DE102010029973A1 - Microbiological production of C4 bodies from sucrose and carbon dioxide - Google Patents

Abstract

The present invention relates to a cell which has been genetically modified in relation to its wild type in such a way that, compared to its wild type, it is able to form more C4 bodies and / or more secondary compounds produced from these C4 bodies from sucrose and carbon dioxide as a carbon source. The invention further relates to a method for producing such a genetically modified cell and a method for producing C4 bodies and / or subsequent compounds produced via these C4 bodies with the aid of these cells. Finally, the invention also relates to the use of the cells for the production of C4 bodies and / or of secondary compounds made from sucrose and carbon dioxide produced via these C4 bodies.

Description

The present invention relates to a cell which has been genetically engineered with respect to its wild type such that it is able to form more C ₄ bodies and / or secondary compounds produced via these C ₄ bodies in comparison to their wild type from sucrose and carbon dioxide. The invention further relates to a method for producing such a genetically modified cell and to a method for producing C ₄ bodies and / or of secondary compounds produced via these C ₄ bodies with the aid of these cells. Finally, the invention also relates to the use of the cells for the production of C ₄ bodies bodies and / or derived from these C ₄ body compounds of sucrose and carbon dioxide.

C ₄ bodies such as succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, epoxy-γ-butyrolactone, butenoic acid, butyrate, butanediol, 1,2 Butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, good, n-butene, cis-2-butene, trans-2-butene, isobutene, butadiene, 1,2-butadiene, 1 , 3-butadiene, 3- and 4-hydroxybutyrolactone, 1-, 2-, and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid and 2- and 3-hydroxyisobutyric acid are nowadays produced mainly from petrochemical raw materials and are important as raw materials for feed industry, agricultural and pharmaceutical products. In addition, these C ₄ bodies are also used as raw materials of the chemical industry for mass-produced products, such as polymers and solvents, or specialty chemicals. Due to the foreseeable shortage of petrochemical raw materials and increasing demand for products based on renewable resources, it is desirable to provide alternatives to the existing petrochemical processes for producing C ₄ bodies based on renewable resources.

The currently pursued approaches are based mainly on microorganisms that produce C ₄ bodies in fermentative processes. In particular, glucose is used as carbon source. The necessary glucose is recovered from starch, which z. B. from maize, potatoes, wheat or other cereals is produced. A disadvantage of these approaches is that glucose is relatively expensive and the corresponding production and use of glucose competes with the use or processing of the starting materials to foods.

The present invention is therefore based on the object to overcome the disadvantages resulting from the prior art.

In particular, the invention has the object to replace glucose as a raw material by a cheaper and available in sufficient quantities alternative.

According to the invention this object is achieved in that sucrose is used as a raw material for the production of C ₄ bodies. Worldwide, sucrose is mostly made from sugar cane (about ¾ of the annual production) and to a lesser extent from sugar beets (about ¼ of the annual production). Due to the high sucrose production from sugarcane in South America and Asia and less competition with the food industry, sucrose is available in large quantities and cheaper than glucose.

A further improvement of the existing processes is that according to the invention, in addition to sucrose, carbon dioxide (CO ₂ ) is used as carbon source. For example, carbon dioxide is a by-product of a number of chemical processes and is therefore comparatively inexpensive to obtain. In addition, by using carbon dioxide, the pollution of the atmosphere with greenhouse gases can be reduced. The prior art has not yet provided technologies for the production of C ₄ bodies from sucrose and carbon dioxide which could compete with existing petrochemical processes. Wild-type organisms, such as Basfia succiniciproducens, Anaerobiospirillum succiniciproducens or Actinobacillus succinogenes, which can convert sucrose to C ₄ bodies are not suitable for biotechnological production, since the yields are low and these organisms require complex nutrient media, due to the associated costs and the effort to make the process uneconomical.

According to the invention, this disadvantage is overcome by providing recombinant cells which can produce large quantities of C ₄ bodies under appropriate culture conditions, starting from sucrose and CO ₂ as carbon sources.

The inventors of the present invention have surprisingly found that the yield of C ₄ bodies by genetic manipulation of suitable cells, in particular a gain of carboxylation reactions that fix carbon dioxide and lead to the formation of C ₄ bodies, a suppression of metabolic pathways that cause the carbon flow from sucrose to fermentation products, which are not C ₄ body, and increasing the sucrose uptake and the Sucrose metabolism can be greatly increased. This approach allows i) organisms which are usually unable to metabolize sucrose, but which are favorable in view of the required culture conditions, and ii) organisms which are naturally capable of metabolizing sucrose but have low efficiency in the Sense of space-time and / or carbon yield in relation to the synthesis of C ₄ bodies have to use as efficient whole-cell catalysts for the production of C ₄ bodies.

In a first aspect, therefore, the present invention relates to a recombinant cell which has been genetically engineered with respect to its wild type to produce more C ₄ bodies and / or over these C ₄ bodies from sucrose and carbon dioxide as carbon sources compared to their wild-type Can form secondary connections.

The term "C ₄ body" as used herein refers to a chemical compound containing four carbon atoms and includes corresponding carboxylic acids, aldehydes, alcohols, sugars, alkanes, alkenes, amines, and derivatives thereof. Exemplary C ₄ bodies within the meaning of the invention include, but are not limited to succinate, Malst, fumarate, oxalacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, epoxy-γ-butyrolactone, Butenoic acid, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, rods, n-butene, cis-2-butene, trans-2-butene, isobutene, Butadiene, 1,2-butadiene, 1,3-butadiene, 3- and 4-hydroxybutyrolactone, 1-, 2- and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid and 2- and 3-hydroxyisobutyric acid. In one embodiment of the invention, the C ₄ body is selected from the group consisting of: Malst and oxaloacetate and / or secondary compounds prepared by enzymatic synthesis from these C ₄ bodies, for example succinate, aspartate, asparagine, threonine, tetrahydrofuran, Butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 3- and 4-hydroxybutyrolactone, 1-, 2-, and tert-butanol, isobutanol, 2- , 3- and 4-hydroxybutyric acid, as well as 2- and 3-hydroxyisobutyric acid, methionine and lysine, as well as compounds prepared via Malst, oxaloacetate and secondary compounds by chemical means. As used herein, the terms of carboxylic acids include both the base form and the acid form, as well as mixtures of these forms. This means that terms such as succinate, Malst, oxaloacetate, fumarate, aspartate, butyrate, 2-, 3- or 4-hydroxybutyric acid and 2- or 3-hydroxyisobutyric acid in each case also succinic acid, malic acid, oxaloacetic acid, fumaric acid, aspartic acid, butyric acid, 2- 3- or 4-hydroxybutyrate and 2- or 3-hydroxyisobutyrate.

The expression "that it is able to form more C ₄ bodies compared to its wild type" also encompasses the theoretical case that the wild-type of the genetically modified cell does not form any C ₄ bodies at all or does not form the desired C ₄ body can or can not produce this compound (s) in a detectable amount and only after the genetic modification detectable amounts of these compounds are formed. Further, it is understood by this term that the corresponding cell is at least 2, 5, 10, 100, or 1000 times the amount in a given period of time, for example, within 2, 4, 8, 12, 24, or 48 hours forms on C ₄ bodies or one or more desired C ₄ bodies as the wild type.

By "wild-type" of a cell is meant herein a cell whose genome is in a state naturally established by evolution. The term is used for both the entire cell and for individual genes. The term "wild-type" therefore does not include, in particular, those cells or genes whose gene sequences have been altered at least in part by humans by means of recombinant methods.

The cells of the invention may be prokaryotes or eukaryotes. These may be mammalian cells (such as human cells), other animal cells (e.g., insect cells), plant cells, or microorganisms such as yeasts, fungi or bacteria, with microorganisms being preferred and bacteria and yeasts being particularly preferred ,

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

Cells preferred according to the invention are those of the genera Aspergillus, Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Actinobacillus, Anaerobiospirillum, Basfia, Wollinella, Fibrobacter, Ruminococcus, Mannheimia, Lactobacillus, Lactococcus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula, Kluveromyces, Saccharomyces , Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium Cupriavidus and carboxydotrophic microorganisms, with Aspergillus nidulans, Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Saccharomyces cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, C orynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Hansenula polymorpha, Ralstonia eutropha, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Pichia pastoris, Thermoanaerobacter kivui, Acetobacterium woodii, Acetoanaerobium notera, Clostridium aceticum, Butyribacterium methylotrophicum , Clostridium acetobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium beijerinckii, Clostridium butyricum, Moorella thermoacetica, Eubacterium limosum, Peptostreptococcus productus, Clostridium ljungdahlii, Clostridium carboxidivorans, Clostridium scatalogenes, Rhodospirillum rubrum, Burkholderia thailandensis and Pseudomonas putida are particularly preferred.

The terms "carbon dioxide" and "CO ₂ " as used herein refer to both the CO ₂ gas, the carbonic acid (H ₂ CO ₃ ) in equilibrium carbon dioxide dissolved in aqueous solution, and the two carbonic acid deprotonation products , Bicarbonate (HCO ₃ ^- ) and carbonate (CO ₃ ^2- ) and their salts.

In one embodiment of the invention, the C ₄ bodies are selected from the group consisting of: succinate, malt, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, epoxy-γ- Butyrolactone, butenoic acid, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, butene, n-butene, cis-2-butene, trans-2-butene, Isobutene, butadiene, 1,2-butadiene, 1,3-butadiene, 3-hydroxybutyrolactone, 4-hydroxybutyrolactone, 1-butanol, 2-butanol, tert-butanol, isobutanol, 2-hydroxybutyric acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid is selected.

In one embodiment, the C ₄ bodies are selected from the group consisting of: Malst, oxaloacetate and from secondary compounds prepared by enzymatic or chemical synthesis via Malst or oxalacetate. The secondary compounds produced by enzymatic synthesis via Malst or oxalacetate can be selected, for example, from the group consisting of: succinate, aspartate, asparagine, threonine, tetrahydrofuran, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4- Butanediol, 2,3-butanediol, 3- and 4-hydroxybutyrolactone, 1-, 2- and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid, 2- and 3-hydroxyisobutyric acid, methionine and lysine.

In one embodiment, the C ₄ bodies are C ₄ carboxylic acids, preferably C ₄ dicarboxylic acids, more preferably succinate. In a further embodiment, the C4 bodies are hydroxycarboxylic acids, more preferably 2-, 3- and / or 4-hydroxybutyric acid and 2- and / or 3-hydroxyisobutyric acid. In another embodiment, the C ₄ bodies are preferably Malst and / or oxaloacetate.

In further embodiments, the recombinant cell is a microbial cell, in particular an Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Methylobacterium extorquens, Ralstonia eutropha, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Clostridium acetobutylicum, saccharoperbutylacetonicum Clostridium, Clostridium beijerinckii, Rhodospirillum rubrum , Burkholderia thailandensis or Pseudomonas putida cell.

For example, the recombinant cell of the present invention may be genetically engineered to accommodate more sucrose compared to its wild-type. Another way, the alternatively or additionally can be used, is a genetic modification of the cell, which allows the recombinant cell to fix more carbon dioxide compared to their wild type.

In one embodiment of the invention, the increase in sucrose uptake is achieved by virtue of the cell having an increased activity of at least one enzyme which catalyzes the transport of sucrose into the cell compared to its wild-type.

The term "at least one" as used herein refers to amounts of ≥ 1, d. H. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30 or more, in particular 1, 2, 3, 4 or 5.

In one embodiment, the recombinant cell of the invention comprises an enzyme E ₁ which catalyzes the transport of sucrose into the cell and the conversion to sucrose-6-phosphate.

By the term "enzyme" or "E _x " as used herein, wherein x is an integer, is meant a protein or protein complex that catalyzes one or more biochemical reactions and / or the transport of certain compounds , For example, by a membrane, is used.

The terms "enhanced activity of an enzyme" or "decreased activity of an enzyme" as used herein preferably refer to increased / decreased intracellular or membrane-bound activity.

The following statements on increasing or decreasing the enzyme activity in cells apply both to the increase / decrease in the activity of the enzyme E ₁ and to all the enzymes mentioned below, the activity of which may optionally be increased or decreased.

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 increases, eliminates repression, prevents inhibition or infiltrates a gene or allele, or manipulates the species to encode a corresponding enzyme with enhanced activity. If necessary, these measures will be combined. Genetically engineered 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 vector which enables expression of the gene. In particular, heterologous expression is achieved 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 relates to the potential for increasing enzyme activity in cells forms part of the disclosure of the present invention.

The expression of the above and all of the enzymes and genes mentioned below can be detected with the aid of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration with appropriate evaluation software in the gel. 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. (Electophoresis, 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. 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 nucleotide exchange (s). These mutations result in genetically engineered cells. Particularly preferred mutants of enzymes are, in particular, also those enzymes which are no longer or at least reduced in comparison to the wild-type enzyme in a feedback-inhibited manner when an increased enzyme activity is desired.

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 the ribosome binding site, which is 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 also cause an 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)) , T Suchiya and Morinaga (Bio / Technology 6, 428-430 (1988)) , at Eikmanns et al. (Gene 102, 93-98 (1991)) , in EP-A-0 472 869 , in US 4,601,893 , at Schwarzer and Puhler (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. 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 may, 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, DM (1985), DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford ; Rodriguez, RL and Denhardt, D.T. (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham ; Goeddel, DV (1990), Systems for heterologous gene expression, Methods Enzymol. 185, 3-7 ; Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York ; Casali, N. and Preston, A. (eds) (2003) E. coli Plasmid Vectors. Humana Press , Suitable plasmids are in particular those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as pZ1 ( Menkel et al., Applied and Environmental Microbiology 64: 549-554 (1989) ), pEKEx1 ( Eikmanns et al., Gene 107: 69-74 (1991) ) or pHS2-I ( 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 hom-thrB 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), pEM1 ( 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 in question.

The above-mentioned methods can analogously achieve a reduction in the enzymatic activity. This is also divided into two strategies, the reduction of expression and / or the inhibition of enzyme activity. To reduce the expression, for example, the corresponding gene can be completely or partially deleted. In addition, transcription can be inhibited or reduced, for example by manipulation of the promoter region or enhancement of repression (genetically or chemically) or reduction of the mRNA half-life. Likewise, at the RNA level, translation can be disturbed or reduced. Numerous techniques are known to the person skilled in the art, for example the RNAi technology or the modification of the DNA sequence in such a way that secondary structures occur at the mRNA level which inhibit or reduce the translation. The enzyme activity can be reduced, for example, by adding inhibitors, by introducing directional or non-directional mutations.

The expression "an activity of an enzyme E _{x which} is increased with respect to its wild type" is preferably always a factor greater than or equal to 2, particularly preferably at least 10, more preferably at least 100, and even more preferably at least 1,000, and most preferably at least 10,000 increased activity of the respective enzyme E _x to understand. 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 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.

Accordingly, under the expression "reduced activity of an enzyme E _x " used below, it is preferable to use a factor of at least 0.5, more preferably at least 0.1, more preferably at least 0.01, even more preferably at least 0.001 and most preferably at least 0.0001 decreased activity. The reduction of the activity of a specific enzyme can be achieved, for example, by targeted mutation, genetic deletion of the gene, by addition of competitive or non-competitive inhibitors or by other measures described above or known to those skilled in the art for reducing the expression and / or function of a particular enzyme respectively.

It is preferred in accordance with the invention for the genetically modified cell to be genetically modified in such a way that it is particularly preferred within a defined time interval, preferably within 2 hours, more preferably within 8 hours, and most preferably within 24 hours, at least 2 times at least 10 times, more preferably at least 100 times, more preferably at least 1,000 times, and most preferably at least 10,000 times more C ₄ body than the wild-type cell. The increase in product formation can be determined, for example, by culturing the cell according to the invention and the wild-type cell separately under the same conditions (same cell density, same nutrient medium, same culture conditions) for a specific time interval in a suitable nutrient medium and then the amount Target product (C ₄ body) is determined in the nutrient medium.

The enzyme E ₁ can be, for example, a phosphoenolpyruvate (PEP) -dependent phosphotransferase system (PTS) enzyme II (sucrose-specific) (EC 2.7.1.69; TCDB classification 4.A.1.2.1, 4.A.1.2.9 or 4.A.1.2.-).

The enzyme E ₁ can be encoded by the scrA gene, for example. The coding nucleotide sequence and its associated protein sequence can be found, for example, in the "Kyoto Encyclopedia of Genes and Genomes" (KEGG database), the National Library of Biotechnology Information (NCBI) databases of the National Library of Medicine (Bethesda, MD), the Protein Data Bank UniProt (cooperation of the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR)) or the nucleotide sequence database of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK) be removed. In a particular case, E ₁ is the E. coli scrA gene (SEQ ID NO: 41) and the resulting protein. In addition, the enzyme E _{1 is} preferably encoded by genes selected from the group of those encoding gene products whose amino acid sequence has a range of at least 100, preferably at least 200, in particular at least 300 amino acids, at least 60%, preferably at least 80%, more preferably at least 95%, most preferably at least 99%, in particular 100% to SEQ ID NO: 58 is identical.

In one embodiment, the enzyme E _{1 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) using RPS-BLAST the presence of the conserved domain "PTS-II-BC-sucr" (TIGR01996 or PssmID 131051) with an E value (English "e-value") less than 1 × 10 ^-5 detected (English "domain hit").

In addition to the enzyme E ₁ , which catalyzes the transport of sucrose into the cell and the conversion to sucrose-6-phosphate, the recombinant cell can also have an increased activity of at least one enzyme E ₂ , E ₃ and E ₄ compared to its wild-type or a combination of these enzymes. E ₂ may be an enzyme which catalyzes the conversion of sucrose-6-phosphate to D-glucose-6-phosphate and D-fructose, E ₃ may be an enzyme which causes the conversion of D-fructose into D-fructose 6-phosphate catalyzes, and E ₄ may be a channel that allows diffusion-dependent sucrose transport into the cell.

In one embodiment, the invention therefore relates to recombinant cells which have an increased activity of at least one of the enzymes E ₂ , E ₃ and E ₄ in comparison to their wild type. In one embodiment, the activity of the enzyme E ₁ and at least one of the enzymes E ₂ , E ₃ and E _{4 is} increased compared to the wild type. In another embodiment, the activity of the enzymes compared to the wild-type is i) E ₁ , E ₂ and E ₃ , ii) E ₂ , E ₃ and E ₄ , iii) E ₂ and E ₃ , iv) E ₃ and E ₄ , v) E ₂ and E ₄ , or vi) E ₁ , E ₃ and E _{4 are} increased. In a further embodiment, the recombinant cell according to the invention has an increased activity of the enzymes E ₁ , E ₂ , E ₃ and E ₄ in comparison to the wild type.

In further embodiments, the enzyme E _{2 may} be a sucrose-6-phosphate fructohydrolase (EC 3.2.1.26), the enzyme E _{3 may} be a fructokinase (EC 2.7.1.4) and / or the enzyme E _{4 may} be a sucrose porin (TCDB classification 1.B.3.1.2) act.

In one embodiment of the invention E _{2 is} encoded by the scrB (E. coli scrB: SEQ ID NO: 42), bfrA, sacA, sacB, cscA, fruA or susH gene (Streptococcus pneumoniae susH: SEQ ID NO: 52), E ₃ is derived from the mac, yajF, mtlZ, rbsK, glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (E.coli scrK: SEQ ID NO: 43) or cscK gene (E. coli ČSČK: SEQ ID NO: 46) encodes and e ₄ is of the ScrY gene (E. coli ScrY: 44: SEQ ID NO) coded.

The nucleotide sequences of the abovementioned genes, their corresponding protein sequences and other genes for the enzymes E ₂ to E ₄ can be taken from among others the KEGG, the NCBI, the UniProt or EMBL database. In a particular case E ₂ , E ₃ and / or E ₄ are E. coli derived genes or the proteins encoded thereby.

In one embodiment, the enzyme E _{2 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 59.

In various embodiments, the enzyme E _{2 is} encoded by genes which are selected from the group of those encoding gene products in their amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (Version 2.20). by means of RPS-BLAST the presence of the conserved domain "scrB_fam" (TIGR01322 or PssmID 162301) with an E-value (English "e-value") smaller 1 × 10 ^{-5 is} found (English "domain hit").

In one embodiment, the enzyme E _{3 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 60 or SEQ ID NO: 63.

In one embodiment, the enzyme E _{3 is} encoded by genes which are selected from the group of those encoding gene products whose amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (Version 2.20) by means of RPS-BLAST Presence of the conserved domain "bac_FRK" (cd01167) is found with an E value (English "e-value") smaller 1 × 10 ^-5 (English "domain hit").

In one embodiment, the enzyme E _{4 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 61.

In one embodiment, the enzyme E _{4 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "maltoporin-like" (cd01346 or PssmID 30073) is found with an E value (English "e-value") smaller 1 × 10 ^-5 (English "domain hit").

In another embodiment of the invention, the recombinant cell is characterized by the fact that the increased sucrose uptake compared to the wild type is brought about by the increased activity of a channel E ₅ which sorbs sucrose into the cell. The present invention also covers embodiments in which this increased sucrose symport is combined with the aforementioned increased activities of the enzymes E ₁ , E ₂ , E ₃ and / or E ₄ .

The channel E ₅ may be, for example, a sucrose permease. In one embodiment, E ₅ is encoded by the cscB gene (for example, E. coli cscB: SEQ ID NO: 45).

In further embodiments, in addition to E ₅ , the recombinant cell may have an increased activity compared to its wild type of at least one of the following enzymes: E ₆ , which catalyzes the conversion of sucrose to D-glucose and D-fructose; and E ₃ , which catalyzes the conversion of D-fructose to D-fructose-6-phosphate. In one embodiment, the activity of both enzymes is increased.

In one embodiment of the invention, the enzyme E _{5 is} a sucrose permease (TCDB classification 2.A.1.5.3), the enzyme E _{3 is} a fructokinase (EC 2.7.1.4) and the enzyme E _{6 is} a -D-fructofuranoside Fructohydrolase (EC 3.2.1.26).

In this connection, the enzyme E _{5 may be} replaced by cscB (for example E.coli cscB: SEQ ID NO: 45), lamB or scrY (for example E.coli scrY: SEQ ID NO: 44), E ₃ by mac, yajF, mtlZ, rbsK, glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (e.g., E. coli scrK: SEQ ID NO: 43) or cscK (e.g., E. coli cscK: SEQ ID NO : 46) and / or E ₆ by cscA (for example E. coli cscA: SEQ ID NO: 47), susH (for example Streptococcus pneumoniae susH: SEQ ID NO: 52), scrB (for example E. coli scrB: SEQ ID NO: 42), rafD, sacA, fruA or bfrA. The nucleotide sequences of the abovementioned genes, their corresponding protein sequence and other genes for the enzymes E ₃ , E ₅ and E ₆ can be taken from among others the KEGG, the NCBI, the UniProt or EMBL database. In a particular case, E ₃ , E ₅ and / or E ₆ are E. coli derived genes and the proteins encoded thereby.

In one embodiment, the enzyme E _{5 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least over a range of at least 100, preferably at least 200, in particular at least 300 amino acids 60%, preferably of at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100% to SEQ ID NO: 62.

In one embodiment, the enzyme E _{5 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) using RPS-BLAST the presence of the conserved domain "LacY_symp" (PFAM domain 01306 or PssmID 110319) with an E value (English "e-value") smaller 1 × 10 ^{-5 is} found (English "domain hit").

In one embodiment, the enzyme E _{6 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 64 or SEQ ID NO: 51.

In one embodiment, the enzyme E _{6 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "SacC" (COG1621 or PssmID 31808) is found with an E value (English "e-value") smaller 1 × 10 ^-5 (English "domain hit").

In a further embodiment, the increased sucrose uptake of the recombinant cell according to the invention is effected by having at least one increased activity of an enzyme complex which transports sucrose into the cell compared to its wild-type.

This sucrose-transporting enzyme complex can be composed, for example, of the enzymes E ₇ , E ₈ and E ₉ , and comprises, for example, a sucrose-specific ABC transporter (TCDB classification 3.A.1.1.-).

In this connection, the enzyme E _{7 can be} replaced by a susT1 gene (for example Streptococcus pneumoniae susT1: SEQ ID NO: 48), E ₈ by a susT2 gene (for example Streptococcus pneumoniae susT2: SEQ ID NO: 49) and / or E ₉ susX gene (for example Streptococcus pneumoniae susX: SEQ ID NO: 50).

Further, the cell having increased sucrose transporter activity may have an increased activity of at least one enzyme E ₁₀ , E ₂ , E ₃ or E ₆ or any combination of these enzymes as compared to its wild-type. The enzyme E ₁₀ catalyses the conversion of sucrose to sucrose-6-phosphate, the enzyme E ₂ the conversion of sucrose-6-phosphate to -D-glucose-6-phosphate and D-fructose, the enzyme E ₃ the reaction from D-fructose to D-fructose-6-phosphate and the enzyme E ₆ the conversion of sucrose to D-glucose and D-fructose.

In one embodiment, the recombinant cell has an increased activity of the enzymes E ₁₀ , E ₂ , E ₃ and E ₆ compared to their wild type.

Enzyme E ₁₀ may be a sucrose kinase, enzyme E _{2 is} a sucrose-6-phosphate fructohydrolase (EC 3.2.1.26), enzyme E _{3 is} a fructokinase (EC 2.7.1.4) and / or enzyme E _{6 is} a -D-fructofuranoside Fructohydrolase (EC 3.2.1.26).

In this connection, the enzyme E _{10 can be} replaced by a sucrose kinase gene, E ₂ by a scrB (for example E. coli scrB: SEQ ID NO: 42), bfrA, sacA, sacB, cscA (for example E. coli cscA: SEQ ID NO: 47), fruA or susH gene and / or E ₃ by a mac, yajF, mtlZ, rbsK, glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (for example E coli scrK: SEQ ID NO: 43) or cscK (for example, E. coli cscK: SEQ ID NO: 46).

The nucleotide sequences of the abovementioned genes, their corresponding protein sequences and other alternative genes for the enzymes E ₂ , E ₃ , E ₇ , E ₈ , E ₉ and E ₁₀ may, inter alia, also the KEGG, the NCBI, the UniProt or EMBL database. In a particular case, E ₂ , E ₃ , and / or E ₁₀ are E. coli and E ₇ , E ₈ and / or E ₉ are Streptococcus pneumoniae genes and the proteins encoded thereby.

In one embodiment, the enzyme E _{7 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 65.

In a further embodiment, the enzyme E _{7 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (Version 2.20) by means of RPS BLAST the presence of the conserved domain "LplB" (COG4209 or PssmID 33938) is found with an E value (English "e-value") smaller 1 × 10 ^-5 (English "domain hit").

In one embodiment, the enzyme E _{8 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 66.

In one embodiment, the enzyme E _{9 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 67.

In one embodiment, the enzyme E _{9 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "UgpB" (COG1653 or PssmID 31839) is found with an E value (English "e-value") smaller than 1 × 10 ^-5 (English "domain hit").

In further embodiments of the invention, two or three of the mentioned ways of manipulating the sucrose uptake pathway may be combined so that the recombinant cell differs from its wild type by the increased activity of two or three sucrose uptake pathways. This can in each case relate to one, several or all enzymes from the described variants. In certain embodiments, therefore, in a recombinant cell according to the invention, the activity of the following enzymes is enhanced over the wild-type:
E ₁ -E ₉ ;
E ₁ -E ₁₀ ;
E ₁ , E ₂ , E ₃ , E ₄ , E ₅ , E ₆ ;
E ₁ , E ₂ , E ₃ , E ₅ , E ₆ ;
E ₁ , E ₂ , E ₃ , E ₇ , E ₈ , E ₉ ;
E ₄ , E ₅ , E ₆ , E ₇ , E ₈ , E ₉ ;
E ₁ , E ₂ , E ₃ , E ₇ , E ₈ , E ₉ ; E ₁₀ ;
E ₃ , E ₄ , E ₆ , E ₇ , E ₈ , E ₉ ; or
E ₃ , E ₆ , E ₇ , E ₈ , E ₉ .

In other embodiments, the activity of at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 enzymes selected from the group of enzymes E ₁ -E _{10 is} increased.

In one embodiment of the invention, the recombinant cell is genetically engineered to have an increased activity of at least one enzyme that catalyzes the fixation of CO ₂ to a C ₃ body as compared to its wild-type. This increased carbon dioxide fixation may be alternative or in addition to an increase in sucrose transport into the cell. The increase in sucrose transport can be accomplished by the techniques outlined above.

The at least one enzyme that catalyzes the fixation of CO ₂ to a C ₃ body may, in certain embodiments of the invention, be selected from the group consisting of the following enzymes:
E ₁₂ , which catalyzes the conversion of pyruvate, ATP and CO ₂ to oxaloacetate, ADP and phosphate;
E ₁₃ , which catalyzes the reaction of phosphoenolpyruvate, H ₂ O and CO ₂ to oxaloacetate and phosphate;
E ₁₄ , which catalyzes the reaction of phosphoenolpyruvate, ADP / GDP / P _i and CO ₂ to oxaloacetate and ATP / GTP / PP _i ;
E ₁₅ , E ₁₆ , E ₁₇ and E ₁₈ , which catalyze the conversion of pyruvate NAD (P) H ⁺ and CO ₂ to malate and NAD (P) ⁺ .

Also encompassed by the present invention are any combination of increased activities of the enzymes E ₁₂ -E ₁₈ and the increased activity of all enzymes E ₁₂ -E ₁₈ .

In one embodiment
E _{12 is} a pyruvate carboxylase (EC 6.4.1.1);
E _{13 is} a phosphoenolpyruvate carboxylase (phosphate: oxaloacetate carboxylase) (EC 4.1.1.31);
E _{14 is} a phosphoenolpyruvate carboxykinase (ATP / GTP / PP _i : oxaloacetate carboxylase) (EC 4.1.1.32, EC 4.1.1.38 or EC 4.1.1.49);
E ₁₅ a malate dehydrogenase ((S) -malate: NAD ⁺ oxidoreductase) (EC 1.1.1.38);
E ₁₆ a malate dehydrogenase ((S) -malate: NAD ⁺ oxidoreductase) (EC 1.1.1.39);
E ₁₇ a malate dehydrogenase ((S) -malate: NADP ⁺ oxidoreductase) (EC 1.1.1.40); and or
E _{18 is} a D-malate dehydrogenase ((R) -malate: NAD ⁺ oxidoreductase) (EC 1.1.1.83).

The enzyme E ₁₂ may preferably be encoded by a gene selected from the group consisting of cgl516, aarI62Cp pyrI, pca, cglO689, pc, pcx, pyc-1, pyc-2, accC-2, pycA, pycA2, pyc, pycB , pycB1, pycB2, accC, accA, oadA, pyr, acc and accC1, with the pyc gene (for example, E. coli pyc: SEQ ID NO: 5) being particularly preferred.

Pyruvate carboxylases preferred according to the invention are also in particular US 6,455,284 . US 6,171,833 . US 6,884,606 . US 6,403,351 . US 6,852,516 and US 6,861,246 described. A particularly preferred pyruvate carboxylase pyc in this context is that mutant which is described in " A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant. ", Ohnishi J et al., Applied Microbiology and Biotechnology, Vol. 58 (2), pp. 217-223 (2002) is described. In this mutant, the amino acid proline at position 458 was replaced by serine. The disclosure of this publication regarding the possibilities of producing pyruvate carboxylase mutants is hereby incorporated by reference and forms part of the disclosure of the present invention. Preferably, pyc is derived from Corynebacterium glutamicum.

The enzyme E ₁₃ may preferably be encoded by a gene selected from the group comprising ppc, capP, pepC and clpA, with the ppc gene being preferred. In one embodiment, ppc is from E. coli (SEQ ID NO: 1).

The enzyme E ₁₄ may preferably be encoded by a gene selected from the group comprising pck, pckG, pckA, pck1, pck2 and pck, the pckA gene being particularly preferred. In a preferred embodiment pckA is from E. coli (SEQ ID NO: 2).

Phosphoenolpyruvate-Carboxylasen preferred according to invention are in particular also in US 4,757,009 . US 4,980,285 . US 5,573,945 . US 6,872,553 and US 6,599,732 described. The disclosure of these references for phosphoenolpyruvate carboxylases is hereby incorporated by reference and forms part of the disclosure of the present invention.

The malate dehydrogenases E ₁₅ , E ₁₆ and E ₁₇ may preferably be encoded by a gene selected from the group comprising me, me1, me2, me3, mae, mae1, mae2, sfcA, sfcA1, maeA, maeB, maeB1, maeB2 , tme, ygkJ, ywkA, ygkJ, malS, ytsJ, mleA, mleS, mez, sce59.10c, 2sc7gll.23, malSI, malS2, dme, maeB1, maeB2, mdh, mdh1, mdh2, dme1 cgi 0120, dme1 cgi 0120 , DME1-cg5889, fl9kl6.27, f6f22.7, t22p22.60, fl8al7.1, mod1, tme, Mao, cgl3007, times and times, whereby (e ₁₅ maeA example, E. coli maeA: SEQ ID NO: 3 ), (e ₁₆ MME1 example, Chlamydomonas reinhardti MME1: SEQ ID NO: 55) as well as E. coli MAEB dme and ₁₇ MAEB (for e: SEQ ID NO: 4) are particularly preferred. In a preferred embodiment, mAb and mAb are from E. coli.

The malate dehydrogenase E ₁₈ may preferably be encoded by a gene selected from the group comprising yeaU, ycsA, ttuC, ttuC1, ttuC2, ttuC3, tdh, leuB, leuB1 and dmlA, with yeaU and dmlA being particularly preferred. In a preferred embodiment, dmlA is from E. coli (SEQ ID NO: 57).

In one embodiment:
E ₁₂ by a pyc gene;
E ₁₃ by a ppc gene;
E ₁₄ by a pckA gene;
E ₁₅ by a mouse gene;
E ₁₆ by a mme1 gene;
E ₁₇ by a mouse gene; and or
E ₁₈ encoded by a dmlA gene.

The abovementioned enzymes can be increased individually, all or in any combination in their activity. In particular, at least one, at least 2, 3, 4, 5, 6, or 7 enzymes of the group E ₁₂ -E _{18 can be increased} in their activity.

The nucleotide sequences of the abovementioned genes, their corresponding protein sequence and other genes for the enzymes E ₁₂ -E ₁₈ can also be taken from the KEGG, NCBI, UniProt or EMBL database.

In one embodiment, the enzyme E _{12 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 68.

In one embodiment, the enzyme E _{12 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "DRE_TIM_PC_TC_5S" (cd07937) or the conserved domain "pyruvate carboxylase" (PRK12999) with an E value (English "e-value") is less than 1 × 10 ^-5 found (English "domain hit") ,

In one embodiment, the enzyme E _{13 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 69.

In one embodiment, the enzyme E _{13 is} encoded by genes selected from the group of those encoding gene products in an amino acid sequence in a search for conserved protein domains contained in the subject amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST Presence of the conserved domain "phosphoenolpyruvate carboxylase" (PssmID 166715 or COG3252 or c14574 or PRK00009) is found with an E value (English "e-value") less than 1 × 10 ^-5 (English "domain hit").

In one embodiment, the enzyme E _{14 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 70.

In one embodiment, the enzyme E _{14 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "PEPCK_ATP" (cd00484) is found with an E value (English "e-value") smaller than 1 × 10 ^-5 (English "domain hit").

In one embodiment, the enzyme E _{15 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 71.

In one embodiment, the enzyme E _{15 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "NAD (P) binding domain of malic enzyme (ME), subgroup 1" (cd05312) with an E value (English "e-value") smaller 1 × 10 ^{-5 is} found (English "domain hit").

In one embodiment, the enzyme E _{16 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 56.

In one embodiment, the enzyme E _{16 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "malate dehydrogenase" (PRK13529) with an E value (English "e-value") is less than 1 × 10 ^{-5 is} found (English "domain hit").

In one embodiment, the enzyme E _{17 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 54.

In one embodiment, the enzyme E _{17 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "NAD (P) binding domain of malic enzyme (ME), subgroup 2" (cd05311) with an E value (English "e-value") smaller 1 × 10 ^{-5 is} found (English "domain hit").

In one embodiment, the enzyme E _{18 is} encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 53.

In one embodiment, the enzyme E _{18 is} encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "Iso_dh Super-family" (c00445 or PssmID 174206) with an E-value (English "e-value") smaller 1 × 10 ^{-5 is} found (English "domain hit").

Furthermore, in a further embodiment of the cells according to the invention, the recombinant cell can additionally be genetically engineered such that it has a reduced carbon flux from sucrose to fermentation products which are not C ₄ bodies compared to their wild type.

This embodiment encompasses recombinant cells according to the invention which, in addition to their wild type, are characterized by producing less C ₁ , C ₂ and / or C _{3 by-} products from sucrose.

The requisite genetic modification may be by manipulation of the recombinant cell, thereby reducing or disrupting the metabolism that produces the fermentation products.

In one embodiment of the invention, the activity of at least one of the following enzymes is reduced compared to the wild-type:
E ₁₉ , E ₂₀ , E ₂₁ and E ₂₂ , which are components of a glucose-specific phosphoenolpyruvate phosphotransferase system and, as such, catalyze the import of glucose and in sum the conversion of phosphoenolpyruvate and glucose to pyruvate and glucose-6-phosphate;
E ₂₃ and E ₂₄ , which catalyze the conversion of pyruvate and CoA to formate and acetyl-CoA;
E ₂₅ , which catalyzes the reaction of pyruvate, H ₂ O and ferricytochrome b ₁ to acetate, CO ₂ and ferrocytochrome b ₁ ;
E ₂₆ , which catalyzes the conversion of S-methylmalonyl CoA to propanoyl CoA and CO ₂ ;
E ₂₇ and E ₂₈ , which catalyze the conversion of acetyl phosphate and ADP / P _i to acetate and ATP / PP _i ;
E ₂₉ , which catalyzes the conversion of acetyl-CoA and P _i (orthophosphate) to acetyl phosphate and CoA;
E ₃₀ , which catalyzes the conversion of pyruvate and NADH to D-lactate and NAD ⁺ ;
E ₃₁ , which catalyzes the conversion of acetyl-CoA to acetaldehyde;
E ₃₂ , which catalyzes the conversion of acetaldehyde to ethanol;
E ₃₃ , which catalyzes the conversion of glycerol phosphate to methylglyoxal and orthophosphate;
E ₃₄ , E ₃₅ and E ₃₆ , which form a complex that catalyzes the conversion of formate to CO ₂ ;
E ₃₇ , which catalyzes the conversion of formate to CO ₂ ;
E ₃₈ , E ₃₉ and E ₄₀ , which form a complex that catalyzes the conversion of formate to CO ₂ ;
E ₄₁ , which catalyzes the reaction of propionyl CoA, H ₂ O and oxaloacetate to 2-methyl citrate and CoA;
E ₄₂ , which catalyzes the conversion of 2-methylcitrate to 2-methyl-cis-aconitate or 2-methyl-trans-aconitate and H ₂ O;
E ₄₃ , which catalyzes the conversion of 2-methyl trans aconitate to 2-methyl cis aconitate;
E ₄₄ , which catalyzes the conversion of 2-methyl cis aconitate to succinate and pyruvate;
E ₄₅ , which catalyzes the conversion of propionyl phosphate and ADP to propionate and ATP;
_E46 , which catalyzes the reaction of propionyl CoA and P _i to propionyl phosphate and CoA;
E ₄₇ , which catalyzes the conversion of pyruvate to acetaldehyde and CO ₂ ;
E ₄₈ , which catalyzes the reaction of pyruvate and CoA 2 oxidized ferredoxins to acetyl-CoA, CO ₂ , and 2 reduced ferredoxins and 2H ⁺ ;
E ₄₉ , which catalyzes the reaction of D-xylulose-5-phosphate and phosphate to acetyl phosphate and D-glyceraldehyde-3-phosphates and H ₂ O;
E ₅₀ , which catalyzes the reaction of (S) -methylmalonyl-CoA and pyruvate to propionyl-CoA and oxaloacetate;
E ₅₁ , which catalyzes the reaction of (S) -methylmalonyl-CoA to succinyl-CoA;
E ₅₂ , which catalyzes the conversion of 2 pyruvate to 2-acetolactate and CO ₂ ;
E ₅₃ , which catalyzes the conversion of 2-acetolactate to acetoin and CO ₂ ;
E ₅₄ , which catalyzes the reaction of acetoin and NAD (P) H ⁺ to butane-2,3-diol and NAD (P) ⁺ ;
E ₅₅ , which catalyzes the reaction of 2 acetyl-CoA to acetoacetyl-CoA;
E ₅₆ , which catalyzes the reaction of acetoacetyl-CoA and NAD (P) H ⁺ to 3-hydroxybutyryl-CoA and NAD (P) ⁺ ;
E ₅₇ , which catalyzes the conversion of crotonyl CoA and H ₂ O to 3-hydroxybutyryl CoA;
E ₅₈ , which catalyzes the reaction of butyraldehyde and NAD (P) H ⁺ to butanol and NAD (P) ⁺ ;
E ₅₉ , which catalyzes the reaction of butyryl-CoA and NAD (P) H ⁺ to buryraldehyde and NAD (P) ⁺ ;
E ₆₀ , which catalyzes the reaction of acetoacetate and H ⁺ to acetone and CO ₂ ;
E ₆₁ , which catalyzes the reaction of acetone and NAD (P) H ⁺ to propanol and NAD (P) ⁺ ;
E ₆₂ , which catalyzes the reaction of acyl-CoA and carboxylic acid to acylate and carboxylic acid CoA and
E ₆₃ , which catalyzes the conversion of pyruvate and NADH into L-lactate and NAD ⁺ .

In certain embodiments, multiple, i. H. at least 2, or all of the aforementioned enzymes are reduced in their activity.

In certain embodiments, / are:
E ₁₉ and E ₂₀ a PEP-dependent phosphotransferase system Enzyme II (EC 2.7.1.69);
E ₂₁ a PEP-dependent phosphotransferase system enzyme I (EC 2.7.3.9);
E ₂₂ a phosphohistidine protein (HPr) -hexose phosphotransferase component of the PEP-dependent phosphotransferase system;
E ₂₃ and E ₂₄ a formate C-acetyltransferase (EC 2.3.1.54);
E ₂₅ a pyruvate: ferricytochrome b ₁ oxidoreductase (EC 1.2.2.2);
E _{26 is} a methylmalonyl-CoA decarboxylase (EC 4.1.1.41);
E ₂₇ an ATP / PP _i : acetate phosphotransferase (EC 2.7.2.1 or EC 2.7.2.1);
E _{28 is} a propionate / acetate kinase (EC 2.7.2.15);
E ₂₉ an acetyl-CoA: phosphate acetyltransferase (EC 2.3.1.8);
E _{30 is} a D-lactate dehydrogenase (EC 1.1.1.28);
E ₃₁ an acetaldehyde dehydrogenase (CoA-acetylating) (EC 1.2.1.10);
E _{32 is} an NAD-dependent alcohol dehydrogenase (EC 1.1.1.1);
E _{33 is} a glycerone phosphate phospholyase (EC 4.2.3.3);
E ₃₄ , E ₃₅ , E ₃₆ , E ₃₇ , E ₃₈ , E ₃₉ and E ₄₀ formate dehydrogenases (EC 1.2.1.2);
E _{41 is} a 2-methylcitrate synthase (EC 2.3.3.5);
E ₄₂ a 2-methylcitrate dehydratase (EC 4.2.1.79 or EC 4.2.1.117);
E _{43 is} a 2-methylaconitate isomerase;
E _{44 is} a methyl isocitrate lyase (EC 4.1.3.30);
E _{45 is} a propionate kinase (EC 2.7.2.15);
E _{46 is} a phosphate propionyltransferase (EC 2.3.1.8);
E ₄₇ a pyruvate decarboxylase (EC 4.1.1.1);
E ₄₈ is a pyruvate: ferredoxin oxidoreductase (EC 1.2.7.1);
E _{49 is} a phosphoketolase (EC 4.1.2.9);
E _{50 is} a methylmalonyl-CoA-carboxytransferase (EC 2.1.3.1);
E _{51 is} a methylmalonyl-CoA mutase (EC 5.4.99.2);
E ₅₂ an acetolactate synthase (EC 2.2.1.6);
E ₅₃ an acetolactate decarboxylase (EC 4.1.1.5);
E ₅₄ a butanediol dehydrogenase (EC 1.1.1.4 or EC 1.1.1.76);
E ₅₅ a thiolase (EC 2.3.1.9);
E _{56 is} a 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.157, EC 1.1.1.35, EC 1.1.1.36 or EC 1.1.1.211);
E ₅₇ a crotonase (EC 4.2.1.17);
E _{58 is} a butanol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2);
E ₅₉ a butyraldehyde dehydrogenase (EC 1.2.1.3, EC 1.2.1.4 or EC 1.2.1.5);
E ₆₀ an acetoacetate decarboxylase (EC 4.1.1.4);
E _{61 is} a propanol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2);
E ₆₂ an acyl-CoA: CoA transferase (EC 2.8.3.-); and or
E ₆₃ an L-lactate dehydrogenase (EC 1.1.1.27).

In one embodiment, the cells according to the invention have a reduced activity compared to the wild type of at least one, preferably at least 2, more preferably at least 3, most preferably at least 5 of said enzymes.

In one embodiment of the invention, the activity of at least one of the enzymes E ₁₉ -E _{22 is} reduced. Such a reduction in enzyme activity may be beneficial as it eliminates the energy-consuming import and activation of glucose, thereby providing the cell with more energy to import and activate sucrose and to fix carbon dioxide.

The enzyme E ₂₆ is preferably encoded by a gene selected from the group comprising ygfG, mmdA, oadB, oadB2, oadB3, SC1C2.16, SC1G7.10, pccBI, mmdB, mmdC and ppcB, the ygfG gene being particularly is preferred. In one embodiment, ygfG is from E. coli.

According to various embodiments of the invention, the cell has a reduced activity of at least one enzyme E ₁₉ -E ₄₅ compared to its wild type, wherein:
E ₁₉ is encoded by a ptsG gene;
E ₂₀ is encoded by a ptsI gene;
E ₂₁ is encoded by a ptsH gene;
E ₂₂ is encoded by a crr gene;
E ₂₃ is encoded by a tdcE gene;
E ₂₄ is encoded by a pflA or pflB gene;
E ₂₅ is encoded by a poxB gene;
E ₂₆ is encoded by a ygfG gene;
E ₂₇ is encoded by a ackA gene;
E ₂₈ is encoded by an ackA or tdcD gene;
E ₂₉ is encoded by a pta gene;
E ₃₀ is encoded by an ldhA gene;
E is encoded by a gene adhE _31;
E ₃₂ is encoded by an adhE gene;
E ₃₃ is encoded by a mgsA gene;
E ₃₄ is encoded by a fdnG gene;
E is encoded by a gene fdnH _35;
E ₃₆ is encoded by a fdnI gene;
E ₃₇ is encoded by a fdhF gene;
E ₃₈ is encoded by a fdoG gene;
E ₃₉ is encoded by a fdoH gene;
E is encoded by a gene fdoI _40;
E ₄₁ is encoded by a prpC gene;
E ₄₂ is encoded by a prpD or acnD gene;
E ₄₃ is encoded by a prpF gene;
E ₄₄ is encoded by a prpB gene;
E ₄₅ is encoded by a tdcD gene
E ₄₆ is encoded by a pta gene;
E ₄₇ is encoded by a pdc gene;
E ₄₈ is encoded by a porA, porB, porC or porD gene;
E ₄₉ is encoded by an xpk1 or xpk2 gene
E ₅₀ is encoded by a methylmalonyl CoA carboxytransferase gene;
E ₅₁ is encoded by a sbm or a mcmA and a mcmB gene;
E ₅₂ is encoded by a gene as il, ilvB, ilvM, ilvN, ilvG, ilvI or ilvH gene;
E ₅₃ is encoded by a alsD gene;
E ₅₄ is encoded by a butBGen;
E ₅₅ is encoded by a thl, thlA, thlB or phaA gene;
E ₅₆ is encoded by a phaB gene;
E ₅₇ is encoded by a crt gene;
E ₅₈ is encoded by an adhE gene;
E ₅₉ is encoded by an adhE, bdhA or bdhB gene;
E ₆₀ is encoded by an adc gene;
E ₆₁ is encoded by an adh gene;
E ₆₂ is encoded by a ctfA and a ctfB or an atoA and an atoD gene and / or
E ₆₃ is encoded by an ldhL gene.

The nucleotide sequences of the abovementioned genes, their corresponding protein sequence and other genes for the enzymes E ₁₉ -E ₆₃ can, inter alia, also be taken from the KEGG, NCBI, UniProt or EMBL database.

In one embodiment of the invention, the recombinant cell which, starting from sucrose and CO ₂ as carbon sources, can produce more C ₄ bodies than the wild type, has a reduced activity of at least one, at least two, at least 3, or their wild-type at least 4 of the enzymes E ₁₉ , E ₂₆ , E ₂₇ , E ₂₉ , E ₃₀ , E ₃₁ , E ₃₂ and E ₄₆ on. In such an embodiment, the enzymes E ₁₉ , E ₂₆ , E ₂₇ , E ₂₉ / E ₄₆ , E ₃₀ and E ₃₁ / E _{32 are} ldhA, adhE, ack, pta, ygfG and ptsG. The cell according to the invention can also have a reduced activity of all enzymes E ₁₉ , E ₂₆ , E ₂₇ , E ₂₉ , E ₃₀ , E ₃₁ , E ₃₂ and E ₄₆ or of all 6 enzymes which are represented by the genes ldhA, adhE, ack, pta, ygfG and ptsG.

In further embodiments, the recombinant cell, which can produce more C ₄ bodies than the wild type from sucrose and CO ₂ as carbon sources, has an increased activity of at least one of the enzymes E ₁ -E ₁₈ and, optionally, one compared to their wild type decreased activity of at least one of the enzymes E ₁₉ -E ₆₃ .

• (i) der Enzyme E₁ bis E₄ und E₁₂ gesteigert und der Enzyme E₁₉, E₂₆, E₂₇, E₂₉ bis E₃₂ und E₄₆ verringert ist;
• (ii) der Enzyme E₃, E₅, E₆ und E₁₂ gesteigert und der Enzyme E₁₉, E₂₆, E₂₇, E₂₉ bis E₃₂ und E₄₅ verringert ist; oder
• (iii) der Enzyme E₂, E₃, E₇, E₈, E₉, E₁₀ und E₁₂ gesteigert und der Enzyme E₁₉, E₂₆, E₂₇, E₂₉ bis E₃₂ und E₄₆ verringert ist.

Certain embodiments of the invention relate to cells in which, compared to the wild type, the activity:

• (i) the enzymes E ₁ to E ₄ and E _{12 are} increased and the enzymes E ₁₉ , E ₂₆ , E ₂₇ , E ₂₉ to E ₃₂ and E _{46 are} reduced;
• (ii) the enzymes E ₃ , E ₅ , E ₆ and E _{12 are} increased and the enzymes E ₁₉ , E ₂₆ , E ₂₇ , E ₂₉ to E ₃₂ and E _{45 are} reduced; or
• (iii) the enzymes E ₂ , E ₃ , E ₇ , E ₈ , E ₉ , E ₁₀ and E _{12 are} increased and the enzymes E ₁₉ , E ₂₆ , E ₂₇ , E ₂₉ to E ₃₂ and E _{46 is} reduced.

In such embodiments, the enzymes E ₁ to E ₁₀ and E ₁₂ to E _{18 may be represented} by the genes scrA, scrB, scrK, scrY, cscB, cscA, cscK, susT1, susT2, susX and pyc, ppc, pckA, maeA, mme1, respectively , maeB and dmlA and the enzymes E ₁₉ , E ₂₀ , E ₂₁ , E ₂₂ , E ₂₃ , E ₂₄ , E ₂₅ , E ₂₇ , E ₂₈ , E ₂₉ , E ₃₀ , E ₃₁ , E ₃₂ , E ₃₃ , E ₄₅ , E ₄₆ , E ₄₇ , E ₄₈ , E ₄₉ , E ₅₀ , E ₅₅ , E ₅₆ , E ₅₇ , E ₅₈ , E ₅₉ , E ₆₀ , E ₆₁ and E ₆₃ ptsG, ptsI, ptsH, crr, tdcE, pflA, pflB, poxB, ack, pta, tdcD, ldhA, adhE, mgsA, ygfG, pdc, porA, porB, porC, porD, xpk1, xpk2, thl, thlA, thlB, phaA, phaB, crt, bdhA, bdhB, adc, adh and ldhL.

In a further aspect, the invention relates to a method for producing a genetically modified cell according to the invention.

In such a method, the increase in the activity of at least one of the above-described enzymes E ₁ -E ₁₈ and, optionally, the reduction of the activity of at least one of the above-described enzymes E ₁₉ -E ₆₃ can be carried out by one of the methods described above. Preferred combinations of enzymes with increased or decreased activity are the above-mentioned combinations.

In a still further aspect, the invention relates to a process for producing at least one C ₄ body and / or at least one secondary compound produced via said C ₄ body, said process comprising incubating a cell according to the invention with a nutrient medium containing sucrose, under conditions which permit the production of at least one C ₄ body and / or at least one sucrose and CO ₂ secondary compound prepared via this C ₄ body.

In a particular embodiment of this process, the medium contains carbon dioxide.

The process according to the invention for producing the at least one C ₄ body and / or at least one secondary compound prepared via this C ₄ body may additionally also comprise isolating the at least one C ₄ body and / or at least one secondary compound produced via this C ₄ body from the nutrient medium.

Finally, in a still further aspect, the invention is directed to the use of the cells according to the invention for the production of at least one C ₄ body and / or at least one sucrose and carbon dioxide secondary compound produced via this C ₄ body as carbon sources.

In certain embodiments of the method according to the invention or the use according to the invention, the C ₄ bodies are selected from the following group: succinate, malt, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, Epoxy-γ-butyrolactone, butenoic acid, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, good, n-butene, cis-2-butene, trans 2-butene, isobutene, butadiene, 1,2-butadiene, 1,3-butadiene, 3-hydroxybutyrolactone, 4-hydroxybutyrolactone, 1-butanol, 2-butanol, tert-butanol, isobutanol, 2-hydroxybutyric acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid.

In one embodiment, the C ₄ bodies are selected from the group consisting of: Malst, oxaloacetate and the follow-up compounds prepared via these C ₄ bodies are selected from the group consisting of secondary compounds prepared by enzymatic or chemical synthesis via Malst or oxalacetate. The secondary compounds produced by enzymatic synthesis via Malst or oxalacetate can be selected, for example, from the group consisting of: succinate, aspartate, asparagine, threonine, tetrahydrofuran, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4- Butanediol, 2,3-butanediol, 3- and 4-hydroxybutyrolactone, 1-, 2- and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid, 2- and 3-hydroxyisobutyric acid, methionine and lysine.

The genetically modified cells according to the invention can be used continuously or discontinuously in the batch process (batch culturing) or in the fed-batch process (feed process) or repeated-fed-batch process (repetitive feed process) for the purpose of producing C ₄ bodies, for example C ₄ carboxylic acids such as succinate, are brought into contact with the nutrient medium and cultured. Also practicable is a semi-continuous process, as described in the GB-A-1009370 is described. A summary of the known cultivation methods is in the textbook of 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) to find. The culture medium to be used must suitably satisfy the requirements of the respective host cell strain. Descriptions of culture media for various microorganisms are in the manual "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C, USA, 1981) contain.

In particular, sucrose and / or carbon dioxide serve as carbon sources in the nutrient media used.

As nitrogen sources, organic nitrogen-containing compounds such as peptones, 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 can be used. The nitrogen sources can be used singly or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.

The culture medium may further contain metal salts, such as. As magnesium sulfate or iron sulfate, which are necessary for the growth of the cells.

Finally, the medium can be further substances such. As amino acids and / or vitamins are added. In addition, suitable precursors can be added to the culture medium. The said feedstocks may be added to the culture in the form of a one-time batch or fed in a suitable manner during the 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, the medium suitable selective substances such. B. antibiotics are added.

The temperature of the culture is usually 20 ° C to 45 ° C, and preferably 25 ° C to 40 ° C.

The purification of the C ₄ body (s) or secondary products from the nutrient solution is preferably carried out continuously, wherein it is further preferred in this context to carry out the production of the C ₄ body by fermentation continuously, so that the entire process of the Production of the C ₄ body can be carried out continuously until its purification from the fermentation broth. For the continuous purification of the C ₄ body from the fermentation broth, this is continuously passed through a device for separating the microorganisms used in the fermentation, preferably via a filter with an exclusion size in a range of 20 to 200 kDa, in which a solid / liquid Separation takes place. It is also conceivable to use a centrifuge, a suitable sedimentation device or a combination of these devices, wherein it is particularly preferred first to separate off at least part of the microorganisms by sedimentation and then to supply the fermentation broth partially freed of the microorganisms to an ultrafiltration or centrifuging device.

The enriched in terms of its C ₄ body portion fermentation product is fed after separation of the microorganisms of a preferably multi-stage separation plant. In this separation plant a plurality of series-connected separation stages are provided, from which each return lines lead, which are returned to the fermentation tank. Furthermore lead out of the respective separation stages derivatives. The individual separation stages can work according to the principle of electrodialysis, reverse osmosis, ultrafiltration or nanofiltration. As a rule, these are membrane separation devices in the individual separation stages. The selection of the individual separation stages results from the nature and extent of the fermentation by-products and substrate residues.

In addition to the removal of the C ₄ body by means of electrodialysis, reverse osmosis, ultrafiltration or nanofiltration, in the course of which an aqueous C ₄ -body solution is obtained, the C ₄ body can also be separated by extraction from the freed from microorganisms fermentation solution in which case, ultimately, the pure C ₄ body can be obtained. For separation of a C ₄ carboxylic acid by extraction, for example, ammonium compounds or amines may be added to the fermentation solution to form an ammonium salt of C ₄ carboxylic acid. This ammonium salt can then be separated from the fermentation solution by adding an organic extractant and then heating the resulting mixture, whereby the ammonium salt accumulates in the organic phase. From this phase, the C ₄ carboxylic acid can then be isolated to obtain the pure C ₄ carboxylic acid, for example by further extraction steps. More details regarding this separation process are the WO-A-02/090312 whose disclosure content is hereby incorporated by reference and forms part of the disclosure of the present application.

Depending on the manner in which the C ₄ body is separated from the fermentation solution, either an aqueous C ₄ -body solution containing from 2 to 90% by weight, preferably from 7.5 to 50% by weight and more preferably from 10 to 25 wt .-% of C ₄ body, or obtained pure C ₄ body.

Furthermore, the C ₄ -carboxylic acids prepared by the process according to the invention can be neutralized before, during or after the purification, it being possible to use bases such as calcium hydroxide or sodium hydroxide for this purpose.

Further embodiments of the invention are contained in the claims and the examples. The following examples serve to illustrate the invention, but the invention is not limited to these specific embodiments.

example 1

Production of E. coli Expression Vectors for the Genes ppc, pck, maeA and maeB from E. coli and pyc from C. glutamicum

For the production of E. coli expression vectors for the genes ppc (SEQ ID NO: 1), pck (SEQ ID NO: 2), mAca (SEQ ID NO: 3) and mAb (SEQ ID NO: 4) from E. coli As well as pyc from C. glutamicum (SEQ ID NO: 5), these genes are amplified from chromosomal DNA of E. coli MG1655 or C. glutamicum ATCC 13032 by PCR and simultaneously via the oligonucleotides used an interface upstream of the respective ribosome binding site and a downstream interface of the stop codon. The preparation of the chromosomal DNA of E. coli MG1655 or C. glutamicum ATCC 13032 is carried out using DNeasy Blood & Tissue Kit (Qiagen, Hilden) according to the manufacturer's instructions. The following oligonucleotides are used in the amplification of the genes ppc, pck, maeA and maeB from E. coli and pyc from C. glutamicum with chromosomal DNA of E. coli MG1655 or C. glutamicum ATCC 13032 as template: E. coil PPC: ppc fw: 5'-ATA GAG CTC AGG GCT ATC AAA CGA TAA GAT GGG GTG-3 '(SEQ ID NO: 6) 5'-ATA CCT GCA GGT TAG CCG GTA TTA CGC ATA CCT GCC G-3 ' ppc rv: (SEQ ID NO: 7) E. coli pck: pck fw: 5'-ATA GGA TCC TTA CTA TTC AGG CAA TAC ATA TTG GCT AAG GA-3 '(SEQ ID NO: 8) pck rv: 5'-ATA CCT GCA GGT CAT TAC AGT TTC GGA CCA GCC GCT AC-3 '(SEQ ID NO: 9) E. coli maeA: maeA-fw: 5'-ATA GAG CTC GTG GAT ATT CAA AAA AGA GTG AGT GAC ATG GAA C-3 '(SEQ ID NO: 10) maeA-rv: 5'-ATA CCT GCA GGT TAG ATG GAG GTA CGG CGG TAG TCG-3 '(SEQ ID NO: 11) E. coli meaB: -fw MAEB: 5'-ATA GAG CTC TAC GTG AAA GGA ACA ACC AAA TGG ATG ACC-3 '(SEQ ID NO: 12) MAEB-rv: 5'-ATA CCT GCA GGT TAC AGC GGT TGG GTT TGC GCT TC-3 '(SEQ ID NO: 13) C. glutamicum pyc: pyc fw: 5'-ATA GAG CTC TGA AAG GAA TAA TTA CTC TAG TGT CGA CTC-3 '(SEQ ID NO: 14) pyc rv: 5'-ATA CCT GCA GGG GTT TAG GAA ACG ACG ACG ATC AAG-3 '(SEQ ID NO: 15)

The following parameters are used for the PCR: 1 ×: initial denaturation, 98 ° C., 0:30 min; 30x: denaturation, 98 ° C, 0:10 min, annealing, 60 ° C, 0:30 min; Elongation, 72 ° C, 3 min; 1 ×: terminal elongation, 72 ° C, 10 min. For amplification, the Phusion ^™ High-Fidelity Master Mix from New England Biolabs (Frankfurt) is used according to the manufacturer's recommendations. Each 10 .mu.l of the PCR reactions are then separated on a 0.8% agarose gel. The carrying out of the PCR, the agarose gel electrophoresis, ethidium bromide staining of the DNA and determination of the PCR fragment sizes are carried out in a manner known to the person skilled in the art.

In all cases, PCR fragments of the expected size can be amplified. For ppc this is 2710 bp, for pck 1686 bp, for maeA 1745 bp, for maeB 3220 bp and for pyc 3466 bp. The PCR products are digested with SacI and SbfI (ppc, maeA, maeB and pyc) or BamHI and SbfI (pck) according to the recommendations of the manufacturer of the Restrictiosendonukleasen (New England Biolabs, Bad Schwalbach) and in the SacI and SbfI ( ppc, maeA, maeB and pyc) and BamHI and SbfI (pck) cut vector pEC-XC99E (SEQ ID NO: 16). pEC-XC99E is an E. coli C. glutamicum shuttle vector, which mediates chloramphenicol resistance in both organisms and carries a ColE1 origin of replication (high copy number:> 500 copies per E. coli cell) and a synthetic trc promoter without ribosome binding site. Ligation and transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) are carried out in a manner known to the person skilled in the art.

The correct insertion of the ppc, pck, maeA, maeB or pyc fragments is checked by a restriction with SacI and SbfI (ppc, maeA, maeB and pyc) or BamHI and SbfI (pck). The authenticity of the inserted fragments is checked by DNA sequencing. The completed E. coli expression vectors are identified as pEC-XC99E-ppc (SEQ ID NO: 17), pEC-XC99E-pck (SEQ ID NO: 18), pEC-XC99E-maeA (SEQ ID NO: 19), pEC- XC99E-maeB (SEQ ID NO: 20) and pEC-XC99E-pyc (SEQ ID NO: 21).

Example 2

Production of E. coli expression vectors for the loci cscA-cscKB from E. coli and scrK-scrYAB from pUR400

For the production of E. coli expression vectors for the loci cscA-cscKB from E. coli DSM 1116 (SEQ ID NO: 22) and scrK-scrYAB from pUR400 (SEQ ID NO: 23), these are obtained from chromosomal DNA of E. coli DSM 1116 or total DNA of E. coli K12 (pUR400) amplified by PCR and introduced simultaneously via the oligonucleotides used each a restriction site at the 5 'or 3' end. The preparation of the chromosomal DNA from E. coli DSM 1116 or total DNA from E. coli K12 (pUR400) is carried out using DNeasy Blonde & Tissue Kit (Qiagen, Hilden) according to the manufacturer's instructions in the amplification of the loci cscA-cscKB from chromosomal DNA of E. coli DSM 1116 or of scrK-scrYAB from total DNA of E. coli K12 (pUR400) as template, the following oligonucleotides are used: E. coli cscA-cscKB: csc-fw: 5'-ATA CAT ATG TTA TTA ACC CAG TAG CCA GAG TGC TCC AT GT-3 '(SEQ ID NO: 24) csc rv: 5'-ATA CTC GAG CTA CTA TAT TGC TGA AGG TAC AGG CGT TTC C-3 '(SEQ ID NO: 25) E. coli scrK-scrYAB: scr-fw: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 26) scr-rv: 5'-ATA GCG GCC GCT TAT TCT ACC ATG CAA GTT CGC AGC-3 '(SEQ ID NO: 27)

The following parameters are used for the PCR: 1 ×: initial denaturation, 98 ° C., 0:30 min; 30x: denaturation, 98 ° C, 0:10 min, annealing, 60 ° C, 0:30 min; Elongation, 72 ° C, 5 min; 1 ×: terminal elongation, 72 ° C, 10 min. For amplification, the Phusion ^™ High-Fidelity Master Mix from New England Biolabs (Frankfurt) is used according to the manufacturer's recommendations. Each 10 .mu.l of the PCR reactions are then aufgtrennt on a 0.8% agarose gel. The carrying out of the PCR, agarose gel electrophoresis, ethidium bromide staining of the DNA and determination of the PCR fragment sizes are carried out in a manner known to the person skilled in the art.

In both cases, PCR fragments of the expected size can be amplified. This is 3907 bp for cscA-cscKB and 5800 bp for scrK-scrYAB. The PCR products are digested with NcoI and NotI (scrK-scrYAB) or NdeI and XhoI (cscA-cscKB) according to the recommendations of the manufacturer of the Restrictiosendonucleases (New England Biolabs, Bad Schwalbach) and in the NcoI and NotI (scrK -scrYAB) or NdeI and XhoI (cscA-cscKB) cut vector pCOLADuet-1 (SEQ ID NO: 28, Merck Biosciences, Nottigham, UK). pCOLADuet-1 is a low copy number (20-40 copies per cell) E. coli vector that mediates kanamycin resistance and carries a ColA origin of replication. Ligation and transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) are carried out in a manner known to the person skilled in the art.

The correct insertion of the cscA-cscKB and scrK-scrYAB fragments is checked by restriction with NcoI and NotI (scrK-scrYAB) or NdeI and XhoI (cscA-cscKB). The authenticity of the inserted fragments is checked by DNA sequencing. The completed E. coli expression vectors are designated pCOLA-csc (SEQ ID NO: 29) and pCOLA-scr (SEQ ID NO: 30), respectively.

Example 3

Chromatographic quantification of educts and products

The chromatographic quantification of sucrose is carried out in the following manner: The quantification of sucrose is carried out by means of high-performance liquid chromatography. For the separation of sugars, an aminophase anion exchange column 5 μm in particle size and 250 × 4.6 mm (Waters Spherisorb NH2, Waters, Eschborn) is used. The mobile phase consists of a mixture of acetonitrile (56% v / v), acetone (26% v / v) and water (16% v / v). The samples are sterile filtered and measured undiluted. The injection volume is 10 μL, the mobile phase flow 2 mL / min and the column temperature 30 ° C. Sucrose is quantified by refractive index detector (Agilent 1200 Series RID, Agilent Technologies, Boeblingen). The reference substance (Sigma-Aldrich, Steinheim) is dissolved in water. There is a linear dependence between the peak area and the substance concentration up to a concentration of 100 g / L. The limit of quantification for sucrose is 1 g / L.

The chromatographic quantification of succinate, acetate, lactate, formate and ethanol was carried out in the following manner: The separation of the organic acids and ethanol is carried out by means of high-performance liquid chromatography. Here is the ion-exclusion column Aminex ^® HPX 87H with the dimensions 300 mm × 7.8 mm (Bio Rad Laboratories, Munich) as the stationary phase used. The mobile phase used is 10 mM sulfuric acid. The column temperature is 40 ° C, the flow rate 0.6 ml / min. The sample is acidified to pH 4 to 5 with 0.5 M sulfuric acid and injected into the column at a volume of 20 μL. Detection is performed using a diode array detector (Agilent 1200 Series DAD, Agilent Technologies, Böblingen) at a wavelength of 190 to 400 nm and a refractive index detector (Agilent 1200 Series RID, Agilent Technologies, Böblingen). The reference substances are measured in concentrations of 0.1 g / L to 20 g / L dissolved in water. The limits of determination for lactate, acetate, succinate and formate are 0.8 g / L, ethanol can be determined up to a concentration of 1 g / L. In the range from 0.8 g / L to 20 g / L there is a linear dependence of the peak areas on the substance concentration.

Example 4

Production of succinate by E. coli strains with expression vectors for the genes ppc, pck, maeA and maeB from E. coli and pyc from C. glutamicum in combination with expression vectors for the loci cscA-cscKB from E. coli and scrK-scrYAB pUR400

For the production of E. coli strains with expression vectors for the genes ppc, pck, maeA and maeB from E. coli and pyc from C. glutamicum in combination with expression vectors for the loci cscA-cscKB from E. coli and scrK-scrYAB from pUR400 Competent cells of E. coli MG1655 are prepared. For this purpose, the strain is grown in Luria-Bertani Bouillon Miller (Merck, Darmstadt) as a 10 mL pre-culture. A 100 mL LB broth is inoculated with 2 mL of preculture and cultured at 37 ° C and 200 rpm in the incubator shaker to an optical density (600 nm) of 0.5. The electrocompetent cells are prepared by washing with a sterile 10% (w / v) glycerol solution as follows: The 100 mL culture is harvested by centrifugation at 4 ° C and 5500 x g for 10 minutes and washed by resuspension in 10% glycerol solution. After another centrifugation and washing step, the pelleted E. coli MG1655 cells are taken up in 0.5 mL of 10% glycerol solution and stored in aliquots of 50 μL at -80 ° C until electroporation.

These are then used sequentially or in combination with the plasmids pEC-XC99E, pEC-XC99E-ppc, pEC-XC99E-pck, pEC-XC99E-maeA, pEC-XC99E-maeB, pEC-XC99E-pyc, pCOLADuet-1, pCOLA- csc and pCOLA-scr and plated on LB plates with chloramphenicol (50 μg / ml) and / or kanamycin (50 μg / ml). Transformants are checked by plasmid preparation and analytical restriction analysis for the presence of the authentic plasmids.

• • E. coli MG1655 (pCOLADuet-1, pEC-XC99E)
• • E. coli MG1655 (pCOLA-csc, pEC-XC99E)
• • E. coli MG1655 (pCOLA-csc, pEC-XC99E-ppc)
• • E. coli MG1655 (pCOLA-csc, pEC-XC99E-pck)
• • E. coli MG1655 (pCOLA-csc, pEC-XC99E-maeA)
• • E. coli MG1655 (pCOLA-csc, pEC-XC99E-maeB)
• • E. coli MG1655 (pCOLA-csc, pEC-XC99E-pyc)
• • E. coli MG1655 (pCOLA-scr, pEC-XC99E)
• • E. coli MG1655 (pCOLA-scr, pEC-XC99E-ppc)
• • E. coli MG1655 (pCOLA-scr, pEC-XC99E-pck)
• • E. coli MG1655 (pCOLA-scr, pEC-XC99E-maeA)
• • E. coli MG1655 (pCOLA-scr, pEC-XC99E-maeB)
• • E. coli MG1655 (pCOLA-scr, pEC-XC99E-pyc)

Thus, the following E. coli strains were generated:

• E. coli MG1655 (pCOLADuet-1, pEC-XC99E)
• E. coli MG1655 (pCOLA-csc, pEC-XC99E)
• E. coli MG1655 (pCOLA-csc, pEC-XC99E-ppc)
• E. coli MG1655 (pCOLA-csc, pEC-XC99E-pck)
• E. coli MG1655 (pCOLA-csc, pEC-XC99E-maeA)
• E. coli MG1655 (pCOLA-csc, pEC-XC99E maeB)
• E. coli MG1655 (pCOLA-csc, pEC-XC99E-pyc)
• E. coli MG1655 (pCOLA-scr, pEC-XC99E)
• E. coli MG1655 (pCOLA-scr, pEC-XC99E-ppc)
• E. coli MG1655 (pCOLA-scr, pEC-XC99E-pck)
• E. coli MG1655 (pCOLA-scr, pEC-XC99E-maeA)
• E. coli MG1655 (pCOLA-scr, pEC-XC99E-maeB)
• E. coli MG1655 (pCOLA-scr, pEC-XC99E-pyc)

These strains are then used to analyze their ability to produce succinate under anaerobic conditions. The procedure is as follows:
The strains undergo a multi-step cultivation process. The preculture for the production of biomass is carried out aerobically in a modified M9 medium which additionally contains yeast extract as complex component. The medium consisting of 38 mM disodium hydrogen phosphate dihydrate (Merck, Darmstadt), 22 mM potassium dihydrogen phosphate (Merck, Darmstadt), 8.6 mM sodium chloride (Merck, Darmstadt), 18.7 mM ammonium chloride (Merck, Darmstadt), 1% ( w / v) yeast extract (Merck, Darmstadt), 2% (w / v) sucrose (Sigma-Aldrich, Steinheim), 0.1 mM calcium chloride dihydrate (Sigma-Aldrich, Steinheim), 1 mM magnesium sulfate heptahydrate (Merck, Darmstadt), 200 mM MOPS (3- (N-morpholino) -propanesulfonic acid, Sigma-Aldrich, Steinheim) and 1% (v / v) MEM vitamin solution (Sigma-Aldrich, Steinheim), with sodium hydroxide to a pH of 7.0 and added to 10 mL of chloramphenicol (50 μg / mL) and / or kanamycin (50 μg / mL) in a 100 mL Erlenmeyer flask with chicane and inoculated with a single colony of the strain to be tested. The cultivation takes place at 37 ° C. and 250 rpm in an incubation shaker. After a culture time of 8 hours, 200 mL of modified M9 medium containing 0.2 mg / L biotin and 0.5 mM IPTG are inoculated with the preculture in a 1000 mL Erlenmeyer flask to give an optical density (600 nm) of 0.2 is reached. After a culture period of at least 12 hours at 37 ° C and 250 rpm, the culture broth is harvested by centrifugation at 3300 g and 4 ° C for 10 minutes. The sedimented bacterial cells are washed with sterile 0.9% sodium chloride solution and then taken up in anaerobic culture medium. The main anaerobic culture is carried out in modified M9 medium without complex components (38 mM disodium hydrogen phosphate dihydrate, 22 mM potassium dihydrogen phosphate, 8.6 mM sodium chloride, 18.7 mM ammonium chloride, 2% (w / v) sucrose, 0.1 mM calcium chloride. Dihydrate, 1mM Magnesium Sulphate Heptahydrate, 200mM MOPS, 1% (v / v) MEM Vitamin Solution, 0.1% (w / v) Sodium bicarbonate, pH 7.0 with sodium hydroxide) with the addition of 0.2 mg / L biotin, 50 g / L magnesium carbonate and 0.5 mM IPTG, and 1 mg / L resazurin as oxygen indicator. 50 ml of medium are placed in 100 ml laboratory glass bottles (Schott, Mainz) and inoculated with the cell suspension so that an optical density (600 nm) of 20 is achieved. The bottles are sealed gas-tight with a cap with stopper, the cultivation is carried out at 37 ° C and 250 rev / min in incubation shaker for a period of 24 hours. Anaerobiosis occurs within the first hour of cultivation and is detected by decolorization of resazurin. During culture, samples of 1 mL are taken sterile with a cannula through the stopper which, after sterile filtration, is analyzed for its sucrose content and organic acid content by means of chromatographic methods described in Example 3.

Example 5

Production of succinate by E. coli strains with deletions in the genes ack-pta, adhE, ldhA and ygfG, in which the gene ptsG was replaced by the scrK-scrYAB locus from the plasmid pUR400 and which contains the gene pyc, coding for a pyruvate carboxylase from Corynebacterium glutamicum, overexpressed.

First, an E. coli strain with deletions in the genes ack-pta (SEQ ID NO: 31), adhE (SEQ ID NO: 32), ldhA (SEQ ID NO: 33), and ygfG (SEQ ID NO: 34) was prepared. and an exchange of the ptsG gene (SEQ ID NO: 35) with the scrK scrYAB locus. E. coli strain MG1655 ack-pta adhE ldhAggfG ptsG :: scrK-scrYAB was generated by methods known to those of skill in the art (e.g., see Datsenko KA, Wanner BL. Proc Natl Acad Sci USA 2000. 97 (12): 6640-5. ). The DNA sequence of the individual loci after deletion or insertion is shown in SEQ ID NO: 36 (ack-pta), SEQ ID NO: 37 (adhE), SEQ ID NO: 38 (ldhA), SEQ ID NO: 39 (ygfG ) and SEQ ID NO: 40 (ptsG :: scrK-scrYAB).

E. coli MG1655 ack-pta adhE IdhA ygfG ptsG :: scrK-scrYAB was subsequently transformed analogously to Example 4 with the plasmids pEC-XC99E and pEC-XC99E-pyc.

The E. coli strains MG1655 ack-pta adhE ldhA ygfG ptsG :: scrK-scrYAB (pEC-XC99E) and ack-pta adhE ldhA ygfG ptsG :: scrK-scrYAB (pEC-XC99E-pyc) were then analogously to Example 4 cultured and quantified the concentration of sucrose, succinate and by-products analogously to Example 3. The results are shown in Tab. 1 and 2. Table 1. Production of succinate with E. coli MG1655 ack-pta adhE ldhA ygfG ptsG :: scrK-scrYAB (pEC-XC99E). Indicated are the concentration of succinate and by-products as well as the product yield (Y _{P / S} ) and the average space-time yield (RZA) in the first 24 hours. Ethanol, lactate, pyruvate and formate could not be detected. t [h] c _sucrose [g / l] c _succinate [g / l] c _acetate [g / l] Y _{P / S} [g / g] RZA [g · l ^-1 · h ^-1 ] 0 20 0 0 0 0 24 9 3.8 1.2 0.19 0.16 Table 2. Production of succinate with E. coli MG1655 ack-pta adhE ldhA ygfG ptsG :: scrK-scrYAB (pEC-XC99E-pyc). Indicated are the concentration of succinate and the by-products acetate, ethanol, lactate and formate as well as the product yield (Y _{P / S} ) and the average space-time yield (RZA) in the first 24 hours. Ethanol, lactate, pyruvate and formate could not be detected. t [h] c _sucrose [g / l] c _succinate [g / l] c _acetate [g / l] Y _{P / S} [g / g] RZA [g · l ^-1 · h ^-1 ] 0 20 0 0 0 0 24 0 15 1.1 0.75 0.6

The disclosure of all documents cited herein is incorporated by reference in its entirety and forms a part of the disclosure of the present application.

The invention is described herein by reference to certain embodiments, but is not limited thereto. In particular, it will be readily apparent to those skilled in the art that various changes may be made in the invention described without departing from the spirit and scope of the invention as defined by the appended claims. The scope of The invention is thus defined by the claims and it is intended that the invention encompass all modifications and changes that fall within the scope of the invention.

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (51)

Recombinant cell which has been altered in such genetically compared to its wild type that it is able to form a result Compounds prepared via this C ₄ -body of sucrose and carbon dioxide as the carbon sources as compared to its wild type more C ₄ -body and / or more. The recombinant cell of claim 1, wherein the C ₄ bodies are selected from the group consisting of succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, epoxy -γ-butyrolactone, butenoic acid, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, butene, n-butene, cis-2-butene, trans-2 Butene, isobutene, butadiene, 1,2-butadiene, 1,3-butadiene, 3-hydroxybutyrolactone, 4-hydroxybutyrolactone, 1-butanol, 2-butanol, tert-butanol, isobutanol, 2-hydroxybutyric acid, 3-hydroxybutyric acid, 4 Hydroxybutyric acid, 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid. The recombinant cell according to claim 1 or 2, wherein the C ₄ bodies are selected from the group consisting of: Malst, oxaloacetate and the derivatives produced via these C ₄ bodies are selected from the group consisting of enzymatic or chemical synthesis via Malst or oxaloacetate produced by followers. The recombinant cell according to claim 3, wherein the secondary compounds produced by enzymatic synthesis via malate or oxaloacetate are selected from the group consisting of: aspartate, asparagine, threonine, tetrahydrofuran, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 3- and 4-hydroxybutyrolactone, 1-, 2- and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid, 2- and 3-hydroxyisobutyric acid, methionine and lysine. The recombinant cell of any one of claims 1 to 4, wherein the cell is a microbial cell. The recombinant cell according to any one of claims 1 to 5, wherein the microbial cell is Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes , Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Methylobacterium extorquens, Ralstonia eutropha, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Clostridium acetobutylicum, saccharoperbutylacetonicum Clostridium, Clostridium beijerinckii, Rhodospirillum rubrum, Burkholderia thailandensis or Pseudomonas putida cell. The recombinant cell according to any one of claims 1 to 6, wherein the cell can take up more sucrose compared to its wild-type. The recombinant cell according to any one of claims 1 to 7, wherein the cell can fix more carbon dioxide compared to its wild type. The recombinant cell of any one of claims 1 to 8, wherein the cell has an increased activity of at least one enzyme that catalyzes the transport of sucrose into the cell compared to its wild-type. The recombinant cell of claim 9, wherein the at least one enzyme comprises an enzyme E1 which catalyzes the transport of sucrose into the cell and the conversion to sucrose-6-phosphate. The recombinant cell according to claim 10, wherein the enzyme E1 is a phosphoenolpyruvate (PEP) -dependent phosphotransferase system (PTS) enzyme II (sucrose-specific) (EC 2.7.1.69; TCDB classification 4.A.1.2.1, 4.A .1.2.9 or 4.A.1.2.-). The recombinant cell of claim 10 or 11, wherein the enzyme E1 is encoded by genes selected from the group of those encoding gene products whose amino acid sequence is over a range of at least 100 amino acids, at least 60% to SEQ ID NO: 58 is identical. The recombinant cell according to at least one of claims 1 to 12, wherein the cell further has an increased activity of at least one enzyme E ₂ , which is the conversion of sucrose-6-phosphate to D-glucose-6-phosphate compared to its wild-type. Catalyzes D-fructose; and / or at least one enzyme E ₃ , which catalyzes the conversion of D-fructose to D-fructose-6-phosphate; and / or at least one channel E ₄ permitting diffusion-dependent sucrose transport into the cell. The recombinant cell of claim 13, wherein the enzyme E _{2 is} a sucrose-6-phosphate fructohydrolase (EC 3.2.1.26); and / or the enzyme E _{3 is} a fructokinase (EC 2.7.1.4); and / or the enzyme E _{4 is} a sucrose porin (TCDB classification 1.B.3.1.2). The recombinant cell of claim 14, wherein E ₂ is encoded by a scrB or susH gene; and / or E ₃ is encoded by a scrK or cscK gene; and / or E ₄ is encoded by a scrY gene. The recombinant cell of claim 9, wherein the at least one enzyme comprises a channel E5 that sorb sucrose into the cell. The recombinant cell of claim 16, wherein the channel E5 is a sucrose permease (TCDB classification 2.A.1.5.3). The recombinant cell of claim 17, wherein the E5 channel is encoded by a cscB gene. The recombinant cell of any one of claims 16 to 18, wherein the cell further comprises an increased activity of at least one enzyme E6, which catalyzes the conversion of sucrose to D-glucose and D-fructose compared to its wild-type; and / or at least one enzyme E3 which catalyzes the conversion of D-fructose to D-fructose-6-phosphate. The recombinant cell of claim 19, wherein the enzyme E _{3 is} a fructokinase (EC 2.7.1.4); and / or the enzyme E _{6 is} a -D-fructofuranoside fructohydrolase (EC 3.2.1.26). The recombinant cell of claim 20, wherein E ₃ is encoded by a scrK or cscK gene; and / or E ₆ is encoded by a cscA gene. The recombinant cell of claim 9, wherein the at least one enzyme comprises an enzyme complex of E7, E8 and E9 which transports sucrose into the cell. The recombinant cell of claim 22, wherein the enzyme complex of E7, E8 and E9 is a sucrose-specific ABC transporter (TCDB classification 3.A.1.1.-). The recombinant cell of claim 22 or 23 wherein E ₇ is a susT1 gene; and / or E ₈ through a susT2 gene; and / or E ₉ is encoded by a susX gene. The recombinant cell of any one of claims 22 to 24, wherein the cell further comprises an increased activity of at least one enzyme E10, which catalyzes the conversion of sucrose to sucrose-6-phosphate, compared to its wild-type; and / or at least one enzyme E2 which catalyzes the conversion of sucrose-6-phosphate to D-glucose-6-phosphate and D-fructose; and / or at least one enzyme E3 which catalyzes the conversion of D-fructose to D-fructose-6-phosphate; having. The recombinant cell of claim 25, wherein the cell has an increased activity of the enzymes E10, E2, E3 and E6 compared to their wild-type. The recombinant cell of claim 26, wherein the enzyme E _{10 is} a sucrose kinase; and or the enzyme E _{2 is} a sucrose-6-phosphate fructohydrolase (EC 3.2.1.26); the enzyme E _{3 is} a fructokinase (EC 2.7.1.4) and / or the enzyme is E ₆ -D-fructofuranoside fructohydrolase (EC 3.2.1.26). The recombinant cell of claim 26 or 27, wherein E ₁₀ is a sucrose kinase gene; and / or E ₂ by a scrB or susH gene; E ₃ is encoded by a scrK or cscK gene and / or E ₆ by a cscA gene The recombinant cell of any one of claims 1 to 28, wherein the cell has an increased activity of at least one enzyme that catalyzes the fixation of CO ₂ to a C ₃ body compared to its wild-type. The recombinant cell of claim 29, wherein the at least one enzyme that catalyzes the fixation of CO ₂ to a C ₃ body is selected from the group consisting of the following enzymes: E ₁₂ , which is the reaction of pyruvate, ATP and CO ₂ catalyses oxaloacetate, ADP and phosphate; E ₁₃ , which catalyzes the reaction of phosphoenolpyruvate, H ₂ O and CO ₂ to oxaloacetate and phosphate; E ₁₄ , which catalyzes the reaction of phosphoenolpyruvate, ADP / GDP / P _i and CO ₂ to oxaloacetate and ATP / GTP / PP _i ; E ₁₅ , E ₁₆ , E ₁₇ and E ₁₈ , which catalyze the conversion of pyruvate NAD (P) H ⁺ and CO ₂ to Malst and NAD (P) ⁺ . The recombinant cell of claim 30, wherein: E _{12 is} a pyruvate carboxylase (EC 6.4.1.1); E _{13 is} a phosphoenolpyruvate carboxylase (phosphate: oxaloacetate carboxylase) (EC 4.1.1.31); E _{14 is} a phosphoenolpyruvate carboxykinase (ATP / GTP / PP _i : oxaloacetate carboxylase) (EC 4.1.1.32, EC 4.1.1.38 or EC 4.1.1.49); E _{15 is} a malate dehydrogenase ((S) -malate: NAD ⁺ oxidoreductase) (EC 1.1.1.38); E _{16 is} a malate dehydrogenase ((S) -malate: NAD ⁺ oxidoreductase) (EC 1.1.1.39); E _{17 is} a malate dehydrogenase ((S) -malate: NADP ⁺ oxidoreductase) (EC 1.1.1.40); and / or E _{18 is} a D-malate dehydrogenase ((R) -malate: NAD ⁺ oxidoreductase) (EC 1.1.1.83). The recombinant cell of claim 31, wherein: E ₁₂ is a pyc gene; E ₁₃ by a ppc gene; E ₁₄ by a pckA gene; E ₁₅ by a mouse gene; E ₁₆ by a mme1 gene; E ₁₇ by a mouse gene; and / or E ₁₈ is encoded by a dmlA gene. The recombinant cell of any one of claims 13 to 15, wherein the cell further has an increased activity of an enzyme E12, which catalyzes the conversion of pyruvate, ATP and CO ₂ to oxaloacetate, ADP and phosphate, compared to its wild-type. The recombinant cell of claim 33, wherein E12 is encoded by a pyc gene. The recombinant cell of any one of claims 1 to 34, wherein the cell has a reduced carbon flux from sucrose to its wild type towards fermentation products other than C ₄ bodies. The recombinant cell of claim 35, wherein less C ₁ , C ₂ and / or C _{3 by-} products are produced from sucrose in the cell as compared to its wild-type. The recombinant cell of claim 35 or 36, wherein the metabolism that produces fermentation products is reduced or disrupted. The recombinant cell according to any one of claims 35 to 37, wherein the cell has a reduced activity of at least one enzyme selected from the group consisting of the following enzymes compared to its wild-type: E ₁₉ , E ₂₀ , E ₂₁ and E ₂₂ , which are components of a glucose-specific phosphoenolpyruvate phosphotransferase system and, as such, catalyze the import of glucose and, in sum, the conversion of phosphoenolpyruvate and glucose to pyruvate and glucose-6-phosphate; E ₂₃ and E ₂₄ , which catalyze the conversion of pyruvate and CoA to formate and acetyl-CoA; E ₂₅ , which catalyzes the reaction of pyruvate, H ₂ O and ferricytochrome b ₁ to acetate, CO ₂ and ferrocytochrome b ₁ ; E ₂₆ , which catalyzes the conversion of S-methylmalonyl CoA to propanoyl CoA and CO ₂ ; E ₂₇ and E ₂₈ , which catalyze the conversion of acetyl phosphate and ADP / P _i to acetate and ATP / PP _i ; E ₂₉ , which catalyzes the conversion of acetyl-CoA and P _i (orthophosphate) to acetyl phosphate and CoA; E ₃₀ , which catalyzes the conversion of pyruvate and NADH to D-lactate and NAD ⁺ ; E ₃₁ , which catalyzes the conversion of acetyl-CoA to acetaldehyde; E ₃₂ , which catalyzes the conversion of acetaldehyde to ethanol; E ₃₃ , which catalyzes the conversion of glycerol phosphate to methylglyoxal and orthophosphate; E ₃₄ , E ₃₅ and E ₃₆ , which form a complex that catalyzes the conversion of formate to CO ₂ ; E ₃₇ , which catalyzes the conversion of formate to CO ₂ ; E ₃₈ , E ₃₉ and E ₄₀ , which form a complex that catalyzes the conversion of formate to CO ₂ ; E ₄₁ , which catalyzes the reaction of propionyl CoA, H ₂ O and oxaloacetate to 2-methyl citrate and CoA; E ₄₂ , which catalyzes the conversion of 2-methylcitrate to 2-methyl-cis-aconitate or 2-methyl-trans-aconitate and H ₂ O; E ₄₃ , which catalyzes the conversion of 2-methyl trans aconitate to 2-methyl cis aconitate; E ₄₄ , which catalyzes the conversion of 2-methyl cis aconitate to succinate and pyruvate; E ₄₅ , which catalyzes the conversion of propionyl phosphate and ADP to propionate and ATP; _E46 , which catalyzes the reaction of propionyl CoA and P _i to propionyl phosphate and CoA; E ₄₇ , which catalyzes the conversion of pyruvate to acetaldehyde and CO ₂ ; E ₄₈ , which catalyzes the reaction of pyruvate and CoA 2 oxidized ferredoxins to acetyl-CoA, CO ₂ , and 2 reduced ferredoxins and 2H ⁺ ; E ₄₉ , which catalyzes the reaction of D-xylulose-5-phosphate and phosphate to acetyl phosphate and D-glyceraldehyde-3-phosphates and H ₂ O; E ₅₀ , which catalyzes the reaction of (S) -methylmalonyl-CoA and pyruvate to propionyl-CoA and oxaloacetate; E ₅₁ , which catalyzes the reaction of (S) -methylmalonyl-CoA to succinyl-CoA; E ₅₂ , which catalyzes the conversion of 2 pyruvate to 2-acetolactate and CO ₂ ; E ₅₃ , which catalyzes the conversion of 2-acetolactate to acetoin and CO ₂ ; E ₅₄ , which catalyzes the reaction of acetoin and NAD (P) H ⁺ to butane-2,3-diol and NAD (P) ⁺ ; E ₅₅ , which catalyzes the reaction of 2 acetyl-CoA to acetoacetyl-CoA; E ₅₆ , which catalyzes the reaction of acetoacetyl-CoA and NAD (P) H ⁺ to 3-hydroxybutyryl-CoA and NAD (P) ⁺ ; E ₅₇ , which catalyzes the conversion of crotonyl CoA and H ₂ O to 3-hydroxybutyryl CoA; E ₅₈ , which catalyzes the reaction of butyraldehyde and NAD (P) H ⁺ to butanol and NAD (P) ⁺ ; E ₅₉ , which catalyzes the reaction of butyryl-CoA and NAD (P) H ⁺ to buryraldehyde and NAD (P) ⁺ ; E ₆₀ , which catalyzes the reaction of acetoacetate and H ⁺ to acetone and CO ₂ ; E ₆₁ , which catalyzes the reaction of acetone and NAD (P) H ⁺ to propanol and NAD (P) ⁺ ; E ₆₂ , which catalyzes the reaction of acyl-CoA and carboxylic acid to acylate and carboxylic acid CoA; and E ₆₃ , which catalyzes the conversion of pyruvate and NADH to L-lactate and NAD ⁺ . The recombinant cell of claim 38, wherein: E ₁₉ and E _{20 are} a PEP-dependent phosphotransferase enzyme II (EC 2.7.1.69) system; E ₂₁ a PEP-dependent phosphotransferase system enzyme I (EC 2.7.3.9); E ₂₂ a phosphohistidine protein (HPr) -hexose phosphotransferase component of the PEP-dependent phosphotransferase system; E ₂₃ and E ₂₄ a formate C-acetyltransferase (EC 2.3.1.54); E ₂₅ a pyruvate: ferricytochrome b ₁ oxidoreductase (EC 1.2.2.2); E _{26 is} a methylmalonyl-CoA decarboxylase (EC 4.1.1.41); E ₂₇ an ATP / PP _i : acetate phosphotransferase (EC 2.7.2.1 or EC 2.7.2.1); E _{28 is} a propionate / acetate kinase (EC 2.7.2.15); E ₂₉ an acetyl-CoA: phosphate acetyltransferase (EC 2.3.1.8); E _{30 is} a D-lactate dehydrogenase (EC 1.1.1.28); E ₃₁ an acetaldehyde dehydrogenase (CoA-acetylating) (EC 1.2.1.10); E _{32 is} an NAD-dependent alcohol dehydrogenase (EC 1.1.1.1); E _{33 is} a glycerone phosphate phospholyase (EC 4.2.3.3); E ₃₄ , E ₃₅ , E ₃₆ , E ₃₇ , E ₃₈ , E ₃₉ and E ₄₀ formate dehydrogenases (EC 1.2.1.2); E _{41 is} a 2-methylcitrate synthase (EC 2.3.3.5); E ₄₂ a 2-methylcitrate dehydratase (EC 4.2.1.79 or EC 4.2.1.117); E _{43 is} a 2-methylaconitate isomerase; E _{44 is} a methyl isocitrate lyase (EC 4.1.3.30); E _{45 is} a propionate kinase (EC 2.7.2.15); E _{46 is} a phosphate propionyltransferase (EC 2.3.1.8); E ₄₇ a pyruvate decarboxylase (EC 4.1.1.1); E ₄₈ is a pyruvate: ferredoxin oxidoreductase (EC 1.2.7.1); E _{49 is} a phosphoketolase (EC 4.1.2.9); E _{50 is} a methylmalonyl-CoA-carboxytransferase (EC 2.1.3.1); E _{51 is} a methylmalonyl-CoA mutase (EC 5.4.99.2); E ₅₂ an acetolactate synthase (EC 2.2.1.6); E ₅₃ an acetolactate decarboxylase (EC 4.1.1.5); E ₅₄ a butanediol dehydrogenase (EC 1.1.1.4 or EC 1.1.1.76); E ₅₅ a thiolase (EC 2.3.1.9); E _{56 is} a 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.157, EC 1.1.1.35, EC 1.1.1.36 or EC 1.1.1.211); E ₅₇ a crotonase (EC 4.2.1.17); E _{58 is} a butanol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2); E ₅₉ a butyraldehyde dehydrogenase (EC 1.2.1.3, EC 1.2.1.4 or EC 1.2.1.5) E ₆₀ an acetoacetate decarboxylase (EC 4.1.1.4); E _{61 is} a propanol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2); E ₆₂ an acyl-CoA: CoA transferase (EC 2.8.3.-); and / or E _{63 is} an L-lactate dehydrogenase (EC 1.1.1.27). The recombinant cell of claim 40, wherein: E ₁₉ is encoded by a ptsG gene; E ₂₀ is encoded by a ptsI gene; E ₂₁ is encoded by a ptsH gene; E ₂₂ is encoded by a crr gene; E ₂₃ is encoded by a tdcE gene; E ₂₄ is encoded by a pflA or pflB gene; E ₂₅ is encoded by a poxB gene; E ₂₆ is encoded by a ygfG gene; E ₂₇ is encoded by a ackA gene; E ₂₈ is encoded by an ackA or tdcD gene; E ₂₉ is encoded by a pta gene; E ₃₀ is encoded by an ldhA gene; E is encoded by a gene adhE _31; E ₃₂ is encoded by an adhE gene; E ₃₃ is encoded by a mgsA gene; E ₃₄ is encoded by a fdnG gene; E is encoded by a gene fdnH _35; E ₃₆ is encoded by a fdnI gene; E ₃₇ is encoded by a fdhF gene; E ₃₈ is encoded by a fdoG gene; E ₃₉ is encoded by a fdoH gene; E is encoded by a gene fdoI _40; E ₄₁ is encoded by a prpC gene; E ₄₂ is encoded by a prpD or acnD gene; E ₄₃ is encoded by a prpF gene; E ₄₄ is encoded by a prpB gene; E ₄₅ encoded by a tdcD gene E ₄₆ is encoded by a pta gene; E ₄₇ is encoded by a pdc gene; E ₄₈ is encoded by a porA, porB, porC or porD gene; E ₄₉ is encoded by an xpk1 or xpk2 gene, E ₅₀ is encoded by a methylmalonyl-CoA carboxytransferase gene; E ₅₁ is encoded by a sbm or a mcmA and a mcmB gene; E ₅₂ is encoded by a gene as il, ilvB, ilvM, ilvN, ilvG, ilvI or ilvH gene; E ₅₃ is encoded by a alsD gene; E ₅₄ is encoded by a butB gene; E ₅₅ is encoded by a thl, thlA, thlB or phaA gene; E ₅₆ is encoded by a phaB gene; E ₅₇ is encoded by a crt gene; E ₅₈ is encoded by an adhE gene; E ₅₉ is encoded by an adhE, bdhA or bdhB gene; E ₆₀ is encoded by an adc gene; E ₆₁ is encoded by an adh gene; E ₆₂ is encoded by a ctfA and a ctfB or an atoA and an atoD gene; and / or E ₆₃ is encoded by an ldhL gene. The recombinant cell according to at least one of claims 13 to 15 or 33 or 34, wherein the cell has a reduced activity compared to its wild type of at least one of the enzymes E19, E20, E21, E22, E23, E24, E25, E26, E27, E28 , E29, E30, E31, E32, E33, E45, E46, E47, E48, E49, E50, E55, E56, E57, E58, E59, E60, E61 and E63. The recombinant cell of claim 42, wherein the enzymes E19, E20, E21, E22, E23, E24, E25, E27, E28, E29, E30, E31, E32, E33, E45, E46, E47, E48, E49, E50, E55, E56, E57, E58, E59, E60, E61 and E63 ptsG, ptsI, ptsH, crr, tdcE, pflA, pflB, poxB, ack, pta, tdcD, ldhA, adhE, mgsA, ygfG, pdc, porA, porB , porC, porD, xpk1, xpk2, th1, thlA, thlB, phaA, phaB, crt, bdhA, bdhB, adc, adh and ldhL. A process for producing a genetically engineered cell according to any one of claims 1 to 42, comprising increasing compared to the wild-type activity of at least one of the enzymes in the cell mentioned in claims 9 to 34. The method of claim 43, further comprising reducing the activity as compared to the wild type of at least one of the enzymes in the cell as claimed in claims 33 to 40. A process for producing at least one C ₄ -Körpers and / or at least one C ₄ -body on this sequence produced compound, said method comprising incubating a cell according to any one of claims 1 to 42 with a nutrient medium containing sucrose, under conditions that the Production of at least one C ₄ body of sucrose and CO ₂ allow comprises. The method of claim 45, wherein the medium further contains carbon dioxide. The method of claim 45 or 46, further comprising isolating the at least one C ₄ body and / or the at least one subsequent compound prepared from this C ₄ body from the nutrient medium. The method according to at least one of claims 45 to 47, wherein the at least one C ₄ body is selected from the group consisting of succinate, Malst, fumarate, oxalacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4- Bionell, hydroxysuccinate, epoxy-γ-butyrolactone, butenoic acid, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, butene, n-butene, cis-2 Butene, trans-2-butene, isobutene, butadiene, 1,2-butadiene, 1,3-butadiene, 3-hydroxybutyrolactone, 4-hydroxybutyrolactone, 1-butanol, 2-butanol, tert-butanol, isobutanol, 2-hydroxybutyric acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid. The method of any one of claims 45 to 48, wherein the C ₄ bodies are selected from the group consisting of: Malst, oxaloacetate, and the recurrent compounds prepared via these C ₄ bodies are selected from the group consisting of by enzymatic or chemical synthesis Follow-up compounds prepared via Malst or oxaloacetate. The method of claim 49, wherein the secondary compounds produced by enzymatic synthesis via malic or oxaloacetate are selected from the group consisting of: aspartate, asparagine, threonine, tetrahydrofuran, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1 , 4-butanediol, 2,3-butanediol, 3- and 4-hydroxybutyrolactone, 1-, 2- and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid, 2- and 3-hydroxyisobutyric acid, methionine and lysine , Use of a cell according to one of claims 1 to 42 for the microbiological production of at least one C ₄ body and / or at least one sucrose and carbon dioxide secondary compound produced via this C ₄ body.