EP2954052A1 - Autotrophic cultivation - Google Patents

Autotrophic cultivation

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Publication number
EP2954052A1
EP2954052A1 EP14701509.3A EP14701509A EP2954052A1 EP 2954052 A1 EP2954052 A1 EP 2954052A1 EP 14701509 A EP14701509 A EP 14701509A EP 2954052 A1 EP2954052 A1 EP 2954052A1
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EP
European Patent Office
Prior art keywords
cells
enzyme
coenzyme
activity
wild
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.)
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Application number
EP14701509.3A
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German (de)
French (fr)
Inventor
Thomas Haas
Markus PÖTTER
Martin DEMLER
Eva-Maria Eckl
Simon Beck
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evonik Operations GmbH
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Evonik Degussa GmbH
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Publication of EP2954052A1 publication Critical patent/EP2954052A1/en
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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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the invention relates to a method comprising an autotrophic cultivation of cells.
  • PRIOR ART In the fermentative production of products, efforts are always made to avoid or minimize the formation of undesirable by-products as far as possible. In general, one can thus increase the product yield and also the purification of the
  • Organisms producing polyhydroxyalkanoate use as precursor 3-hydroxyalkanyl-CoA.
  • This metabolic product can serve as an excellent starting substance, in particular to be metabolized by artificially inserted metabolic pathways to other, high-quality organic substances.
  • the object of the invention was to provide a process which reduces by-product formation in fermentation processes and thus increases the yield of monomer units of the polyhydroxyalkanoate reduced in the biosynthesis.
  • Precultivation which in particular serves for the generation of cell mass, is carried out under autotrophic conditions, followed by a further autotrophic cultivation, which preferably serves for product formation over at least a part-time range of the cultivation.
  • An advantage of the present invention is that unwanted by-products such as acetate, lactate, pyruvate, succinate, alanine, valine, butanol, butyrate, malate, acetone, 2-methyl-propionic acid or 3-methyl-butyrate, especially acetate, pyruvate and acetone, are formed significantly reduced.
  • the present invention advantageously increases the space-time yield.
  • Yet another advantage of the present invention is that the desired product can be more easily isolated.
  • Yet another advantage of the present invention is that one can operate completely independently of complex carbon sources, such as sugars.
  • Yet another advantage of the present invention is that it binds carbon dioxide, which could otherwise be problematic as a greenhouse gas.
  • the method according to the invention comprises the method steps
  • under autotrophic conditions in the context of the present invention is to be understood as culturing conditions in which the carbon source available to the cells contains at least 90% by weight, based on carbon atoms, of inorganic carbon.
  • a wild-type cell is preferably referred to as a cell whose genome is in a state as naturally produced by evolution, and is used for both the entire cell and for individual genes. fall therefore in particular not such cells or genes whose Gene sequences have been altered at least in part by humans by recombinant methods.
  • reduced polyhydroxyalkanoate synthesis compared to wild-type in the context of the present invention is meant that the cultured cells, when cultured under the same conditions as the wild-type, produce less polyhydroxyalkanoate per cell than the wild-type.
  • the cells used in the method according to the invention are genetically engineered, therefore recombinant cells. They can be prokaryotes or eukaryotes. These may be mammalian cells (such as human cells), plant cells, or microorganisms such as yeasts, fungi or bacteria, with microorganisms being most preferred and bacteria and yeasts being most preferred. It is preferred according to the invention that the cells used are selected from acetogenic bacteria or oxyhydrogen bacteria.
  • explosive gas bacterium a bacterium capable of growing chemolithoautotrophically and of H 2 and C0 2 in the presence of oxygen
  • Oxy-gas bacteria preferably used according to the invention are selected from the genera Achromobacter, Acidithiobacillus, Acidovorax, Alcaligenes, Anabena, Ancyclobacter, Aquifex, Arthrobacter, Azospirillum, Bacillus, Bradyrhizobium, Cupriavidus, Derxia, Helicobacter,
  • Mycobacterium Nocardia, Oligotropha, Paracoccus, Pelomonas, Polaromonas, Pseudomonas, Pseudonocardia, Rhizobium, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Seliberia, Streptomyces, Thiocapsa, Variovorax, Xanthobacter, Wautersia, cupriavidus being particularly preferred especially from the species Cupriavidus necator (also known as Ralstonia eutropha, Wautersia eutropha, Alcaligenes eutrophus, Hydrogenomonas eutropha), Achromobacter ruhlandii, Acidithiobacillus ferrooxidans, Acidovorax facilis, Alcaligenes hydrogenophilus, Alcaligenes latus, Anabena cylindrica, Anabena oscillaroides, Ana
  • Herbaspirillum autotrophicum Hydrogenobacter hydrogenophilus, Hydrogenobacter thermophilus, Hydrogenobaculum acidophilum, Hydrogenophaga flava, Hydrogenophaga palleronii, Hydrogenophaga pseudoflava, Hydrogenophaga taeniospirales, Hydrogeneophilus thermoluteolus, Hydrogenothermus marinus, Hydrogenovibrio marinus, Ideonella sp. 0-1, Kyrpidia tusciae, Metallosphaera sedula, Myobacterium gordonae, Nocardia autotrophica, Oligotropha carboxidivorans, Paracoccus denitrificans, Pelomonas saccharophila,
  • Polaromonas hydrogenivorans Pseudomonas hydrogenovora, Pseudomonas thermophila, Rhizobium japonicum, Rhodococcus opacus, Rhodopseudomonas palustris, Seliberia carboxydohydrogena, Thiocapsa roseopersicina, Variovorax paradoxus, Xanthobacter autrophicus, Xanthobacter flavus, Cupriavidus necator is particularly preferred, in particular from the strains Cupriavidus necator H 16, Cupriavidus necator H1 or Cupriavidus necator Z-1.
  • polyhydroxyalkanoate Preferably, the polyhydroxyalkanoate whose synthesis is reduced, polyhydroxybutyrate.
  • the reduced compared to the wild type polyhydroxyalkanoate synthesis can preferably be achieved by a genetic modification, so that compared to the wild-type cell reduced activity of at least one enzyme, which is the conversion of 3-hydroxyalkanyl coenzyme A to polyhydroxyalkanoate, preferably 3-hydroxybutyryl Coenzyme A too
  • this is preferably a factor reduced by a factor of at least 0.5, more preferably of at least 0.1, more preferably of at least 0.01, even more preferably of at least 0.001, and most preferably of at least 0.0001.
  • the phrase "decreased activity” also does not include any detectable activity ("zero activity”).
  • the reduction of the activity of a particular Enzyme can be carried out, for example, by targeted mutation or by other measures known in the art for reducing the activity of a particular enzyme. Methods for reducing enzymatic activities in microorganisms are known to the person skilled in the art, these include insertion of foreign DNA into the gene coding for the target enzyme, deletion of at least parts of the gene coding for the target enzyme,
  • RNA interference siRNA
  • antisense RNA modification (insertion, deletion or point mutations) of
  • regulatory sequences such as promoters and terminators or of
  • foreign DNA is to be understood as any DNA sequence which is "foreign” to the gene (and not to the organism), that is to say endogenous DNA sequences may also function as “foreign DNA” in this connection.
  • Polyhydroxyalkanoate is preferably a polyhydroxyalkanoate synthase, more preferably a polyhydroxybutyrate synthase.
  • this enzyme is preferably encoded by the genes phbC or phaC, with phaC being particularly preferred.
  • the method is used in method step A) under autotrophic conditions, thus under conditions in which the carbon source available to the cells to at least 90 wt .-%, preferably 95 wt .-%, particularly preferably 99 wt .-% based on carbon atoms, inorganic carbon ,
  • inventive method step A) is used, in particular in the form of substances supplied, selected from the group comprising, preferably consisting of carbonates, carbon dioxide and carbon monoxide, carbon dioxide being particularly preferred. It is also possible to use mixtures of the carbon sources in process step A).
  • the medium in which the cells are contained is containing a gas
  • the cells are preferably multiplied by at least 10-fold, more preferably by at least 100-fold, in particular by at least 1000-fold, based on the number of cells per volume of the total culture.
  • the cell density X in method step A is preferably at least 1 ⁇ 10 5 , more preferably at least 1 ⁇ 10 7 , in particular at least 1 ⁇ 10 8 cells / ml of total culture.
  • step B) of the method according to the invention dilution of the cells with medium takes place.
  • the medium used can be the same as used in process step 1, but it can also be a different one.
  • the cells are under autotrophic conditions, thus under conditions in which the carbon source available to the cells to at least 90 wt .-%, preferably 95 wt .-%, particularly preferably 99 wt .-% based on carbon atoms , inorganic carbon contains, increased.
  • Carbon which is used in process step C) according to the invention is supplied in particular in the form of substances selected from the group comprising, preferably consisting of carbonates, carbon dioxide and carbon monoxide, carbon dioxide being particularly preferred.
  • carbonates preferably consisting of carbonates, carbon dioxide and carbon monoxide, carbon dioxide being particularly preferred.
  • Carbon sources are used.
  • Process step C) is advantageously and thus preferably used for at least part of the time range for product production.
  • Process step C) synthesized by the cells target products whose formation on the
  • the desired product can be reacted, wherein as a "chemical compound" those with or without coenzyme A thioester functionalization should be considered equivalent and thus the thioester forming or cleaving enzymes are not counted.
  • a preferred target product is 2-hydroxyisobutyric acid.
  • the cell used in the method according to the invention has an increased compared to their wild type activity of the enzyme egg, which catalyzes the conversion of 3-hydroxybutyryl-coenzyme A to 2-hydroxyisobutyryl-coenzyme A.
  • the enzyme Ei is preferably a hydroxyl isobutyryl-CoA mutase, an isobutyryl-CoA mutase (EC 5.4.99.13) or a
  • Methylmalonyl-CoA mutase (EC 5.4.99.2), each preferably a coenzyme B12-dependent mutase.
  • the enzyme Ei is preferably those enzymes which are derived from the
  • DQ436456.1 of at least 60%, preferably of at least 80%, more preferably of at least 95%, most preferably at least 99% at the amino acid level, determined according to the blastp algorithm with an expect threshold of 10, a word size of 3, a blosum62 matrix with gap costs of existence: 1 1 and extension: 1 and a conditional compositional score matrix adjustment.
  • enhanced activity of an enzyme as used above in connection with the enzyme Ei and in the following statements in connection with the enzymes E 2 , etc., is preferably to be understood as an increased intracellular activity the enzyme activity in cells apply both to the increase in the activity of the enzyme E- ⁇ and for all the enzymes mentioned below, the activity of which may optionally be increased.
  • an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence or gene sequences which code for the enzyme, using a strong promoter, changing the codon usage of the gene, in various ways the half-life of the mRNA or of the enzyme that increases
  • the gene coding for the enzyme E x overexpressed, resulting in increased activity through overexpression.
  • Genetically modified cells according to the invention are produced, for example, by transformation, transduction, conjugation or a combination of these methods with a vector which contains the desired gene, an allele of this gene or parts thereof and a promoter which enables the expression of the gene.
  • Heterologous expression is particularly by integration of the gene or alleles in the chromosome of the cell or a
  • Protein separations between wild type and genetically engineered cell can be determined.
  • a common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001).) Protein concentration can also be assessed by Western blot hybridization with an antibody specific for the protein to be detected (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989) and subsequent optical evaluation with appropriate software for concentration determination (Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 1 1 1: 2630-2647)
  • the activity of DNA-binding proteins can be measured by DNA band shift assays (also referred to as gel retardation) (Wilson et al., (2001) Journal of Bacteriology, 183: 2151-2155.)
  • the intracellular enzymatic activities can be determined by various methods described (Donahue et al., (2000) Journal of Bacteriology 182 (19): 5624-5627, Ray et al., (2000) Journal of Bacteriology 182 (8): 2277-2284, Freedberg et (1973) Journal of Bacteriology 1 15 (3): 816-823).
  • mutations can be generated either undirected by classical methods, such as by UV irradiation or by mutagenic chemicals, or specifically by genetic engineering methods such as deletion (s), insertion (s) and or
  • Nucleotide substitution results in altered cells.
  • Particularly preferred mutants of enzymes are, in particular, also those enzymes which are no longer or at least less feedback-inhibitable in comparison with the wild-type enzyme.
  • RNA polymerase For example, one increases the copy number of the corresponding genes or mutates the promoter and regulatory region or the ribosome binding site, which is located upstream of the structural gene.
  • expression cassettes act, which are installed upstream of the structural gene.
  • Inducible promoters also make it possible to increase expression at any time.
  • the enzyme gene can be assigned as regulatory sequences but also so-called “enhancers", which have an improved interaction between RNA polymerase and
  • DNA also cause increased gene expression. Measures to extend the lifetime of mRNA also improve expression. Furthermore, by
  • genes or gene constructs are either present in plasmids with different copy numbers or are integrated and amplified in the chromosome. Alternatively, a further
  • episomal plasmids are used, for example. In principle, all embodiments which are available to the person skilled in the art for this purpose are suitable as plasmids or vectors. Such plasmids and
  • Vectors can, for. B. the brochures of the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL be removed. Further preferred plasmids and vectors can be found in: Glover, D.M. (1985), DNA cloning: a practical approach, Vol. I-Ill, IRL Press Ltd. , Oxford; Rodriguez, R.L. and Denhardt, D.T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D.V. (1990), Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J .; Fritsch, E.F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.
  • the plasmid vector containing the gene to be amplified is then passed through
  • Conjugation or transformation into the desired strain is described, for example, in Schwarzerbach et al., Applied and Environmental Microbiology 60: 756-759 (1994). Methods for transformation are described, for example, in Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio / Technology 7: 1067-1070 (1989), and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994). After homologous recombination by means of a cross-over event, the resulting strain contains at least two copies of the gene of interest.
  • an increased activity of an enzyme E x which is increased in comparison with its wild type, is preferably always a factor greater than or equal to at least 2, particularly preferably at least 10, more preferably at least 100, moreover even more preferably of at least 1, 000 and most preferably of at least 10,000 increased activity of the respective enzyme E x
  • the cell according to the invention comprises "an activity of an enzyme E x increased compared to its wild type, in particular also a cell whose wild type has no or at least no detectable activity of this enzyme E x and which only after increasing the enzyme activity, for example by overexpression, a
  • the term "overexpression” or the expression “increase in expression” used in the following also encompasses the case that a starting cell, for example a wild-type cell, has no or at least no detectable expression and only by recombinant methods a detectable Synthesis of the enzyme E x is induced. It can furthermore be advantageous if the cells used in the method according to the invention have an increased activity of an enzyme E 2 compared to their wild-type, which inhibits the Reaction of acetoacetyl-coenzyme A catalyzed to 3-hydroxybutyryl-coenzyme A have.
  • the enzyme E 2 is preferably an enzyme selected from the group comprising:
  • This enzyme is preferably encoded by the genes selected from the group consisting of phaB, phbB, fabG, phbN1, phbB2 or, with phaB, phbB being particularly preferred.
  • the nucleotide sequence of these genes may be, for example, the "Kyoto
  • the process according to the invention preferably has a process step D) purification of the target product.
  • a process step D purification of the target product.
  • Embodiments should be limited.
  • Cupriavidus necator H16 PHB1 pBBR1 MCS-2 :: icmA-icmB (lac) (hereinafter called RITA).
  • the RITA strain is transformed with an expression vector for icmA and icmB from Aquincola tertiaricarbonis. A detailed generation of the strain is described in Example 2 of WO2009156214.
  • the medium was used according to Vollbrecht, consisting of 2 g / l (NH 4 ) 2 HP0 4 , 2.1 g / l KH 2 P0 4 , 0.2 g / l MgS0 4 , 0.01 g / l CaCl 2 , 6 mg / l FeCl 3 , 0.05 mg / l Titriplex III, 0.02 mg / l FeSO 4 ⁇ 7 H 2 O, 1 g / l ZnSO 4 ⁇ 7 H 2 O, 0.3 g / l MnCl 2 x 4 H 2 0, 3 g / l H 3 B0 3 , 2 Mg / l CoCl 2 x 6 H 2 O, 0.1 pg / l CuCl 2 x 2 H 2 O, 0.2 Mg 1 NiCl 2 x 6 H 2 0 and 0.3 Mg l Na 2 M04 x 2 H 2 0.
  • Heterotrophic preculture (method step A) but heterotrophic, not according to the invention):
  • the preculture was carried out in 500 ml shake flasks with 50 ml of medium per strain in duplicate.
  • the preculture was inoculated with a single colony from an agar plate.
  • the incubation was carried out at 30 ° C and 150 rpm for 28.5 hours.
  • the cultures were centrifuged for 10 min at 4500 x g.
  • pellets were resuspended in 2 ml of medium and added to the main autotrophic culture (see there).
  • the preculture was carried out in 250 ml pressure-resistant coated Schott bottles with 50 ml of medium.
  • Bottle cap had a gassing frit, an exhaust filter and a sampling tube.
  • the bottles were rinsed 3 times with N 2 and 3 times with oxyhydrogen before seeding.
  • the experiment took place at 0.8 bar overpressure, 28 ° C and 150 rpm.
  • the fumigation took place with oxyhydrogen gas
  • Composition 4% 0 2 , 6% C0 2 and 90% H 2 Composition 4% 0 2 , 6% C0 2 and 90% H 2 .
  • the preculture was inoculated with a single colony from an agar plate.
  • the cultivation period was 143 hours.
  • the cultures were centrifuged for 10 min at 4500 x g.
  • the pellets were resuspended in 2 ml of medium and added to the main autotrophic culture (see there).
  • the main culture was carried out in pressure-resistant coated 250 ml Schott bottles with 50 ml of medium.
  • the bottle cap had a gassing frit, an exhaust filter and a sampling tube.
  • the bottles were rinsed 3 times with N 2 and 3 times with oxyhydrogen before seeding.
  • the experiment took place at 0.8 bar overpressure, 28 ° C and 150 rpm, each strain in duplicate. It is inoculated to a start OD of 0.5.
  • the fumigation was carried out with oxyhydrogen of composition 4% 0 2 , 6% C0 2 and 90% H 2 .
  • the sampling took place after 25 hours.
  • the cultures were centrifuged off at 4500 ⁇ g for 10 min and the supernatant was analyzed by means of NMR. Observations:

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Abstract

The invention relates to a method comprising the method steps: A) propagating cells that have been genetically modified in such a manner that they have a reduced polyhydroxyalkanoate synthesis compared to their wild type, in a medium under autotrophic conditions to form a cell density of X cells/litre; B) diluting at least one part of the cells to a cell density of 0.001 X to 0.5 X, preferably 0.01 X to 0.3 X, particularly preferably 0.05 X to 0.2 X in a medium; and C) propagating the at least one part of the cells under autotrophic conditions.

Description

Autotrophe Kultivierung  Autotrophic cultivation
Gebiet der Erfindung Field of the invention
Gegenstand der Erfindung ist ein Verfahren umfassend eine autotrophe Kultivierung von Zellen. Stand der Technik Bei der fermentativen Herstellung von Produkten ist man stets bemüht, die Bildung von unerwünschten Nebenprodukten möglichst zu vermeiden oder auf ein Minimum zu reduzieren. In der Regel kann man so die Produktausbeute erhöhen und auch die Aufreinigung des The invention relates to a method comprising an autotrophic cultivation of cells. PRIOR ART In the fermentative production of products, efforts are always made to avoid or minimize the formation of undesirable by-products as far as possible. In general, one can thus increase the product yield and also the purification of the
Endproduktes erleichtern. Zellen, die als Kohlenstoffquelle anorganischen Kohlenstoff verwenden, um organische Facilitate the final product. Cells that use inorganic carbon as a carbon source to produce organic
Moleküle zu synthetisieren sind insbesondere in Form von Knallgasbakterien und acetogenen Organismen bekannt.  To synthesize molecules are known in particular in the form of explosive gas bacteria and acetogenic organisms.
Organismen, die Polyhydroxyalkanoat produzieren, benutzen als Vorstufe 3-Hydroxyalkanyl- CoA. Dieses Stoffwechselprodukt kann als hervorragende Ausgangssubstanz dienen, um insbesondere auch durch künstlich eingefügte Stoffwechselwege zu weiteren, hochwertigen organischen Substanzen verstoffwechselt zu werden.  Organisms producing polyhydroxyalkanoate use as precursor 3-hydroxyalkanyl-CoA. This metabolic product can serve as an excellent starting substance, in particular to be metabolized by artificially inserted metabolic pathways to other, high-quality organic substances.
Insbesondere das Knallgasbakterium Cupriavidus necator wurde in den letzten Jahren ausführlich charakterisiert und untersucht.  In particular, the oxyhydrogen bacterium Cupriavidus necator has been extensively characterized and studied in recent years.
In Vollbrecht et al., Excretion of Metabolites by Hydrogen Bacteria I. Autotrophic and  In Vollbrecht et al., Excretion of Metabolites by Hydrogen Bacteria I. Autotrophic and
Heterotrophic Fermentations, European J. Appl. Microbiol. Biotechnol. 6,145-155 (1978) wurde die Nebenproduktbildung verschiedener Cupriavidus necator Stämme analysiert. Heterotrophic Fermentations, European J. Appl. Microbiol. Biotechnol. 6,145-155 (1978), the by-product formation of various Cupriavidus necator strains was analyzed.
Aufgabe der Erfindung war es, ein Verfahren bereitzustellen, welches die Nebenproduktbildung in Fermentationsverfahren reduziert und somit die Ausbeute an Monomereinheiten der in der Biosynthese verminderten Polyhydroxyalkanoats erhöht. The object of the invention was to provide a process which reduces by-product formation in fermentation processes and thus increases the yield of monomer units of the polyhydroxyalkanoate reduced in the biosynthesis.
Beschreibung der Erfindung Description of the invention
Überraschenderweise wurde gefunden, dass das im Folgenden beschriebene Verfahren die gestellte Aufgabe zu lösen vermag. Gegenstand der vorliegenden Erfindung ist daher ein Verfahren, bei dem bereits die Surprisingly, it has been found that the method described below is able to solve the stated problem. The subject of the present invention is therefore a method in which the
Vorkultivierung, welche insbesondere der Generierung von Zellmasse dient, unter autotrophen Bedingungen durchgeführt wird, gefolgt von einer weiteren autotrophen Kultivierung, welche bevorzugt mindestens über einen Teilzeitbereich der Kultivierung hinweg der Produktbildung dient. Precultivation, which in particular serves for the generation of cell mass, is carried out under autotrophic conditions, followed by a further autotrophic cultivation, which preferably serves for product formation over at least a part-time range of the cultivation.
Ein Vorteil der vorliegenden Erfindung liegt darin, dass unerwünschte Nebenprodukte, wie beispielweise Acetat, Lactat, Pyruvat, Succinat, Alanin, Valin, Butanol, Butyrat, Malat, Aceton, 2-Methyl-Propionsäure oder 3-Methyl-Butyrat, insbesondere Acetat, Pyruvat und Aceton, deutlich vermindert gebildet werden. An advantage of the present invention is that unwanted by-products such as acetate, lactate, pyruvate, succinate, alanine, valine, butanol, butyrate, malate, acetone, 2-methyl-propionic acid or 3-methyl-butyrate, especially acetate, pyruvate and acetone, are formed significantly reduced.
Somit erhöht die vorliegende Erfindung vorteilhaft die Raum-Zeit-Ausbeute.  Thus, the present invention advantageously increases the space-time yield.
Noch ein Vorteil der vorliegenden Erfindung ist, dass das gewünschte Produkt leichter isoliert werden kann.  Yet another advantage of the present invention is that the desired product can be more easily isolated.
Noch ein Vorteil der vorliegenden Erfindung ist es, dass man komplett unabhängig von komplexen Kohlenstoffquellen, wie beispielsweise Zucker agieren kann.  Yet another advantage of the present invention is that one can operate completely independently of complex carbon sources, such as sugars.
Noch ein Vorteil der vorliegenden Erfindung ist es, dass Kohlendioxid gebunden wird, welches ansonsten als Klimagas problematisch sein könnte.  Yet another advantage of the present invention is that it binds carbon dioxide, which could otherwise be problematic as a greenhouse gas.
Das erfindungsgemäße Verfahren umfasst die Verfahrensschritte The method according to the invention comprises the method steps
A) Vermehren von Zellen, welche derart gentechnisch verändert wurden, dass sie verglichen zu ihrem Wildtyp eine verminderte Polyhydroxyalkanoat-Synthese aufweisen, in einem Medium unter autotrophen Bedingungen bis zu einer Zelldichte X Zellen/Liter,  A) Increasing cells genetically engineered to have reduced polyhydroxyalkanoate synthesis compared to their wild type in a medium under autotrophic conditions up to a cell density X cells / liter,
B) Verdünnen mindestens eines Teils der Zellen auf eine Zelldichte von 0,001 X bis 0,5 X, bevorzugt 0,01 X bis 0,3 X, besonders bevorzugt 0,1 X bis 0,2 X in einem Medium und B) diluting at least a portion of the cells to a cell density of 0.001 X to 0.5 X, preferably 0.01 X to 0.3 X, more preferably 0.1 X to 0.2 X in a medium and
C) Vermehren des mindestens einen Teils der Zellen unter autotrophen Bedingungen. C) increasing the at least part of the cells under autotrophic conditions.
Unter dem Begriff„unter autotrophen Bedingungen" im Zusammenhang mit der vorliegenden Erfindung sind Kultivierungsbedingungen zu verstehen, bei denen die den Zellen verfügbare Kohlenstoffquelle zu mindestens 90 Gew.-%, bezogen auf Kohlenstoffatome, anorganischen Kohlenstoff enthält. The term "under autotrophic conditions" in the context of the present invention is to be understood as culturing conditions in which the carbon source available to the cells contains at least 90% by weight, based on carbon atoms, of inorganic carbon.
Unter einem„Wildtyp" einer Zelle wird vorzugsweise eine Zelle bezeichnet, deren Genom in einem Zustand vorliegt, wie er natürlicherweise durch die Evolution entstanden ist. Der Begriff wird sowohl für die gesamte Zelle als auch für einzelne Gene verwendet. Unter den Begriff „Wildtyp" fallen daher insbesondere nicht solche Zellen bzw. solche Gene, deren Gensequenzen zumindest teilweise durch den Menschen mittels rekombinanter Verfahren verändert worden sind. A "wild-type" cell is preferably referred to as a cell whose genome is in a state as naturally produced by evolution, and is used for both the entire cell and for individual genes. fall therefore in particular not such cells or genes whose Gene sequences have been altered at least in part by humans by recombinant methods.
Unter dem Begriff„verglichen zum Wildtyp verminderte Polyhydroxyalkanoat-Synthese" im Zusammenhang mit der vorliegenden Erfindung ist zu verstehen, dass die betrachteten Zellen, wenn unter gleichen Bedingungen kultiviert wie der Wildtyp, weniger Polyhdroxyalkanoat pro Zellen bilden als der Wildtyp.  By the term "reduced polyhydroxyalkanoate synthesis compared to wild-type" in the context of the present invention is meant that the cultured cells, when cultured under the same conditions as the wild-type, produce less polyhydroxyalkanoate per cell than the wild-type.
Angegebene Prozente (%) sind, wenn nicht anders angegeben, Massenprozente.  Specified percentages (%) are percentages by weight unless otherwise specified.
Die in dem erfindungsgemäßen Verfahren eingesetzten Zellen sind gentechnisch veränderte, daher rekombinante Zellen. Sie können Prokaryonten oder Eukaryonten sein. Dabei kann es sich um Säugetierzellen (wie etwa Zellen aus dem Menschen), um pflanzliche Zellen oder um Mikroorganismen wie Hefen, Pilze oder Bakterien handeln, wobei Mikroorganismen besonders bevorzugt und Bakterien und Hefen am meisten bevorzugt sind. Es ist erfindungsgemäß bevorzugt, dass die eingesetzten Zellen aus acetogene Bakterien oder Knallgasbakterien ausgewählt sind. The cells used in the method according to the invention are genetically engineered, therefore recombinant cells. They can be prokaryotes or eukaryotes. These may be mammalian cells (such as human cells), plant cells, or microorganisms such as yeasts, fungi or bacteria, with microorganisms being most preferred and bacteria and yeasts being most preferred. It is preferred according to the invention that the cells used are selected from acetogenic bacteria or oxyhydrogen bacteria.
Unter dem Begriff„Knallgasbakterium" ist ein Bakterium zu verstehen, das in der Lage ist chemolithoautotroph zu wachsen und aus H2 und C02 in Gegenwart von Sauerstoff By the term "explosive gas bacterium" is meant a bacterium capable of growing chemolithoautotrophically and of H 2 and C0 2 in the presence of oxygen
Kohlenstoffgerüste mit mehr als einem C-Atom aufzubauen, wobei der Wasserstoff oxidiert und der Sauerstoff als terminaler Elektronenakzeptor benutzt wird. Es können erfindungsgemäß sowohl solche Bakterien eingesetzt werden, die von Natur aus Knallgasbakterien darstellen, oder aber auch Bakterien, welche durch gentechnische Modifikation zu Knallgasbakterien wurden, wie beispielsweise eine E. coli Zelle, die durch rekombinantes Einfügen der notwendigen Enzyme in die Lage versetzt wurde, aus H2 und C02 in Gegenwart von Sauerstoff Kohlenstoffgerüste mit mehr als einem C-Atom aufzubauen, wobei der Wasserstoff oxidiert und der Sauerstoff als terminaler Elektronenakzeptor benutzt wird. Bevorzugt handelt es sich bei den im erfindungsgemäßen Verfahren eingesetzten Knallgasbakterien um solche, welche bereits als Wildtyp Knallgasbakterien darstellen. Build carbon skeletons with more than one carbon atom, whereby the hydrogen is oxidized and the oxygen is used as a terminal electron acceptor. According to the invention, it is possible to use both bacteria which are naturally oxyhydrogen bacteria, or also bacteria which have been converted into oxyhydrogen bacteria by genetic modification, for example an E. coli cell which has been made capable of recombinant insertion of the necessary enzymes. from H 2 and C0 2 in the presence of oxygen to build carbon skeletons with more than one carbon atom, wherein the hydrogen is oxidized and the oxygen is used as a terminal electron acceptor. The oxyhydrogen bacteria used in the process according to the invention are preferably those which already represent oxyhydrogen bacteria as wild-type.
Erfindungsgemäß bevorzugt eingesetzte Knallgasbakterien sind ausgewählt aus den Gattungen Achromobacter, Acidithiobacillus, Acidovorax, Alcaligenes, Anabena, Ancyclobacter, Aquifex, Arthrobacter, Azospirillum, Bacillus, Bradyrhizobium, Cupriavidus, Derxia, Helicobacter, Oxy-gas bacteria preferably used according to the invention are selected from the genera Achromobacter, Acidithiobacillus, Acidovorax, Alcaligenes, Anabena, Ancyclobacter, Aquifex, Arthrobacter, Azospirillum, Bacillus, Bradyrhizobium, Cupriavidus, Derxia, Helicobacter,
Herbaspirillum, Hydrogenobacter, Hydrogenobaculum, Hydrogenophaga,, Hydrogenophilus, Hydrogenothermus, Hydrogenovibrio, Ideonella sp. 01, Kyrpidia, Metallosphaera, Herbaspirillum, Hydrogenobacter, Hydrogenobaculum, Hydrogenophaga, Hydrogenophilus, Hydrogenothermus, Hydrogenovibrio, Ideonella sp. 01, Kyrpidia, Metallosphaera,
Mycobacterium, Nocardia, Oligotropha, Paracoccus, Pelomonas, Polaromonas, Pseudomonas, Pseudonocardia, Rhizobium, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Seliberia, Streptomyces, Thiocapsa, Variovorax, Xanthobacter, Wautersia, wobei Cupriavidus besonders bevorzugt ist, besonders aus den Spezies Cupriavidus necator (alias Ralstonia eutropha, Wautersia eutropha, Alcaligenes eutrophus, Hydrogenomonas eutropha), Achromobacter ruhlandii, Acidithiobacillus ferrooxidans, Acidovorax facilis, Alcaligenes hydrogenophilus, Alcaligenes latus, Anabena cylindrica, Anabena oscillaroides, Anabena sp., Anabena spiroides, Ancyclobacter vacuolatus, Aquifex aeolicus, Aquifex pyrophilus, Arthrobacter strain 11X, Bacillus schlegelii, Mycobacterium, Nocardia, Oligotropha, Paracoccus, Pelomonas, Polaromonas, Pseudomonas, Pseudonocardia, Rhizobium, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Seliberia, Streptomyces, Thiocapsa, Variovorax, Xanthobacter, Wautersia, cupriavidus being particularly preferred especially from the species Cupriavidus necator (also known as Ralstonia eutropha, Wautersia eutropha, Alcaligenes eutrophus, Hydrogenomonas eutropha), Achromobacter ruhlandii, Acidithiobacillus ferrooxidans, Acidovorax facilis, Alcaligenes hydrogenophilus, Alcaligenes latus, Anabena cylindrica, Anabena oscillaroides, Anabena sp., Anabena spiroides, Ancyclobacter vacuolatus, Aquifex aeolicus, Aquifex pyrophilus, Arthrobacter strain 11X, Bacillus schlegelii,
Bradyrhizobium japonicum, Derxia gummosa, Escherichia coli, Heliobacter pylori, Bradyrhizobium japonicum, Derxia gummosa, Escherichia coli, Heliobacter pylori,
Herbaspirillum autotrophicum, Hydrogenobacter hydrogenophilus., Hydrogenobacter thermophilus, Hydrogenobaculum acidophilum, Hydrogenophaga flava, Hydrogenophaga palleronii, Hydrogenophaga pseudoflava, Hydrogenophaga taeniospirales, Hydrogeneophilus thermoluteolus, Hydrogenothermus marinus, Hydrogenovibrio marinus, Ideonella sp. 0-1, Kyrpidia tusciae, Metallosphaera sedula, Myobacterium gordonae, Nocardia autotrophica, Oligotropha carboxidivorans, Paracoccus denitrificans, Pelomonas saccharophila, Herbaspirillum autotrophicum, Hydrogenobacter hydrogenophilus, Hydrogenobacter thermophilus, Hydrogenobaculum acidophilum, Hydrogenophaga flava, Hydrogenophaga palleronii, Hydrogenophaga pseudoflava, Hydrogenophaga taeniospirales, Hydrogeneophilus thermoluteolus, Hydrogenothermus marinus, Hydrogenovibrio marinus, Ideonella sp. 0-1, Kyrpidia tusciae, Metallosphaera sedula, Myobacterium gordonae, Nocardia autotrophica, Oligotropha carboxidivorans, Paracoccus denitrificans, Pelomonas saccharophila,
Polaromonas hydrogenivorans, Pseudomonas hydrogenovora, Pseudomonas thermophila, Rhizobium japonicum, Rhodococcus opacus, Rhodopseudomonas palustris, Seliberia carboxydohydrogena, Thiocapsa roseopersicina, Variovorax paradoxus, Xanthobacter autrophicus, Xanthobacter flavus, wobei Cupriavidus necator besonders bevorzugt ist, insbesondere aus den Stämmen Cupriavidus necator H 16, Cupriavidus necator H1 oder Cupriavidus necator Z-1 . Polaromonas hydrogenivorans, Pseudomonas hydrogenovora, Pseudomonas thermophila, Rhizobium japonicum, Rhodococcus opacus, Rhodopseudomonas palustris, Seliberia carboxydohydrogena, Thiocapsa roseopersicina, Variovorax paradoxus, Xanthobacter autrophicus, Xanthobacter flavus, Cupriavidus necator is particularly preferred, in particular from the strains Cupriavidus necator H 16, Cupriavidus necator H1 or Cupriavidus necator Z-1.
Besonders bevorzugt wird der Stamm Cupriavidus necator H 16 PHB-4, DSM 541 eingesetzt. Die erfindungsgemäß eingesetzten Zellen weisen aufgrund von mindestens einer  Particular preference is given to using the strain Cupriavidus necator H 16 PHB-4, DSM 541. Due to at least one of the cells used according to the invention
gentechnischen Veränderung eine verglichen zu ihrem Wildtyp verminderte genetic modification reduced compared to their wild type
Polyhydroxyalkanoat-Synthese auf. In diesem Zusammenhang ist es insbesondere bevorzugt, dass die erfindungsgemäß eingesetzten Zellen keine nachweisbaren Mengen an Polyhydroxyalkanoate synthesis on. In this connection, it is particularly preferred that the cells used according to the invention do not have any detectable amounts
Polyhydroxyalkanoat synthetisieren. Bevorzugt ist das Polyhydroxyalkanoat, dessen Synthese vermindert ist, Polyhydroxybutyrat. Synthesize polyhydroxyalkanoate. Preferably, the polyhydroxyalkanoate whose synthesis is reduced, polyhydroxybutyrate.
Die verglichen zum Wildtyp verminderte Polyhydroxyalkanoat-Synthese lässt sich bevorzugt durch eine gentechnische Veränderung erreichen, so dass die verglichen zum Wildtyp der Zelle verminderte Aktivität mindestens eines Enzyms, welches die Umsetzung von 3-Hydroxyalkanyl- Coenzym A zu Polyhydroxyalkanoat, bevorzugt von 3-Hydroxybutyryl-Coenzym A zu  The reduced compared to the wild type polyhydroxyalkanoate synthesis can preferably be achieved by a genetic modification, so that compared to the wild-type cell reduced activity of at least one enzyme, which is the conversion of 3-hydroxyalkanyl coenzyme A to polyhydroxyalkanoate, preferably 3-hydroxybutyryl Coenzyme A too
Polyhydroxybutyrat katalysiert. Polyhydroxybutyrate catalysed.
Unter der verwendeten Formulierung„verminderte Aktivität eines Enzyms" wird  Under the phrase "decreased activity of an enzyme" is used
dementsprechend vorzugsweise eine um einen Faktor von mindestens 0,5, besonders bevorzugt von mindestens 0,1 , darüber hinaus bevorzugt von mindestens 0,01 , darüber hinaus noch mehr bevorzugt von mindestens 0,001 und am meisten bevorzugt von mindestens 0,0001 verminderte Aktivität verstanden. Die Formulierung„verminderte Aktivität" beinhaltet auch keine detektierbare Aktivität („Aktivität von null"). Die Verminderung der Aktivität eines bestimmten Enzyms kann beispielsweise durch gezielte Mutation oder durch andere, dem Fachmann bekannte Maßnahmen zur Verminderung der Aktivität eines bestimmten Enzyms erfolgen. Verfahren zur Verminderung von enzymatischen Aktivitäten in Mikroorganismen sind dem Fachmann bekannt, diese umfassen Insertion von Fremd-DNA in das Gen kodierend für das Zielenzym, Deletion mindestens von Teilen des Gens kodierend für das Zielenzym, Accordingly, it is to be understood that this is preferably a factor reduced by a factor of at least 0.5, more preferably of at least 0.1, more preferably of at least 0.01, even more preferably of at least 0.001, and most preferably of at least 0.0001. The phrase "decreased activity" also does not include any detectable activity ("zero activity"). The reduction of the activity of a particular Enzyme can be carried out, for example, by targeted mutation or by other measures known in the art for reducing the activity of a particular enzyme. Methods for reducing enzymatic activities in microorganisms are known to the person skilled in the art, these include insertion of foreign DNA into the gene coding for the target enzyme, deletion of at least parts of the gene coding for the target enzyme,
Punktmutationen in der Gensequenz kodierend für das Zielenzym, RNA-Interferenz (siRNA), antisense-RNA oder Modifikation (Insertion, Deletion oder Punktmutationen) von  Point mutations in the gene sequence coding for the target enzyme, RNA interference (siRNA), antisense RNA or modification (insertion, deletion or point mutations) of
regulatorischen Sequenzen, wie etwa Promotoren und Terminatoren oder von regulatory sequences, such as promoters and terminators or of
Ribosomenbindestellen, welche das Gen kodierend für das Zielenzym flankieren. Unter Fremd- DNA ist in diesem Zusammenhang jegliche DNA-Sequenz zu verstehen, die dem Gen (und nicht dem Organismus)„fremd" ist, d.h. auch endogene DNA-Sequenzen können in diesem Zusammenhang als„Fremd-DNA" fungieren. Ribosome binding sites flanking the gene encoding the target enzyme. In this context, foreign DNA is to be understood as any DNA sequence which is "foreign" to the gene (and not to the organism), that is to say endogenous DNA sequences may also function as "foreign DNA" in this connection.
Bei dem Enzym, welches die Umsetzung von 3-Hydroxyalkanyl-Coenzym A zu  In the enzyme, which involves the reaction of 3-hydroxyalkanyl-coenzyme A too
Polyhydroxyalkanoat handelt es sich vorzugsweise um eine Polyhydroxyalkanoat Synthase, besonders bevorzugt um eine Polyhydroxybutyrat Synthase. Dieses Enzym wird insbesondere vorzugsweise von den Genen phbC oder phaC kodiert, wobei phaC besonders bevorzugt ist. Polyhydroxyalkanoate is preferably a polyhydroxyalkanoate synthase, more preferably a polyhydroxybutyrate synthase. In particular, this enzyme is preferably encoded by the genes phbC or phaC, with phaC being particularly preferred.
Das Verfahren wird in Verfahrensschritt A) unter autotrophen Bedingungen, somit unter Bedingungen bei denen die den Zellen verfügbare Kohlenstoffquelle zu mindestens 90 Gew.-%, bevorzugt 95 Gew.-%, insbesondere bevorzugt 99 Gew.-% bezogen auf Kohlenstoffatome, anorganischen Kohlenstoff enthält. Der anorganische Kohlenstoff, der in dem The method is used in method step A) under autotrophic conditions, thus under conditions in which the carbon source available to the cells to at least 90 wt .-%, preferably 95 wt .-%, particularly preferably 99 wt .-% based on carbon atoms, inorganic carbon , The inorganic carbon contained in the
erfindungsgemäßen Verfahrensschritt A) eingesetzt wird, wird insbesondere in der Form von Substanzen zugeführt, ausgewählt aus der Gruppe umfassend, bevorzugt bestehend aus Carbonaten, Kohlendioxid und Kohlenmonoxid, wobei Kohlendioxid besonders bevorzugt ist. Es können in Verfahrensschritt A) auch Mischungen der Kohlenstoffquellen eingesetzt werden. inventive method step A) is used, in particular in the form of substances supplied, selected from the group comprising, preferably consisting of carbonates, carbon dioxide and carbon monoxide, carbon dioxide being particularly preferred. It is also possible to use mixtures of the carbon sources in process step A).
Im Fall, dass in dem erfindungsgemäßen Verfahren Knallgasbakterien eingesetzt werden, ist es bevorzugt, das Verfahren in Gegenwart von Wasserstoff durchzuführen. Insbesondere wird in Verfahrensschritt A) und C) das Medium, in dem die Zellen enthalten sind mit einem Gas enthaltend In the event that explosive gas bacteria are used in the process according to the invention, it is preferred to carry out the process in the presence of hydrogen. In particular, in method step A) and C), the medium in which the cells are contained is containing a gas
H2, C02 und 02, H 2 , C0 2 and 0 2 ,
bevorzugt in einem Gewichtsverhältnis von 20 bis 95 zu 2 bis 45 zu 1 bis 35, insbesondere von 70 bis 95 zu 4 bis 15 zu 1 bis 10, preferably in a weight ratio of from 20 to 95 to 2 to 45 to 1 to 35, in particular from 70 to 95 to 4 to 15 to 1 to 10,
in Kontakt gebracht. In Verfahrensschritt A) werden die Zellen bevorzugt um mindestens das 10-fache, besonders bevorzugt um mindestens das 100-fache, insbesondere um mindestens das 1000-fache bezogen auf die Zellanzahl pro Volumen der Gesamtkultur vermehrt. Die Zelldichte X in Verfahrensschritt A beträgt bevorzugt mindestens 1 x105, besonders bevorzugt mindestens 1 x107, insbesondere mindestens 1 x108 Zellen/ml Gesamtkultur. brought into contact. In method step A), the cells are preferably multiplied by at least 10-fold, more preferably by at least 100-fold, in particular by at least 1000-fold, based on the number of cells per volume of the total culture. The cell density X in method step A is preferably at least 1 × 10 5 , more preferably at least 1 × 10 7 , in particular at least 1 × 10 8 cells / ml of total culture.
In Verfahrensschritt B) des erfindungsgemäßen Verfahrens findet eine Verdünnung der Zellen mit Medium statt. Das eingesetzte Medium kann das gleiche sein, wie in Verfahrensschritt 1 verwendet, es kann aber auch ein davon verschiedenes sein. In method step B) of the method according to the invention, dilution of the cells with medium takes place. The medium used can be the same as used in process step 1, but it can also be a different one.
In Verfahrenschritt C) des erfindungsgemäßen Verfahrens werden die Zellen unter autotrophen Bedingungen, somit unter Bedingungen bei denen die den Zellen verfügbare Kohlenstoffquelle zu mindestens 90 Gew.-%, bevorzugt 95 Gew.-%, insbesondere bevorzugt 99 Gew.-% bezogen auf Kohlenstoff atome, anorganischen Kohlenstoff enthält, vermehrt. Der anorganische In method step C) of the method according to the invention, the cells are under autotrophic conditions, thus under conditions in which the carbon source available to the cells to at least 90 wt .-%, preferably 95 wt .-%, particularly preferably 99 wt .-% based on carbon atoms , inorganic carbon contains, increased. The inorganic one
Kohlenstoff, der in dem erfindungsgemäßen Verfahrenschritt C) eingesetzt wird, wird insbesondere in der Form von Substanzen zugeführt, ausgewählt aus der Gruppe umfassend, bevorzugt bestehend aus Carbonaten, Kohlendioxid und Kohlenmonoxid, wobei Kohlendioxid besonders bevorzugt ist. Es können in Verfahrensschritt C) auch Mischungen der Carbon which is used in process step C) according to the invention is supplied in particular in the form of substances selected from the group comprising, preferably consisting of carbonates, carbon dioxide and carbon monoxide, carbon dioxide being particularly preferred. In method step C) it is also possible to use mixtures of
Kohlenstoffquellen eingesetzt werden. Carbon sources are used.
Verfahrensschritt C) wird vorteilhaft und somit bevorzugt mindestens über einen Teilzeitbereich hinweg zur Produktproduktion genutzt. Process step C) is advantageously and thus preferably used for at least part of the time range for product production.
In einer bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens werden in  In a preferred embodiment of the method according to the invention are in
Verfahrensschritt C) durch die Zellen Zielprodukte synthetisiert, dessen Bildung über dasProcess step C) synthesized by the cells target products whose formation on the
Zwischenprodukt 3-Hydroxyalkanyl-CoA, insbesondere 3-Hydroxybutyryl-Coenzym A, erfolgt. Der Begriff„Zwischenprodukt" definiert in diesem Zusammenhang eine chemische Verbindung, die durch den Einsatz mindestens eines Enzymes auf enzymatischem Weg zu dem Intermediate 3-hydroxyalkanyl-CoA, in particular 3-hydroxybutyryl-coenzyme A, takes place. The term "intermediate" in this context defines a chemical compound which can be obtained by the use of at least one enzyme enzymatically to the
gewünschten Produkt umgesetzt werden kann, wobei als„chemische Verbindung" solche mit oder ohne Coenzym A-Thioester-Funktionalisierung als gleichwertig betrachtet werden sollen und somit die Thioester bildenden bzw. -spaltenden Enzyme nicht mitgezählt werden. the desired product can be reacted, wherein as a "chemical compound" those with or without coenzyme A thioester functionalization should be considered equivalent and thus the thioester forming or cleaving enzymes are not counted.
Es kann vorteilhaft sein, wenn die Zellen entsprechend des gewünschten Zielproduktes durch genetische Modifikation derart verändert wurden, dass das Zielprodukt verstärkt durch die Zellen gebildet wird. Im Zusammenhang mit der vorliegenden Erfindung ist ein bevorzugtes Zielprodukt 2- Hydroxyisobuttersäure. Für dieses Zielprodukt ist es bevorzugt, wenn die im erfindungsgemäße Verfahren eingesetzte Zelle eine im Vergleich zu ihrem Wildtyp erhöhte Aktivität des Enzyms E-i aufweist, welches die Umsetzung von 3-Hydroxybutyryl-Coenzym A zu 2-Hydroxyisobutyryl- Coenzym A katalysiert. Bei dem Enzym E-i handelt es sich vorzugsweise um eine Hydroxyl- Isobutyryl-CoA Mutase, um eine Isobutyryl-CoA Mutase (EC 5.4.99.13) oder um eine It may be advantageous if the cells have been modified according to the desired target product by genetic modification such that the target product is increasingly formed by the cells. In the context of the present invention, a preferred target product is 2-hydroxyisobutyric acid. For this target product, it is preferred if the cell used in the method according to the invention has an increased compared to their wild type activity of the enzyme egg, which catalyzes the conversion of 3-hydroxybutyryl-coenzyme A to 2-hydroxyisobutyryl-coenzyme A. The enzyme Ei is preferably a hydroxyl isobutyryl-CoA mutase, an isobutyryl-CoA mutase (EC 5.4.99.13) or a
Methylmalonyl-CoA Mutase (EC 5.4.99.2), jeweils bevorzugt um eine Coenzym B12-abhängige Mutase. Methylmalonyl-CoA mutase (EC 5.4.99.2), each preferably a coenzyme B12-dependent mutase.
Bei dem Enzym Ei handelt es sich bevorzugt um solche Enzyme, die sich aus den  The enzyme Ei is preferably those enzymes which are derived from the
Mikroorganismen Aquincola tertiaricarbonis L108, DSM18028, DSM18512, Methylibium petroleiphilum PM1 , Methylibium sp. R8, Xanthobacter autotrophicus Py2, Rhodobacter sphaeroides (ATCC 17029), Nocardioides sp. JS614, Marinobacter algicola DG893, Microorganisms Aquincola tertiaricarbonis L108, DSM18028, DSM18512, Methylibium petroleiphilum PM1, Methylibium sp. R8, Xanthobacter autotrophicus Py2, Rhodobacter sphaeroides (ATCC 17029), Nocardioides sp. JS614, Marinobacter algicola DG893,
Sinorhizobium medicae WSM419, Roseovarius sp. 217, Pyrococcus furiosus DSM 3638 isolieren lassen, besonders bevorzugt um die in PCT/EP2007/052830 beschriebenen Coenzym B12-abhängigen Mutase, sowie Enzyme, die in mindestens einer ihrer Teilsequenz eine Sequenzidentität zu der Aminosäuresequenz der kleinen bzw. großen Untereinheit der in PCT/EP2007/052830 beschriebenen Mutase (accession number DQ436457.1 und Sinorhizobium medicae WSM419, Roseovarius sp. 217, Pyrococcus furiosus DSM 3638, particularly preferably the coenzyme B12-dependent mutase described in PCT / EP2007 / 052830, as well as enzymes which in at least one of their partial sequence have a sequence identity to the amino acid sequence of the small or large subunit of PCT / EP2007 / 052830 described (accession number DQ436457.1 and
DQ436456.1 ) von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt mindestens 99% auf Aminosäureebene aufweisen, bestimmt nach dem blastp Algorithmus mit einem expect threshold von 10, einer word size von 3, einer blosum62 Matrix mit gap costs von existence: 1 1 und extension: 1 und einem conditional compositional score matrix adjustment. DQ436456.1) of at least 60%, preferably of at least 80%, more preferably of at least 95%, most preferably at least 99% at the amino acid level, determined according to the blastp algorithm with an expect threshold of 10, a word size of 3, a blosum62 matrix with gap costs of existence: 1 1 and extension: 1 and a conditional compositional score matrix adjustment.
Der Begriff„gesteigerte Aktivität eines Enzyms", wie er vorstehend im Zusammenhang mit dem Enzym Ei und in den nachfolgenden Ausführungen im Zusammenhang mit den Enzymen E2 usw. verwendet wird, ist vorzugsweise als gesteigerte intrazelluläre Aktivität zu verstehen. Die nun folgenden Ausführungen zur Erhöhung der Enzymaktivität in Zellen gelten sowohl für die Erhöhung der Aktivität des Enzyms E-ι als auch für alle nachfolgend genannten Enzyme, deren Aktivität gegebenenfalls erhöht werden kann. The term "enhanced activity of an enzyme" as used above in connection with the enzyme Ei and in the following statements in connection with the enzymes E 2 , etc., is preferably to be understood as an increased intracellular activity the enzyme activity in cells apply both to the increase in the activity of the enzyme E-ι and for all the enzymes mentioned below, the activity of which may optionally be increased.
Grundsätzlich lässt sich eine Steigerung der enzymatischen Aktivität dadurch erzielen, dass man die Kopienzahl der Gensequenz bzw. der Gensequenzen erhöht, welche für das Enzym kodieren, einen starken Promotor verwendet, die Kodonnutzung des Gens verändert, auf verschiedene Art und Weise die Halbwertszeit der mRNA oder des Enzyms erhöht, die In principle, an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence or gene sequences which code for the enzyme, using a strong promoter, changing the codon usage of the gene, in various ways the half-life of the mRNA or of the enzyme that increases
Regulation der Expression des Gens modifiziert oder ein Gen oder Allel nutzt, das für ein entsprechendes Enzym mit einer gesteigerten Aktivität kodiert und gegebenenfalls diese Maßnahmen kombiniert. Insbesondere wird somit das für das Enzym Ex kodierende Gen überexpremiert, was zu einer gesteigerten Aktivität durch Überexpresssion führt. Modifies regulation of the expression of the gene or uses a gene or allele which codes for a corresponding enzyme with an increased activity and optionally combines these measures. In particular, thus, the gene coding for the enzyme E x overexpressed, resulting in increased activity through overexpression.
Erfindungsgemäß gentechnisch veränderte Zellen werden beispielsweise durch Transformation, Transduktion, Konjugation oder eine Kombination dieser Methoden mit einem Vektor erzeugt, der das gewünschte Gen, ein Allel dieses Gens oder Teile davon und einen die Expression des Gens ermöglichenden Promotor enthält. Die heterologe Expression wird insbesondere durch Integration des Gens oder der Allele in das Chromosom der Zelle oder einem Genetically modified cells according to the invention are produced, for example, by transformation, transduction, conjugation or a combination of these methods with a vector which contains the desired gene, an allele of this gene or parts thereof and a promoter which enables the expression of the gene. Heterologous expression is particularly by integration of the gene or alleles in the chromosome of the cell or a
extrachromosomal replizierenden Vektor erzielt. scored extrachromosomally replicating vector.
Einen Überblick über die Möglichkeiten zur Erhöhung der Enzym-Aktivität in Zellen am Beispiel der Pyruvat-Carboxylase gibt DE-A-100 31 999, die hiermit als Referenz eingeführt wird und deren Offenbarungsgehalt hinsichtlich der Möglichkeiten zur Erhöhung der Enzym-Aktivität in Zellen einen Teil der Offenbarung der vorliegenden Erfindung bildet.  An overview of the possibilities for increasing the enzyme activity in cells using the example of the pyruvate carboxylase is given in DE-A-100 31 999, which is hereby incorporated by reference, and the disclosure of which relates to the possibilities of increasing the enzyme activity in cells of the disclosure of the present invention.
Die Expression der vorstehend und aller nachfolgend genannten Enzyme bzw. Gene ist mit Hilfe von 1 - und 2-dimensionaler Proteingelauftrennung und anschließender optischer The expression of the above and of all the enzymes and genes mentioned below is carried out with the aid of 1- and 2-dimensional protein gel separation and subsequently optical
Identifizierung der Proteinkonzentration mit entsprechender Auswertesoftware im Gel nachweisbar. Wenn die Erhöhung einer Enzymaktivität ausschließlich auf einer Erhöhung der Expression des entsprechenden Gens basiert, so kann die Quantifizierung der Erhöhung der Enzymaktivität in einfacher Weise durch einen Vergleich der 1 - oder 2-dimensionalen Identification of the protein concentration with appropriate evaluation software detectable in the gel. If the enhancement of an enzyme activity is based solely on an increase in the expression of the corresponding gene, the quantification of the increase in the enzyme activity can be easily accomplished by comparing the 1- or 2-dimensional
Proteinauftrennungen zwischen Wildtyp und gentechnisch veränderter Zelle bestimmt werden. Eine gebräuchliche Methode zur Präparation der Proteingele bei coryneformen Bakterien und zur Identifizierung der Proteine ist die von Hermann et al. (Electrophoresis, 22: 1712.23 (2001 ) beschriebene Vorgehensweise. Die Proteinkonzentration kann ebenfalls durch Western-Blot- Hybridisierung mit einem für das nachzuweisende Protein spezifischen Antikörper (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989) und anschließender optische Auswertung mit entsprechender Software zur Konzentrationsbestimmung (Lohaus und Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 1 1 1 : 2630-2647) analysiert werden. Die Aktivität von DNA-bindenden Proteinen kann mittels DNA-Band-Shift-Assays (auch als Gelretardation bezeichnet) gemessen werden (Wilson et al. (2001 ) Journal of Bacteriology, 183: 2151 -2155). Die Wirkung von DNA-bindenden Proteinen auf die Expression anderer Gene kann durch verschiedene gut beschriebene Methoden des Reportergen-Assays nachgewiesen werden (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Protein separations between wild type and genetically engineered cell can be determined. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001).) Protein concentration can also be assessed by Western blot hybridization with an antibody specific for the protein to be detected (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989) and subsequent optical evaluation with appropriate software for concentration determination (Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 1 1 1: 2630-2647) The activity of DNA-binding proteins can be measured by DNA band shift assays (also referred to as gel retardation) (Wilson et al., (2001) Journal of Bacteriology, 183: 2151-2155.) The effect of DNA binding proteins to the expression of other genes can be detected by various well-described methods of the reporter gene assay (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989). Die intrazellulären enzymatischen Aktivitäten können nach verschiedenen beschriebenen Methoden (Donahue et al. (2000) Journal of Bacteriology 182 (19): 5624-5627; Ray et al. (2000) Journal of Bacteriology 182 (8): 2277-2284; Freedberg et al. (1973) Journal of Bacteriology 1 15 (3): 816-823) bestimmt werden. Sofern in den nachfolgenden Ausführungen keine konkreten Methoden zur Bestimmung der Aktivität eines bestimmten Enzyms angegeben werden, erfolgt die Bestimmung der Steigerung der Enzymaktivität und auch die Bestimmung der Verminderung einer Enzymaktivität vorzugsweise mittels der in Hermann et al., Electophoresis, 22: 1712-23 (2001 ), Lohaus et al., Biospektrum 5 32-39 (1998), Lottspeich, Angewandte Chemie 1 1 1 : 2630-2647 (1999) und Wilson et al., Journal of Bacteriology 183: 2151 -2155 (2001 ) beschriebenen Methoden. Laboratory Press, Cold Spring Harbor, NY USA, 1989). The intracellular enzymatic activities can be determined by various methods described (Donahue et al., (2000) Journal of Bacteriology 182 (19): 5624-5627, Ray et al., (2000) Journal of Bacteriology 182 (8): 2277-2284, Freedberg et (1973) Journal of Bacteriology 1 15 (3): 816-823). Unless in the following explanations concrete methods for the determination of Activity of a particular enzyme, the determination of the increase in the enzyme activity and also the determination of the reduction of an enzyme activity, preferably by means of the in Hermann et al., Electophoresis, 22: 1712-23 (2001), Lohaus et al., Biospektrum 5 32 -39 (1998), Lottspeich, Angewandte Chemie 1 1 1: 2630-2647 (1999) and Wilson et al., Journal of Bacteriology 183: 2151-2155 (2001).
Wird die Erhöhung der Enzymaktivität durch Mutation des endogenen Gens bewerkstelligt, so können derartige Mutationen entweder nach klassischen Methoden ungerichtet erzeugt werden, wie etwa durch UV-Bestrahlung oder durch mutationsauslösende Chemikalien, oder gezielt mittels gentechnologischer Methoden wie Deletion(en), Insertion(en) und/oder  If the increase in enzyme activity is accomplished by mutation of the endogenous gene, such mutations can be generated either undirected by classical methods, such as by UV irradiation or by mutagenic chemicals, or specifically by genetic engineering methods such as deletion (s), insertion (s) and or
Nukleotidaustausch(e). Durch diese Mutationen werden veränderte Zellen erhalten. Besonders bevorzugte Mutanten von Enzymen sind insbesondere auch solche Enzyme, die nicht mehr oder zumindest im Vergleich zum Wildtyp-Enzym vermindert feedback-inhibierbar sind. Nucleotide substitution (s). These mutations result in altered cells. Particularly preferred mutants of enzymes are, in particular, also those enzymes which are no longer or at least less feedback-inhibitable in comparison with the wild-type enzyme.
Wird die Erhöhung der Enzymaktivität durch Erhöhung der Synthese eines Enzyms Is the increase in enzyme activity by increasing the synthesis of an enzyme
bewerkstelligt, so erhöht man beispielsweise die Kopienzahl der entsprechenden Gene oder mutiert die Promotor- und Regulationsregion oder die Ribosomenbindungsstelle, die sich stromaufwärts des Strukturgens befindet. In gleicher Weise wirken Expressionskassetten, die stromaufwärts des Strukturgens eingebaut werden. Durch induzierbare Promotoren ist es zusätzlich möglich, die Expression zu jedem beliebigen Zeitpunkt zu steigern. Des Weiteren können dem Enzym-Gen als regulatorische Sequenzen aber auch sogenannte "Enhancer" zugeordnet sein, die über eine verbesserte Wechselwirkung zwischen RNA-Polymerase undFor example, one increases the copy number of the corresponding genes or mutates the promoter and regulatory region or the ribosome binding site, which is located upstream of the structural gene. In the same way, expression cassettes act, which are installed upstream of the structural gene. Inducible promoters also make it possible to increase expression at any time. Furthermore, the enzyme gene can be assigned as regulatory sequences but also so-called "enhancers", which have an improved interaction between RNA polymerase and
DNA ebenfalls eine erhöhte Genexpression bewirken. Durch Maßnahmen zur Verlängerung der Lebensdauer der mRNA wird ebenfalls die Expression verbessert. Weiterhin wird durch DNA also cause increased gene expression. Measures to extend the lifetime of mRNA also improve expression. Furthermore, by
Verhinderung des Abbaus des Enzymproteins ebenfalls die Enzymaktivität verstärkt. Die Gene oder Genkonstrukte liegen dabei entweder in Plasmiden mit unterschiedlicher Kopienzahl vor oder sind im Chromosom integriert und amplifiziert. Alternativ kann weiterhin eine Preventing the degradation of the enzyme protein also enhances the enzyme activity. The genes or gene constructs are either present in plasmids with different copy numbers or are integrated and amplified in the chromosome. Alternatively, a further
Überexpression der betreffenden Gene durch Veränderung der Medienzusammensetzung und Kulturführung erreicht werden. Anleitungen hierzu findet der Fachmann unter anderem bei Martin et al. (Bio/Technology 5, 137-146 (1987)), bei Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya und Morinaga (Bio/Technology 6, 428-430 (1988)), bei Eikmanns et al. (Gene 102, 93- 98 (1991 )), in EP-A-0 472 869, im US 4,601 ,893, bei Schwarzer und Pühler (Bio/Technology 9, 84-87 (1991 ), bei Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), bei LaBarre et al. (Journal of Bacteriology 175, 1001 -1007 (1993)), in WO-A-96/15246, bei Malumbres et al. (Gene 134, 15-24 (1993), in JP-A-10-229891 , bei Jensen und Hammer (Biotechnology and Bioengineering 58, 191 -195 (1998)) und in bekannten Lehrbüchern der Genetik und Molekularbiologie. Die vorstehend beschriebenen Maßnahmen führen ebenso wie die Mutationen zu gentechnisch veränderten Zellen. Zur Erhöhung der Expression der jeweiligen Gene werden zum Beispiel episomale Plasmide eingesetzt. Als Plasmide bzw. Vektoren kommen im Prinzip alle dem Fachmann für diesen Zweck zur Verfügung stehenden Ausführungsformen in Frage. Derartige Plasmide und Overexpression of the genes in question can be achieved by changing the composition of the medium and culture. For instructions on this, the skilled person will find, inter alia, in Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in EP-A-0 472 869, in US 4,601,893, in Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991), in Reinscheid et al. Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al., (Journal of Bacteriology 175, 1001-1007 (1993)), WO-A-96/15246, Malumbres et al., Gene 134 , 15-24 (1993), JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), and well-known textbooks on genetics and molecular biology the mutations to genetically modified cells. To increase the expression of the respective genes, episomal plasmids are used, for example. In principle, all embodiments which are available to the person skilled in the art for this purpose are suitable as plasmids or vectors. Such plasmids and
Vektoren können z. B. den Broschüren der Firmen Novagen, Promega, New England Biolabs, Clontech oder Gibco BRL entnommen werden. Weitere bevorzugte Plasmide und Vektoren können gefunden werden in: Glover, D. M. (1985), DNA cloning: a practical approach, Vol. I-Ill, IRL Press Ltd. , Oxford; Rodriguez, R.L. und Denhardt, D. T (eds) (1988) , Vectors : a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V. (1990), Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, E. F. und Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York. Vectors can, for. B. the brochures of the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL be removed. Further preferred plasmids and vectors can be found in: Glover, D.M. (1985), DNA cloning: a practical approach, Vol. I-Ill, IRL Press Ltd. , Oxford; Rodriguez, R.L. and Denhardt, D.T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D.V. (1990), Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J .; Fritsch, E.F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.
Der Plasmidvektor, der das zu amplifizierende Gen enthält, wird anschließend durch  The plasmid vector containing the gene to be amplified is then passed through
Konjugation oder Transformation in den gewünschten Stamm überführt. Die Methode der Konjugation ist beispielsweise bei Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994) beschrieben. Methoden zur Transformation sind beispielsweise bei Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican und Shivnan, Bio/Technology 7: 1067-1070 (1989) und Tauch et al., FEMS Microbiology Let-ters 123: 343- 347 (1994) beschrieben. Nach homologer Rekombination mittels eines„cross-over"-Ereignisses enthält der resultierende Stamm mindestens zwei Kopien des betreffenden Gens. Conjugation or transformation into the desired strain. The method of conjugation is described, for example, in Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994). Methods for transformation are described, for example, in Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio / Technology 7: 1067-1070 (1989), and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994). After homologous recombination by means of a cross-over event, the resulting strain contains at least two copies of the gene of interest.
Unter der vorstehend und in den nachfolgenden Ausführungen verwendeten Formulierung„eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität eines Enzyms Ex" ist vorzugsweise stets eine um einen Faktor von mindestens 2, besonders bevorzugt von mindestens 10, darüber hinaus bevorzugt von mindestens 100, darüber hinaus noch mehr bevorzugt von mindestens 1 .000 und am meisten bevorzugt von mindestens 10.000 gesteigerte Aktivität des jeweiligen Enzyms Ex zu verstehen. Weiterhin umfasst die erfindungsgemäße Zelle, welche„eine gegenüber ihrem Wildtyp gesteigerte Aktivität eines Enzyms Ex" aufweist, insbesondere auch eine Zelle, deren Wildtyp keine oder zumindest keine nachweisbare Aktivität dieses Enzyms Ex aufweist und die erst nach Erhöhung der Enzymaktivität, beispielsweise durch Überexpression, eine The expression "an increased activity of an enzyme E x, which is increased in comparison with its wild type, is preferably always a factor greater than or equal to at least 2, particularly preferably at least 10, more preferably at least 100, moreover even more preferably of at least 1, 000 and most preferably of at least 10,000 increased activity of the respective enzyme E x Furthermore, the cell according to the invention comprises "an activity of an enzyme E x increased compared to its wild type, in particular also a cell whose wild type has no or at least no detectable activity of this enzyme E x and which only after increasing the enzyme activity, for example by overexpression, a
nachweisbare Aktivität dieses Enzyms Ex zeigt. In diesem Zusammenhang umfasst der Begriff „Überexpression" oder die in den nachfolgenden Ausführungen verwendete Formulierung „Erhöhung der Expression" auch den Fall, dass eine Ausgangszelle, beispielsweise eine Wildtyp-Zelle, keine oder zumindest keine nachweisbare Expression aufweist und erst durch rekombinante Verfahren eine nachweisbare Synthese des Enzyms Ex induziert wird. Es kann weiterhin vorteilhaft sein, wenn die in dem erfindungsgemäßen Verfahren eingesetzten Zellen eine verglichen zu ihrem Wildtyp gesteigerte Aktivität eines Enzyms E2, welches die Umsetzung von Acetoacetyl-Coenzym A zu 3-Hydroxybutyryl-Coenzym A katalysiert, aufweisen. detectable activity of this enzyme E x shows. In this context, the term "overexpression" or the expression "increase in expression" used in the following also encompasses the case that a starting cell, for example a wild-type cell, has no or at least no detectable expression and only by recombinant methods a detectable Synthesis of the enzyme E x is induced. It can furthermore be advantageous if the cells used in the method according to the invention have an increased activity of an enzyme E 2 compared to their wild-type, which inhibits the Reaction of acetoacetyl-coenzyme A catalyzed to 3-hydroxybutyryl-coenzyme A have.
Bei dem Enzym E2 handelt es sich vorzugsweise um ein Enzym ausgewählt aus der Gruppe enthaltend: The enzyme E 2 is preferably an enzyme selected from the group comprising:
eine 3-Hydroxyacyl-CoA Dehydrogenase (EC 1 .1 .1.35), a 3-hydroxyacyl-CoA dehydrogenase (EC 1 .1 .1.35),
eine Acetoacetyl-Coenzym A Reduktase (EC 1 .1 .1 .36), an acetoacetyl-coenzyme A reductase (EC 1 .1 .1 .36),
eine Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase ((EC 1 .1 .1.21 1 ) und a long-chain 3-hydroxyacyl-CoA dehydrogenase ((EC 1 .1 .1.21 1) and
eine 3-Hydroxybutyryl-Coenzym A-Dehydrogenase (EC 1 .1 .1.157). a 3-hydroxybutyryl-coenzyme A dehydrogenase (EC 1 .1 .1.157).
Dieses Enzym wird vorzugsweise von den Genen kodiert ausgewählt aus der Gruppe bestehend aus phaB, phbB, fabG, phbN1 , phbB2 oder, wobei phaB, phbB besonders bevorzugt sind. Die Nukleotidsequenz dieser Gene können beispielsweise der„Kyoto This enzyme is preferably encoded by the genes selected from the group consisting of phaB, phbB, fabG, phbN1, phbB2 or, with phaB, phbB being particularly preferred. The nucleotide sequence of these genes may be, for example, the "Kyoto
Encyclopedia of Genes and Genomes" (KEGG-Datenbank), den Datenbanken des National Center for Biotechnology Information (NCBI) der National Library of Medicine (Bethesda, MD, USA) oder der Nukleotidsequenz-Datenbank der European Molecular Biologies Laboratories (EMBL, Heidelberg, Deutschland bzw. Cambridge, UK) entnommen werden. Encyclopedia of Genes and Genomes "(KEGG database), the National Library of Biotechnology Information (NCBI) databases of the National Library of Medicine (Bethesda, MD, USA) or the nucleotide sequence database of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK).
Das erfindungsgemäße Verfahren weist bevorzugt einen Verfahrensschritt D) Aufreinigung des Zielproduktes auf. In den nachfolgend aufgeführten Beispielen wird die vorliegende Erfindung beispielhaft beschrieben, ohne dass die Erfindung, deren Anwendungsbreite sich aus der gesamten Beschreibung und den Ansprüchen ergibt, auf die in den Beispielen genannten The process according to the invention preferably has a process step D) purification of the target product. In the examples given below, the present invention is described by way of example, without the invention, whose scope of application is apparent from the entire description and the claims, referred to in the examples
Ausführungsformen beschränkt sein soll. Embodiments should be limited.
Beispiele: Examples:
Vergleich heterotrophe/autotrophe Kultivierung versus autotrophe/autotrophe Kultivierung von Cupriavidus necator H 16 PHB-4 DSM 541 Comparison of heterotrophic / autotrophic culture versus autotrophic / autotrophic culture of Cupriavidus necator H 16 PHB-4 DSM 541
Es wurden zwei Cupriavidus necator H 16 PHB-4 DSM 541 eingesetzt: Two Cupriavidus necator H 16 PHB-4 DSM 541 were used:
Cupriavidus necator H 16 PHB-4 DSM 541 und Cupriavidus necator H 16 PHB-4 DSM 541 and
Cupriavidus necator H16 PHB^l pBBR1 MCS-2::icmA-icmB(lac) (im Folgenden RITA genannt). Der RITA Stamm ist mit einem Expression vektor für icmA und icmB aus Aquincola tertiaricarbonis transformiert. Eine detaillierte Generierung des Stammes ist in Beispiel 2 der WO2009156214 beschrieben. In dem Verfahren wurde das Medium nach Vollbrecht eingesetzt, bestehend aus 2 g/l (NH4)2HP04, 2,1 g/l KH2P04, 0,2 g/l MgS04, 0,01 g/l CaCI2, 6 mg/l FeCI3, 0,05 mg/l Titriplex III, 0,02 mg/l FeS04 x 7 H20, 1 g/l ZnS04 x 7 H20, 0,3 g/l MnCI2 x 4 H20, 3 g/l H3B03, 2 Mg/l CoCI2 x 6 H20, 0,1 pg/l CuCI2 x 2 H20, 0,2 Mg l NiCI2 x 6 H20 und 0,3 Mg l Na2M04 x 2 H20. Cupriavidus necator H16 PHB1 pBBR1 MCS-2 :: icmA-icmB (lac) (hereinafter called RITA). The RITA strain is transformed with an expression vector for icmA and icmB from Aquincola tertiaricarbonis. A detailed generation of the strain is described in Example 2 of WO2009156214. In the process, the medium was used according to Vollbrecht, consisting of 2 g / l (NH 4 ) 2 HP0 4 , 2.1 g / l KH 2 P0 4 , 0.2 g / l MgS0 4 , 0.01 g / l CaCl 2 , 6 mg / l FeCl 3 , 0.05 mg / l Titriplex III, 0.02 mg / l FeSO 4 × 7 H 2 O, 1 g / l ZnSO 4 × 7 H 2 O, 0.3 g / l MnCl 2 x 4 H 2 0, 3 g / l H 3 B0 3 , 2 Mg / l CoCl 2 x 6 H 2 O, 0.1 pg / l CuCl 2 x 2 H 2 O, 0.2 Mg 1 NiCl 2 x 6 H 2 0 and 0.3 Mg l Na 2 M04 x 2 H 2 0.
Bei plasmidtragenden Stämmen wurde 1 ml Kanamycin der Konzentration 300 g/m\ in  In plasmid-carrying strains, 1 ml kanamycin concentration 300 g / m \ in
Verfahrensschritt A) und in Verfahrensschritt C) zusätzlich 120 g/\ Coenzym B12 zugegeben. Der pH wurde mit NaOH (aq) auf 6,8 eingestellt. Zu der heterotrophen Vorkultur wurden 10 g/l Fructose zugegeben. Process step A) and in process step C) additionally 120 g / \ coenzyme B12 was added. The pH was adjusted to 6.8 with NaOH (aq). To the heterotrophic preculture was added 10 g / l fructose.
Heterotrophe Vorkultur (Verfahrensschritt A) aber heterotroph, nicht erfindungsgemäß): Heterotrophic preculture (method step A) but heterotrophic, not according to the invention):
Die Vorkultur erfolgte in 500 ml Schüttelkolben mit je 50 ml Medium, pro Stamm im Doppelansatz. Die Vorkultur wurde mit einer Einzelkolonie von einer Agarplatte angeimpft. Die Inkubation erfolgte bei 30 °C und 150 rpm für 28,5 Stunden. The preculture was carried out in 500 ml shake flasks with 50 ml of medium per strain in duplicate. The preculture was inoculated with a single colony from an agar plate. The incubation was carried out at 30 ° C and 150 rpm for 28.5 hours.
Die Kulturen wurden für 10 min bei 4500 x g zentrifugiert. The cultures were centrifuged for 10 min at 4500 x g.
Die Pellets wurden in 2 ml Medium resuspendiert und der autotrophen Hauptkultur (siehe dort) zugeführt.  The pellets were resuspended in 2 ml of medium and added to the main autotrophic culture (see there).
Autotrophe Vorkultur (Verfahrensschritt A): Autotrophic preculture (step A):
Die Vorkultur erfolgte in 250 ml druckfest beschichteten Schottflaschen mit je 50 ml Medium. DerThe preculture was carried out in 250 ml pressure-resistant coated Schott bottles with 50 ml of medium. Of the
Flaschenverschluss besaß eine Begasungsfritte, einen Abluftfilter und ein Probenahmerohr. Bottle cap had a gassing frit, an exhaust filter and a sampling tube.
Die Flaschen wurden vor dem Animpfen 3fach mit N2 und 3fach mit Knallgas gespült. Der Versuch fand bei 0,8 bar Überdruck, 28 °C und 150 rpm statt. Die Begasung erfolgte mit Knallgas derThe bottles were rinsed 3 times with N 2 and 3 times with oxyhydrogen before seeding. The experiment took place at 0.8 bar overpressure, 28 ° C and 150 rpm. The fumigation took place with oxyhydrogen gas
Zusammensetzung 4 % 02, 6 % C02 und 90 % H2. Die Vorkultur wurde mit einer Einzelkolonie von einer Agarplatte angeimpft. Die Kultivierungsdauer betrug 143 Stunden. Composition 4% 0 2 , 6% C0 2 and 90% H 2 . The preculture was inoculated with a single colony from an agar plate. The cultivation period was 143 hours.
Die Kulturen wurden für 10 min bei 4500 x g zentrifugiert. Die Pellets wurden in 2 ml Medium resuspendiert und der autotrophen Hauptkultur (siehe dort) zugeführt.  The cultures were centrifuged for 10 min at 4500 x g. The pellets were resuspended in 2 ml of medium and added to the main autotrophic culture (see there).
Autotrophe Hauptkultur (Verfahrensschritt C): Autotrophic main culture (step C):
Die Hauptkultur erfolgte in druckfest beschichteten 250 ml Schottflaschen mit 50 ml Medium. Der Flaschenverschluss besaß eine Begasungsfritte, einen Abluftfilter und ein Probenahmerohr. The main culture was carried out in pressure-resistant coated 250 ml Schott bottles with 50 ml of medium. The bottle cap had a gassing frit, an exhaust filter and a sampling tube.
Die Flaschen wurden vor dem Animpfen 3fach mit N2 und 3fach mit Knallgas gespült. Der Versuch fand bei 0,8 bar Überdruck, 28 °C und 150 rpm statt, je Stamm im Doppelansatz. Es wird auf eine StartOD von 0,5 angeimpft. Die Begasung erfolgte mit Knallgas der Zusammensetzung 4% 02, 6% C02 und 90% H2. Die Probenentnahme erfolgte nach 25 Stunden. Hierzu wurden die Kulturen 10 min bei 4500 x g abzentrifugiert und der Überstand wurde mittels NMR analysiert. Messwerte: The bottles were rinsed 3 times with N 2 and 3 times with oxyhydrogen before seeding. The experiment took place at 0.8 bar overpressure, 28 ° C and 150 rpm, each strain in duplicate. It is inoculated to a start OD of 0.5. The fumigation was carried out with oxyhydrogen of composition 4% 0 2 , 6% C0 2 and 90% H 2 . The sampling took place after 25 hours. For this purpose, the cultures were centrifuged off at 4500 × g for 10 min and the supernatant was analyzed by means of NMR. Observations:
Diese Experimente zeigen, dass durch das erfindungsgemäße Verfahren die Ausbeute an 3- Hydroxybutyrat (3HB) bzw. der davon direkt abgeleiteten Substanz 2-Hydroxyisobuttersäure (2-HIB) zunimmt. Die akkumuliert reduzierte Nebenproduktsbildung geht also zu Gunsten der Zunahme von 3HB und 2-HIB. These experiments show that the yield of 3-hydroxybutyrate (3HB) or of the directly derived substance 2-hydroxyisobutyric acid (2-HIB) increases as a result of the process according to the invention. The accumulated reduced by-product formation thus goes in favor of the increase of 3HB and 2-HIB.

Claims

Ansprüche claims
Verfahren umfassend die Verfahrensschritte Method comprising the method steps
A) Vermehren von Zellen, welche derart gentechnisch verändert wurden, dass sie  A) Increasing cells that have been genetically engineered to
verglichen zu ihrem Wildtyp eine verminderte Polyhydroxyalkanoat-Synthese aufweisen, in einem Medium unter autotrophen Bedingungen bis zu einer Zelldichte X Zellen/Liter,  have reduced polyhydroxyalkanoate synthesis compared to their wild type, in a medium under autotrophic conditions up to a cell density X cells / liter,
B) Verdünnen mindestens eines Teils der Zellen auf eine Zelldichte von 0,001 X bis 0,5 X, bevorzugt 0,01 X bis 0,3 X, besonders bevorzugt 0,05 X bis 0,2 X in einem Medium und  B) diluting at least a portion of the cells to a cell density of 0.001 X to 0.5 X, preferably 0.01 X to 0.3 X, more preferably 0.05 X to 0.2 X in a medium and
C) Vermehren des mindestens einen Teils der Zellen unter autotrophen Bedingungen.  C) increasing the at least part of the cells under autotrophic conditions.
Verfahren gemäß Anspruch 1 , Method according to claim 1,
dadurch gekennzeichnet,  characterized,
dass die eingesetzten Zellen aus acetogene Bakterien oder Knallgasbakterien ausgewählt sind.  the cells used are selected from acetogenic bacteria or oxyhydrogen bacteria.
Verfahren gemäß Anspruch 1 oder 2, Method according to claim 1 or 2,
dadurch gekennzeichnet,  characterized,
dass das Polyhydroxyalkanoat, dessen Synthese vermindert ist, Polyhydroxybutyrat ist.  the polyhydroxyalkanoate whose synthesis is reduced is polyhydroxybutyrate.
Verfahren gemäß mindestens einem der vorherigen Ansprüche, Method according to at least one of the preceding claims,
dadurch gekennzeichnet,  characterized,
dass die Zelle eine verglichen um Wildtyp verminderte Aktivität Polyhydroxybutyrat Synthase kodiert von den Genen phbC oder phaC aufweist.  in that the cell has a reduced activity compared to wild-type polyhydroxybutyrate synthase encoded by the genes phbC or phaC.
Verfahren gemäß mindestens einem der vorherigen Ansprüche, Method according to at least one of the preceding claims,
dadurch gekennzeichnet,  characterized,
dass die den Zellen verfügbare Kohlenstoffquelle zu mindestens 90 Gew.-%, bezogen auf Kohlenstoffatome, Kohlendioxid enthält.  that the carbon source available to the cells contains at least 90% by weight, based on carbon atoms, of carbon dioxide.
6. Verfahren gemäß mindestens einem der vorherigen Ansprüche, 6. Method according to at least one of the preceding claims,
dadurch gekennzeichnet, dass Knallgasbakterien eingesetzt werden, und in Verfahrensschritt A) und C) das Medium mit einem Gas enthaltend H2, C02 und 02 in einem Gewichtsverhältnis von 20 bis 95 zu 2 bis 45 zu 1 bis 35, insbesondere von 70 bis 95 zu 4 bis 15 zu 1 bis 10 n Kontakt gebracht wird. characterized, that oxyhydrogen bacteria are used, and in process step A) and C) the medium with a gas containing H 2 , C0 2 and O 2 in a weight ratio of 20 to 95 to 2 to 45 to 1 to 35, in particular from 70 to 95 to 4 is brought to 15 to 1 to 10 n contact.
7. Verfahren gemäß mindestens einem der vorherigen Ansprüche, 7. Method according to at least one of the preceding claims,
dadurch gekennzeichnet,  characterized,
dass die Zellen in Verfahrensschritt C) Zielprodukt synthetisieren, dessen Bildung über das Zwischenprodukt 3-Hydroxyalkanyl-CoA, insbesondere 3-Hydroxybutyryl-Coenzym A, erfolgt.  that the cells in process step C) synthesize the target product whose formation takes place via the intermediate 3-hydroxyalkanyl-CoA, in particular 3-hydroxybutyryl-coenzyme A.
8. Verfahren gemäß Anspruch 7, 8. The method according to claim 7,
dadurch gekennzeichnet,  characterized,
dass in Verfahrensschritt C) durch die Zellen 2-Hydroxyisobuttersäure synthetisiert wird und die Zelle, eine im Vergleich zu ihrem Wildtyp erhöhte Aktivität des Enzyms E-i aufweisen, welches die Umsetzung von 3-Hydroxybutyryl-Coenzym A zu  in method step C) the cells synthesize 2-hydroxyisobutyric acid and the cell has an increased activity of the enzyme E-i compared to its wild-type, which increases the conversion of 3-hydroxybutyryl-coenzyme A to
2-Hydroxyisobutyryl-Coenzym A katalysiert.  2-hydroxyisobutyryl coenzyme A catalyzes.
9. Verfahren gemäß mindestens einem der vorherigen Ansprüche, 9. Method according to at least one of the preceding claims,
dadurch gekennzeichnet,  characterized,
dass die in dem erfindungsgemäßen Verfahren eingesetzten Zellen eine verglichen zu ihrem Wildtyp gesteigerte Aktivität eines Enzyms E2, welches die Umsetzung von that the cells used in the method according to the invention compared to their wild type increased activity of an enzyme E 2 , which is the implementation of
Acetoacetyl-Coenzym A zu 3-Hydroxybutyryl-Coenzym A katalysiert, aufweisen. 10. Verfahren gemäß mindestens einem der vorherigen Ansprüche,  Acetoacetyl-coenzyme A catalyzed to 3-hydroxybutyryl-coenzyme A. 10. The method according to at least one of the preceding claims,
dadurch gekennzeichnet,  characterized,
dass es den Verfahrensschritt  that it is the procedural step
D) Aufreinigung des Zielproduktes  D) Purification of the target product
umfasst.  includes.
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