DE102007059248A1 - Recombinant cell capable of converting bicarbonate to organic compounds utilizes a 16-step metabolic pathway starting with acetyl coenzyme A - Google Patents

Abstract

Recombinant cell capable of converting bicarbonate to organic compounds with at least two carbon atoms utilizes a 16-step metabolic pathway starting with acetyl coenzyme A. Recombinant cell capable of converting bicarbonate to organic compounds with at least two carbon atoms utilizes the following metabolic pathway: acetyl coenzyme A (plus bicarbonate) to malonyl coenzyme A to malonate semialdehyde to 3-hydroxypropionate to 3-hydroxypropionyl coenzyme A to acryloyl coenzyme A to propionyl coenzyme A (plus bicarbonate) to (S)-methylmalonyl coenzyme A to (R)-methylmalonyl coenzyme A to succinate semialdehyde to 4-hydroxybutyric acid to 4-hydroxybutyryl coenzyme A to crotonyl coenzyme A to 3-hydroxybutyryl coenzyme A to acetoacetyl coenzyme A to acetyl coenzyme A (2 equivalents). Independent claims are also included for: (1) producing organic compounds with at least two carbon atoms by contacting a recombinant cell as above with a bicarbonate-containing nutrient medium; (2) producing acrylic acid or acrylate esters by producing 3-hydroxypropionic acid as above, dehydrating it and optionally esterifying the resulting acrylic acid; (3) producing methacrylic acid or methacrylate esters by producing 3-hydroxyisobutyric acid as above, dehydrating it and optionally esterifying the resulting methacrylic acid; (4) producing poly(meth)acrylic acid or poly(meth)acrylate esters by radical polymerization of (meth)acrylic acid or (meth)acrylate esters produced as above.

Description

The The present invention relates to a recombinant cell, a method for producing a recombinant cell by this method available recombinant cell, the use of a recombinant Cell, a process for producing an organic compound having at least two carbon atoms, a process for the preparation of acrylic acid, a process for the production of methacrylic acid and a process for producing poly (meth) acrylic acid or poly (meth) acrylic esters.

organic Connections are important industrial products. So be For example, acrylic acid and methacrylic acid Production of polymers, amino acids such as glutamate as food additives or alcohols such as 1,2-propanediol or 1,3-propanediol used as a solvent.

The large-scale production of such organic compounds either by purely chemical means (in the case of acrylic acid for example, by catalytic gas phase oxidation of propylene), or but at least in partial steps on biotechnological way (in the case the acrylic acid, for example by fermentation reaction of carbohydrates by recombinant microorganisms to form of 3-hydroxypropionic acid, which subsequently chemically dehydrated to acrylic acid). raw materials for the large-scale production of organic Compounds are thus either hydrocarbons, which as products incurred in oil or natural gas refining, or Biomass, such as carbohydrates.

It is known that global warming mainly caused by carbon dioxide gas, which is caused by human Acting of factories, industrial plants, thermal power plants, Automobiles etc. is launched. The reduction of the Carbon dioxide gas emissions or the recovery of carbon dioxide gas, which occurs in the atmosphere is thus considered one of the most important environmental protection measures.

Various methods have been proposed to reduce the emission or content of carbon dioxide gas. Sun strikes GB-A-1263231 to concentrate CO ₂ in an electrolytic concentrator. After the removal of water, CO ₂ is reacted with hydrogen gas generated in an electrolytic cell into carbon and water in a CO ₂ reduction reactor. The disadvantage of this in the GB-A-1263231 However, among other things, it consists in the fact that it allows only the production of carbon from carbon dioxide, but not the selective production of industrially interesting, organic compounds such as acrylic acid or methacrylic acid. Moreover, that requires in the GB-A-1263231 first described the concentration of CO ₂ in electrolytic concentrators, so that this method is very complex and therefore uneconomical.

Of the Present invention was based on the object resulting from the State of the art resulting disadvantages in connection with the carbon dioxide fixation to overcome.

Especially The present invention was based on the object, a method specify fixed with the carbon dioxide from the atmosphere and from this purpose specifically industrially relevant, organic Compounds can be formed. Also, this should be Procedures include as few process steps and In addition, operated in a cost effective manner can be.

a Contribute to the solution of the above-mentioned tasks a recombinant cell which is able to exploit a metabolic pathway in which acetyl-CoA and bicarbonate malonyl-coenzyme A, from malonyl-coenzyme A malonate semialdehyde, from malonate semialdehyde 3-hydroxypropionate, from 3-hydroxypropionate 3-hydroxypropionyl-coenzyme A, from 3-hydroxypropionyl-coenzyme A acryloyl-coenzyme A, from acryloyl-coenzyme A propionyl coenzyme A, from propionyl coenzyme A and bicarbonate (S) -methylmalonyl coenzyme A, from (S) -methylmalonyl-coenzyme A (R) -methylmalonyl-coenzyme A, from (R) -methylmalonyl-coenzyme A succinate semialdehyde, from succinate semialdehyde 4-hydroxybutyric acid, from 4-hydroxybutyric acid 4-hydroxybutyryl-coenzyme A, from 4-hydroxybutyryl-coenzyme A crotonyl-coenzyme A, from crotonyl-coenzyme A 3-hydroxybutyryl-coenzyme A, from 3-hydroxybutyryl-coenzyme A acetoacetyl-coenzyme A and acetoacetyl-coenzyme A two equivalents Acetyl coenzyme A can be formed from bicarbonate organic To form compounds with at least two carbon atoms.

Surprisingly, it has been discovered that a completely new metabolic pathway discovered in Metallosphaera sedula, comprising one equivalent of acetyl-coenzyme A and two equivalents of hydrogencar first of all, malonyl coenzyme A, malonate semialdehyde, 3-hydroxypropionic acid, 3-hydroxypropionyl coenzyme A, acrylyl coenzyme A, propionyl coenzyme A and methylmalonyl coenzyme A according to the 3-hydroxypropionate cycle succinylate known from Chloroflexus aurantiacus. Coenzyme A is, which then unlike the known 3-hydroxypropionate cycle via malyl coenzyme A to acetyl coenzyme A and glyoxylate, but on succinate semialdehyde, 4-hydroxybutyric acid, 4-hydroxybutyryl coenzyme A, crotonyl coenzyme A , 3-hydroxybutyryl-coenzyme A and acetoacetyl-coenzyme A are converted to two equivalents of acetyl-coenzyme A (see 1 ). By means of this newly discovered metabolic pathway, two equivalents of bicarbonate (which are introduced in the reaction of acetyl-coenzyme A to malonyl-coenzyme A and in the reaction of propionyl-coenzyme A to methylmalonyl-coenzyme A) can be converted to acetyl-coenzyme A. , from which then corresponding organic compounds having more than two carbon atoms can be formed via further reaction steps.

The Formulation "taking advantage of a metabolic pathway, in which acetyl-CoA and bicarbonate malonyl-coenzyme A, from Malonyl coenzyme A malonate semialdehyde, from malonate semialdehyde 3-hydroxypropionate, from 3-hydroxypropionate 3-hydroxypropionyl-coenzyme A, from 3-hydroxypropionyl-coenzyme A acryloyl-coenzyme A, from acryloyl-coenzyme A propionyl-coenzyme A, from propionyl-coenzyme A and bicarbonate (S) -methylmalonyl-coenzyme A, from (S) -methylmalonyl-coenzyme A (R) -methylmalonyl-coenzyme A, from (R) -methylmalonyl-coenzyme A succinate semialdehyde, from succinate semialdehyde 4-hydroxybutyric acid, from 4-hydroxybutyric acid 4-hydroxybutyryl-coenzyme A, from 4-hydroxybutyryl-coenzyme A crotonyl-coenzyme A, from crotonyl-coenzyme A 3-hydroxybutyryl-coenzyme A, from 3-hydroxybutyryl-coenzyme A acetoacetyl-coenzyme A and from acetoacetyl-coenzyme A two equivalents of acetyl-coenzyme A "are preferably to be understood that in the cell by recombinant methods the activity at least one of the enzymes of this metabolic pathway, for example by overexpression, is increased, so that one over the wild type increased turnover via this metabolic pathway he follows.

In In this connection it is particularly preferred that the recombinant Cell compared to their wild type so by recombinant Procedure was changed, that compared to her Wild-type more organic compounds with at least two carbon atoms from bicarbonate, especially before given more 3-hydroxypropionic acid or 3-hydroxyisobutyric acid from bicarbonate can.

The Formulation "that they are more compared to their wild type organic compounds having at least two carbon atoms Bicarbonate, more preferably more 3-hydroxypropionic acid or 3-hydroxyisobutyric acid from bicarbonate "also concerns the case that the wild type of the genetically engineered changed cell no organic at all Compounds having at least two carbon atoms from bicarbonate, but at least no detectable amounts of these organic compounds from bicarbonate and only after the recombinant Change detectable amounts of these components formed can be.

Under A "wild-type" cell is preferably a cell whose genome is in a state as naturally originated through evolution. The term is used both for used the entire cell as well as for individual genes. The term "wild type" therefore does not apply in particular to such Cells or genes whose gene sequences are at least partially modified by humans using recombinant techniques have been.

there it is particularly preferred according to the invention that the recombinant cell is altered to be in a defined time interval, preferably within 24 hours, at least 2 times, more preferably at least 10 times, above In addition, preferably at least 100 times, moreover more preferably at least 1,000 times, and most preferably at least 10,000 times more organic compounds with at least two carbon atoms, more preferably more 3-hydroxypropionic acid or 3-hydroxyisobutyric acid, from bicarbonate forms as the wild type of the cell. The increase the product formation can be determined, for example, by that the fiction, contemporary cell and the wild-type cell each separately under the same conditions (same cell density, same culture medium, same culture conditions) for a certain time interval in a suitable nutrient medium cultivated and then the amount of target product (organic compound with at least two carbon atoms) in the nutrient medium is determined.

The recombinant cells of the invention may be prokaryotes or eukaryotes. These may be mammalian cells (such as human cells), plant cells or microorganisms such as yeasts, fungi or bacteria, microorganisms being particularly preferred and Bak Yeasts and yeasts are the most preferred.

Particularly suitable bacteria, yeasts or fungi are those bacteria, yeasts or fungi which are deposited as bacterial, yeast or fungal strains in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany. Bacteria suitable according to the invention belong to the genera under
http://www.dsmz.de/species/bacteria.htm
Yeasts which are suitable according to the invention belong to those genera which are listed under
http://www.dsmz.de/species/yeasts.htm
are listed and suitable fungi according to the invention are those under
http://www.dsmz.de/species/fungi.htm
are listed.

According to a particularly preferred embodiment of the recombinant cell according to the invention, this is derived from a wild-type CO ₂ -autotrophic cell, in particular from a wild-type CO ₂ -autotrophic bacterial cell, wherein a CO ₂ -autotrophic wild-type cell or a CO ₂ - Autotrophic wild-type bacterial cell is preferably understood a cell whose wild-type is already able to use CO ₂ as a carbon source for the formation of organic building materials.

Particularly according to the invention preferred cells are derived from Archeae bacteria, in particular from Archeae bacteria of the genera Metallosphera, Sulfolobus or Archaeoglubus, most preferred of Archeal bacteria of the species Metallosphaera hakonensis, Metallosphaera prunae, Metallosphaera sedula, Sulfolobus acidocaldarius, Sulfolobus brierleyi, Sulfolobus hakonensis, Sulfolobus metallicus, sulfolobus shibatae, sulfolobus solfataricus, sulfolobus tokodaii, Archaeoglubus fulgidus, Archaeoglubus profundus or Archaeoglubus veneficus. In addition to these Archeae bacteria can become the cells also of bacteria of the genus Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Lactobacillus, Lactococcus, Candida, Pichia, Kluveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Burkholderia and Clostridium.

• – eines Enzyms E₁, welches die Umsetzung von Carbonat und Acetyl-Coenzym A zu Malonyl-Coenzym A katalysiert;
• – eines Enzyms E₂, welches die Umsetzung von Malonyl-Coenzym A zu Malonatsemialdehyd katalysiert;
• – eines Enzyms E₃, welches die Umsetzung von Malonatsemialdehyd zu 3-Hydroxypropionsäure katalysiert;
• – eines Enzyms E₄, welches die Umsetzung von 3-Hydroxypropionsäure zu 3-Hydroxypropionyl-Coenzym A katalysiert;
• – eines Enzyms E₅, welches die Umsetzung von 3-Hydroxypropionyl-Coenzym A zu Acryloyl-Coenzym A katalysiert;
• – eines Enzyms E₆, welches die Umsetzung von Acryloyl-Coenzym A zu Propionyl-Coenzym A katalysiert;
• – eines Enzyms E₇, welches die Umsetzung von Carbonat und Propionyl-Coenzym A zu (S)-Methylmalonyl-Coenzym A katalysiert;
• – eines Enzyms E₈, welches die Umsetzung von (S)-Methylmalonyl-Coenzym A zu (R)-Methylmalonyl-Coenzym A katalysiert;
• – eines Enzyms E₉, welches die Umsetzung von (R)-Methylmalonyl-Coenzym A zu Succinyl-Coenzym A katalysiert;
• – eines Enzyms E₁₀, welches die Umsetzung von Succinyl-Coenzym A zu Succinatsemialdehyd katalysiert;
• – eines Enzyms E₁₁, welches die Umsetzung von Succinat-Semialdehyd zu 4-Hydroxybuttersäure katalysiert;
• – eines Enzyms E₁₂, welches die Umsetzung von 4-Hydroxybuttersäure zu 4-Hydroxybutyryl-Coenzym A katalysiert;
• – E₁₃ aufweist, welches die Umsetzung von 4-Hydroxybutyryl-Coenzym A zu Crotonyl-Coenzym A katalysiert;
• – eines Enzyms E₁₄, welches die Umsetzung von Crotonyl-Coenzym A zu (S)-3-Hydroxybutyryl-Coenzem A katalysiert;
• – eines Enzyms E₁₅, welches die Umsetzung von (S)-3-Hydroxybutyryl-Coenzem A zu Acetoacetyl-Coenzym A katalysiert;
• – eines Enzyms E₁₆, welches die Umsetzung von Acetoacetyl-Coenzym A zu zwei Molekülen Acetyl-Coenzym A katalysiert.

According to a preferred embodiment of the recombinant cell according to the invention, this has an increased activity of at least one of the following enzymes E ₁ to E ₁₆ in comparison to their wild type:

• An enzyme E ₁ which catalyzes the conversion of carbonate and acetyl coenzyme A to malonyl coenzyme A;
• An enzyme E ₂ which catalyzes the conversion of malonyl coenzyme A to malonate semialdehyde;
• An enzyme E ₃ which catalyzes the conversion of malonate semialdehyde to 3-hydroxypropionic acid;
• An enzyme E ₄ which catalyzes the conversion of 3-hydroxypropionic acid to 3-hydroxypropionyl-coenzyme A;
• An enzyme E ₅ which catalyzes the conversion of 3-hydroxypropionyl-coenzyme A into acryloyl-coenzyme A;
• An enzyme E ₆ which catalyzes the reaction of acryloyl-coenzyme A into propionyl-coenzyme A;
• An enzyme E ₇ which catalyzes the conversion of carbonate and propionyl coenzyme A to (S) -methylmalonyl coenzyme A;
• An enzyme E ₈ which catalyzes the conversion of (S) -methylmalonyl-coenzyme A to (R) -methylmalonyl-coenzyme A;
• An enzyme E ₉ which catalyzes the conversion of (R) -methylmalonyl-coenzyme A to succinyl-coenzyme A;
• An enzyme E ₁₀ which catalyzes the conversion of succinyl-coenzyme A to succinic semialdehyde;
• An enzyme E ₁₁ which catalyzes the conversion of succinate semialdehyde to 4-hydroxybutyric acid;
• An enzyme E ₁₂ which catalyzes the conversion of 4-hydroxybutyric acid to 4-hydroxybutyryl-coenzyme A;
• - E ₁₃ , which catalyzes the reaction of 4-hydroxybutyryl-coenzyme A to crotonyl-coenzyme A;
• An enzyme E ₁₄ which catalyzes the conversion of crotonyl-coenzyme A to (S) -3-hydroxybutyryl-coenzyme A;
• An enzyme E ₁₅ which catalyzes the conversion of (S) -3-hydroxybutyryl-coenzyme A to acetoacetyl-coenzyme A;
• - An enzyme E ₁₆ , which is the reaction of acetoacetyl-coenzyme A to two molecules of acetyl-Coen catalyzed A zym.

Of particular importance in this newly discovered metabolic pathway is that enzyme E ₁₃ which catalyzes the conversion of 4-hydroxybutyryl-coenzyme A into crotonyl-coenzyme A.

The following statements on increasing the enzyme activity in cells apply both to the increase in the activity of the enzyme E ₁₃ and to all the enzymes mentioned below, the activity of which may optionally be increased, in particular also for the enzymes E ₁ to E ₁₂ and E ₁₄ to E ₁₆ .

in principle can be an increase in enzymatic activity Achieve by the copy number of the gene sequence or the Increases gene sequences encoding the enzyme, use a strong promoter or use a gene or allele, that for a corresponding enzyme with an increased activity coded and if necessary combined these measures. Genetically modified according to the invention Cells are transformed, for example, by transformation, transduction, Conjugation or a combination of these methods with a vector generates the desired gene, an allele of this gene or parts thereof and one that enables the expression of the gene Vector contains. The heterologous expression is particularly by Integration of the gene or alleles into the chromosome of the cell or an extrachromosomally replicating vector.

An overview of the possibilities for increasing the enzyme activity in cells using the example of pyruvate carboxylase gives DE-A-100 31 999 , which is hereby incorporated by reference, and the disclosure of which with regard to the possibilities of increasing the enzyme activity in cells forms part of the disclosure of the present invention.

The expression of the above and all of the enzymes or genes mentioned below can be detected in the gel with the aid of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration with appropriate evaluation software. If the increase in enzyme activity is based solely on an increase in the expression of the corresponding gene, the quantification of the increase in enzyme activity can be determined in a simple manner by comparing the 1- or 2-dimensional protein separations between wild type and genetically modified cell. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that of Hermann et al. (Electrophoresis, 22: 1712.23 (2001) described procedure. The protein concentration can also be determined 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 111: 2630-2647 ) to be analyzed. The activity of DNA-binding proteins can be measured by DNA band-shift assays (also called gel retardation) ( Wilson et al. (2001) Journal of Bacteriology, 183: 2151-2155 ). The effect of DNA-binding proteins on the expression of other genes can be demonstrated 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, 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 al. (1973) Journal of Bacteriology 115 (3): 816-823 ). Unless specific methods for the determination of the activity of a particular enzyme are specified in the following, the determination of the increase in the enzyme activity and also the determination of the reduction of an enzyme activity is preferably carried out by means of in Hermann et al., Electrophoresis, 22: 1712-23 (2001) . Lohaus et al., Biospektrum 5 32-39 (1998) . Lottspeich, Angewandte Chemie 111: 2630-2647 (1999) and Wilson et al., Journal of Bacteriology 183: 2151-2155 (2001) described methods.

Becomes the increase of enzyme activity by mutation of the endogenous gene, such Mutations generated either undirected by classical methods be such as by UV irradiation or by mutagenic Chemicals, or specifically by means of genetic engineering methods such as Deletion (s), insertion (s) and / or nucleotide exchange (s). By These mutations will be genetically engineered cells receive. Particularly preferred mutants of enzymes are in particular even those enzymes that are no longer or at least compared to Wild-type enzyme reduces feedback are inhibitable.

If the increase in enzyme activity is accomplished by increasing the expression of an enzyme, for example, one increases the copy number of the corresponding genes or mutates the promoter and regulatory region or ribosome binding site 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, it is also possible to associate with the enzyme gene as regulatory sequences so-called "enhancers" which likewise bring about increased gene expression via an improved interaction between RNA polymerase and DNA. Measures to extend the lifetime of m-RNA also improve expression. Furthermore, by preventing degradation of the enzyme protein, enzyme activity is also enhanced. The genes or gene constructs are either present in plasmids with different copy numbers or are integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can be achieved by changing the composition of the medium and culture. Instructions for this, the expert finds among others Martin et al. (Bio / Technology 5, 137-146 (1987)) , at Guerrero et al. (Gene 138, 35-41 (1994) ) Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)) , at Eikmanns et al. (Gene 102, 93-98 (1991)) , in EP-A-0 472 869 , in the US 4,601,893 , at Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991) , at Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) , at LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)) , in WO-A-96/15246 , at Malumbres et al. (Gene 134, 15-24 (1993) , in JP-A-10-229891 , at Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) and in known textbooks of genetics and molecular biology. The measures described above as well as the mutations lead to genetically modified cells.

To increase the expression of the respective genes, episomal plasmids are used, for example. Suitable plasmids are in particular those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as pZl ( Menkel et al., Applied and Environmental Microbiology 64: 549-554 (1989) ), pEKEx1 ( Eikmanns et al., Gene 107: 69-74 (1991) ) or pHS2-1 ( Sonnen et al., Gene 107: 69-74 (1991) ) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such as those based on pCG4 ( US 4,489,160 ) or pNG2 ( Serwold-Davis et al., FEMS Microbiology Letters 66: 119-124 (1990) ) or pAG1 ( US 5,158,891 ) can be used in the same way.

Also suitable are those plasmid vectors, by means of which one can apply the method of gene amplification by integration into the chromosome, as it is for example of Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994)) for duplication or amplification of the homthrB operon. In this method, the complete gene is cloned into a plasmid vector which can be replicated in a host (typically Escherichia coli) but not in Corynebacterium glutamicum. As vectors, for example, pSUP301 ( Simon et al., Bio / Technology 1: 784-791 (1983) ), pK18mob or pK19mob ( Schäfer et al., Gene 145: 69-73 (1994) ), pGEM-T (Promega Corporation, Madison, Wisconsin, USA), pCR2.1-TOPO ( Shuman, Journal of Biological Chemistry 269: 32678-84 (1994) (), PCR ^® Blunt (Invitrogen, Groningen, The Netherlands), Pemi Schrumpf et al., Journal of Bacteriology 173: 4510-4516) ) or pBGS8 ( Spratt et al., Gene 41: 337-342 (1986) ) in question. The plasmid vector containing the gene to be amplified is then converted by conjugation or transformation into the desired strain of Corynebacterium glutamicum. The method of conjugation is for example at Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994) described. Methods for transformation are for example 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) described. After homologous recombination by means of a cross-over event, the resulting strain contains at least two copies of the gene of interest.

The expression "an activity of an enzyme E _{x which} is increased in relation to its wild-type" is preferably always an additional factor of at least 2, more preferably of at least 10, more preferably of at least 100, and moreover 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 which "exhibits an activity of an enzyme E _x which is increased compared to its wild type" comprises in particular also a cell whose wild-type has no or at least no detectable activity of this enzyme E _x and only after increasing the enzyme activity, for example by overexpression, a 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 Expression of the enzyme E _{x is} induced.

Recombinant cells which are particularly preferred according to the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E ₁ , E ₂ , E ₃ , E ₄ , E ₅ , E ₆ , E ₇ , E ₈ , E ₉ , E ₁₀ , E ₁₁ , E ₁₂ , E ₁₃ , E ₁₄ , E ₁₅ , E ₁₆ , E ₁ E ₂ , E ₁ E ₃ , E ₁ E ₄ , E ₁ E ₅ , E ₁ E ₆ , E ₁ E ₇ , E ₁ E ₈ , E ₁ E ₉ , E ₁ E ₁₀ , E ₁ E ₁₁ , E ₁ E ₁₂ , E ₁ E ₁₃ , E ₁ E ₁₄ , E ₁ E ₁₅ , E ₁ E ₁₆ , E ₂ E ₃ , E ₂ E ₄ , E ₂ E ₅ , E ₂ E ₆ , E ₂ E ₇ , E ₂ E ₈ , E ₂ E ₉ , E ₂ E ₁₀ , E ₁ E ₁₁ , E ₂ E ₁₂ , E ₂ E ₁₃ , E ₂ E ₁₄ , E ₂ E ₁₅ , E ₂ E ₁₆ , E ₃ E ₄ , E ₃ E ₅ , E ₃ E ₆ , E ₃ E ₇ , E ₃ E ₈ , E ₃ E ₉ , E ₃ E ₁₀ , E ₃ E ₁₁ , E ₃ E ₁₂ , E ₃ E ₁₃ , E ₃ E ₁₄ , E ₃ E ₁₅ , E ₃ E ₁₆ , E ₄ E ₅ , E ₄ E ₆ , E ₄ E ₇ , E ₄ E ₈ , E ₄ E ₉ , E ₄ E ₁₀ , E ₄ E ₁₁ , E ₄ E ₁₂ , E ₄ E ₁₃ , E ₄ E ₁₄ , E ₄ E ₁₅ , E ₄ E ₁₆ , E ₅ E ₆ , E ₅ E ₇ , E ₅ E ₈ , E ₅ E ₉ , E ₅ E ₁₀ , E ₅ E ₁₁ , E ₅ E ₁₂ , E ₅ E ₁₃ , E ₅ E ₁₄ , E ₅ E ₁₅ , E ₅ E ₁₆ , E ₆ E ₇ , E ₆ E ₈ , E ₆ E ₉ , E ₆ E ₁₀ , E ₆ E _{1 1} , E ₆ E ₁₂ , E ₆ E ₁₃ , E ₆ E ₁₄ , E ₆ E ₁₅ , E ₆ E ₁₆ , E ₇ E ₈ , E ₇ E ₉ , E ₇ E ₁₀ , E ₇ E ₁₁ , E ₇ E ₁₂ , E ₇ E ₁₃ , E ₇ E ₁₄ , E ₇ E ₁₅ , E ₇ E ₁₆ , E ₈ E ₉ , E ₈ E ₁₀ , E ₈ E ₁₁ , E ₈ E ₁₂ , E ₈ E ₁₃ , E ₈ E ₁₄ , E ₈ E ₁₅ , E ₈ E ₁₆ , E ₉ E ₁₀ , E ₉ E ₁₁ , E ₉ E ₁₂ , E ₉ E ₁₃ , E ₉ E ₁₄ , E ₉ E ₁₅ , E ₉ E ₁₆ , E ₁₀ E ₁₁ , E ₁₀ E ₁₂ , E ₁₀ E ₁₃ , E ₁₀ E ₁₄ , E ₁₀ E ₁₅ , E ₁₀ E ₁₆ , E ₁₁ E ₁₂ , E ₁₁ E ₁₃ , E ₁₁ E ₁₄ , E ₁₁ E ₁₅ , E ₁₁ E ₁₆ , E ₁₂ E ₁₃ , E ₁₂ E ₁₄ , E ₁₂ E ₁₅ , E ₁₂ E ₁₆ , E ₁₃ E ₁₄ , E ₁₃ E ₁₅ , E ₁₃ E ₁₆ , E ₁₄ E ₁₅ , E ₁₄ E ₁₆ , E ₁₅ E ₁₆ , E ₁ E ₂ E ₇ , E ₁ E ₃ E ₇ , E ₁ E ₄ E ₇ , E ₁ E ₅ E ₇ , E ₁ E ₆ E ₇ , E ₁ E ₈ E ₇ , E ₁ E ₉ E ₇ , E ₁ E ₁₀ E ₇ , E ₁ E ₁₁ E ₇ , E ₁ E ₁₂ E ₇ , E ₁ E ₁₃ E ₇ , E ₁ E ₁₄ E ₇ , E ₁ E ₁₅ E ₇ , E ₁ E ₁₆ E ₇ , E ₁ E ₂ E ₇ E ₁₃ , E ₁ E ₃ E ₇ E ₁₃ , E ₁ E ₄ E ₇ E ₁₃ , E ₁ E ₅ E ₇ E ₁₃ , E ₁ E ₆ E ₇ E ₁₃ , E ₁ E ₈ E ₇ E ₁₃ , E ₁ E ₉ E ₇ E ₁₃ , E ₁ E ₁₀ E ₇ E ₁₃ , E ₁ E ₁₁ E ₇ E ₁₃ , E ₁ E ₁₂ E ₇ E ₁₃ , E ₁ E ₁₄ E ₇ E ₁₃ , E ₁ E ₁₅ E ₇ E ₁₃ , E ₁ E ₁₆ E ₇ E ₁₃ , E ₁ E ₂ E ₃ E ₁₃ , E ₁ E ₂ E ₃ E ₁₃ , E ₁ E ₂ E ₃ E ₇ E ₁₃ and E ₁ E ₂ E ₃ E ₄ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ E ₁₂ E ₁₄ E ₁₅ E ₁₆ , where E ₁₃ , E ₁ E ₁₃ E ₇ and E ₁ E ₂ E ₃ E ₁₃ and E ₁ E ₂ E ₃ E ₇ E _{13 are} particularly preferred.

In this context, it is further preferred that the enzyme
E _{1 is} an acetyl-coenzyme A carboxylase (EC 6.4.1.2),
E ₂ a malonyl-coenzyme A reductase (EC 1.2.1.18),
E _{3 is} a malonate semialdehyde reductase (EC 1.1.1.59),
E _{4 is} a 3-hydroxypropionyl synthetase (EC 6.2.1.-),
E _{5 is} a 5,3-hydroxypropionyl-coenzyme A dehydratase (EC 4.2.1.17),
E ₆ an acryloyl-coenzyme A reductase (EC 1.3.99.3),
E _{7 is} a propionyl-coenzyme A carboxylase (EC 6.4.1.3),
E ₈ a methylmalonyl-coenzyme A-epimerase (EC 5.1.99.1)
E _{9 is} a methylmalonyl-coenzyme A mutase (EC 5.4.99.2),
E _{10 is} a succinyl-coenzyme A reductase,
E _{11 is} a succinic semialdehyde reductase (EC 1.1.1.61),
E _{12 is} a 4-hydroxybutyryl-coenzyme A synthetase
E _{13 is} a 4-hydroxybutyryl-coenzyme A dehydratase,
E ₁₄ a crotonyl-coenzyme A hydratase (EC 4.2.1.17),
E _{15 is} a 3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35), and
E ₁₆ an acetoacetyl-coenzyme A-β-ketothiolase (2.3.1.9)
is.

The enzyme E ₁ is preferably encoded by genes selected from the group consisting of acaca, acacb, f5j5.21, fl5c21.2, t8p21.5, acc1, aar071wp, accA, accB, accC, accD, accC1 and accC2, with accA , accC and accD are most preferred. An example of a suitable acetyl-coenzyme A carboxylase is, inter alia, that of Rhizobium leguminosarum which is derived from the DNA sequence according to SEQ. ID. 01 is coded and which the amino acid sequence according to SEQ. 02 has.

The enzyme E ₂ is preferably encoded by the ioID gene.

The enzymes E ₅ and E ₁₄ are preferably selected from the group comprising echS1, ehhadh, hadha, echs1-prov, cg4389, cg4389, cg6543, cg6984, cg8778, ech-1, ech-2, ech-3, ech- 4, ech-5, ech-6, ech-7, FCAALL.314, fcaall.21, fox2, ecil, eci2, paaF, paaG, yfcX, fadB, faoA, rpfF, phaA, phaB, echA1, echA2, echA3, echA4, echA5, echA6, echA7, echA8, echA9, echA9, echA10, echA11, echA12, echA13, echA14, echA15, echA16, echA17, echA18, echA19, echA20, echA21, fad-1, fad-2, fad-3, dcaE, hcaA, fadJ, rsp0671, rsp0035, rsp0648, rsp0647, rs03234, rs03271, rs04421, rs04419, rs02820, rs02946, paaG1, paaG2, paaG3, ech, pksH, ydbS, eccH1, eccH2, pimF, fabJ1, fabJ2, caiD2, ysiB, yngF, yusL, fucA, cg10919, scf41.23, scd10.16, sck13.22, scp8.07c, stbac16h6.14, sc5f2a.15, sc6a5.38, hbd-1, hbd-2, hdb-3, hdb-4, hdb-5, hdb-6, hdb-7, hdb-8, hdb-9, hdb-10, fad-1, fad-2, fad-3, fad-4, fad-5, paaF 1, paaF-2, paaF-3, paaF-4, paaF-5, paaF-6, paaF-7 and crt.

The enzyme E ₆ is preferably selected from the group consisting of acadl, acadm, acad10, acad11, acadmpro, acadl-prov, mgc81873, cg12262, cg4703, cg4860, f3e22.5, afl213wp, acdC, fadE13, acd-1 acd-2, acd-3, acd-4, acd-5, acd-6, acd-7, acd-8, acd-9, acd-10, acd-11, acd-12, acd, fadE1, fadE2 , stringE3, stringE4, stringE5, stringE6, stringE7, stringE13, stringE14, stringE15, stringE16, stringE17, stringE18, stringE19, stringE20, stringE21, stringE22, stringE23, stringE26, stringE27, stringE30, stringE31, stringE33, stringE35, stringE38, stringE45, stringE , caiA, aidB, RSp0036, RS03588, mmgC, acdA-3, bcd, acdA, acdH1, acdH2, acdH3, aidB, acdI and acdH.

The enzyme E ₇ is preferably selected from the group consisting of pCCA, pCCB, LOC699844, MGC68650, LOC582365, LOC592281, pcca-1, pccb-1, gnyA, gnyB, accA, accA2, accB, accD1, accD2, accD3, accD4 , accD5, accD6, mccA, cg10707, cg10708, cg12870, dtsR, dtsR1, dtsR2 and yngE. An example of a suitable propionyl-coenzyme A carboxylase is, inter alia, that from Rhizobium leguminosarum which is derived from the DNA sequence according to SEQ. ID. 03 is coded and which the amino acid sequence according to SEQ. 04 has.

The enzyme E ₈ is preferably encoded by the mcee gene.

The enzyme E ₉ is preferably encoded by genes selected from the group consisting of mut, mutA, mutB, sbm, sbmA, sbmB, sbm5, bhbA, mcmA, mcmA1, mcmA2, mcmB, mcm1, mcm2, mcm3, icmA, meaA1 and meaA2 ,

The enzyme E ₁₀ is preferably selected from genes consisting of the group consisting of aldH5F1, uga2, ald3, ald4, ald3, uga22, gabD, attK, attK1, attK2, XOO2614, gabD-1, gabD-2, gabD3, gabD4, gabD5, gabD6, gave gab, thmS1, thmS2, ssdH, gaveDch, gaveDc, ypf10097, gaveDf1, gaveDf2, ssdA, sucD, cg10051, cg10480, 2SCG58.04 and SCD63.12.

The enzyme E ₁₁ is preferably encoded by genes selected from the group comprising gbd, gbd1, gbd2, abfH and 4hbD.

The enzyme E ₁₃ , which catalyzes the conversion of 4-hydroxybutyryl-coenzyme A into crotonyl-coenzyme A, is preferably a 4-hydroxybutyryl-coenzyme A dehydratase. An enzyme E _{13 which} is particularly suitable according to the invention is, for example, a Sulfolobus solfataricus 4-hydroxybutyryl-coenzyme A dehydratase, which is derived from the DNA sequence according to SEQ. ID. 05 is coded and which the amino acid sequence according to SEQ. 06, or a Sulfolobus tokodaii 4-hydroxybutyryl-coenzyme A dehydratase derived from the DNA sequence according to SEQ. ID. 07 is coded and which the amino acid sequence according to SEQ. 08 has.

The enzyme E ₁₅ is preferably selected from genes selected from the group consisting of hibadh, cg15093, cg15093, cg4747, mwL2.23, t13k14.90, f19b15.150, hibA, ygbJ, mmsB, mmsB, garR, tsar, mmsB-1, mmsB-2, yfjR, ykwC, ywjF, hibD, glxR, SCM1.40c, hibD, ehhand, hadh2, hadhsc, hsd17B4, loc488110, had, mgC81885, hadh2-prov, cg3415, cg7113, ech-1, ech-8, ech-9, ard-1, yfcX, fadB, faoA, fadB2x, hbd-1, hbd-2, hbd-3, hbd-4, hbd-5, hbd-6, hbd-7, hbd-8, hbd- 9, hbd-10, fadJ, rs04421, rs02946, rs05766, bbsD, bbsC, fadB1, fadB2, fadB5, hbdA, pimF, fabJ-1, fabJ, scbac19f3.11, sci35.13, scbac8d1.10c, sc5f2a.15, sc6a5.38, fadC2, fadC4, fadC5, fadC6, had and paaH encoded.

The enzyme E ₁₆ is preferably selected from a gene selected from the group consisting of acaT1, acaT2, RCJMB04_34i5, MGC69098, MGC81403, MGC81256, MGC83664, LOC759502, cat-1, ACAT2 / EMB1276, erg10, atoB, yqeF, th1A, XOO1778, fadAx , phbA-1, phaA, phbA-2, atoB-2, bktB, tioL, thlA, paaJ, phbAch, phbAf, pimB, pcaF, mmGA, yhfS, th1, vraB, mvaC, thiL, fAdA, th1A1, th1A2, th1A3 , paaJ, fadA1, fadA2, fadA3, fadA4, fadA5, fadA6, fadA7, fadA8, fadA9, fadA10, fadA11, fadA12, cg12392, pcaF, catF, SC8F4.03, thiL1, and thiL2.

The gene sequences of the genes mentioned above in connection with the enzymes E ₁ , E ₂ , E ₅ , E ₆ to E ₁₁ and E ₁₄ to E ₁₆ can, inter alia, the KEGG database ("Kyoto Encyclopedia of Genes and Genomes") be removed.

According to the invention, it is particularly preferred to use a recombinant cell whose wild-type is already capable of acetyl-coenzyme A via malonyl-coenzyme A, malonate semialdehyde, 3-hydroxypropionate, 3-hydroxypropionyl-coenzyme A, acryloyl-coenzyme A, propionyl-coenzyme A, methylmalonyl coenzyme A, succinyl coenzyme A, succinate semialdehyde and 4-hydroxybutyric acid to 4-hydroxybutyryl coenzyme A implement (ie their wild type already has a detectable activity of the enzymes E ₁ to E ₁₂ ) and which is also able to To convert crotonyl-coenzyme A via 3-hydroxybutyryl-coenzyme A and acetoacetyl-coenzyme A to two equivalents of acetyl-coenzyme A (the wild type thus already has a detectable activity of the enzymes E ₁₄ to E ₁₆ ), and in this cell the activity of Enzyme E ₁₃ or the activity of the enzymes E ₁ , E ₇ and E ₁₃ increase.

As already explained at the outset, the recombinant cell according to the invention, in which the activity of at least one of the enzymes E ₁ to E _{16 is} increased, is capable of converting from two equivalents of bicarbonate to one eq to produce valent acetyl coenzyme A. This C ₂ carbon unit can now serve as a starting material for the preparation of organic compounds having at least two carbon atoms by the recombinant cell.

Preferably, these organic compounds having at least two carbon atoms are a C ₃ compound, a C ₄ compound, a C ₅ compound, a C ₆ compound, a C ₇ compound, a C ₈ compound, a C ₉ compound, a C ₁₀ compound or a compound with more than 10 carbon atoms. Among these compounds, particularly preferred are organic compound selected from the group consisting of a carboxylic acid, a dicarboxylic acid, a hydroxycarboxylic acid, an amine, an amino acid, a ketone and an alcohol.

When Examples of organic compounds, which by the can be produced according to the invention cell, in particular compounds are selected from the Group consisting of acrylic acid, methacrylic acid, 3-hydroxypropionic acid, 3-hydroxyisobutyric acid, Lysine, glutamate, methionine, phenylalanine, aspartic acid, Tryptophan, threonine, butanol, 1,2-propanediol, 1,3-propanediol, 2,3-butanediol, 1,4-butanediol, 1,6-hexanediol, glycerol, sorbitol, manitol, xylitol, Arabinitol, glucose, acetone and dihydroxyacetone.

If the recombinant cell according to the invention to provide organic compounds of bicarbonate, already as intermediates in the in the 1 can be formed, it may be sufficient to increase the activity of those enzymes which acetyl coenzyme A, which by the in 1 shown pathway is formed from two equivalents of bicarbonate, implement this corresponding compound. However, by means of the recombinant cells according to the invention organic compounds are to be provided which are not directly available in the 1 In addition to increasing the activity of one or more of the enzymes E ₁ to E ₁₆ , it may also be necessary to increase the activity of enzymes involved in the formation of such compounds from acetyl coenzyme A or from any intermediates in the 1 involved metabolic pathway are involved.

According to a first, particular variant of the recombinant cell according to the invention, the latter is capable of forming hydrogencarbonate from 3-hydroxypropionic acid. Since 3-hydroxypropionic acid as an intermediate in the in the 1 According to this first variant of the recombinant cell according to the invention, it is particularly preferred if it has an increased activity of the following enzymes or enzyme combinations: E ₁ , E ₂ , E ₃ , E ₁ E ₁₃ , E ₂ E ₁₃ , E ₃ E ₁₃ , E ₁ E ₂ E ₁₃ , E ₁ E ₃ E ₁₃ , E ₂ E ₃ E ₁₃ , E ₁ E ₂ E ₇ E ₁₃ , E ₁ E ₃ E ₇ E ₁₃ , E ₂ E ₃ E ₇ E ₁₃ , E ₁ E ₂ , E ₁ E ₃ , E ₂ E ₃ and E ₁ E ₂ E ₃ E ₇ E ₁₃ , where as enzymes E ₁ , E ₂ , E ₃ , E ₇ and E _{13 are} those which are already have been described above.

The term "3-hydroxypropionic acid" as used above, as well as the term "3-hydroxyisobutyric acid" used below, always describes the corresponding C ₃ - or C ₄ -carboxylic acid in the form in which they are formed after formation by the corresponding microorganisms as a function of the pH Value is present. The term thus always includes the pure acid form (3-hydroxypropionic acid or 3-hydroxyisobutyric acid), the pure base form (3-hydroxypropionate or 3-hydroxyisobutyrate) and mixtures of protonated and deprotonated form of the acid.

• – eines Enzyms E₁₇, welches die Umsetzung von Propionyl-Coenzym A zu Methylmalonatsemialdehyd katalysiert;
• – eines Enzyms E₁₈, welches die Umsetzung von Methylmalonatsemialdehyd zu 3-Hydroxyisobuttersäure katalysiert.

According to a second, particular variant of the recombinant cell according to the invention, the latter is capable of forming hydrogencarbonate from 3-hydroxyisobutyric acid. In connection with this second variant of the recombinant cell according to the invention, it is particularly preferred that this, in addition to an increased activity of one or more of the enzymes E ₁ to E _16, also has an increased activity of at least one of the enzymes E ₁₇ and E ₁₈ :

• An enzyme E ₁₇ which catalyzes the conversion of propionyl-coenzyme A to methylmalonate semialdehyde;
• An enzyme E ₁₈ which catalyzes the conversion of methylmalonate semialdehyde to 3-hydroxyisobutyric acid.

In this connection it is particularly preferred that the enzyme
E _{17 is} a methylmalonate semialdehyde dehydrogenase (EC 1.2.1.27), and
E _{18 is} a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a 3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35)
is.

Suitable genes for the enzyme E ₁₇ are preferably selected from the group consisting of aldh6al, cg17896, t22c12.10, ald6, putA1, mmsA, mmsA-1, mmsA-2, mmsA-3, mmsA-4, msdA, io1A and iolAB.

Suitable genes for the enzyme E ₁₈ are selected from the group consisting of hibadh, cg15093, cg15093, cg4747, mwL2.23, t13k14.90, fl9b15.150, hibA, ygbJ, mmsB, mmsB, garR, tsar, mmsB-1, mmsB-2, yfjR, ykwC, ywjF, hibD, g1xR, SCM1.40c, hibD, ehhand, hadh2, hadhsc, hsd17B4, loc488110, had, mgC81885, hadh2-prov, cg3415, cg7113, ech-1, ech-8, ech-9, ard-1, yfcX, fadB, faoA, fadB2x, hbd-1, hbd-2, hbd-3, hbd-4, hbd-5, hbd-6, hbd-7, hbd-8, hbd- 9, hbd-10, fadJ, rs04421, rs02946, rs05766, bbsD, bbsC, fadB1, fadB2, fadB5, hbdA, pimF, fabJ-1, fabJ, scbac19f3.11, sci35.13, scbac8d1.10c, sc5f2a.15, sc6a5.38, fadC2, fadC4, fadC5, fadC6, had and paaH. Further suitable 3-Hudroxuisobuturat dehudrogenases are, for example, in Bannerjee et al. (1970), J. Biol. Chem, 245, pp. 1828-1835 . Steele et al. (1992), J. Biol. Chem., 267, pages 13,585 to 13,592 . Harris et al. (1988), J. Biol. Chem., 263, pages 327 to 331 . Harris et al., Biochim. Biophys. Acta, 1645 (1), pages 89-95 . Hawes et al. (2000), Methods Enzymol., 324, pages 218-228 . Harris et al., J. Biol. Chem., 275 (49), pages 38,780 to 38,786 . Rougraff et al. (1988), J. Biol. Chem., 263 (1), pages 327 to 331 . Robinson et al., J. Biol. Chem., 225, pp. 511-521 . Hawes et al. (1995), Biochemistry, 34, pp. 4,231-4,237 . Hasegawa J. (1981), Agric. Biol. Chem., 45, pp. 2,805-2814, Hawes et al. (1996), FERS Lett., 389, pages 263 to 267 . Hawes et al. (1996), Enzymology and Molecular Biology of Carbonyl Metabolism, Plenum Press, New York, pages 395 to 402 . Adams et al. (1994), Structure, 2, pp. 651-668 . Zhang et al. (1999), Biochemistry, 38, pages 11.231 to 11.238 . Mirny et al., (1999), J. Mol. Biol., 291, pages 177 to 196 and Lokanath et al. (2005), J Mol Biol. , described. The disclosure of these references is hereby incorporated by reference and forms part of the disclosure of the present invention.

The nucleotide sequences of the abovementioned genes for the enzymes E ₁₇ and E ₁₈ can also be taken from the KEGG database, among others.

Since propionyl-coenzyme A is already used as an intermediate in the 1 In addition to increasing the activity of one or more of the enzymes E ₁₇ and E ₁₈ , the activity of one or more of the enzymes E ₁ to E ₆ may also be offered in particular in this second, particular variant of the recombinant cell according to the invention increase.

According to a third, particular variant of the recombinant cell according to the invention, the latter is able to form pyrogen from bicarbonate. In connection with this third variant of the recombinant cell according to the invention, it is particularly preferred that in addition to an increased activity of one or more of the enzymes E ₁ to E ₁₆ also has an increased activity of an enzyme E ₁₉ , which is the reaction of acetyl coenzyme A and Catalyses carbon dioxide to pyruvate, which is preferably a pyruvate synthase (EC 1.2.7.1) in this enzyme E ₁₉ . In particular, genes for a suitable pyruvate synthase can be selected from the group consisting of yccM, padE, padG, padI, padF, porA, porB, porC, porD, porG, nifJ, forA1, forA2, forB1, forB2, forG1, forG2, porA_1, PorA_2, PorB_1, PorB_2, PorC-1, PorD-1, PorD-2, PorG-1, PorG_2, PorD-like, PorA-like and PorB-like. The nucleotide sequences of the abovementioned genes for the enzyme E ₁₉ can also be taken from the KEGG database.

A contribution to the solution of the abovementioned objects is also provided by a method for producing a recombinant cell which is capable of forming from bicarbonate organic compounds having at least two carbon atoms, comprising the step of increasing the activity of one or more of the enzymes E ₁ to E ₁₆ in the cell. Also, the recombinant cell obtainable by this method makes a contribution to the solution of the above-mentioned objects.

a further contribution to the solution of the aforementioned tasks in particular also makes use of a recombinant cell for the targeted production of organic compounds from bicarbonate taking advantage of a metabolic pathway in which acetyl-CoA and bicarbonate malonyl coenzyme A, malonyl coenzyme A malonate semialdehyde, from malonate semialdehyde 3-hydroxypropionate, from 3-hydroxypropionate 3-hydroxypropionyl-coenzyme A, from 3-hydroxypropionyl-coenzyme A acryloyl-coenzyme A, from acryloyl-coenzyme A propionyl-coenzyme A, from propionyl-coenzyme A and bicarbonate (S) -methylmalonyl-coenzyme A, from (S) -methylmalonyl-coenzyme A (R) -methylmalonyl coenzyme A, from (R) -methylmalonyl-coenzyme A succinate semialdehyde, from succinic semialdehyde 4-hydroxybutyric acid, from 4-hydroxybutyric acid 4-hydroxybutyryl-coenzyme A, from 4-hydroxybutyryl-coenzyme A crotonyl-coenzyme A, from crotonyl-coenzyme A 3-hydroxybutyryl-coenzyme A, from 3-hydroxybutyryl-coenzyme A acetoacetyl-coenzyme A and acetoacetyl-coenzyme A two equivalents Acetyl-coenzyme A are formed.

The Formulation "for the targeted production of organic compounds From bicarbonate "is to be understood that the cell in the Intention is used to make organic compounds with at least produce two carbon atoms from bicarbonate. Therefore includes the targeted production of such organic compounds in particular also bringing the inventive invention recombinant cell with a bicarbonate-containing nutrient medium and at least one purification step, by means of which the organic compounds after their production by the cells can be isolated.

preferred In turn, cells and organic compounds are those cells and organic compounds already related in the beginning with the recombinant cells according to the invention have been mentioned, wherein as organic compounds 3-hydroxypropionic acid and 3-hydroxyisobutyric acid are particularly preferred.

a Contribute to the solution of the above-mentioned tasks in particular also a process for the production of organic Compounds having at least two carbon atoms, comprising the Process step of bringing into contact a inventive Cell with a hydrogen carbonate containing nutrient medium, preferably with a nutrient medium into which a carbon dioxide-containing Gas introduced with at least partial formation of bicarbonate is, under conditions, among which from the bicarbonate organic Compounds having at least two carbon atoms are formed, and optionally purification of the organic compounds with at least two carbon atoms from the nutrient medium.

The recombinant cells according to the invention can continuously or discontinuously in the batch process (sentence culturing) or in the fed-batch process (feed process) or repeated fed-batch V experienced (repetitive feed process) for the purpose of producing organic compounds having at least two carbon atoms of bicarbonate with the culture medium in contact and thus cultivated. Also conceivable is a semi-continuous process, as described in the GB-A-1009370 is described. A summary of known cultivation methods can be found in the textbook by Chmiel ( "Bioprocessing Technology 1. Introduction to Bioprocess Engineering" (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the textbook of Storhas ( "Bioreactors and Peripheral Facilities", Vieweg Verlag, Braunschweig / Wiesbaden, 1994 ).

The culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981) contain.

When Carbon source, which optionally in addition to the bicarbonate can be added, carbohydrates such as. Glucose, Sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such. Soybean oil, Sunflower oil, peanut oil and coconut oil, fatty acids such as Palmitic acid, stearic acid and linoleic acid, Alcohols such. As glycerol and methanol, hydrocarbons such Methane, amino acids such as L-glutamate or L-valine or organic Acids such. As acetic acid can be used.

When Nitrogen source can be organic nitrogen-containing compounds such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, Soybean meal and urea or inorganic compounds such as Ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate are used. The nitrogen sources can used singly or as a mixture.

When Phosphorus source can phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing ones Salts are used. The culture medium must continue to salts of Metals include such. As magnesium sulfate or iron sulfate, the necessary for growth. Finally, you can essential growth factors such as amino acids and vitamins in addition used for the above-mentioned substances. The culture medium In addition, suitable precursors may be added become. The stated feedstocks can be used to culture in Formed as a one-off approach or as appropriate be fed during cultivation.

For pH control of the culture, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid are suitably used. To control the foam development antifoams such. B. fatty acid polyglycol esters are used. To maintain the stability of plasmids can the medium suitable nete selective substances such. B. antibiotics are added. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures such. For example, air is introduced into the culture. The temperature of the culture is usually 20 ° C to 45 ° C, and preferably 25 ° C to 40 ° C.

The bicarbonate is preferably added to the nutrient medium by the introduction of carbon dioxide-containing gas. As carbon dioxide-containing gas pure carbon dioxide can be used. However, carbon dioxide-rich gas mixtures, in particular gas mixtures having a CO ₂ content of at least 50% by volume, more preferably at least 75% by volume and most preferably at least 90% by volume, are conceivable and preferred according to the invention. As gas mixtures come here in particular the exhaust gases of internal combustion engines and coal power plants into consideration. The exhaust gases from breweries or Müllverbren nungsanlagen can serve as a starting material for suitable carbon dioxide-rich gas mixtures.

According to one particularly preferred embodiment of the invention Process for the preparation of an organic compound with at least Two carbon atoms are used in cells capable are 3-hydroxypropionic acid or 3-hydroxyisobutyric acid to produce from bicarbonate.

Also are embodiments of the method according to the invention for the preparation of organic compounds having at least two Carbon atoms conceivable in which not the organic compounds with at least two carbon atoms themselves, but only precursor compounds for the preparation of these organic compounds with at least two carbon atoms by the inventive recombinant cell are formed. These precursor compounds can then by means of further, optionally recombinant Cells, which may even co-culture with the inventive Recombinant cells are cultured, taken up and in the desired converted organic compound having at least two carbon atoms become. Such a procedure could be, for example in the third, particular variant of the invention recombinant cell capable of producing pyruvate of two equivalents To form bicarbonate and one equivalent of carbon dioxide, to offer. Because pyruvate could be from other cells in more Target products are transferred.

The Purification of the organic compound having at least two carbon atoms is preferably carried out by means of methods known to those skilled in the Purification of target products from fermentation solutions. This reconciliation can, as well as the production of the organic Compounds by the cells according to the invention itself, continuously or discontinuously. Usually The purification begins with a separation of the first cells according to the invention from the nutrient medium, in the case of a continuous purification, the fermentation broth continuously over a filter with an exclusion size is conducted in a range of 20 to 200 kDa, in the a solid / liquid separation takes place. It is also possible the use of a centrifuge, a suitable sedimentation device or a combination of these devices, with particular preference is, at least part of the microorganisms first separated by sedimentation and then the by The microorganisms partially freed fermentation broth an ultrafiltration or centrifugation device.

The further purification of the target product (organic compound with at least two carbon atoms) depends on the type of Target product from, but usually takes place in several Plates. Conceivable here is the use of known in the art Separators, such as separators, according to the principle Electrodialysis, reverse osmosis, ultrafiltration or Nanofiltration work.

• IA) Herstellung von 3-Hydroxypropionsäure durch das vorstehend beschriebene Verfahren zur Herstellung organischer Verbindungen mit mindestens zwei Kohlenstoffatomen, bei dem Zellen eingesetzt werden, die in der Lage sind, 3-Hydroxypropionsäure aus Hydrogencarbonat zu bilden, sowie gegebenenfalls Neutralisation der gegebenenfalls aufgereinigten 3-Hydroxypropionsäure;
• IB) Dehydratisierung der 3-Hydroxypropionsäure unter Bildung von Acrylsäure sowie gegebenenfalls Veresterung der Acrylsäure.

A contribution to the solution of the abovementioned objects is also provided by a process for the preparation of acrylic acid or acrylic esters, comprising the process steps

• IA) Preparation of 3-hydroxypropionic acid by the process described above for the preparation of organic compounds having at least two carbon atoms, in which cells are used which are capable of forming 3-hydroxypropionic acid from bicarbonate, and optionally neutralization of the optionally purified 3-hydroxypropionic acid ;
• IB) dehydration of the 3-hydroxypropionic acid to form acrylic acid and optionally esterification of the acrylic acid.

• IIA) Herstellung von 3-Hydroxyisobuttersäure durch das vorstehend beschriebene Verfahren zur Herstellung organischer Verbindungen mit mindestens zwei Kohlenstoffatomen, bei dem Zellen eingesetzt werden, die in der Lage sind, 3-Hydroxyisobuttersäure aus Hydrogencarbonat zu bilden, sowie gegebenenfalls Neutralisation der gegebenenfalls aufgereinigten 3-Hydroxyisobuttersäure;
• IIB) Dehydratisierung der 3-Hydroxyisobuttersäure unter Bildung von Methacrylsäure sowie gegebenenfalls Veresterung der Methacrylsäure.

A contribution to the solution of the abovementioned objects is also made by a process for the preparation of methacrylic acid or methacrylic acid esters, comprising the process steps

• IIA) Preparation of 3-hydroxyisobutyric acid by the process described above for producing organic compounds having at least two carbon atoms, in which cells are used which are capable of forming 3-hydroxyisobutyric acid from bicarbonate, and optionally neutralization of the optionally purified 3-hydroxyisobutyric acid ;
• IIB) dehydration of the 3-hydroxyisobutyric acid to form methacrylic acid and optionally esterification of methacrylic acid.

According to the Process steps IB) and IIB) is the 3-hydroxypropionic acid or the 3-hydroxyisobutyric acid to form acrylic acid or methacrylic dehydrated, this either the pure hydroxycarboxylic acid isolated from the fermentation solution or in the workup of the fermentation solution isolated aqueous hydroxycarboxylic acid solution can be used, this optionally still before Dehydration, for example by distillation, optionally in the presence of a suitable entraining agent.

The dehydration can in principle be carried out in the liquid phase or in the gas phase. Furthermore, it is preferred according to the invention that the dehydration takes place in the presence of a catalyst, wherein the type of catalyst used depends on whether a gas phase or a liquid phase reaction is carried out. The catalysts used are preferably inorganic acid such as, for example, sulfuric acid or phosphoric acid, it being advantageous to use support materials such as natural or synthetic silicatic substances, in particular mordenite, montmorillonite, acidic zeolites, oxidic or siliceous substances, for example Al ₂ O ₃ , TiO ₂ ; Oxides and mixed oxides, such as γ-Al ₂ O ₃ and ZnO-Al ₂ O ₃ mixed oxides of heteropolyacids, which are impregnated with inorganic acids.

• IIIA) Herstellung von (Meth)acrylsäure durch die vorstehend beschriebenen Verfahren zur Herstellung von Acrylsäure bzw. Methacrylsäure;
• IIIB) radikalische Polymerisation der (Meth)Acrylsäure,

wobei gegebenenfalls die Carboxylgruppen der (Meth)Acrylsäure bzw. die Carboxylatgruppe des (Meth)Acrylates vor oder nach der radikalischen Polymerisation zumindest teilweise verestert werden können.A contribution to the solution of the abovementioned objects is also made by a process for the preparation of poly (meth) acrylic acid or poly (meth) acrylic acid esters, comprising the process steps

• IIIA) Preparation of (meth) acrylic acid by the above-described processes for the preparation of acrylic acid or methacrylic acid;
• IIIB) radical polymerization of (meth) acrylic acid,

where appropriate, the carboxyl groups of the (meth) acrylic acid or the carboxylate group of the (meth) acrylate can be at least partially esterified before or after the radical polymerization.

EXAMPLE

1. Preparation of an expression vector

It was a shuttle vector pA, which is based on the plasmid pRNI, according to the method described by Berkner et al. in "Small Multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-) archaea ", Nucleic Acids Research, Vol. 35 (12), pages 1 to 12 (2007). In this shuttle vector became the gene for hydroxybutyryl-coenzyme A dehydratase from Sulfolobus solfataricus (SEQ. ID. 05) in analogy to the in Berkner et al. procedure described for the pyrEF gene inserted.

2. Transformation of sulfolobus solfataricus cells

To Methylation of the shuttle vector according to the Berkner et al. described methods were the vector according to the in Berkner et al. described electro-surgery procedure in sulfolobus solfataricus cells introduced.

3. Cultivation of the recombinant cell

The cells according to the invention thus obtained were cultured in Brock's Basal Salt Medium at a pH of 3.5, in which CO ₂ gas was continuously injected. Sequences

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

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Claims (19)

A recombinant cell that is able to taking advantage of a metabolic pathway in which acetyl-CoA and bicarbonate malonyl coenzyme A, from malonyl coenzyme A malonate semialdehyde, from Malonate semialdehyde 3-hydroxypropionate, from 3-hydroxypropionate 3-hydroxypropionyl coenzyme A, from 3-hydroxypropionyl-coenzyme A acryloyl-coenzyme A, from acryloyl-coenzyme A propionyl coenzyme A, from propionyl coenzyme A and bicarbonate (S) -methylmalonyl coenzyme A, from (S) -methylmalonyl-coenzyme A (R) -methylmalonyl-coenzyme A, from (R) -methylmalonyl-coenzyme A succinate semialdehyde, from succinate semialdehyde 4-hydroxybutyric acid, from 4-hydroxybutyric acid 4-hydroxybutyryl-coenzyme A, from 4-hydroxybutyryl-coenzyme A crotonyl-coenzyme A, from crotonyl-coenzyme A 3-hydroxybutyryl-coenzyme A, from 3-hydroxybutyryl-coenzyme A acetoacetyl-coenzyme A and acetoacetyl-coenzyme A two equivalents Acetyl coenzyme A can be formed from bicarbonate organic To form compounds with at least two carbon atoms. The recombinant cell according to claim 1, wherein the cell has an increased activity of at least one of the following enzymes E ₁ to E ₁₆ compared to its wild-type: an enzyme E ₁ which converts carbonate and acetyl coenzyme A to malonyl coenzyme A catalyzes; An enzyme E ₂ which catalyzes the conversion of malonyl coenzyme A to malonate semialdehyde; An enzyme E ₃ which catalyzes the conversion of malonate-semialdehyde to 3-hydroxypropionic acid; An enzyme E ₄ which catalyzes the conversion of 3-hydroxypropionic acid to 3-hydroxypropionyl-coenzyme A; An enzyme E ₅ which catalyzes the conversion of 3-hydroxypropionyl-coenzyme A into acryloyl-coenzyme A; An enzyme E ₆ which catalyzes the reaction of acryloyl-coenzyme A into propionyl-coenzyme A; An enzyme E ₇ which catalyzes the conversion of carbonate and propionyl coenzyme A to (S) -methylmalonyl coenzyme A; An enzyme E ₈ which catalyzes the conversion of (S) -methylmalonyl-coenzyme A to (R) -methylmalonyl-coenzyme A; An enzyme E ₉ which catalyzes the conversion of (R) -methylmalonyl-coenzyme A to succinyl-coenzyme A; An enzyme E ₁₀ which catalyzes the conversion of succinyl-coenzyme A to succinic semialdehyde; An enzyme E ₁₁ which catalyzes the conversion of succinate semialdehyde to 4-hydroxybutyric acid; An enzyme E ₁₂ which catalyzes the conversion of 4-hydroxybutyric acid to 4-hydroxybutyryl-coenzyme A; - E ₁₃ , which catalyzes the reaction of 4-hydroxybutyryl-coenzyme A to crotonyl-coenzyme A; An enzyme E ₁₄ which catalyzes the conversion of crotonyl-coenzyme A to (S) -3-hydroxybutyryl-coenzyme A; An enzyme E ₁₅ which catalyzes the conversion of (S) -3-hydroxybutyryl-coenzyme A to acetoacetyl-coenzyme A; An enzyme E ₁₆ which catalyzes the conversion of acetoacetyl-coenzyme A into two molecules of acetyl-coenzyme A. The recombinant cell according to claim 2, wherein the enzyme E _{1 is} an acetyl-coenzyme A carboxylase (EC 6.4.1.2), E _{2 is} a malonyl-coenzyme A reductase (EC 1.2.1.18), E _{3 is} a malonate semialdehyde reductase (EC 1.1.1.59), E ₄ a 3-hydroxypropionyl synthetase (EC 6.2.1.-), E ₅ a 5.3-hydroxypropionyl-coenzyme A dehydratase (EC 4.2.1.17), E ₆ an acryloyl-coenzyme A- Reductase (EC 1.3.99.3), E ₇ a propionyl-coenzyme A carboxylase (EC 6.4.1.3), E ₈ a methylmalonyl-coenzyme A-epimerase (EC 5.1.99.1) E ₉ a methylmalonyl-coenzyme A mutase (EC 5.4.99.2), e ₁₀ is a succinyl-coenzyme a reductase, e ₁₁ is a succinic semialdehyde reductase (EC 1.1.1.61), e ₁₂ is a 4-hydroxybutyryl-coenzyme a synthetase e ₁₃ is a 4-hydroxybutyryl-coenzyme a-dehydratase , E ₁₄ a crotonyl-coenzyme A hydratase (EC 4.2.1.17), E ₁₅ a 3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35), and E ₁₆ an acetoacetyl-coenzyme A-β-ketothiolase (2.3. 9/1) is. The recombinant cell according to any one of claims 2 or 3, wherein the cell has an increased activity of the following enzymes E ₁ and E ₇ compared to their wild type: the enzyme E ₁ , which is the reaction of carbonate and acetyl coenzyme A to malonyl Coenzyme A catalyzes; - The enzyme E ₇ , which catalyzes the conversion of propionyl-coenzyme A to (S) -methylmalonyl-coenzyme A. The recombinant cell of any one of the preceding claims, wherein the organic compound is a C ₃ compound, a C ₄ compound, a C ₅ compound, a C ₆ compound, a C ₇ compound, a C ₈ compound, a C ₉ compound, a C ₁₀ compound or a compound having more than 10 carbon atoms. The recombinant cell of claim 5, wherein said organic compound is selected from the group consisting from a carboxylic acid, a dicarboxylic acid, a Hydroxycarboxylic acid, an amine, an amino acid, a ketone and an alcohol. The recombinant cell of claim 5 or 6, wherein the organic compound is selected from the group consisting of acrylic acid, methacrylic acid, 3-hydroxypropionic acid, 3-hydroxyisobutyric acid, lysine, glutamate, methionine, phenylalanine, Aspartic acid, tryptophan, threonine, butanol, 1,2-propanediol, 1,3-propanediol, 2,3-butanediol, 1,4-butanediol, 1,6-hexanediol, glycerol, sorbitol, Manitol, xylitol, arabinitol, glucose, acetone or dihydroxyacetone. The recombinant cell according to one of the preceding Claims wherein the cell is capable of bicarbonate To form 3-hydroxypropionic acid. The recombinant cell according to any one of claims 1 to 7, wherein the cell is capable of hydrogencarbonate 3-hydroxyisobutyric acid to build. A method for producing a recombinant cell capable of producing from bicarbonate organic compounds having at least two carbon atoms, comprising the step of increasing the activity of one or more of the enzymes E ₁ to E ₁₆ defined in claim 2 in the cell. A recombinant cell, obtainable by a method according to claim 10. Use of a recombinant cell for targeted Production of organic compounds from bicarbonate under Utilization of a metabolic pathway in which acetyl-CoA and bicarbonate malonyl-coenzyme A, from malonyl-coenzyme A malonate semialdehyde, from malonate semialdehyde 3-hydroxypropionate, from 3-hydroxypropionate 3-hydroxypropionyl-coenzyme A, from 3-hydroxypropionyl-coenzyme A acryloyl-coenzyme A, from acryloyl-coenzyme A propionyl coenzyme A, from propionyl coenzyme A and bicarbonate (S) -methylmalonyl coenzyme A, from (S) -methylmalonyl-coenzyme A (R) -methylmalonyl-coenzyme A, from (R) -methylmalonyl-coenzyme A succinate semialdehyde, from succinate semialdehyde 4-hydroxybutyric acid, from 4-hydroxybutyric acid 4-hydroxybutyryl-coenzyme A, from 4-hydroxybutyryl-coenzyme A crotonyl-coenzyme A, from crotonyl-coenzyme A 3-hydroxybutyryl-coenzyme A, from 3-hydroxybutyryl-coenzyme A acetoacetyl-coenzyme A and acetoacetyl-coenzyme A two equivalents Acetyl-coenzyme A are formed. The use of claim 12, wherein the recombinant Cell for the targeted production of 3-hydroxypropionic acid or 3-hydroxyisobutyric acid is used. A process for the production of organic compounds having at least two carbon atoms, comprising the process step contacting a recombinant cell according to any one of Claims 1 to 9 or 11 with a bicarbonate-containing Nutrient medium under conditions, among which from the bicarbonate formed organic compounds having at least two carbon atoms be, and optionally purification of the organic compounds with at least two carbon atoms from the nutrient medium. The method of claim 14, wherein the recombinant Cell used according to claim 8 and 3-hydroxypropionic acid is formed from the hydrogencarbonate. The method of claim 14, wherein the recombinant Cell used according to claim 9 and 3-hydroxyisobutyric acid is formed from the hydrogencarbonate. A process for the production of acrylic acid or acrylic acid esters, comprising the process steps IA) Preparation of 3-hydroxypropionic acid by a process according to claim 15 and optionally neutralization of the 3-hydroxypropionic acid; IB) Dehydration of 3-hydroxypropionic acid with formation of acrylic acid and optionally esterification of acrylic acid. A process for the production of methacrylic acid or methacrylic acid esters, comprising the process steps IIA) Preparation of 3-hydroxyisobutyric acid by a process according to claim 16 and optionally neutralization of the 3-hydroxyisobutyric acid; IIB) Dehydration of 3-hydroxyisobutyric acid with formation of methacrylic acid and optionally esterification of Methacrylic acid. A process for producing poly (meth) acrylic acid or poly (meth) acrylic acid esters, comprising the process steps IIIA) Preparation of (meth) acrylic acid by a process according to claim 17 or 18; IIIB) radical polymerization of (Meth) acrylic acid, where appropriate, the carboxyl groups the (meth) acrylic acid or the carboxylate group of the (meth) acrylate before or after the radical polymerization at least partially can be esterified.