.S. Patent Application No.
  • 13/127,825 causes disruption of nucleic acids, the cost is high since large amount of nuclease are required, and the efficiency of degradation is low because the cell wall of the host cell blocks the access to nucleic acids by the nuclease when added in vitro.
  • the transgenic approach such as the one reported in U.S. Patent Application No. 10/607,903 allows the nuclease to be co-expressed with the host cell and thus gain access to the host nucleic acids in vivo.
  • co-expressing a nuclease in a cell without any protection mechanism, such as methylation will significantly stress the cell, resulting in weakened cell growth and reduced production of end product.
  • the problem underlying the present invention is therefore to provide a cost-saving way to degrade the nucleic acids of a host cell that produces biological compounds and fine chemicals, especially on an industrial production scale.
  • a further problem underlying the present invention is to provide a method in which the nucleic acids degradation process is controllable and does not impede the production of the desired end product(s) in the host cell.
  • thermophilic nucleases can be used for degrading nucleic acid in vivo and/or in situ in a controlled manner.
  • This invention relates to a novel method for degrading nucleic acids of a host organism where the host organism is modified or transformed with a heterologous thermophilic nuclease gene.
  • Thermophilic nuclease is latent at temperatures at which the cell culture is grown normally to produce the desired product, but can be selectively activated at a higher temperature and thus degrades the nucleic acids of the host organism once activated.
  • the activation can be triggered at any desired time point, such as at the harvest time of the cell culture when product formation is already complete.
  • the application of this inventive process has the benefit of acting in vivo and/or in situ and thus improving the efficiency of the enzymatic reaction and avoids the drawbacks and problems of the existing physical or chemical methods. It has thus been made possible, by application of the disclosed invention, to obtain biological compounds and fine chemicals in the form of intact cells where the active nucleic acids content of the cell, especially recombinant DNAs, is degraded and inactivated.
  • the practice of this invention is broadly applicable to both microorganisms and higher eukaryotic cell cultures, and particularly industrial strains used in a large scale production of commercial end products.
  • One aspect of the invention relates to a method for degrading nucleic acids in vivo and/or in situ, where the method comprises: a) introducing the gene of a thermophilic nuclease into a host cell; b) growing the host cell at a temperature at which the thermophilic nuclease is latent; and c) degrading the nucleic acids of the host cell by changing the temperature in step (b) to a temperature at which the thermophilic nuclease is active.
  • the present invention also provides a genetically modified cell which comprises a heterologous nucleic acid sequence encoding the thermophilic nuclease gene described above, wherein the genetically modified cell produces one or more end products, and the end products produced by the genetically modified cell are not the above thermophilic nuclease.
  • the genetically modified cell is created by introducing the heterologous thermophilic nuclease gene described above into a host cell.
  • This host cell can be either a cell which has not been genetically modified, or a cell which has been genetically modified.
  • This host cell before being modified with the heterologous thermophilic nuclease gene, produces one or more end products wherein said one or more end products are not the thermophilic nuclease.
  • the above mentioned host cell may be introduced first with thermophilic nuclease gene to create a genetically modified cell, and the genetically modified cell is subsequently genetically engineered to produce one or more other end products.
  • Another aspect of the invention relates to the use a thermophilic nuclease for degrading the nucleic acids of a host cell in vivo and/or in situ, wherein the thermophilic nuclease is heterologous to the host cell.
  • Yet another aspect of the invention relates to a process for the production of a biomass product which is free of active nucleic acid molecules, wherein the process comprises: a) introducing a gene of a thermophilic nuclease into a host cell wherein the host cell is and/or produces the biomass product; b) fermenting the host cell at a temperature at which the thermophilic nuclease is latent; c) degrading the nucleic acids of the cell by changing the temperature in step (b) to a temperature at which the thermophilic nuclease is active; and d) recovering the biomass product, wherein the order for performing steps c) and step d) may be exchanged.
  • the nuclease mentioned in the above aspects of invention is a DNA- degrading nuclease and the nucleic acids of the host cell are DNA.
  • the DNA of the host cell contains recombinant DNA.
  • the nuclease is a RNA-degrading nuclease and the nucleic acids the host cell are RNA.
  • the temperature in step (a) of the above process invention and the method invention is optimal for the growth of the host cell.
  • the degradation is conducted at a temperature that is within about ⁇ 5 °C of the optimum temperature of said thermophilic nuclease. In a specific embodiment, the degradation is conducted at the optimum temperature of said thermophilic nuclease.
  • the DNA-degrading nuclease is Taql nuclease.
  • the degradation is conducted at a temperature ranging between about 60 °C and about 70 °C. In a specific embodiment, the degradation is conducted at 65 °C.
  • the host cell is from a plant. In another embodiment, the host cell is from an animal. In yet another embodiment, the host cell is from a microorganism. In one embodiment, the microorganism is selected from a group consisting of: yeast, fungi, algae, and bacteria. In another embodiment, the microorganism is selected from a group consisting of: Yarrowia, Bacillus, Escherichia, Pseudomonas, Candida, Hansenula, Saccharomyces, Mortierella, Schizosaccharomyces, Aspergillus, Fusarium, Trichoderma, Crypthecodinium, Schizochytrium, and Thraustochytrium. In a specific embodiment, the Yarrowia species is Yarrowia lipolytica.
  • the host cell produces one or more end products that is not the thermophilic nuclease.
  • the end product is selected from the group consisting of: phytoene, lycopene, beta-carotene, alpha-carotene, beta-cryptoxanthin, lutein, zeaxanthin, astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin, violaxanthin, and adonixanthin.
  • the gene of the thermophilic nuclease is codon optimized to match the codon usage bias of the host cell.
  • the codon optimized thermophilic nuclease gene comprises the nucleic acid sequence of SEQ ID NO:l .
  • the invention relates to the use of Taql nuclease for degrading recombinant DNA of a Yarrowia lipolytica strain in vivo and/or in situ.
  • SEQ ID NO: l is the DNA sequence encoding Taql nuclease from Thermus aquaticus, as optimized for expression in Yarrowia lipolytica, and includes restriction sites for cloning and a 5' ribosomal binding site.
  • SEQ ID NO:2 is the non-optimized DNA sequence encoding Taql nuclease, as isolated from Thermus aquaticus .
  • SEQ ID NO:3 is the amino acid sequence of Taql nuclease, as deduced from SEQ ID NO: l .
  • SEQ ID N0:4 is the DNA sequence of the heterologous carB open reading frame harbored by a Yarrowia lipolytica strain.
  • SEQ ID NO:4 carB gene
  • SEQ ID NOs:5 and 6 are the 5' and 3' PCR primers for a 529 bp fragment within SEQ ID NO:4.
  • SEQ ID NO:5 primer M04641 : caatctgttgcctccctgc
  • SEQ ID NO:6 primer M04642: atcctttgtgctgggacgg
  • SEQ ID NO: 7 is the DNA sequence of the heterologous crtZ-Dc open reading frame harbored by a Yarrowia lipolytica strain.
  • Fig. 1 shows the time course of DNA degradation activity in Y. lipolytica strains with or without the taql nuclease gene.
  • Fig. 2 shows DNA degradation activity at different temperatures in Y. lipolytica strains with or without the taql nuclease gene.
  • Fig. 3 shows the completeness of DNA degradation based on PCR result.
  • Fig. 4 shows the production of astaxanthin and its precursors in Y. lipolytica strains with and without the taql nuclease gene.
  • Fig. 5 shows the production of zeaxanthin and its precursors in a Y. lipolytica strain which has the taql nuclease gene.
  • Fig. 6 shows the time course for degradation of DNA in Y. lipolytica strains producing astaxathin and zeaxanthin in fermentors.
  • the present invention provides a method of degrading nucleic acids in vivo and/or in situ using a heterologous thermophilic nuclease.
  • the method is intended to degrad4 the
  • thermophilic nuclease gene is introduced into the organism in which nucleic acids are intended to be degraded.
  • the thermophilic nuclease is latent at the normal growth temperature of the organism, but can be activated by raising the
  • thermophilic nuclease is used to inactivate the biological activity of the nucleic acids of the host organism.
  • the term "inactivate the biological activity of nucleic acids” is considered to refer to the degradation of nucleic acids by the present invention.
  • nucleic acid is recombinant DNA.
  • nucleic acid will be cleaved into units which are 500 base pairs or less.
  • thermophilic nuclease is produced by the host cell rather than being added exogenously, and thus degrades the nucleic acids of the host cell in vivo and/or in situ.
  • in vivo refers to the degradation of nucleic acids inside the host cell by the thermophilic nuclease produced by the cell, which may or may not be viable at the time of degradation.
  • m situ refers to the degradation of nucleic acids by the thermophilic nuclease produced by the host cell after the cell wall of the host cell is broken (fully or partially).
  • an in situ degradation can be performed at the end of the fermentation process where cells are lysed to release their contents. Degradation can be performed immediately after the cells are lysed or after some additional processing of the lysed broth has already occurred.
  • nucleic acid denotes single stranded, double-stranded, or partially double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • thermophilic nuclease gene generally refers to a nucleic acid encoding a polypeptide, optionally including certain regulatory elements that may affect expression of one or more gene products (i.e., RNA or protein).
  • a thermophilic nuclease gene refers to the open reading frame (ORF) encoding a polypeptide of the thermophilic nuclease, and also to nucleic acid sequences which encode the ORF of thermophilic nuclease together with certain promoters which are placed upstream of the ORF to affect the expression of nuclease gene and/or a region for termination of transcription.
  • heterologous refers to a gene or a polypeptide that does not naturally occur in the organism in which it is being expressed. It is understood that, where a heterologous polypeptide is to be expressed in a host cell, it will often be desirable to utilize nucleic acid sequences encoding the polypeptide that have been adjusted to accommodate codon preferences of the host cell and/or to link the encoding sequences with regulatory elements active in the host cell.
  • a gene sequence encoding a given polypeptide is altered to conform more closely with the codon preference of a species related to the host cell.
  • Such embodiments are advantageous when the gene sequence encoding a given polypeptide is difficult to optimize to conform to the codon preference of the host cell due to experimental (e.g., cloning) and/or other reasons.
  • the gene sequence encoding a given polypeptide is optimized even when such a gene sequence is derived from the host cell itself (and thus is not
  • a gene sequence encoding a polypeptide of interest may be codon optimized for expression in a given host cell even though such a gene sequence is isolated from the host cell strain.
  • the gene sequence may be further optimized to account for codon preferences of the host cell.
  • the (non-heterologous) gene sequence might not be codon optimized but instead be linked to a regulatory element other than its own (whether that regulatory element comes from another gene in the host or from another species).
  • the "host cell” means any cell type that is susceptible to introduction of a nucleic acid construct or expression vector.
  • the means of introduction of the nucleic acid construct or expression vector can be in the form of transformation, transfection, transduction, and the like.
  • the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • the host cells produce one or more end products which are desired biological compounds or fine chemicals. It is preferred that the host cell is an industrial strain.
  • the host cell can be a genetically modified cell or a natural cell which was non-genetically modified.
  • a genetically modified host cell refers to a host cell which has been modified, engineered, or manipulated, often to overexpress certain biological compounds or fine chemicals.
  • the host cell may be any cell useful in the recombinant production of biological compounds and/or fine chemicals in a prokaryote or a eukaryote.
  • biological compounds is known in the art and includes compounds which are the building blocks of an organism.
  • biological compounds include, but are not restricted to: proteins, polypeptides, amino acids, nucleic acids, nucleotides, carbohydrates, and lipids.
  • fine chemical is known in the art and includes compounds which are produced by an organism and are used in various branches of industry such as, for example but not restricted to, the pharmaceutical industry, the agriculture, cosmetics, food and feed industries. These compounds include organic acids such as, for example, tartaric acid, itaconic acid and diaminopimelic acid, lipids, saturated and unsaturated fatty acids (e.g. arachidonic acid), diols (e.g. propanediol and butanediol), aromatic compounds (e.g. aromatic amines, vanillin and indigo), carotenoids, vitamins and cofactors.
  • organic acids such as, for example, tartaric acid, itaconic acid and diaminopimelic acid
  • lipids saturated and unsaturated fatty acids (e.g. arachidonic acid), diols (e.g. propanediol and butanediol), aromatic compounds (e.g. aromatic amines, vanillin and indigo), caroten
  • vitamins include vitamins, carotenoids, cofactors and nutraceuticals and therefore need to take them in, although they are easily synthesized by other organisms such as bacteria.
  • These molecules are either biologically active molecules per se or precursors of biologically active substances which serve as electron carriers or intermediates in a number of metabolic pathways. These compounds have, besides their nutritional value, also a significant industrial value as coloring agents, antioxidants and catalysts or other processing aids.
  • vitamin is known in the art and includes nutrients which are required by an organism for normal functioning, but cannot be synthesized by this organism itself.
  • the group of vitamins may include cofactors and nutraceutical compounds.
  • cofactor includes non-protein compounds which are necessary for the occurrence of normal enzymatic activity.
  • These compounds may be organic or inorganic; the cofactor molecules of the invention are preferably organic.
  • the term "nutraceutical” includes food additives which promote health in organisms and animals, especially in humans. Examples of such molecules are vitamins, antioxidants and likewise certain lipids (e.g. polyunsaturated fatty acids).
  • Preferred fine chemicals or biosynthetic products which can be produced in organisms of the genus Yarrowia are carotenoids such as, for example, phytoene, lycopene, beta-carotene, alpha-carotene, beta-cryptoxanthin, lutein, zeaxanthin, astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin, violaxanthin and adonixanthin.
  • carotenoids such as, for example, phytoene, lycopene, beta-carotene, alpha-carotene, beta-cryptoxanthin, lutein, zeaxanthin, astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin, violaxanthin and adonixanthin.
  • the prokaryotic host cell may be any gram-positive or gram-negative bacterium.
  • Gram- positive bacteria include, but are not limited to, Bacillus, Brevibacillus, Clostridium, Geobacillus, Lactobacillus, Lactococcus, Paenibacillus, and Streptomyces.
  • Gram-negative bacteria include, but are not limited to E. coli and Pseudomonas.
  • the bacterial host cell may be any Bacillales cell including, but not limited to, Bacillus amyloliquefaciens, Brevibacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lentus, Bacillus licheniformis, Geobacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
  • the bacterial host cell may also be any Streptomyces cell including, but not limited to,
  • Streptomyces achromogenes Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
  • the host cell may also be a eukaryote, such as a mammalian, insect, plant, algal, or fungal cell.
  • the host cell may be a fungal cell.
  • "Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et ah, In, Ainsworth and Bisby 's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et ah, 1995, supra, page 171) and all mitosporic fungi (Hawksworth et ah, 1995, supra).
  • the fungal host cell may be a yeast cell.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series ⁇ o. 9, 1980).
  • the yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
  • the algal host cell may be a Crypthecodinium, Schizochytrium, and Thraustochytrium cell such as a Crypthecodinium cohnii, Schizochytrium sp. or Thraustochytrium sp. cell.
  • the fungal host cell may be a filamentous fungal cell.
  • "Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
  • the filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
  • the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zona
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al, 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al, 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
  • thermophilic nuclease gene construct is inserted into a host specific vector which allows optimal gene expression in the host.
  • Vectors are well known in "Cloning Vectors" (Pouwels et al, Eds., Elsevier, Amsterdam-New York-Oxford, 1985). Vectors are to be understood as meaning not only plasmids, but all other vectors known to the skilled worker such as, for example, phages, viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, plasmids, cosmids, and linear or circular DNA. These vectors can be replicated autonomously in the host organism or chromosomally.
  • the vectors according to the invention allow the generation of recombinant organisms which are transformed, for example, with at least one vector according to the invention and which can be employed for producing the thermophilic nuclease.
  • the above-described recombinant constructs according to the invention are advantageously introduced into the genome of the host organism and expressed. It is preferred to use usual cloning and transfection methods known to the skilled worker in order to bring about expression of the thermophilic nucleic acids in the expression system in question. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al, Eds., Wiley Interscience, New York 1997.
  • the modified host cell contains a plasmid carrying the
  • thermophilic nuclease gene replicates autonomously in the host organism.
  • DNA is considered to be "recombinant” if it results from the application of Recombinant DNA Techniques.
  • recombinant techniques include but are not limited to cloning, mutagenesis, transformation, etc.
  • Recombinant DNA Techniques are disclosed, for example, in Sambrook, et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York (1989) and in Ausubel FM, et al, Current Protocols in Molecular Biology, Wiley, New York (1998).
  • nuclease denotes an enzyme that effects hydrolytic cleavage of the ester bond between the 5 '-phosphate group of a nucleotide and the 3 '-hydroxy group of the adjacent nucleotide in a nucleic acid and therefore accomplishes the degradation of a DNA or RNA molecule.
  • Nucleases are known from numerous organisms. Nucleases cleave either RNA or DNA to smaller units or even to their monomers.
  • thermophilic nuclease according to the present invention is a DNA-degrading nuclease
  • the nucleic acid which the thermophilic nuclease degrades is DNA.
  • the DNA being degraded is a recombinant DNA.
  • inactivation of DNA inactivation of any nucleic acid molecule can be achieved. This includes inactivation of RNA or nucleic acid-protein conjugates such as ribozymes.
  • DNA-degrading nuclease means a nuclease which is able to cleave DNA molecules into smaller units or monomers. Such enzyme is also termed “DNase” in the state of art.
  • RNA- degrading nuclease is a nuclease that is able to cleave RNA molecules into smaller units or monomers. Such enzymes are also termed “RNase” in the state of art.
  • the DNA nuclease can be an endonuclease or an exonuclease.
  • An endonuclease is an enzyme that degrades a nucleic acid chain from a position inside the chain.
  • an exonuclease is an enzyme that degrades a nucleic acid chain from one or both ends of the chain. It is preferred for the nuclease to be an endonuclease.
  • Thermophilic nuclease means a nuclease which has its optimal activity at a temperature of 45°C or higher. Said optimal activity is observed at a temperature that is optimal for the activity of the nuclease.
  • the optimal temperature may be determined for the nuclease in an in vitro assay, or in an in vivo assay where the nuclease is expressed in the recombinant host according to the present invention, using a suitable substrate and under assay conditions (in terms of medium composition, assay time, etc.) that are relevant for the intended application, as determined by a person skilled in the art.
  • the temperature optimum of a nuclease such as TaqI is typically determined in vitro using the purified TaqI nuclease.
  • a fixed quantity of enzyme can be incubated with one microgram of lambda DNA in a fifty microliter reaction held at a range of temperatures (typically from 25 to 85 °C) for one hour.
  • the optimum temperature is the one at which the most digestion is observed (for example, as assayed by gel electrophoresis of the resulting lambda DNA TaqI restriction fragments).
  • one unit of enzyme is defined as the amount of protein required to fully digest one microgram of lambda DNA in a fifty microliter reaction in one hour at the optimum temperature.
  • the optimum temperature is the temperature at which the quantity of protein required to achieve full digestion is the lowest.
  • a defined ratio of enzyme to DNA substrate (as used in in vitro assays) cannot be guaranteed. Instead, a more pragmatic approach is taken.
  • the in vitro defined optimum can be used as a guide. Nuclease acid digestion by the nuclease is examined at and around the in vitro optimal temperature. The optimal temperature is the one at which the most digestion is observed. For some nucleases, the in vivo optimal temperature is a range of temperatures rather than a single point, as complete digestion of genomic DNA can be observed throughout the temperature range.
  • thermophilic nucleases used in the present invention include but not limited to: isolation from thermophiles, modification of homologous nuclease to acquire thermophilic activity by rational enzyme engineering and/or directed evolution.
  • thermophiles are heat-loving organisms with an optimum growth temperature of 50°C or higher.
  • the majority of cells grows at a lower temperature and cannot survive at the high temperature preferred by thermophiles.
  • common microbes grow at a temperature between 25 and 40 °C
  • mammalian cell cultures grow at around 37 °C
  • plant and insect cultures grow at their respective low temperatures which were known in the art.
  • thermophilic nucleases lose activity quickly outside their optimum temperature and have low to no activity at the temperature at which common microbes and cell cultures grow.
  • the levels of nuclease activity of each thermophilic nuclease at a low temperature are different and thus have to be determined individually.
  • a thermophilic nuclease is considered latent at a low temperature if the nuclease activity is reduced to less than 20% of the peak activity at the optimum reaction temperature of the nuclease (either commonly known or can be determined experimentally under relevant conditions).
  • the method of the invention is amenable to a wide range of thermophilic nucleases. Any nuclease which is latent at the normal growth temperature of the host microorganisms or eukaryotic cell cultures but is highly active at its own optimum reaction temperature is suitable for use in the present invention.
  • thermophilic nucleases are less suited for the present invention than others. For example, some thermophilic nucleases have low level nuclease activities at low temperatures between 25 and 37 °C. Such residual activities at the growth temperature of the host cell may constrain the growth of the host cells and thus hinder the production of their end product. Some thermophilic nucleases may not have strong nuclease activity at their optimum temperature and thus are inefficient in DNA degradation. Thus, a screening process may be performed to select those thermophilic nucleases which work best in the present invention.
  • thermophilic nuclease candidates are those whose nuclease activities are low enough at the normal growth temperature so as not to impose any significant stress on the host organism, and are high enough at the optimum reaction temperature to perform efficient degradation of the nucleic acids of the host.
  • thermophilic nuclease according to the invention can be used advantageously in degrading nucleic acids of the host cell in a controlled manner.
  • the present invention discloses that a heterologous thermophilic nuclease gene is introduced into a host cell and thus is used to degrade the nucleic acid of a host cell.
  • the thermophilic nuclease gene is introduced into a host cell whose nucleic acids are to be degraded. The host cell is grown at its optimum growth temperature at which the heterologous thermophilic nuclease is latent.
  • the activity of the thermophilic nuclease in the host call can be activated by switching the temperature to one which is optimal for the nuclease activity.
  • the thermophilic nuclease By activating the thermophilic nuclease, the nucleic acids of the host cell are degraded by the nuclease, thus eliminating activities of the nucleic acids of the host cell.
  • thermophilic nuclease gene instead of using a heterologous thermophilic nuclease gene, the method according to the present invention could use a homologus nuclease of the host cell which has been made thermophilic using methods such as rational enzyme engineering and/or directed evolution.
  • thermophilic nuclease is introduced into a host cell and thus is used to degrade the nucleic acid of a host cell.
  • two or more thermophilic nuclease is introduced into a host cell and thus is used to degrade the nucleic acid of a host cell.
  • thermophilic nuclease are introduced into a host cell.
  • thermophilic nucleases may, for example, enhance the efficiency of the degradation of the nucleic acid.
  • the above method of nucleic acid degradation can be carried out at any scale ranging from lab bench to commercial scale fermentation.
  • DNA of the host cell especially recombinant DNA
  • the present invention provides a process for degrading the recombinant DNA in the biomass and thus inactivating any recombinant DNA.
  • the biomass can be, for example, cells harvested from cultivation of microorganism or higher eukaryotic cell culture.
  • the invention provides a fermentation process by which the recombinant DNA in the biomass is inactivated.
  • the methodological steps for such process are exemplified as follows: First, the gene encoding the thermophilic nuclease according to the invention is introduced into the host cell. Integration of a nucleic acid construct containing the gene of a thermophilic nuclease into the host cell can take place intrachromosomally or extrachromosomally. Second, the host cell is grown in a fermentor at a temperature which is optimal for the growth of the host cell. Third, at the end of the fermentation process, the temperature of the fermentor is raised to the optimum temperature of the thermophilic nuclease and thus activates the nuclease.
  • the higher temperature is maintained for a period of time to allow sufficient degradation of the DNA (recombinant or not) in the host cell. Subsequently, the biomass is collected and further processed to isolate the end product. In this embodiment, the amount of end product is not affected by the action of the nuclease or the heat treatment used to activate the nuclease.
  • the method disclosed in the present invention can also be used for degrading DNA in partially purified products.
  • the activation of the nuclease can be withheld until after the biomass is collected and further processed to obtain partially purified end products.
  • the thermophilic nuclease is then activated to degrade the DNA contained in the partially purified end products, which can then be further purified, if desired.
  • the process according to the invention can be used in many other technical fields in addition to fermentation.
  • the process according to the invention can be used for inactivating the DNA of the host cell during vaccine productions.
  • the process comprises the steps of: (a) introducing the gene of a thermophilic DNA nuclease into a host cell culture and thus creating a new host strain; (b) inoculating the population of the new host strain with a virus; (c) culturing the population of host cells so as to allow the virus to replicate, wherein the culturing is conducted at a temperature at which the thermophilic nuclease is latent; (d) degrading the DNA of the host cells by changing the temperature in step (c) to a temperature at which the
  • thermophilic DNA nuclease is active; (e) collecting the produced virus thereby providing a viral harvest, and (f) isolating the virus.
  • the order of steps (d) through (f) can be exchangeable.
  • the virus can be, for example, influenza virus. However, any virus or viral antigen can be produced according to the invention.
  • thermophilic nuclease according to the invention can be introduced into a genetically modified crop.
  • the crop is grown in the field at temperatures which normally do not exceed 50 °C.
  • After the crop is harvested, it can be, for example, processed at the optimal temperature of the above thermophilic nuclease in order to degrade the crop DNA.
  • the process according to the invention can also be used for reducing viscosity in cell lysate. It is known in the art that when cells are lysed, DNA is released from the cell and thus causes the cell lysate to become viscous. This hinders the further processing of cell lysate.
  • the thermophilic DNA degrading nuclease works by cleaving DNA into small oligonucleotide fragments and thus removing the viscosity caused by the DNA content in a controlled manner.
  • thermophilic nuclease that is used according to the invention can be, for example, Taql endonuclease from Thermus aquaticus. Taql has an optimum temperature of 65 °C.
  • Other thermophilic nucleases can be tested for their suitability of being used in the present invention.
  • additional thermophilic nucleases include: Tsp509I which has an optimum temperature of 65 °C, Mwol which has an optimum temperature of 60 °C, Phol which has an optimum temperature of 75 °C, BsaJI which has an optimum temperature of 60 °C, and BspQI which has an optimum temperature of 50°C.
  • thermophilic nuclease any existing thermophilic nuclease known in the state of art or any existing thermophilic nuclease known in the state of art or any existing thermophilic nuclease known in the state of art or any existing thermophilic nuclease known in the state of art or any
  • thermophilic nuclease yet to be identified can be used according to the invention, as long as such thermophilic nuclease has high nucleic acid cleavage activity at its optimum reaction temperature but is latent at the lower temperature at which the host microorganisms or cell cultures normally grow.
  • the temperature for degrading nucleic acid is the temperature known to have the optimal activity of the specific thermophilic nuclease.
  • one preferred temperature for degrading nucleic acids of the host cell using thermophilic nuclease Taql is 65 °C because Taql is known to be most active at 65 °C.
  • the optimum temperature according to the present invention is not limited to a single temperature point. According to the present invention, it is also preferred that the temperature for degrading nucleic acid is any temperature which is within a range of ⁇ 5 °C of the known optimum temperature of the thermophilic nuclease.
  • a preferred temperature is any temperature which falls between 60 °C and 70 °C. It is particularly preferred that a temperature which falls within ⁇ 1 °C, ⁇ 2 °C, ⁇ 3 °C, or ⁇ 4 °C of the known optimum temperature of the thermophilic nuclease is the temperature for activating the thermophilic nuclease.
  • the phrase "optimum temperature" of a thermophilic nuclease as used in the present invention refers to a temperature which is within ⁇ 5 °C of the known optimum temperature of the thermophilic nuclease.
  • the nucleic acid degradation process according to the present invention can be carried out relatively quickly and thus saves energy cost.
  • the process of nucleic acid degradation is carried out at the optimum temperature of the thermophilic nuclease for the duration of less than 48 hours, less than 24 hours, less than 1 hour, less than 40 minutes, less than 30 minutes, or less than 20 minutes.
  • the optimum temperature is broadly defined as including any temperature which is within a range of ⁇ 5 °C of the known optimum temperature of the thermophilic nuclease.
  • a preferred temperature is the lowest temperature in the temperature range.
  • the present invention can be used in any host cell.
  • host cells include microorganisms and higher eukaryotic cells such as agricultural crops or animal cells in a tissue.
  • the microorganisms can be for example prokaryotic cells, such as bacteria or archaebacteria, or eukaryotic cells, such as yeasts, lower or higher fungi, algae, or protozoa.
  • microorganisms are bacterial cells, fungal cells or yeast, or microalgal cells.
  • the organism whose nucleic acids can be degraded according to the method of the present invention is a microbe from a genus such as, but not limited to, Yarrowia, Bacillus, Escherichia, Pseudomonas, Candida, Hansenula,
  • the host cell in accordance with the present invention is Yarrowia lipolytica.
  • Advantages of Y. lipolytica include, for example, tractable genetics and molecular biology, availability of genomic sequence, suitability to various cost- effective growth conditions, and ability to grow to high cell density. There is already extensive commercial experience with Y. lipolytica.
  • Escherichia coli is useful because it is the most commonly used bacterial strain. It has been regularly used as a carrier strain for recombinant DNA.
  • Saccharomyces cerevisiae is also a useful host cell in accordance with the present invention, particularly due to its experimental tractability and the extensive experience that researchers have accumulated with the organism.
  • the host cell in accordance with the present invention also includes higher eukaryotic cells.
  • the term "higher eukaryotic cell” means a eukaryotic cell of a high state of development, such as those which occur for example in animal or plant organisms. Higher eukaryotic cells do not perform all vital biochemical and metabolic functions independently and thus are grown as cell culture.
  • the higher eukaryotic cell in accordance with the present invention comprises, inter alia, all cells from a mammal, a plant, an insect, or an avian. According to one
  • the higher eukaryotic cell is from a mammal. According to another embodiment of the invention, the higher eukaryotic cell is from a plant.
  • the degradation of the nucleic acids of the host cell is carried out without affecting the other cellular components of the host cell.
  • the degradation of the nucleic acids of the host cell is carried out without affecting the production of the biological compounds and/or fine chemicals which the host cell is engineered to produce.
  • the host cell is a Yarrowia strain and the biological compounds which are genetically engineered to produce include: phytoene, lycopene, beta- carotene, alpha-carotene, beta-cryptoxanthin, lutein, zeaxanthin, astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin, violaxanthin and adonixanthin.
  • the Yarrowia strain is a genetically modified Yarrowia strain.
  • the genetically modified Yarrowia strain cell introduced with a thermophilic nuclease gene is Yarrowia lipolytica strain ML 12924 and the biological compound produced is astaxanthin.
  • the genetically modified Yarrowia strain cell introduced with a thermophilic nuclease gene is Yarrowia lipolytica strain ML 12921 and the biological compound produced is zeaxanthin.
  • the genetically modified Yarrowia strain cell introduced with a thermophilic nuclease gene is Yarrowia lipolytica strain ML12805 and the biological compound produced is lycopene.
  • codon optimization is performed in order to enhance the heterologous nuclease gene in the host cell. It is known in the art that the expression of non- native genes is hampered by the existence of variation in their respective codon usage pattern compared to the host organism. To overcome these problems, codon optimization according to the present invention is performed on the thermophilic nuclease gene in order to match the host codon usage before the gene is introduced into the host cell. This process encompasses the replacement of rare codons within the DNA sequence of the nuclease gene in order to closely match the host codon usage bias while retaining 100% identity or near 100% identity to the original amino acid sequence. This process of codon optimization also allows for the
  • thermophilic nuclease of the present invention the codon usage pattern is altered from that typical of the thermophilic nuclease gene to modify the codons without altering the coded amino acid sequence.
  • the nucleic acid sequences for many thermophilic nuclease genes are known.
  • thermophilic nuclease gene segments were converted to sequences having identical translated sequences but with alternative codon usage.
  • the codon usage pattern of the native thermophilic nuclease Taql (SEQ ID NO:2) is optimized to the codon bias of Y. lipolytica.
  • the resulting codon modified Taql gene is specified in SEQ ID NO: 1.
  • the amino acid sequence encoded by SEQ ID NO: l and SEQ ID NO:2 is specified in SEQ ID NO:3.
  • codon modified taql gene illustrated in SEQ ID NO: 1 serves only as one example of many possible codon modified taql genes. It is clear to a skilled person that any suitable codon modification methods could be used to modify the thermophilic nuclease gene according to the present invention, and the resulting sequence from using different codon modified methods may vary. Having now generally described this invention, it will become more readily understood by referencing the following specific examples which are included herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
  • Table 1 Yarrowia lipolytica strains.
  • Yarrowia strains ML8195, ML12805, ML11956, ML12924, ML11218 and ML12921 were constructed by the introduction of heterologous genes under the control of the constitutive promoters (for example, TEF1), coupled with several generations of crossbreeding, starting with ML350 and ATCC201249 as described in U.S. Patent No. 7,851,199 B2.
  • the GGS gene and the truncated HMG gene (“H G-tr”) were derived from Yarrowia lipolytica sequences
  • the crtW gene was synthesized to encode the carotene ketolase of Parvularcula bermudensis.
  • the crtZ gene was amplified from Xanthobacter autotrophicus (Xa), or synthesized to encode the carotene hydroxylase from Enterobacter pulveris (Ep) or Enterobacteriaceae bacterium DC404 (Dc) (SEQ ID NO:7). These genes are sometimes but not always associated with auxotrophic markers (URA3, LEU2, URA2, LYS1, ADEl) or a loxP site, remnant of a Hyg R (hygromycin resistance) marker.
  • Example 1 Production of plasmids for strain construction.
  • Plasmids were generated for expression of Taql thermophilic nucleases as described in Table 2.
  • a codon optimized taql nuclease ORF sequence was synthesized de novo based on the sequence of the taql nuclease gene of Thermus aquaticus (SEQ ID NO:2), using the Y. lipolytica codon bias as specified in SEQ ID NO: l .
  • This codon-optimized taql nuclease ORF was cleaved using Nhel and Mlul and ligated to pMB5082 cut with Nhel and Mlul to produce pMB6722.
  • the resulting encoded Taql protein of pMB6722 is specified in SEQ ID NO:3.
  • a second plasmid the same taql nuclease gene sequence was excised from plasmid pMB6722 and cloned into a different plasmid backbone, pMB6157, to produce a new plasmid, pMB6736. Plasmid constructions were performed based on basic molecular biology and DNA manipulation procedures. All basic molecular biology and DNA manipulation procedures described in this and other examples are generally performed according to Sambrook et al. or Ausubel et al. (Sambrook J, Fritsch EF, Maniatis T (eds). 1989. Molecular Cloning: A
  • the gene encoding Taqi nuclease was introduced into a host strain of Y. lipolytica.
  • the Taqi nuclease activity in the host strain was tested.
  • Plasmid pMB6722 was cleaved with Xbal and transformed into host strain ML8195 (Table 1).
  • Yarrowia strain ML8195 is a strain which was previously genetically modified to produce lycopene.
  • Transformants introduced with the heterologous taqi nuclease gene were selected on YNB glutamate plates; one such colony was selected and designated as Yarrowia strain ML12805.
  • ML12805 was incubated for 3 days at 30 °C at 250 rpm in 20 mL YPD medium in a 125 mL flask. The cultures were split and 1.5 mL aliquots incubated at 65 °C or 25 °C for 5 min to 24 hr.
  • the pellet was resuspended in 0.2 mL Smash and Grab Solution (1% SDS, 2% Triton- X100, 100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA) and 0.2 mL Phenol-Chloroform- Isoamyl Alcohol, and 0.3 g glass beads were added.
  • the tubes were vortexed for 5 min using a multi-tube holder.
  • 0.2 mL TE pH 8.0 was added and centrifuged for 5 min.
  • the aqueous phase was transferred to a new tube and 1.0 mL cold ethanol was added and mixed to precipitate the DNA. After centrifugation for 5 min, the supernatant was removed.
  • the pellet was resuspended in 0.4 mL TE with 75 ⁇ g/mL RNaseA and incubated at room temperature for 5 min. 10 4 M ammonium acetate and 1.0 mL ethanol was added. After incubation on ice for 5 minutes and centrifugation for 10 minutes, the supernatant was discarded. The pellet was washed with 0.5 mL 70% ethanol and centrifuged 10 min. The supernatant was discarded and the pellet air dried, then resuspended in 100 TE. The resulting DNA from the above extraction process was analyzed by electrophoresis on 0.8% agarose gel in lx TAE at 100 V for 1.5 h.
  • the host strain of Y. lipolytica with no taql nuclease gene did not display DNA degradation activity at either 65 °C or 25 °C.
  • lipolytica that harbors the taql nuclease gene (ML 12805) displayed DNA degradation activity at 65 °C but not at 25 °C. The degradation activity was observed as early as 20 minutes after the cell culture was incubated at 65 °C. The ML 12805 strain did not display any DNA degradation activity at 25 °C even after being incubated for 24 hours, showing that Taql nuclease in the Y. lipolytica strain is latent at 25 °C.
  • Y. lipolytica strains ML 12805 and ML8195 were grown for 3 days as described in Example 2. 1.5 mL aliquots of ML12805 and ML8195 cells were incubated at temperatures of 25 °C, 30 °C, 40 °C, 50 °C and 65 °C for 1.5 h and DNA was extracted and analyzed as described in Example 2. As shown in Fig. 2, no DNA degradation was observed at temperatures of 25 °C, 30 °C, 40 °C, and 50 °C, in both strains ML12805 and ML8195. DNA degradation was observed at 65 °C, only in strain ML12805, as was shown in Example 2. The results show that Taql nuclease is latent at temperatures equal to or below 50 °C.
  • Example 4 PCR analysis of in vivo Tagl-digested gDNA
  • the carB gene is 1740 nucleotides in length and encodes phytoene dehydrogenase, an enzyme in the carotenoid biosynthetic pathway.
  • carB has been introduced into Y. lipolytica to enable production of carotenoids.
  • the presence of the carB gene was examined in both strains ML8195 and ML 12805 at both 25 °C and 65 °C. Serial dilutions of genomic DNA from ML12805 incubated at 25 °C or 65 °C for 24 hours were prepared. PCR was performed on the above samples using primers designed for amplifying a 529 bp fragment of the carB gene.
  • the sequence of primer M04641 is specified in SEQ ID NO:5.
  • the sequence of primer M04642 is specified in SEQ ID NO:6.
  • the DNA fragment of the carB gene between oligos M04641 and M04642 contains two Taql sites.
  • PCR was performed under the conditions described below.
  • 1 ⁇ of diluted gDNA was combined with 0.5 iL of each primer (10 ⁇ stock solution), 0.5 water and 22.5 of Platinum Taq Super Mix.
  • the reaction parameters were 1 cycle at 94 °C for 2 min followed by 45 cycles at 94 °C for 30 s, 58 °C for 30 s and 72 °C for 60 s.
  • a final single cycle of 72 °C for 5 min finished the PCR in a MJ Research PTC-225 Peltier Thermal Cycler.
  • the entire reaction plus lx loading dye was loaded onto a 2% agarose in lxTAE gel with 0.5 ⁇ g/ml ethidium bromide and run at 100 V for 1.5 h.
  • Fig. 3 shows that for the Y. lipolytica strain in which the taql nuclease gene was introduced (ML 12805), a 529 bp PCR product of carB was observed in the sample which was incubated at 25 °C but not in the sample which was incubated at 65 °C.
  • Fig. 3 also illustrates that foreign DNA in strain ML12805 can be degraded to a size of less than 500 nucleotides, since no noticeable band of carB DNA of 529 bp was observed in the gel.
  • Example 5a Astaxanthin production in a Y. lipolytica strain harboring the taql
  • a strain of Y. lipolytica which produces astaxanthin (ML 11956) was used.
  • a second strain (ML 12924) was constructed by introducing the taql nuclease gene (MB6736) into strain ML 11956. The amount of astaxanthin and its precursors in both strains was measured. Measurements were made at different time points during the cell growth period and after the host strains were incubated at 65 °C.
  • Strains ML 11956 and ML 12924 were grown in a fermentor using a fed-batch process. Samples were taken at different time points during the fermentation process and carotenoid analysis was performed according to the methods described previously in U.S. Patent No.
  • Fig. 4 shows that the amount of astaxanthin increased as strain ML 11956 grew and the production eventually reached a plateau. The amount of astaxanthin did not change after ML11956 was incubated at 65°C. In comparison, strain ML12924 behaved in the same manner as strain ML 11956. The amount of astaxanthin produced by strain ML 12924 was the same as in strain ML 11956 throughout the fermentation period and after incubation at 65°C. The amount of astaxanthin precursors produced also remained unchanged in strains ML 11956 and ML 12924 before and after incubation at 65°C. This result shows that neither the introduction of the taql nuclease gene nor activation of its activity affected the amount of astaxanthin produced by Y. lipolytica.
  • Example 5b Zeaxanthin production in a Y.lipolytica strain harboring the taql
  • thermophilic nuclease gene In this example, the production of zeaxanthin in a host strain of Y. lipolytica with the taql nuclease gene was examined.
  • a strain of Y. lipolytica which produces zeaxanthin (ML11218) was transformed with a taql nuclease gene (MB6736).
  • ML112128 zeaxanthin
  • MB6736 taql nuclease gene
  • Strain ML 12921 was grown in a fermentor using a fed-batch process. Samples were taken at different time points during the fermentation process and carotenoid analysis was performed according to the methods described previously in U.S. Patent No. 7,851,199 B2.
  • Fig. 5 shows that the amount of zeaxanthin increased as strain ML 12921 grew and the production eventually reached a plateau.
  • the amount of zeaxanthin produced did not change after the host cells were incubated at 65°C.
  • the amount of zeaxanthin precursors also remained the same after the host cells were incubated at 65 °C.
  • zeaxanthin production was similar to ML 12921 in all the conditions tested. This result shows that activation of the Taql nuclease activity does not affect the production of zeaxanthin in Y. lipolytica.
  • strains ML11956, ML12924, and ML12921 were grown in fermentors.
  • strains ML11956 and ML12924 samples were taken at 46, 121 and 236 h post inoculation.
  • strain ML 12921 samples were taken at 46 and 69 h post inoculation.
  • the temperature was raised to 65 °C and strains were incubated at this temperature for 6 hours. Samples were taken at 0 min, 10 min, 30 min, 2 h and 6 h after the shift to the higher temperature. DNA was extracted and analyzed as described in Example 2. As shown in Fig.

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Abstract

La présente invention concerne l'utilisation d'une nucléase thermophile pour dégrader des acides nucléiques intracellulairement, la nucléase thermophile étant hétérologue à la cellule hôte et étant produite par l'hôte plutôt que d'être ajoutée par voie exogène. La présente invention concerne en outre une cellule génétiquement modifiée qui a été produite selon le procédé susmentionné. La présente invention est particulièrement avantageuse pour inactiver l'activité biologique de l'ADN recombiné dans une biomasse.
PCT/US2014/026317 2013-03-15 2014-03-13 Utilisation de nucléases thermophiles pour la dégradation d'acides nucléiques Ceased WO2014151723A1 (fr)

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Citations (2)

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