EP1733029A2 - Pufa-pks gene aus ulkenia - Google Patents

Pufa-pks gene aus ulkenia

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Publication number
EP1733029A2
EP1733029A2 EP05751638A EP05751638A EP1733029A2 EP 1733029 A2 EP1733029 A2 EP 1733029A2 EP 05751638 A EP05751638 A EP 05751638A EP 05751638 A EP05751638 A EP 05751638A EP 1733029 A2 EP1733029 A2 EP 1733029A2
Authority
EP
European Patent Office
Prior art keywords
pufa
orf
pks
seq
particularly preferably
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP05751638A
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German (de)
English (en)
French (fr)
Inventor
Thomas Kiy
Markus Luy
Matthias RÜSING
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Lonza AG
Original Assignee
Nutrinova Nutrition Specialties and Food Ingredients GmbH
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Publication of EP1733029A2 publication Critical patent/EP1733029A2/de
Ceased legal-status Critical Current

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    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C12N15/11—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
    • 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

Definitions

  • the invention describes genes which code for sequences specific to polyketide synthases (PKS).
  • PKS polyketide synthases
  • the PKS synthesized from it is characterized by its enzymatic ability to produce PUFAs (polyunsaturated fatty acids).
  • the invention further includes the identification of the corresponding DNA sequences and the use of the nucleotide sequences for the production of recombinant or transgenic organisms.
  • PUFAs polyunsaturated fatty acids
  • PUFAs are understood as meaning polyunsaturated long-chain fatty acids with a chain length> C12 and at least two double bonds.
  • PUFA PUFA
  • omega-3 n-3
  • omega-6 n-6 fatty acids
  • They are important components of the cell membrane, where they are in the form of lipids, especially phospholipids.
  • PUFAs also serve as standard stages of important molecules in humans and animals, such as, for example, prostaglandins, leukotrienes and prostacyclins (Simopoulos, AP Essential fatty acids in health and chronic disease. Am. J Clin. 1999utr. 1999 (70) pp. 560-569).
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • An important step in omega-6 fatty acids is ERA (arachidonic acid), which occurs in filamentous fungi, for example, but can also be isolated from animal tissues such as the liver and kidney.
  • DHA and ERA occur side by side in human breast milk.
  • PUFA are essential for humans in terms of adequate development, especially for the developing brain, tissue formation and its repair.
  • DHA is an important component of human cell membranes, especially those of the nerves. It plays an important role in the maturation of brain function and is essential for the development of eyesight.
  • Omega-3 PUFAs such as DHA and EPA are used as food supplements, since a balanced diet with sufficient DHA supply is advantageous for the prophylaxis of certain diseases (Simopoulos, AP Essential fatty acids in health and chronic disease American Journal of Clinical ⁇ utrition 1999; 70: 560S-569S).
  • certain diseases Simopoulos, AP Essential fatty acids in health and chronic disease American Journal of Clinical ⁇ utrition 1999; 70: 560S-569S.
  • DHA fatty acids
  • EPA Omega-3 PUFAs
  • Microorganisms which are suitable for obtaining n-3 PUFA can be found, for example, in the bacteria under the Vibrio genus (e.g. Vibrio marinus) or under the Dinoflagellates (Dinophyta), in particular the Crypthecodinium genus, such as C. cohnii or among the Stramenopiles (or Labyrinthulomycota), such as the Pinguiophyceae such as Glossomastix, Phaeomonas, Pinguiochrysis, Pinguiococcus and Polypodochysis.
  • Vibrio genus e.g. Vibrio marinus
  • Dinoflagellates Dinophyta
  • Crypthecodinium genus such as C. cohnii or among the Stramenopiles (or Labyrinthulomycota)
  • Pinguiophyceae such as Glossomastix, Phaeomonas, Pinguioch
  • Thraustochytriales Thraustchytriidea
  • the oils obtained from commercially known PUFA sources such as plants or animals are often characterized by a very heterogeneous composition.
  • the oils obtained in this way have to be subjected to complex cleaning processes in order to be able to enrich one or more PUFAs.
  • the supply of PUFA from such sources is still subject to uncontrollable fluctuations. Diseases and weather influences can reduce both animal and plant yields.
  • DHA is present in amounts of approximately 50% of the total fat content of the cell and they can be cultivated relatively inexpensively in large fermenters.
  • Another advantage of microorganisms is a composition of the oils obtained from them that is limited to a few components.
  • PUFAs such as docosahexaenoic acid (DHA; 22: 6, n-3) and eicosapentaenoic acid (EPA; 20: 5, n-3).
  • the conventional biosynthetic route for producing long-chain PUFA in eukaryotic organisms begins with the delta-6 desaturation of linoleic acid (LA; 18: 2, n-6) and alphalinolenic acid (ALA; 18: 3, n-3). It results in the synthesis of gammalinolenic acid (GLA; 18: 3, n-6) from linoleic acid and octadecatetraenoic acid (OTA; 18: 4, n-3) from alphalinolenic acid.
  • LA delta-6 desaturation of linoleic acid
  • ALA alphalinolenic acid
  • GLA gammalinolenic acid
  • OTA octadecatetraenoic acid
  • This desaturation step is followed by an elongation step for both the n-6 and the n-3 fatty acid and a delta-5 desaturation, resulting in arachidonic acid (ERA; 20: 4, n-6) and eicosapentaenoic acid (EPA; 20: 5, n-3).
  • ERA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • eicosapentaenoic acid EPA; 20: 5, n-3
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • the so-called speaker pathway is independent of delta-4 desaturation. It consists of two successive elongation steps by 2 carbon units each to tetracosapentaenoic acid (24: 5, n-3) and a subsequent delta-6 desaturation to tetracosahexaenoic acid (24: 6, n-3).
  • docosahexaenoic acid is formed by shortening by two carbon units as a result of peroxisomal ⁇ -oxidation (Speaker, H.
  • the alternative C20 PUFA synthesis consists in an elongation of the C18 fatty acids, linoleic acid (LA; 18: 2, n-6) and alphalinolenic acid (ALA; 18: 3, n-3) by two carbon units each.
  • eicosadienoic acid (20: 2, n-6) and eicosatrienoic acid (20: 3, n-3) are then subjected to delta-8 desaturation followed by delta-5 desaturation in arachidonic acid (ERA; 20: 4, n- 6) or eicosapentaenoic acid (EPA; 20: 5, n-3) (Sayanova and Napier, Eicosapentaenoic acid: biosynthetic routes and the potential for synthesis in transgenic plants.
  • ERA arachidonic acid
  • EPA eicosapentaenoic acid
  • PUFA-producing microorganisms include marine representatives of the gamma proteobacteria and some species of the Cytophaga-Flavobacterium-Bacteroides group and so far a eukaryotic protist, Schizochytrium sp. ATCC 20888 (Metz et al. 2001, Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293: 290-293). They synthesize long-chain PUFA via so-called polyketide synthases (PKS).
  • PKS polyketide synthases
  • PKS PKS are large enzymes that catalyze the synthesis of secondary metabolites consisting of ketide units (Wallis, GW, Watts, JL and Browse, J. Polyunsaturated fatty acid synthesis: what will they think of next? Trends in Biochemical Sciences 27 (9) ( 2002) pp. 467-473).
  • the synthesis of the polyketides involves a series of enzymatic reactions which are analogous to those of the fatty acid synthesis (Hopwood & Sherman Annu. Rev. Genet. 24 (1990) pp. 37-66; Katz & Donadio Annu. Rev. of Microbiol. 47 (1993 ) Pp. 875-912).
  • PUFA-PKS characterized in that it a. at least one of the amino acid sequences shown in SEQ ID NOs 6 (ORF 1), 7 (ORF 2), 8 and / or 80 (ORF 3) and homologous sequences thereto with at least 70%, preferably 80%, particularly preferably at least 90% and very particularly preferably at least 99%, most preferably 100% sequence homology which have the biological activity of at least one domain of the PUFA-PKS, or b.
  • Isolated PUFA-PKS according to claim 1 with 10 or more ACP domains.
  • the invention relates to such a PUFA-PKS which has at least one amino acid sequence with at least 70%, preferably at least 80%, particularly preferably at least 90% and very particularly preferably at least 99% identity to at least 500 directly consecutive amino acids of the sequences SEQ ID NO 6 (ORF 1), 7 (ORF 2) and / or 8 and / or 80 (ORF 3).
  • the invention relates to an amino acid sequence with at least 70%, preferably at least 80%, particularly preferably at least 90% and very particularly preferably at least 99%) identity to at least 500 directly successive amino acids of the sequences SEQ ID NO 6 (ORF 1 ), 7 (ORF 2) and / or 8 and / or 80 (ORF 3).
  • the invention relates to an isolated DNA molecule coding for a PUFA-PKS according to one of the preceding claims.
  • This is preferably characterized in that it encodes an amino acid sequence which is at least 70% identical to at least 500 directly consecutive amino acids of the sequences SEQ ID NO 6 (ORF 1), 7 (ORF 2), 8 and / or 80 (ORF 3 ).
  • the present invention relates to such an isolated DNA molecule which has at least 70%, preferably at least 80%, particularly preferably at least 90% and very particularly preferably at least 95% identity with at least 500 consecutive nucleotides from the SEQ ID NOs 3, 4, 5 and / or 9 has.
  • the invention relates to a recombinant DNA molecule comprising one of the DNA molecules described above, which is functional with at least one DNA sequence which controls the transcription, preferably selected from the group consisting of SEQ ID NOs 3, 4 and 5 and / or 9, or parts thereof of at least 500 nucleotides and functional variants thereof.
  • the invention relates to a recombinant host cell comprising a recombinant DNA molecule described above.
  • the invention relates to a recombinant host cell which endogenously expresses the PUFA PKS according to the invention with at least 10 ACP domains.
  • the invention relates to a method for producing oil containing PUFA, preferably DHA, comprising the cultivation of such a recombinant host cell, and the oil thus produced.
  • the invention relates to a process for the production of biomass containing PUFA, preferably DHA, comprising the cultivation of such a recombinant host cell, and the biomass produced in this way. Therefore, in a further preferred aspect, the invention also relates to a recombinant biomass according to claim 15, comprising a nucleic acid according to claim 8 and / or an amino acid sequence according to claim 1 or parts of at least 500 consecutive amino acids homologous thereto.
  • the invention relates to the use of individual enzyme domains from the PUFA-PKS comprising SEQ ID NOs 6, 7, 8 and / or 80, shown in SEQ ID NOs 32, 33, 34, 45, 58, 59 , 60, 61, 72, 74 and / or 77 for the production of artificial polyketides, for example polyketide antibiotics and / or new, modified fatty acids.
  • identity in the case of nucleic acids means identical base pairs at the respective position of the strands to be compared.
  • gaps are possible.
  • the programs blastn and fasta represent one possibility for calculating the identity values in%.
  • homology also includes, for example, conservative exchanges in the amino acid sequence which do not significantly influence the function or structure of the protein. Such homology values are also calculated by programs known to those skilled in the art, such as blastp, Matrix PAM30, gap penalties: 9, extension: 1 (Altschul et al., NAR 25, 3389-3402).
  • sequence information of PUFA-PKS genes from Ulkenia sp. is provided by the nucleic acid and amino acid sequences defined in SEQ ID NOs 3 to 5 and / or 9.
  • SEQ ID NOs 1 and 2 represent the entire genomic DNA sequence on the two cosmids isolated here (see Examples 2 and 3).
  • the invention further comprises a method for the homologous and heterologous transformation of host organisms with nucleic acids according to the invention for the production of high-purity PUFAs.
  • the isolated open reading frames in the syngeneic and in the transgenic organism preferably lead to the production of PUFA, in particular DHA, EPA and DPA.
  • the PUFAs produced are preferably in the form of biomass or as oil.
  • the chromosomal sequence information provides insight into the location and arrangement of the individual PUFA-PKS genes. It was completely surprising that the cluster as such, as it is known from prokaryotic PUFA-PKS nerds such as Shewanella, Photobacterium or Moritella, no longer exists.
  • the initially identified Cosmid (Seq ID No. 1) showed that the linear arrangement of the individual ORFs is interrupted in Ulkenia and that the reading direction of individual ORFs is opposite ( Figure 1). This may be the result of massive gene rearrangements. As a result of the rearrangements, the individual ORFs also showed significantly greater distances from one another.
  • the two ORFs 1 and 2 are spaced about 13 kb apart.
  • the third ORF could only be identified on another cosmid (Seq ID No. 2), whereby no partial identities between the two cosmids (Seq ID No. 1 and 2) could be found (FIG. 1).
  • ORF 3 from Ulkenia sp. is no longer spatially close to the two ORFs 1 and 2.
  • the PUFA gene cluster as is known from the prokaryotic representatives mentioned above, is found in the eukaryote Ulkenia sp. does not exist anymore.
  • the location and arrangement of the individual PUFA-PKS genes of the protist Schizochytrium on the genome has been determined in part (WO 02/083870) and also shows an opposite orientation of the two ORFs A and B.
  • ORF 1 from Ulkenia sp. contains, on the one hand, a so-called beta-ketoacyl synthase domain (Seq ID No. 14 and 32), which is identified by the motif (DXAC) (Seq ID No.
  • This motif for the active center of the enzyme domain in Ulkenia ORF 1 can be extended in a preferred form to a range of 17 amino acids (GMNCVVDAACASSLIAV) (Seq ID No. 11 and 29).
  • the ketoacyl synthase domain can be divided into an N-terminal (Seq ID No. 10 and 28) and a C-terminal (Seq ID No.
  • the biological function of the beta-ketoacyl synthase domain is Catalysis of the condensation reaction within the fatty acid or PKS synth ese Elongation determined acyl group bound via a thioester bond to the cysteine residue of the active center of the enzyme domain and transferred in several steps to the carbon atom 2 of the fflelonyl group on the acyl carrier protein, with the release of CO 2 .
  • the beta-ketoacyl synthase domain is followed by a malonylCoA-ACP transferase domain (Seq ID No. 15 and 33). This domain catalyzes the transfer of MalonylCoA to the 4 ⁇ -phosphopantethein residue on the acyl carrier protein (ACP).
  • MalonylCoA-ACP transferase domains also transfer methyl or ethyl malonate to the ACP, whereby they can insert branches into the otherwise linear carbon chain.
  • linker region After a linker region there follows an alanine-rich sequence section (Seq ID No. 16 and 34) which contains 10 repetitions of an acyl carrier protein domain (ACP domain) (17-26 and 35-44).
  • ACP domain acyl carrier protein domain
  • linker regions consisting primarily of alanines and prolines.
  • Each of the ACP domains is characterized by a binding motif for a 4 -phosphopantethein molecule (LGXDS (L / I)). The 4 "-phosphopantethein molecule is bound to the conserved serine within the motif.
  • the ACP domains serve as carriers of the growing fatty acid or polyketide chain via the 4'-phosphopantethein residue.
  • a sequence with partial identities to ketoreductases follows (Seq ID no. 27 and 45)
  • the biological function of this domain is the NADPH-dependent reduction of 3-ketoacyl-ACP compounds, which represents the first reduction reaction in fatty acid biosynthesis, and this reaction also frequently takes place in polyketide synthesis (see also FIG. 3).
  • ORF 2 from Ulkenia sp. (Seq ID No. 4 and 7) also begins with a beta-ketoacyl synthase domain (Seq ID No. 50 and 58) by the motif (DXAC) (Seq ID No.
  • This motif for the active center of the enzyme domain in Ulkenia ORF 2 can be extended in a preferred form to a range of 17 amino acids (PLHYSVDAACATALYVL) (Seq ID No. 47 and 55).
  • the entire beta-ketoacyl synthase domain can be divided into an N-terminal (Seq ID No. 46 and 54) and a C-terminal (Seq ID No. 49 and 57) section.
  • the biological activity of this domain corresponds to the beta-ketoacyl synthase domain described in ORF 1.
  • Ketosynthases play a key role in the elongation cycle and show a higher substrate specificity than other enzymes in fatty acid synthesis.
  • acetyl group can then bind to the active center of a beta-ketoacyl synthase domain and thus represents the so-called priming molecule of the initial condensation reaction.
  • CLF-homologous sequences can also be found as loading domains in modular PKS systems. Domains with CLF sequence properties are present in all PUFA-PKS systems available to date. This is followed by an acyl transferase domain (Seq ID No. 52 and 60). This domain catalyzes a number of acyl transfers, such as the transfer of acyl to CoenzymA or to ACP domains.
  • the final domain from ORF 2 shows partial identities to oxidoreductases (Seq ID No.
  • ORF 3 from Ulkenia sp. (Seq ID No. 5 and 8) consists of two dehydrase / isomerase domains (Seq ID No. 66, 68, 72 and 74). Both domains contain an "active site" histidine with a directly adjacent cysteine (Seq ID No. 67 and 73 and Seq ID No. 69 and 75).
  • the biological function of these domains is the insertion of trans double bonds in the fatty acid or polyketide molecule with elimination of H 2 O and the subsequent conversion of the double bond into the cis-isomeric form.
  • the second dehydrase / isomerase domain merges into an alanine-rich region (Seq ID No. 70 and 76) without any known function, but this may be This is followed by an enoyl reductase domain (Seq ID Nos. 71 and 77) with high partial identity to the enoyl reductase domain from Ulkenia already present in ORF 2. Its biological function corresponds to the enoyl reductase domain already described above (see also FIG. 2).
  • the promoter sequence is preferably 2000 bp (sequence ID No. 62) before the start ATG codon. most preferably lOOObp before the start.
  • the termination sequence for ORF 1 is preferably 2000 bp (sequence ID No. 63) after the stop codon TAA. 1500 bp are particularly preferred, very particularly preferred lOOObp after the stop.
  • a potential termination signal for the mRNA synthesis of ORF 1, with the base sequence AATAAA, is 412 bp after the stop codon TAA.
  • the preferred promoter sequence is 2000 bp (sequence ID No. 64) before the start ATG codon.
  • the termination sequence for ORF 2 is preferably 2000 bp (sequence ID No. 65) after the stop codon TAA.
  • a potential termination signal for the mRNA synthesis of ORF 2, with the base sequence AATAAA, is 1650 bp after the stop codon TAA.
  • For ORF 3 from Ulkenia sp. are preferably 2000 bp (sequence ID No. 78) as the promoter sequence before the start ATG codon. These are particularly preferably 1500 bp, very particularly preferably 100 bp before the start.
  • the termination sequence for ORF 3 is preferably 2000 bp (sequence ID No. 79) after the stop codon TAA.
  • a potential termination signal for the mRNA synthesis of ORF 3, with the base sequence AATAAA, is 4229 bp after the stop codon TAA.
  • PUFA such as DHA
  • the nucleic acid sequences according to the invention can be used to increase the production of PUFA, for example by using the number of PUFA-PKS genes within the PUFA-producing organism to increase the number.
  • individual nucleic acid segments such as the sequence segments coding for the ACP domains, can of course also be duplicated in a homologous but also heterologous production organism.
  • the ACP domains as binding sites for the cofactor 4-phosphapanthetein essential for PUFA synthesis, are particularly suitable for increasing production.
  • the use of different regulatory elements such as promoters, terminators and enhancer elements can also lead to an increase in production in genetically modified PUFA producers. Genetic modifications within individual sequence sections can lead to changes in the structure of the resulting product and thus to the production of different PUFAs.
  • the similarity of PUFA synthases to polyketide synthases enables the construction of mixed systems. This so-called combinatorial biosynthesis allows the production of new, artificial bioactive substances.
  • PUFA-PKS units for example, new ones are conceivable Polyketide antibiotics, produced in transgenic microorganisms by a mixed system of PKS and PUFA-PKS units.
  • hosts suitable for the heterologous expression of the PUFA genes present here are, for example, yeasts such as Saccharomyces cerevisiae and Pichia Pastoris or filamentous fungi such as Aspergillus nidulans and Acremonium chysogenum.
  • Plants producing PUFA can be generated by inserting the genes according to the invention into, for example, soybean, rapeseed, sunflower, flax or other, preferably oil-rich, plants.
  • Additional effective genes such as 4-phosphopanthetein transferases can also be used for effective heterologous expression of the PUFA genes.
  • host-specific promoter / operator systems can be used for enhanced or inducible gene expression.
  • a variety of prokaryotic expression systems can be used for heterologous PUFA production.
  • Expression vectors can be constructed which, in addition to the corresponding PUFA genes, also contain promoters, ribosome binding sites and transcription terminators.
  • the promoter / operator region of the E. coli tryptophan biosynthesis and promoters of the lambda phage may be mentioned as examples of such regulatory elements in E. coli.
  • Selectable markers such as resistance to ampicillin, tetracycline or chloramphenicol, can also be used on the corresponding vectors.
  • Very suitable vectors for the transformation of E. coli are pBR322, pCQV2 and the pUC plasmid and their derivatives. These plasmids can contain both viral and bacterial elements. Any strain derived from E. coli K12 such as JM 101, JM109, RR1, HB101, DH1 or AG1 can be used as the E. coli host strain. Of course, all other common prokaryotic expression systems for heterologous PUFA production are also conceivable (see also Sambrook et al.). The use of oil-forming bacteria as host systems is also conceivable. Mammalian, plant and insect cells, but also fungi such as yeasts can be used as eukaryotic expression systems.
  • transcription initiation elements from genes of enzymes from glycolysis can be used. These include regulatory elements of alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglycerate kinase etc. However, regulatory elements from genes such as acid phosphatase, lactase, metallothionein or glucoamylase can also be used. Here too, promoters find one enhanced or inducible expression allow use. Promoters inducible by galactose (GAL1, GAL7 and GAL10) are also of particular interest (Lue et al. 1987 Mol. Cell. Biol. 7, p.
  • the 3 "termination sequence preferably also comes from a yeast. Since nucleotide sequences directly around the start codon (ATG) influence gene expression in yeast, efficient translation initiation sequences from the yeast are also preferred. In cases in which yeast plasmids are used, these contain one Yeast replication origin and a selection marker. This selection marker is preferably an auxotrophy marker such as LEU, TRP or HIS.
  • yeast plasmids are the so-called YRps (Yeast Replicating plasmids), YCps (Yeast Centromere plasmids) and YEps (Yeast Episomal plasmids)
  • the origin of replication are the YIps (Yeast Integrating plasmids), which are used to integrate the transformed DNA into the genome.
  • the plasmids ⁇ YES2 and pYX424 and the pPICZ plasmids are of particular interest. If filamentous fungi such as Aspergillus nidulans are used as heterologous PUFA producers, come also Prom otoren from the corresponding organism for use.
  • the gpc 4 promoter can be used for enhanced expression and the alcA promoter for inducible expression.
  • Helper plasmids such as pHELP (Ballance, DJ. And Turner G. (1985) Development of a high-frequency transforming vector for Aspergillus nidulans. Gene 36, 321-331) and selectable markers such as ura, bio or paba are preferably used for the transformation of filamentous fungi used. 3 'regulatory elements made of filamentous fungi are also preferred.
  • PUFA can be produced in insect cells using the baculovirus expression system. Such expression systems are commercially available, for example, from Clonetech or Invitrogen.
  • Vectors such as the Ti plasmid from Agrobacterium or whole viruses such as Cauliflower mosaic virus (CaMV), gemini virus, Tomato golden mosaic virus or tobacco mosaic virus (TMV) can be used for the transformation of plants.
  • a preferred promoter is, for example, the 35S promoter from CaMV.
  • Other possibilities for the transformation of plants are the calcium phosphate method, the polyethylene glycol method, microinjection, electroporation or lipofection of protoplasts.
  • the transformation by bombardment with DNA-loaded microparticles (gene gun) is also preferred.
  • An alternative PUFA production in plants results from the transformation of chloroplasts. Proteins can be transported in choroplasts, for example, by N-terminal leaders.
  • leader peptis comes from the small subunit of ribulose bisphosphate carboxylase, but leader peptides of other chloroplastic proteins can also be used. Another possibility is the stable transformation of the chloroplast genome. Above all, biolistic but also other methods come into question (Blowers et al. Plant Cell 1989 1 pp. 123-132, Kline et al. Nature 1987 327 pp. 70-73 and Shrier et al. Embo J. 4 pp.
  • FIG. 25 Commercially available expression systems can also be used for mammalian cells, for example viral and non-viral transformations and expression systems such as the lentiviral or adenoviral systems or the T-Rex system from Invitrogen can be used for the targeted integration of DNA into mammalian cells
  • the Flp-In system is also suitable from Invitrogen.
  • the nucleic acid and amino acid sequences on which the method according to the invention is based are described below with the aid of a few examples. The sequences and the invention are, however, not restricted to these examples. Brief description of the figures: FIG the location of the PUFA-PKS genes from Ulkenia sp. on the genome Furthermore, the individual domains are de r PUFA-PKS encoded by these genes.
  • FIG. 2 shows a comparison of ORF 2 and ORF 3 from Ulkenia sp. with the corresponding homologous ORFs from Moritella marina (GehBank accession no .: AB025342.1), Photobacterium profundum SS9 (GenBank accession no .: AF409100), Shewanella sp.
  • FIG. 3 shows a comparison of ORF 1 from Ulkenia sp. with the corresponding homologous ORFs from Moritella marina (GenBank accession no .: AB025342.1), Photobacterium profundum SS9 (GenBank accession no .: AF409100), Shewanella sp.
  • FIG. 4 contains a sequence comparison of ORF 1 from Ulkenia sp. with ORF A from schizochytrium. The degree of partial identity of both sequences is approximately 81.5%.
  • FIG. 5 contains a sequence comparison of ORF 2 from Ulkenia sp. with ORF B from schizochytrium. The degree of partial identity of both sequences is approximately 75.9%.
  • FIG. 6 contains a sequence comparison of ORF 3 from Ulkenia sp. with ORF C from schizochytrium. The degree of partial identity of both sequences is approximately 80.0%.
  • FIG. 7 describes a sequence comparison carried out with FASTAX of the PCR product described in Example 1 with database sequences (Swiss-PROT All library).
  • FIG. 8 shows a vector map of Cosmid SuperCosI (Stratagene), which was used to produce the cosmid bank from Example 2.
  • FIG. 9 describes a sequence comparison carried out with BLASTX of the PCR product described in Example 3 with database sequences (Swiss-PROT All library).
  • DHI medium 50g / L glucose; 12.5g / L yeast extract; 16.65g / L Tropic Marin; pH 6.0
  • DHI medium 50g / L glucose; 12.5g / L yeast extract; 16.65g / L Tropic Marin; pH 6.0
  • SAM 2179 inoculated xmd cultivated for 48h at 28 ° C and 150rpm.
  • the cells were then washed twice with sterile tap water, centrifuged xmd, the cell sediment frozen at -85 ° C. For further processing, the cell sediment was then transferred to a mortar and ground into a fine powder with a pestle under liquid nitrogen.
  • the PCR primers MOF1 and MOR1 were used as motif-specific oligonucleotides.
  • MOF1 5 '- CTC GGC ATT GAC TCC ATC - 3 (Seq ID No. 81)
  • MOR1 5 "- GAG AAT CTC GAC ACG CTT - 3 V (Seq ID No. 82)
  • the genomic DNA from Ulkenia as described under 1.1 sp. 2179 was diluted 1: 100.
  • PCR reaction mixture (1 x buffer (Sigma); dNTPs (200 ⁇ M each); MOF1 (20pmol), MOR1 (20pmol) and 2.5U Taq- DNA polymerase (Sigma) was transferred and the PCR was performed under The following conditions were carried out: initial denaturation 94 ° C for 3 minutes, followed by 30 cycles with 94 ° C for 1 minute each, 55 ° C for 1 minute, 72 ° C for 1 minute. Finally 8min 72 ° C. The PCR products were then analyzed by gel electrophoresis and fragments of the appropriate size were incorporated into the vector pCR2.1 TOPO via T / A cloning (Invitrogen). After transformation of E. coli TOP10F ", plasmid DNA was isolated (Qiaprep Spin, QIAGEN) and sequenced.
  • the enzyme was then heat inactivated at 65 ° C. for 20 minutes and the cut cosmid was dephosphorylated with SAP (shrimp alkaline phosphatase; Röche) according to the manufacturer. Here too, the enzyme was deactivated by heating the reaction mixture to 65 ° C. for 20 minutes. Xbal cleaved and dephosphorylated Supercos I cosmid was then completely cleaved with BamHI for several hours at 37 ° C. The cut Cos id DNA was then precipitated with phenol / chloroform, precipitated with ethanol and subsequently included in H 2 O est.
  • SAP shrimp alkaline phosphatase
  • ligation For the ligation, 1 ⁇ g of Xbal xmd BamHI digested cosmid DNA and 3.5 ⁇ l Sau3AI digested genomic DNA in a volume of 20 ⁇ l were combined and ligated with T4 ligase (Biolabs) according to the manufacturer for several hours. About 1/7 of the ligation batch was then packaged in phage using the Gigapack III XL Packaging Extract (Stratagene), according to the manufacturer. These were then used to transfect E. coli XLl-Blue MR.
  • PCR primers were used as PUFA-PKS-specific oligonucleotides:
  • CFOR1 5 ⁇ - GTC GAG AGT GGC CAG TGC GAT - 3 ⁇ (Seq ID No. 85)
  • CREV3 5-AAA GTG GCA GGGAAA GTA CCA-3 '(SeqIDNr.86)
  • the genomic DNA from Ulkenia sp. 2179 was diluted 1:10. 2 ⁇ l of this dilution were then transferred to a 50 ⁇ l volume of PCR reaction mixture (1 ⁇ buffer (Sigma); dNTPs (200 ⁇ m each); CFOR1 (20 pmol), CREV3 (20 pmol) and 2.5U Taq DNA polymerase (Sigma) PCR was carried out under the following conditions: initial denaturation at 94 ° C. for 3 minutes, followed by 30 cycles with 94 ° C. for 1 minute each, 60 ° C. for 1 minute, 72 ° C. for 1 minute, and finally 8 minutes at 72 ° C.
  • PCR products were then run through Gel electrophoresis was analyzed and fragments of the appropriate size were incorporated into the vector pCR2.1 TOPO via T / A cloning (Invitrogen) After transformation of E. coli TOP10F ', plasmid DNA was isolated (Qiaprep Spin, QIAGEN) and partially sequenced.

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CN105219789B (zh) 2014-06-27 2023-04-07 联邦科学技术研究组织 包含二十二碳五烯酸的提取的植物脂质
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CN108753810B (zh) * 2018-05-22 2021-06-18 昆明理工大学 一种转录调节蛋白基因orf2的用途
JPWO2020032261A1 (ja) * 2018-08-10 2021-08-10 協和発酵バイオ株式会社 エイコサペンタエン酸を生産する微生物及びエイコサペンタエン酸の製造法
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