WO2011154503A1 - Préparation micro biologique de corps en c4 à partir de saccharose et de dioxyde de carbone - Google Patents

Préparation micro biologique de corps en c4 à partir de saccharose et de dioxyde de carbone Download PDF

Info

Publication number
WO2011154503A1
WO2011154503A1 PCT/EP2011/059619 EP2011059619W WO2011154503A1 WO 2011154503 A1 WO2011154503 A1 WO 2011154503A1 EP 2011059619 W EP2011059619 W EP 2011059619W WO 2011154503 A1 WO2011154503 A1 WO 2011154503A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
encoded
catalyzes
enzyme
coa
Prior art date
Application number
PCT/EP2011/059619
Other languages
German (de)
English (en)
Inventor
Steffen Schaffer
Nicolas Rudinger
Liv Reinecke
Original Assignee
Evonik Degussa Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evonik Degussa Gmbh filed Critical Evonik Degussa Gmbh
Publication of WO2011154503A1 publication Critical patent/WO2011154503A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01038—Malate dehydrogenase (oxaloacetate-decarboxylating) (1.1.1.38)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/0104—Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) (1.1.1.40)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01083—D-Malate dehydrogenase (decarboxylating) (1.1.1.83)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/05—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with a quinone or similar compound as acceptor (1.2.5)
    • C12Y102/05001—Pyruvate dehydrogenase (quinone) (1.2.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01054—Formate C-acetyltransferase (2.3.1.54), i.e. pyruvate formate-lyase or PFL
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01004—Fructokinase (2.7.1.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01069—Protein-Npi-phosphohistidine-sugar phosphotransferase (2.7.1.69), i.e. sucrose phosphotransferase system II
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/03—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C12Y207/03009—Phosphoenolpyruvate-protein phosphotransferase (2.7.3.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01031—Phosphoenolpyruvate carboxylase (4.1.1.31)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01032—Phosphoenolpyruvate carboxykinase (GTP) (4.1.1.32)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01038—Phosphoenolpyruvate carboxykinase (diphosphate) (4.1.1.38)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01049—Phosphoenolpyruvate carboxykinase (ATP) (4.1.1.49)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
    • C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
    • C12Y604/01001—Pyruvate carboxylase (6.4.1.1)

Definitions

  • the present invention relates to a cell which has been genetically engineered with respect to its wild type such that it produces more C 4 bodies and / or over these C 4 bodies compared to their wild type of sucrose and carbon dioxide as the carbon source
  • the invention further relates to a process for the production of such a genetically modified cell and to a process for the production of 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 -body such as succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-Bionell, hydroxysuccinate, epoxy-v-butyrolactone, butenoic acid, butyrate, butanediol, 1, 2 Butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol,
  • C 4 bodies Feed industry, agriculture and the pharmaceutical industry of importance.
  • 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 raw materials, it is desirable alternatives to the existing petrochemical processes for the production of C 4 bodies
  • the currently pursued approaches are based mainly on microorganisms that produce C 4 bodies in fermentative processes.
  • glucose is used as carbon source.
  • the necessary glucose is recovered from starch, which z. From corn,
  • the present invention is therefore based on the object to overcome the disadvantages resulting from the prior art.
  • the invention has the object to replace glucose as a raw material by a cheaper and available in sufficient quantities alternative.
  • sucrose is used as a raw material for the production of C 4 bodies.
  • sucrose is mostly made from sugar cane (about% of annual production) and to a lesser extent from sugar beet (about% of 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.
  • carbon dioxide CO 2
  • carbon dioxide is a by-product of a number of chemical processes and is therefore comparatively inexpensive to obtain.
  • carbon dioxide is a by-product of a number of chemical processes and is therefore comparatively inexpensive to obtain.
  • Carbon dioxide reduce the pollution of the atmosphere with greenhouse gases.
  • Anaerobiospirillum succiniciproducens or Actinobacillus succinogenes which are able to convert sucrose into C 4 bodies, are not suitable for biotechnological production because the yields are low and these organisms require complex nutrient media which, due to the associated costs and effort, render the process uneconomic ,
  • Sucrose and C0 2 as carbon sources can produce large quantities of C 4 bodies.
  • the inventors of the present invention have surprisingly found that the yield of C 4 bodies is fixed by genetic manipulation of suitable cells, in particular an enhancement of carboxylation reactions, the carbon dioxide and formation lead from C 4 -Körpern, a suppression of metabolic pathways that cause the carbon flux of sucrose, to fermentation products that are not C 4 -body, and increasing uptake of sucrose and the sucrose metabolism can be greatly increased.
  • This approach allows i) organisms that are usually unable
  • 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.
  • C 4 body refers to a chemical
  • Compound containing four carbon atoms 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, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, epoxy-v-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-butad
  • the C 4 body is selected from the group consisting of: malate 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 and 2- and 3-hydroxyisobutyric acid, methionine and lysine as well as malate, oxaloacetate and secondary compounds produced by chemical means compounds.
  • succinate aspartate
  • asparagine threonine
  • tetrahydrofuran Butyrate
  • butanediol 1, 2-butan
  • carboxylic acids include both the base form and the acid form, as well as mixtures of these forms.
  • succinate, malate, oxaloacetate, fumarate, aspartate, butyrate, 2-, 3- or 4-hydroxybutyric acid and 2- or 3-hydroxyisobutyric acid also each include succinic, malic, oxalacetic, fumaric, aspartic, butyric, 2-, 3- or 4-hydroxybutyrate and 2- or 3-hydroxyisobutyrate.
  • 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 or that this compound (s) can not be produced in a detectable amount and only after the genetic modification verifiable amounts of these compounds are formed.Furthermore, this expression detects that the corresponding cell in a fixed period, for example within 2, 4 , 8, 12, 24 or 48 hours, at least the 2-, 5-, 10-, 100- or 1000-fold amount of C 4 bodies or one or more desired C 4 bodies forms like the wild type.
  • a "wild-type” cell is referred to herein as a cell whose genome is in a state as naturally evolved. 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.
  • 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 which can be found at http://www.dsmz.de/microorganisms/bacteria_catalogue.php are listed.
  • Yeasts which are suitable according to the invention belong to the genera listed under http://www.dsmz.de/microorganisms/yeast_catalogue.php.
  • Fungi suitable according to the invention are those listed at http://www.dsmz.de/microorganisms/fungus_catalogue.php.
  • Preferred cells according to the invention are those of the genera Aspergillus,
  • Fibrobacter succinogenes Ruminococcus flavefaciens, A. aerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Saccharomyces cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, Corynebacterium glutamicum,
  • Paracoccus versutus Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Pichia pastoris, Thermoanaerobacter kivui, Acetobacterium woodii, Acetoanaerobium notera, Clostridium aceticum, acetobutylicum Butyribacterium methylotrophicum, Clostridium saccharoperbutylacetonicum Clostridium, Clostridium beijerinckii, butyricum Clostridium, Moor Ella thermoacetica, Eubacterium limosum, Peptostreptococcus productus, Clostridium ljungdahlii , Clostridium carboxidivorans, Clostridium scatalogenes, Rhodospirillum rubrum, Burkholderia thailandensis and Pseudomonas putida are particularly preferred.
  • carbon dioxide and “C0 2 " as used herein refer to both the gas C0 2 , the carbonic acid (H 2 C0 3 ) which is in equilibrium with dissolved carbon dioxide in aqueous solution, and the two carbonic acid deprotonation products , Bicarbonate (HC0 3 " ) and carbonate (C0 3 2" ) and their salts.
  • the C 4 bodies are selected from the group consisting of:
  • the C 4 bodies are selected from the group consisting of: malate, oxaloacetate and derived compounds produced by enzymatic or chemical synthesis via malate or oxalacetate.
  • the secondary compounds produced by enzymatic synthesis via malate 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 C 4 bodies are C 4 carboxylic acids, preferably C 4 dicarboxylic acids, more preferably succinate.
  • the C 4 bodies are hydroxycarboxylic acids, more preferably 2-, 3- and / or 4-hydroxybutyric acid and 2- and / or 3-hydroxyisobutyric acid.
  • the C 4 bodies are preferably malate and / or oxaloacetate.
  • 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, Clostridium
  • saccharoperbutylacetonicum Clostridium beijerinckii, Rhodospirillum rubrum, Burkholderia thailandensis or Pseudomonas putida cell.
  • the recombinant cell of the present invention may be genetically engineered to accommodate more sucrose compared to its wild-type.
  • Another possibility, which may alternatively or additionally be used, is a genetic modification of the cell which allows the recombinant cell to fix more carbon dioxide compared to its wild-type.
  • 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.
  • At least one refers to amounts of> 1, ie 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.
  • the recombinant cell of the invention comprises an enzyme E-i, which catalyzes the transport of sucrose into the cell and the conversion to sucrose-6-phosphate.
  • 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.
  • enhanced activity of an enzyme or “decreased activity of an enzyme” as used herein preferably refer to increased / decreased intracellular or membrane-bound activity.
  • increased 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.
  • 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.
  • heterologous expression is achieved by integration of the gene or alleles into the chromosome of the cell or an extrachromosomally replicating vector.
  • Protein separations between wild type and genetically engineered cell can be determined.
  • a common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001)).
  • 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
  • DNA-binding proteins can be measured by DNA band shift assays (also referred to as gel retardation) (Wilson et al., (2001) Journal of Bacteriology, 183: 2151-2155).
  • DNA band shift assays also referred to as gel retardation
  • 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 (1973) Journal of Bacteriology 1 15 (3): 816-823). Unless specific methods for determining the activity of a specific 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 are preferably carried out by means of the methods described in Hermann et al. (Electophoresis, 22: 1712-23 (2001)), Lohaus et al.
  • Nucleotide substitution results in genetically engineered cells.
  • Particularly preferred mutants of enzymes are, in particular, also those enzymes which no longer diminish or at least in comparison with the wild-type enzyme
  • 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.
  • expression cassettes act, which are installed upstream of the structural gene.
  • Inducible promoters also make it possible to increase expression at any time.
  • 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, a further
  • Vectors can, for. B. the brochures of the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL be removed. Further preferred plasmids and vectors can be found in: Glover, 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.
  • 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-l ( Sonnen et al., Gene 107: 69- 74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
  • plasmid vectors such as those based on pCG4 (US 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66: 1 19-124 (1990)) or pAG1 (US 5,158,891) can be used in the same way be used. Also suitable are those plasmid vectors with the aid of which one can apply the method of gene amplification by integration into the chromosome, as described for example by Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994)) for duplication or amplification of the homodB operon.
  • the complete gene is cloned into a plasmid vector which can be replicated in a host (typically Escherichia coli) but not in Corynebacterium glutamicum.
  • vectors include 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 (shrink et al, Journal of. Bacteriology 173: 4510-4516)) or pBGS8 (Spratt et al., Gene 41: 337-342 (1986)).
  • 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, the
  • Transcription for example, by the manipulation of the promoter region or enhancement of repression (genetically or chemically) or the reduction of the mRNA half-life, be inhibited or reduced.
  • 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 that it is intended for Secondary structure formation occurs at the mRNA level, which inhibit or reduce translation.
  • the enzyme activity can be reduced, for example, by adding inhibitors, by introducing directional or non-directional mutations.
  • 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 of at least 1, 000 and most preferably of at least 10,000 increased activity of the respective enzyme E x
  • the cell according to the invention comprises "an activity of an enzyme E x increased compared to its wild type, in particular also a cell whose Wild type has no or at least no detectable activity of this enzyme E x and only after increasing the enzyme activity, for example by overexpression, a
  • detectable activity of this enzyme E x shows.
  • 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.
  • the genetically modified cell is genetically modified in such a way that it is metabolized in a defined time interval, preferably within 2 hours, more preferably within a defined time interval 8 hours n and most preferably within 24 hours, at least 2 times, more preferably at least 10 times, more preferably at least 100 times, even more preferably at least I.OOO times, and most preferably at least 10,000 times more C 4 body forms than the wild type of the 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, for example, a phosphoenolpyruvate (PEP) -dependent
  • PPS Phosphotransferase system
  • the enzyme E-1 can be coded, for example, by the scrA gene.
  • the coding nucleotide sequence and the 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, USA), the protein database UniProt (cooperation of the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SI B) and the Protein Information Resource (PI R)) or the nucleotide sequence database of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany and Cambridge, In a particular case, egg is the scrA gene (SEQ ID NO: 41) derived from E.
  • the enzyme Ei is preferably encoded by genes derived from the Group of those are selected which encode gene products whose amino acid sequence over a range of at least 100, preferably at least 200, in particular minde 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.
  • the enzyme E-1 is encoded by genes which are selected from the group of those encoding gene products in 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 "PTS-II BC-sucr" (TIGR01996 or PssmI D 131051) with an E value (English “e-value”) less than 1 x 10 "5 detected (English” domain hit ”) , In addition to the enzyme egg, which facilitates the transport of sucrose into the cell and the
  • the recombinant cell may further have an increased compared to their wild type activity of at least one enzyme E 2 , E 3 and E 4 or a combination of these enzymes.
  • E 2 can be an enzyme that the
  • E 3 may be an enzyme that catalyzes the conversion of D-fructose to D-fructose-6-phosphate
  • E 4 may be a channel that allows diffusion-dependent sucrose transport into the cell.
  • 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.
  • the activity of the enzyme Ei and at least one of the enzymes E 2 , E 3 and E 4 is increased compared to the wild type.
  • the activity of the enzymes compared to the wild-type is i) Ei, 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) Ei, E 3 and E 4 increased.
  • the recombinant cell according to the invention has an increased activity of the enzymes Ei, E 2 , E 3 and E 4 in comparison with the wild type.
  • 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 is a sucrose porin (TCDB Classification 1 .B.3.1 .2).
  • 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 (£ coli ⁇ S ⁇ K. SEQ ID NO: 46) encodes and e 4 is of the ScrY gene (£ coli ScrY. 44: SEQ ID NO) coded.
  • Protein sequences as well as other genes for the enzymes E 2 to E 4 can also be taken from the KEGG, NCBI, UniProt or EMBL database.
  • E 2 , E 3 and / or E 4 are genes derived from E. coli or the proteins encoded thereby.
  • 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.
  • 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 contained in the relevant amino acid sequence
  • 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.
  • 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
  • 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.
  • 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
  • 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 Ei, E 2 , E 3 and / or E 4 .
  • the channel E 5 may be, for example, a sucrose permease.
  • E 5 is encoded by the cscB gene (for example, E. coli cscB: SEQ ID NO: 45).
  • 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.
  • E 6 which catalyzes the conversion of sucrose to D-glucose and D-fructose
  • E 3 which catalyzes the conversion of D-fructose to D-fructose-6-phosphate.
  • 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).
  • 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 (for example, E. coli scrK: SEQ ID NO: 43) or cscK (for example, E.
  • coli cscK SEQ ID NO: 46
  • 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 of £. coli-derived genes and the proteins encoded thereby.
  • 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 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: 62.
  • 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 1 10319) with an E value (English “e-value”) is smaller than 1 x 10 "5 found (English” domain hit ").
  • 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.
  • 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) with an E value (English “e-value") smaller 1 x 10 "5 is found (English” domain hit ").
  • 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, for example, from the
  • Enzymes E 7 , E 8 and E 9 and includes, for example, a sucrose-specific ABC transporter (TCDB classification 3.A.1 .1.-).
  • 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
  • E 9 by a susX gene
  • 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 catalyzes the
  • sucrose-6-phosphate 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 conversion of D-fructose to - D-fructose 6-phosphate and the enzyme E 6 the conversion of sucrose to D-glucose and D-fructose.
  • 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).
  • 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
  • cscK for example, E. coli cscK: SEQ ID NO: 46
  • E 2 , E 3 , E 7 , E 8 , E 9 and E 10 can also be found in the KEGG, NCBI, UniProt or EMBL database.
  • 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.
  • 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.
  • 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 been searched for conserved in the relevant amino acid sequence
  • Protein domains in the NCBI CDD (Version 2.20) by means of RPS-BLAST the presence of the conserved domain "LplB” (COG4209 or PssmID 33938) with an E-value (English “e-value") smaller 1 x 10 "5 is found (English "Domain hit”).
  • 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.
  • 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%, especially preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 67.
  • 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) with an E value (English “e-value”) is less than 1 x 10 "5 is found (English" domain hit ").
  • embodiments in a recombinant cell according to the invention increase the activity of the following enzymes over the wild-type:
  • the activity is at least one, preferably
  • the recombinant cell is genetically engineered to have an increased activity of at least one enzyme that catalyzes the fixation of C0 2 to a C 3 body compared to its wild-type.
  • This heightened 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 C0 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 C0 2 to oxaloacetate, ADP and phosphate;
  • E 15 , E 16 , E 17 and E 18 which catalyze the reaction of pyruvate NAD (P) H + and C0 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 .
  • any combination of increased activities of the enzymes E 12 -E 18 and the increased activity of all enzymes E 12 -E 18 are also encompassed by the present invention.
  • E 13 is a phosphoenolpyruvate carboxylase (phosphate: oxaloacetate carboxylase) (EC 4.1 .1 .31);
  • E 14 is a phosphoenolpyruvate carboxykinase (ATP / GTP / PPi: 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);
  • 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 comprising cgl516, aarl62Cppyrl, pca, cgl0689, 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.
  • a gene selected from the group comprising cgl516, aarl62Cppyrl, pca, cgl0689, pc, pcx, pyc-1, pyc-2, accC-2, pycA, pycA2, pyc, pycB, pyc
  • Pyruvate carboxylases preferred according to the invention are also described in particular in US Pat. Nos. 6,455,284, 6,171,833, 6,884,606, 6,403,351, 6,852,516 and 6,861,246.
  • a pyruvate carboxylase pyc which is particularly preferred in this context is that mutant which is known in the novel methodology employing Corynebacterium glutamicum genome
  • pyc is derived
  • 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.
  • 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, pckI, pck2 and pck, the pckA gene being particularly preferred.
  • pckA is from E. coli (SEQ ID NO: 2).
  • Phosphoenolpyruvate carboxylases preferred according to the invention are also described in particular in US Pat. Nos. 4,757,009, 4,980,285, 5,573,945, 6,872,553 and 6,599,732.
  • 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, mael, mae2, sfcA, sfcA1, maeA, maeB, maeB1, maeB2 , tme, yqkJ, ywkA, yqkJ, malS, ytsJ, mleA, mleS, mez, sce59.10c, 2sc7gll.23, malSI, malS2, dme, maeBl, maeB2, mdh, mdh1, mdh2, dmel cgi 0120, dmel cgi 0120 , dme1 -cg5889, fl9kl6.27, f6f22.7, t22p22.60, fl8al7.1, mod1, tme, mao, cgl300
  • E. coli maeA SEQ ID NO: 3
  • E 16 mmel for example Chlamydomonas reinhardti mmel: SEQ ID NO: 55
  • E 17 maeB for example E. coli maeB : SEQ ID NO: 4
  • mouse and mouse 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.
  • dmlA is derived from £. coli (SEQ ID NO: 57).
  • the aforementioned enzymes may be individually, all or in any combination increased in their activity.
  • 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.
  • Protein sequence and other genes for the enzymes E 12 -E 18 can also be found in the KEGG, the NCBI, the UniProt or EMBL database.
  • 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.
  • 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") smaller than 1 x 10 "5 is found (English” domain hit ”) ,
  • 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.
  • 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) with an E value (English “e-value") less than 1 x 10 "5 is found (English” domain hit ").
  • 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.
  • 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) with an E value (English “e-value") smaller 1 x 10 "5 is found (English” domain hit ").
  • 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.
  • 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 x 10 "5 is found (English” domain hit”).
  • 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.
  • 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") smaller 1 x 10 "5 is found (English” domain hit ").
  • 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 range of at least 100, preferably at least 200, in particular at least 300 amino acids have a sequence identity of at least 60%, preferably of at least 80%, more preferably of at least 95%, most preferably of at least 99%, in particular of 100% to SEQ ID NO: 54.
  • 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" (cd0531 1) with an E value (English “e-value”) smaller 1 x 10 "5 is found (English” domain hit ").
  • 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.
  • 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 "lso_dh super-family" (c00445 or PssmID 174206) with an E-value (English “e-value") smaller 1 x 10 "5 is found (English” domain hit ").
  • 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 d, 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.
  • the activity is at least one of the following
  • Ei9, E 2 o, E21 and E22 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 B-phosphate;
  • E 2 3 and E 2 4 which catalyze the reaction of pyruvate and CoA to formate and acetyl-CoA;
  • E 2 which catalyzes the reaction of pyruvate, H 2 0 and ferricytochrome bi to acetate, C0 2 and ferrocytochrome bi;
  • E 2 6 which catalyzes the reaction of S-methylmalonyl-CoA to propanoyl-CoA and C0 2 ;
  • E 2 7 and E 2 8 which catalyze the reaction of acetyl phosphate and ADP / P, to acetate and ATP / PP;
  • E 42 which catalyzes the conversion of 2-methyl citrate 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;
  • E 48 which catalyzes the reaction of pyruvate and CoA 2 oxidized ferredoxins to acetyl CoA, C0 2 , and 2 reduced ferredoxins and 2 H + ;
  • E51 which catalyzes the reaction of (S) -methylmalonyl-CoA to succinyl-CoA
  • E 52 which catalyzes the conversion of 2 pyruvate to 2-acetolactate and C0 2 ;
  • E 6 o which catalyzes the reaction of acetoacetate and H + to acetone and C0 2
  • E 6 i which catalyzes the reaction of acetone and NAD (P) H + to propanol and NAD (P) + ;
  • E 6 which catalyzes the conversion of pyruvate and NADH to L-lactate and NAD + .
  • also several, ie at least 2, or all of the aforementioned enzymes may be reduced in their activity.
  • E 19 and E 2 o a PEP-dependent phosphotransferase system enzyme II (EC 2.7.1 .69);
  • E 2 i is a PEP-dependent phosphotransferase system Enzyme I (EC 2.7.3.9);
  • E 2 2 is a phosphohistidine protein (HPr) -hexose phosphotransferase component of the
  • E 2 3 and E 2 4 a formate C-acetyltransferase (EC 2.3.1.54);
  • E 26 is a methylmalonyl-CoA decarboxylase (EC 4.1 .1 .41);
  • E 27 an ATP / PPi: 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 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 .1 17);
  • 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 a phosphate-propionyltransferase (EC 2.3.1 .8)
  • E 47 is 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 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 .1.21 1);
  • 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 6 i is a propanol dehydrogenase (EC 1 .1 .1.1 or EC 1 .1 .1.2);
  • E 6 2 an acyl-CoA: CoA transferase (EC 2.8.3.-); and or
  • E 6 3 an L-lactate dehydrogenase (EC 1.1 .1 .27).
  • 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.
  • the activity of at least one of the enzymes E19-E22 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 2 6 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, where the ygfG Gene is particularly preferred.
  • ygfG is from £. coli.
  • 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 2 o is encoded by a ptsl gene
  • E21 is encoded by a ptsH gene
  • E22 is encoded by a crr gene
  • E 2 3 is encoded by a tdcE gene
  • E 24 is encoded by a pflA or pflB gene
  • E 2 5 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 2 9 is encoded by a pta gene
  • E 30 is encoded by an IdhA 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 fdnl 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 fdol 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 xpkl 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, ilvB, ilvM, ilvN, ilvG, ilvl or ilvH gene;
  • E 53 is encoded by a alsD gene
  • E 54 is encoded by a butBGen
  • E 55 is encoded by a thI, thIA, thIB 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 6 o is encoded by an ade gene;
  • E 6 i is encoded by an adh gene
  • E 6 2 is encoded by a ctfA and a ctfB or an atoA and an atoD gene and / or E 6 3 is encoded by an IdhL gene.
  • Protein sequence and other genes for the enzymes E 19 -E 6 3 can be found, inter alia, the KEGG, the NCBI, the UniProt or EMBL database.
  • the recombinant cell which, starting from sucrose and C0 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 2 6, E 2 7, E 29 , E 30 , E 31 , E 32 and E 46 on.
  • the enzymes E 19 , E 26 , E 27 , E 29 / E 46 , E 30 and E 31 / E 32 are IdhA, adhE, ack, pta, ygfG and ptsG.
  • the cell according to the invention can also have a reduced activity of all the 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 IdhA, adhE, ack, pta, ygfG and ptsG.
  • the recombinant cell of sucrose and C0 2 can produce 4 -body than wild type as carbon sources more C, increased compared with its wild type activity of at least one of the enzymes EE 18 and, optionally, a reduced activity of at least one of the enzymes E 19 -E 63 .
  • Certain embodiments of the invention relate to cells in which, compared to the wild type, the activity:
  • 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 or pyc, ppc, pckA, maeA, mm, maeB and dmlA and the enzymes E 19 , E 20 , E 2 i, E 22 , E 23 , E 24 , E 25 , E 27 , E 28 , E 29 , E 30 , E 3 i, E 32 , E 33 , E 45 , E 4 6, E 47, E 4 8, E 49, E 5 O, E 55 , E 5 6, E 57, E 5 8, E 59, EQO, E ⁇ i and ⁇ 3 ptsG, ptsl, ptsH, crr, tdcE, pflA, pflB, poxB, ack, pta,
  • the invention relates to a method for producing a genetically modified cell according to the invention.
  • the increase of the activity of at least one of the above-described enzymes EE 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.
  • the invention relates to a process for producing at least one C 4 body and / or at least one prepared over this C 4 body
  • Sequential compound the method comprising incubating a cell according to the invention with a nutrient medium containing sucrose under conditions which include the production of at least one C 4 body and / or at least one sucrose and C0 2 secondary compound produced via this C 4 body allow, includes.
  • 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.
  • 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.
  • the C 4 bodies are selected from the following group: succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, Epoxy-v-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
  • the C 4 bodies are selected from the group consisting of: malate, oxaloacetate and the derivatives produced via these C 4 bodies are selected from the group consisting of secondary compounds prepared by enzymatic or chemical synthesis via malate or oxalacetate.
  • the secondary compounds produced by enzymatic synthesis via malate or oxalacetate can be selected, for example, from the group consisting of: succinate, aspartate, asparagine, threonine,
  • the genetically modified cells of the invention can be used continuously or discontinuously in the batch process (batch culturing) or in the fed-batch process
  • sucrose and / or carbon dioxide serve as carbon sources in the nutrient media used.
  • 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.
  • the nitrogen sources can be used singly or as a mixture.
  • a phosphorus source can phosphoric acid, potassium dihydrogen phosphate or
  • Dipotassium hydrogen phosphate or the corresponding sodium-containing salts are used.
  • 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.
  • the medium may contain other substances, e.g. Amino acids and / or vitamins are added.
  • suitable precursors can be added to the culture medium.
  • the stated feedstocks can be used for culture in the form of a unique approach
  • Phosphoric acid or sulfuric acid used in a suitable manner.
  • Foaming can anti-foaming agents such.
  • B. fatty acid polyglycol esters are used.
  • 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, it being further preferred in this context to carry out the production of the C 4 body by fermentation continuously, so that the entire process from the production of the C 4 body to its purification from the fermentation broth can be carried out continuously.
  • 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, it being particularly preferred that at least some of the microorganisms pass through first
  • 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.
  • a 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.
  • Separation stages can operate on 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.
  • 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.
  • ammonium compounds or amines may be added to the fermentation solution to form an ammonium salt of C 4 carboxylic acid.
  • Ammonium salt can then be separated from the fermentation solution by adding an organic extractant and then heating the mixture thus obtained, 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
  • C 4 -carboxylic acids prepared by the process according to the invention can still be neutralized before, during or after the purification, it being possible to use bases such as calcium hydroxide or sodium hydroxide for this purpose.
  • glutamicum SEQ ID NO: 5
  • these genes are made from chromosomal DNA of £. coli MG1655 or C. glutamicum ATCC 13032 were amplified by PCR and introduced simultaneously via the oligonucleotides used an interface upstream of the respective ribosome binding site and an interface downstream of the stop codon.
  • Chromosomal DNA of E. coli MG 1655 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 MG 1655 or C. glutamicum ATCC 13032 as template.
  • E. coli pck pck-fw: 5'-ATA GGA TCC TTA CTA TTC AGG CAA TAC ATA TTG GCT AAG
  • 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.
  • PCR products are digested with Sac ⁇ and Sbfi ⁇ ppc, maeA, maeB and pyc) and / or bamHI and Sbfi ⁇ pck) according to the recommendations of the manufacturer of the Restriktiosendonukleasen (New England Biolabs, bath Schwalbach) and in those with Sac ⁇ and Sbfi ⁇ ppc, maeA, maeB and pyc) or ßamHI and Sbfi ⁇ pck) cut vector pEC-XC99E (SEQ ID NO: 16) ligated.
  • pEC-XC99E is a coli coli C. g / yfam / ci / m shuttle vector present in both organisms
  • Chloramphenicol resistance and a ColE1 origin of replication (high
  • the correct insertion of the ppc, pck, maeA, maeB or pyc fragments is checked by a restriction with Sac ⁇ and Sbfi ⁇ ppc, maeA, maeB and pyc) or ßamHI and Sbfi ⁇ pck).
  • the authenticity of the inserted fragments is checked by DNA sequencing.
  • the completed £ co // 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).
  • SEQ ID NO: 17 The completed £ co // 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).
  • Coli cscA-cscKB csc-fw: 5'-ATA CAT ATG TTA TTA ACC CAG TAG CCA GAG TGC TCC AT GT-
  • Coli coli scrK-scrYAB scr-fw 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA T
  • PCR products are labeled with ⁇ / col and Not ⁇ (scrK-scrYAB) or Nde ⁇ and Xho ⁇ (cscA-cscKB) according to the
  • pCOLADuet-1 (SEQ ID NO : 28; Merck Biosciences; Nottigham, UK).
  • pCOLADuet-1 is a £. co // vector of low copy number (20-40 copies per cell) that mediates kanamycin resistance and carries a ColA origin of replication.
  • the chromatographic quantification of sucrose is carried out in the following way: The quantification of sucrose is carried out by means of high-performance liquid chromatography carried out.
  • the quantification of sucrose is carried out by means of high-performance liquid chromatography carried out.
  • For the separation of sugars is an anion exchange column with
  • 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 ⁇ _, 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, Böblingen). The
  • Reference substance (Sigma-Aldrich, Steinheim) is measured 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.
  • Ion exclusion column Aminex® HPX 87H with the dimensions 300 mm x 7.8 mm (Bio Rad Laboratories, Kunststoff) as a stationary phase use. As the mobile phase becomes 10 mM
  • Sulfuric acid used.
  • the column temperature is 40 ° C, the flow rate 0.6 ml / min.
  • the sample is acidified with 0.5 M sulfuric acid to a pH of 4 to 5 and injected into the column with a volume of 20 ⁇ .
  • 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.
  • lactate, acetate, succinate and formate are 0.8 g / L
  • ethanol can be determined up to a concentration of 1 g / L.
  • concentration of 1 g / L In the range of 0.8 g / L to 20 g / L there is a linear dependence of the peak areas of the
  • 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 xg 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 10% glycerol solution and stored in aliquots of 50 L at -80 ° C until electroporation.
  • Coli coli 1655 (pCOLADuet-1, pEC-XC99E)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E-ppc)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E-pck)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E-maeA)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E-maeB)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E-pyc)
  • Coli coli 1655 (pCOLA-scr, pEC-XC99E)
  • Coli coli 1655 (pCOLA-scr, pEC-XC99E-ppc)
  • Coli coli 1655 (pCOLA-scr, pEC-XC99E-maeA)
  • Coli coli 1655 (pCOLA-scr, pEC-XC99E-maeB)
  • E. coli MG1655 (pCOLA-scr, pEC-XC99E-pyc)
  • strains are then used to analyze their ability to produce succinate under anaerobic conditions.
  • the procedure is as follows: The strains are subjected to a multi-stage 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), 0.1mM calcium chloride dihydrate (Sigma-Aldrich, Steinheim), 1mM
  • modified M9 medium with 0.2 mg / L biotin and 0.5 mM IPTG in a 1000 ml Erlenmeyer flask with chicane inocultivated with the preculture so that an optical density (600 nm) of 0.2 is achieved.
  • 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
  • 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 gas-tight with a cap with a stopper closed, 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.
  • samples of 1 ml_ sterile are withdrawn through the stopper with a cannula 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 was the production of succinate by E. co // 'strains with deletions in the genes ack-pta, adhE, IDHA and ygfG, in which the gene is replaced by the ptsG SCRk-scrYAB locus from the plasmid pUR400 and which the Gene pyc, coding for a
  • a pound. co // strain with deletions in the genes ack-pta (SEQ ID NO: 31), adhE (SEQ ID NO: 32), IdhA (SEQ ID NO: 33) and ygfG (SEQ ID NO: 34) and an exchange of the pisG gene (SEQ ID NO: 35) is constructed by the scrK scrYAB locus.
  • the E. coli strain MG1655 ack-pta adhE IdhAggfG ptsG :: scrK-scrYAB was generated by methods known to the person skilled in the art (eg see Datsenko KA, Wanner BL Proc Natl Acad Sei USA 2000. 97 ( 12): 6640-5)..
  • SEQ ID NO: 36 (ack-pta), SEQ ID NO: 37 (adhE), SEQ ID NO: 38 (IdhA), SEQ ID NO: 39 (ygfG ) and SEQ ID NO: 40 ⁇ ptsGwscrK-scrYAB).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne une cellule qui a été génétiquement modifiée par rapport à son type sauvage de sorte qu'elle permet une production en corps en C4 et/ou en composés successeurs obtenus à partir de ces corps en C4, à partir de saccharose et de dioxyde de carbone en tant que source de carbone, qui est accrue vis-à-vis de son type sauvage. L'invention a également pour objet un procédé pour produire une telle cellule génétiquement modifiée et un procédé pour préparer des corps en C4 et/ou des composés successeurs obtenus à partir de ces corps en C4, au moyen de ces cellules. L'invention concerne finalement l'utilisation des cellules pour préparer des corps en C4 et/ou des composés successeurs obtenus à partir des ces corps en C4, à partir de saccharose et de dioxyde de carbone.
PCT/EP2011/059619 2010-06-11 2011-06-09 Préparation micro biologique de corps en c4 à partir de saccharose et de dioxyde de carbone WO2011154503A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102010029973.1 2010-06-11
DE102010029973A DE102010029973A1 (de) 2010-06-11 2010-06-11 Mikrobiologische Herstellung von C4-Körpern aus Saccharose und Kohlendioxid

Publications (1)

Publication Number Publication Date
WO2011154503A1 true WO2011154503A1 (fr) 2011-12-15

Family

ID=44356275

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2011/059619 WO2011154503A1 (fr) 2010-06-11 2011-06-09 Préparation micro biologique de corps en c4 à partir de saccharose et de dioxyde de carbone

Country Status (2)

Country Link
DE (1) DE102010029973A1 (fr)
WO (1) WO2011154503A1 (fr)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102876760A (zh) * 2012-10-22 2013-01-16 南京工业大学 枯草芽孢菌在高产腺苷且联产乙偶姻中的应用
DE102012201360A1 (de) 2012-01-31 2013-08-01 Evonik Industries Ag Zellen und Verfahren zur Herstellung von Rhamnolipiden
WO2013134167A1 (fr) * 2012-03-05 2013-09-12 E. I. Du Pont De Nemours And Company Variants de transporteurs polypeptidiques de saccharose
WO2013142033A1 (fr) * 2012-03-20 2013-09-26 Metabolix, Inc. Micro-organismes génétiquement modifiés pour la production de poly-4-hydroxybutyrate
FR3002774A1 (fr) * 2013-03-04 2014-09-05 Agronomique Inst Nat Rech Levures mutantes ayant une production accrue de lipides et d'acide citrique
US8871488B2 (en) 2010-06-18 2014-10-28 Butamax Advanced Biofuels Llc Recombinant host cells comprising phosphoketolases
WO2015158716A1 (fr) * 2014-04-16 2015-10-22 Novamont S.P.A. Procédé de production de 1,4-butanediol
US9725746B2 (en) 2012-12-21 2017-08-08 Evonik Degussa Gmbh Producing amines and diamines from a carboxylic acid or dicarboxylic acid or a monoester thereof
US9765366B2 (en) 2012-02-22 2017-09-19 Evonik Degussa Gmbh Biotechnological method for producing butanol and butyric acid
US9765370B2 (en) 2012-04-02 2017-09-19 Evonik Degussa Gmbh Method for aerobically producing alanine or a compound produced using alanine
US9850192B2 (en) 2012-06-08 2017-12-26 Cj Cheiljedang Corporation Renewable acrylic acid production and products made therefrom
US9914941B2 (en) 2011-11-09 2018-03-13 Amyris, Inc. Production of acetyl-coenzyme a derived isoprenoids
WO2018091525A1 (fr) 2016-11-15 2018-05-24 Danmarks Tekniske Universitet Cellules bactériennes à tolérance améliorée aux diacides
US10174353B2 (en) 2014-05-26 2019-01-08 Evonik Degussa Gmbh Methods of producing rhamnolipids
CN110651037A (zh) * 2017-05-19 2020-01-03 巴斯夫欧洲公司 用于产生有机化合物的方法
CN111139193A (zh) * 2019-12-05 2020-05-12 天津科技大学 一种低产高级醇和强降解苹果酸的葡萄汁酵母菌株及其应用
US10786064B2 (en) 2010-02-11 2020-09-29 Cj Cheiljedang Corporation Process for producing a monomer component from a genetically modified polyhydroxyalkanoate biomass

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102012101153A1 (de) * 2012-02-14 2013-08-14 Thyssenkrupp Uhde Gmbh Verfahren zur biotechnologischen herstellung von organischen säuren durch mindestens einen hefestamm
CN103103147B (zh) * 2012-07-18 2014-11-12 大连理工大学 一株同时具有反硝化和铁还原功能的善变副球菌菌株、培养方法及其应用

Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1009370A (en) 1962-12-18 1965-11-10 Ajinomoto I I A process for the production of 1-glutamic acid by fermentation
US4489160A (en) 1981-08-26 1984-12-18 Kyowa Hakko Kogyo Co., Ltd. Plasmid pCG2
US4601893A (en) 1984-02-08 1986-07-22 Pfizer Inc. Laminate device for controlled and prolonged release of substances to an ambient environment and method of use
US4757009A (en) 1983-08-29 1988-07-12 Ajinomoto Co., Inc. Recombinant DNA having a phosphoenol pyruvate carboxylase gene inserted therein, bacteria carrying said recombinant DNA and a process for producing amino acids using said bacteria
US4980285A (en) 1984-10-04 1990-12-25 Ajinomoto Co., Inc. Method for producing L-amino acids
EP0472869A2 (fr) 1990-08-30 1992-03-04 Degussa Ag Plasmides de corynebacterium glutamicum et vecteurs plasmidiques dérivés
US5158891A (en) 1984-08-21 1992-10-27 Asahi Kasei Kogyo Kabushiki Kaisha Plasmid containing a gene for tetracycline resistance and DNA fragments derived therefrom
WO1996015246A1 (fr) 1994-11-11 1996-05-23 Forschungszentrum Jülich GmbH Adn regulant l'expression genique dans des bacteries du genre corynebacterium
US5573945A (en) 1994-01-10 1996-11-12 Ajinomoto Co., Inc. Mutant and method for producing L-glutamic acid by fermentation
JPH10229891A (ja) 1997-02-20 1998-09-02 Mitsubishi Rayon Co Ltd マロン酸誘導体の製造法
US6171833B1 (en) 1998-12-23 2001-01-09 Massachusetts Institute Of Technology Pyruvate carboxylase from corynebacterium glutamicum
DE10031999A1 (de) 1999-09-09 2001-04-19 Degussa Verfahren zur fermentativen Herstellung von D-Pantothensäure unter Verwendung coryneformer Bakterien
US6455284B1 (en) 1998-04-13 2002-09-24 The University Of Georgia Research Foundation, Inc. Metabolically engineered E. coli for enhanced production of oxaloacetate-derived biochemicals
WO2002090312A1 (fr) 2001-05-07 2002-11-14 Cargill, Incorporated Procede de preparation d'acides carboxyliques et de leurs derives
US6599732B1 (en) 1999-06-29 2003-07-29 Archer-Daniels-Midland Company Regulation of carbon assimilation
US6852516B2 (en) 1999-01-13 2005-02-08 Ajinomoto Co., Inc. Method for producing L-glutamic acid by fermentation
US6861246B2 (en) 1999-07-07 2005-03-01 Degussa Ag L-lysine-producing corynebacteria and process for the preparation of lysine
US6872553B2 (en) 1999-10-20 2005-03-29 Degussa Ag Nucleotide sequences which code for the pck gene
WO2008137402A1 (fr) * 2007-05-02 2008-11-13 E. I. Du Pont De Nemours And Company Procédé de production de 1-butanol
WO2009078687A2 (fr) * 2007-12-18 2009-06-25 Korea Advanced Institute Of Science And Technology Micro-organisme recombinant possédant la capacité d'utiliser le saccharose en tant que source de carbone

Patent Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1009370A (en) 1962-12-18 1965-11-10 Ajinomoto I I A process for the production of 1-glutamic acid by fermentation
US4489160A (en) 1981-08-26 1984-12-18 Kyowa Hakko Kogyo Co., Ltd. Plasmid pCG2
US4757009A (en) 1983-08-29 1988-07-12 Ajinomoto Co., Inc. Recombinant DNA having a phosphoenol pyruvate carboxylase gene inserted therein, bacteria carrying said recombinant DNA and a process for producing amino acids using said bacteria
US4601893A (en) 1984-02-08 1986-07-22 Pfizer Inc. Laminate device for controlled and prolonged release of substances to an ambient environment and method of use
US5158891A (en) 1984-08-21 1992-10-27 Asahi Kasei Kogyo Kabushiki Kaisha Plasmid containing a gene for tetracycline resistance and DNA fragments derived therefrom
US4980285A (en) 1984-10-04 1990-12-25 Ajinomoto Co., Inc. Method for producing L-amino acids
EP0472869A2 (fr) 1990-08-30 1992-03-04 Degussa Ag Plasmides de corynebacterium glutamicum et vecteurs plasmidiques dérivés
US5573945A (en) 1994-01-10 1996-11-12 Ajinomoto Co., Inc. Mutant and method for producing L-glutamic acid by fermentation
WO1996015246A1 (fr) 1994-11-11 1996-05-23 Forschungszentrum Jülich GmbH Adn regulant l'expression genique dans des bacteries du genre corynebacterium
JPH10229891A (ja) 1997-02-20 1998-09-02 Mitsubishi Rayon Co Ltd マロン酸誘導体の製造法
US6455284B1 (en) 1998-04-13 2002-09-24 The University Of Georgia Research Foundation, Inc. Metabolically engineered E. coli for enhanced production of oxaloacetate-derived biochemicals
US6171833B1 (en) 1998-12-23 2001-01-09 Massachusetts Institute Of Technology Pyruvate carboxylase from corynebacterium glutamicum
US6403351B1 (en) 1998-12-23 2002-06-11 Archer Daniels Midland Company Pyruvate carboxylase polypeptide from Corynebacterium glutamicum
US6884606B2 (en) 1998-12-23 2005-04-26 Massachusetts Institute Of Technology Pyruvate carboxylase from Corynebacterium glutamicum
US6852516B2 (en) 1999-01-13 2005-02-08 Ajinomoto Co., Inc. Method for producing L-glutamic acid by fermentation
US6599732B1 (en) 1999-06-29 2003-07-29 Archer-Daniels-Midland Company Regulation of carbon assimilation
US6861246B2 (en) 1999-07-07 2005-03-01 Degussa Ag L-lysine-producing corynebacteria and process for the preparation of lysine
DE10031999A1 (de) 1999-09-09 2001-04-19 Degussa Verfahren zur fermentativen Herstellung von D-Pantothensäure unter Verwendung coryneformer Bakterien
US6872553B2 (en) 1999-10-20 2005-03-29 Degussa Ag Nucleotide sequences which code for the pck gene
WO2002090312A1 (fr) 2001-05-07 2002-11-14 Cargill, Incorporated Procede de preparation d'acides carboxyliques et de leurs derives
WO2008137402A1 (fr) * 2007-05-02 2008-11-13 E. I. Du Pont De Nemours And Company Procédé de production de 1-butanol
WO2009078687A2 (fr) * 2007-12-18 2009-06-25 Korea Advanced Institute Of Science And Technology Micro-organisme recombinant possédant la capacité d'utiliser le saccharose en tant que source de carbone

Non-Patent Citations (46)

* Cited by examiner, † Cited by third party
Title
"E. coli Plasmid Vectors", 2003, HUMANA PRESS
"Manual of Methods for General Bacteriology", 1981, AMERICAN SOCIETY FOR BACTERIOLOGY
"Methods Enzymol.", vol. 185, 1988, article "Vectors : a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V. (1990), Systems for heterologous gene expression", pages: 3 - 7
CHMIEL: "Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik", 1991, GUSTAV FISCHER VERLAG
DONAHUE ET AL., JOURNAL OF BACTERIOLOGY, vol. 182, no. 19, 2000, pages 5624 - 5627
DUNICAN, SHIVNAN, BIO/TECHNOLOGY, vol. 7, 1989, pages 1067 - 1070
EIKMANNS ET AL., GENE, vol. 102, 1991, pages 93 - 98
EIKMANNS ET AL., GENE, vol. 107, 1991, pages 69 - 74
FREEDBERG ET AL., JOURNAL OF BACTERIOLOGY, vol. 115, no. 3, 1973, pages 816 - 823
GLOVER, D. M.: "DNA cloning: a practical approach", vol. 1-111, 1985, IRL PRESS LTD.
GUERRERO ET AL., GENE, vol. 138, 1994, pages 35 - 41
H. LIN ET AL: "Increasing the Acetyl-CoA Pool in the Presence of Overexpressed Phosphoenolpyruvate Carboxylase or Pyruvate Carboxylase Enhances Succinate Production in Escherichia coli", BIOTECHNOLOGY PROGRESS, vol. 20, no. 5, 1 October 2004 (2004-10-01), pages 1599 - 1604, XP055006788, ISSN: 8756-7938, DOI: 10.1021/bp049843a *
HERMANN ET AL., ELECTOPHORESIS, vol. 22, 2001, pages 1712 - 23
HERMANN ET AL., ELECTROPHORESIS, vol. 22, 2001, pages 1712.23
JAHREIS K ET AL: "Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON, DC; US, vol. 184, no. 19, 1 October 2002 (2002-10-01), pages 5307 - 5316, XP002984526, ISSN: 0021-9193, DOI: 10.1128/JB.184.19.5307-5316.2002 *
JENSEN, HAMMER, BIOTECHNOLOGY AND BIOENGINEERING, vol. 58, 1998, pages 191 - 195
JIAN WANG ET AL: "Succinate production from different carbon sources under anaerobic conditions by metabolic engineered Escherichia coli strains", METABOLIC ENGINEERING, vol. 13, no. 3, 1 May 2011 (2011-05-01), pages 328 - 335, XP055010835, ISSN: 1096-7176, DOI: 10.1016/j.ymben.2011.03.004 *
JIAN WANG ET AL: "Succinate production from sucrose by metabolic engineered escherichia coli strains under aerobic conditions", BIOTECHNOLOGY PROGRESS, vol. 27, no. 5, 1 September 2011 (2011-09-01), pages 1242 - 1247, XP055010836, ISSN: 8756-7938, DOI: 10.1002/btpr.661 *
LABARRE ET AL., JOURNAL OF BACTERIOLOGY, vol. 175, 1993, pages 1001 - 1007
LOHAUS ET AL., BIOSPEKTRUM, vol. 5, 1998, pages 32 - 39
LOHAUS, MEYER, BIOSPEKTRUM, vol. 5, 1989, pages 32 - 39
LOTTSPEICH, ANGEWANDTE CHEMIE, vol. 111, 1999, pages 2630 - 2647
MALUMBRES ET AL., GENE, vol. 134, 1993, pages 15 - 24
MARTIN ET AL., BIO/TECHNOLOGY, vol. 5, 1987, pages 137 - 146
MENKEL ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 64, 1989, pages 549 - 554
OHNISHI J ET AL.: "A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine- producing mutant.", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 58, no. 2, 2002, pages 217 - 223, XP002289984, DOI: doi:10.1007/s00253-001-0883-6
RAY ET AL., JOURNAL OF BACTERIOLOGY, vol. 182, no. 8, 2000, pages 2277 - 2284
REINSCHEID ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 60, 1994, pages 126 - 132
SAMBROOK ET AL.: "Molecular Cloning: a Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK, J., FRITSCH, E. F., MANIATIS, T.: "Molecular cloning: a laboratory manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SCHÄFER ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 60, 1994, pages 756 - 759
SCHÄFER ET AL., GENE, vol. 145, 1994, pages 69 - 73
SCHMID K ET AL: "PLASMID-MEDIATED SUCROSE METABOLISM IN ESCHERICHIA COLI K12: MAPPING OF THE SCR GENES OF PUR400", MOLECULAR MICROBIOLOGY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 2, no. 1, 1 January 1988 (1988-01-01), pages 1 - 08, XP001024290, ISSN: 0950-382X, DOI: 10.1111/J.1365-2958.1988.TB00001.X *
SCHRUMPF ET AL., JOURNAL OF BACTERIOLOGY, vol. 173, pages 4510 - 4516
SCHWARZER, PUHLER, BIO/TECHNOLOGY, vol. 9, 1991, pages 84 - 87
SERWOLD-DAVIS ET AL., FEMS MICROBIOLOGY LETTERS, vol. 66, 1990, pages 119 - 124
SHUMAN, JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, 1994, pages 32678 - 84
SIMON ET AL., BIO/TECHNOLOGY, vol. 1, 1983, pages 784 - 791
SONNEN ET AL., GENE, vol. 107, 1991, pages 69 - 74
SPRATT ET AL., GENE, vol. 41, 1986, pages 337 - 342
STORHAS: "Bioreaktoren und periphere Einrichtungen", 1994, VIEWEG VERLAG
TAUCH ET AL., FEMS MICROBIOLOGY LETTERS, vol. 123, 1994, pages 343 - 347
THIERBACH ET AL., APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 29, 1988, pages 356 - 362
TSUCHIYA, MORINAGA, BIO/TECHNOLOGY, vol. 6, 1988, pages 428 - 430
WILSON ET AL., JOURNAL OF BACTERIOLOGY, vol. 183, 2001, pages 2151 - 2155
X. ZHANG ET AL: "Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 106, no. 48, 1 December 2009 (2009-12-01), pages 20180 - 20185, XP055010824, ISSN: 0027-8424, DOI: 10.1073/pnas.0905396106 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10786064B2 (en) 2010-02-11 2020-09-29 Cj Cheiljedang Corporation Process for producing a monomer component from a genetically modified polyhydroxyalkanoate biomass
US10006058B2 (en) 2010-06-18 2018-06-26 Butamax Advanced Biofuels Llc Recombinant host cells comprising phosphoketalase
US8871488B2 (en) 2010-06-18 2014-10-28 Butamax Advanced Biofuels Llc Recombinant host cells comprising phosphoketolases
US9914941B2 (en) 2011-11-09 2018-03-13 Amyris, Inc. Production of acetyl-coenzyme a derived isoprenoids
DE102012201360A1 (de) 2012-01-31 2013-08-01 Evonik Industries Ag Zellen und Verfahren zur Herstellung von Rhamnolipiden
US9765366B2 (en) 2012-02-22 2017-09-19 Evonik Degussa Gmbh Biotechnological method for producing butanol and butyric acid
WO2013134167A1 (fr) * 2012-03-05 2013-09-12 E. I. Du Pont De Nemours And Company Variants de transporteurs polypeptidiques de saccharose
CN104245723A (zh) * 2012-03-05 2014-12-24 纳幕尔杜邦公司 变体蔗糖转运蛋白多肽
WO2013142033A1 (fr) * 2012-03-20 2013-09-26 Metabolix, Inc. Micro-organismes génétiquement modifiés pour la production de poly-4-hydroxybutyrate
US9765370B2 (en) 2012-04-02 2017-09-19 Evonik Degussa Gmbh Method for aerobically producing alanine or a compound produced using alanine
US9850192B2 (en) 2012-06-08 2017-12-26 Cj Cheiljedang Corporation Renewable acrylic acid production and products made therefrom
CN102876760B (zh) * 2012-10-22 2014-07-30 南京工业大学 枯草芽孢菌在高产腺苷且联产乙偶姻中的应用
CN102876760A (zh) * 2012-10-22 2013-01-16 南京工业大学 枯草芽孢菌在高产腺苷且联产乙偶姻中的应用
US9725746B2 (en) 2012-12-21 2017-08-08 Evonik Degussa Gmbh Producing amines and diamines from a carboxylic acid or dicarboxylic acid or a monoester thereof
FR3002774A1 (fr) * 2013-03-04 2014-09-05 Agronomique Inst Nat Rech Levures mutantes ayant une production accrue de lipides et d'acide citrique
WO2014136028A1 (fr) * 2013-03-04 2014-09-12 Institut National De La Recherche Agronomique Levures mutantes ayant une production accrue de lipides et d'acide citrique
WO2015158716A1 (fr) * 2014-04-16 2015-10-22 Novamont S.P.A. Procédé de production de 1,4-butanediol
US10227616B2 (en) 2014-04-16 2019-03-12 Novamont S.P.A. Process for the production of 1,4-butanediol
US10174353B2 (en) 2014-05-26 2019-01-08 Evonik Degussa Gmbh Methods of producing rhamnolipids
WO2018091525A1 (fr) 2016-11-15 2018-05-24 Danmarks Tekniske Universitet Cellules bactériennes à tolérance améliorée aux diacides
CN110651037A (zh) * 2017-05-19 2020-01-03 巴斯夫欧洲公司 用于产生有机化合物的方法
CN111139193A (zh) * 2019-12-05 2020-05-12 天津科技大学 一种低产高级醇和强降解苹果酸的葡萄汁酵母菌株及其应用

Also Published As

Publication number Publication date
DE102010029973A1 (de) 2011-12-15

Similar Documents

Publication Publication Date Title
WO2011154503A1 (fr) Préparation micro biologique de corps en c4 à partir de saccharose et de dioxyde de carbone
EP2175024B1 (fr) Fabrication microbiologique d'acides 3-hydroxypropion
EP2152659B1 (fr) Procédé de production d'acide méthacrylique ou d'esters d'acide méthacrylique
US10100342B2 (en) Method for producing 3-hydroxypropionic acid and other products
EP2291530B1 (fr) Cellule recombinante produisant de l'acide 2-hydroxyisobutyrique
Wang et al. Anaerobic fermentation for production of carboxylic acids as bulk chemicals from renewable biomass
Joo et al. Production of 5-aminovaleric acid in recombinant Corynebacterium glutamicum strains from a Miscanthus hydrolysate solution prepared by a newly developed Miscanthus hydrolysis process
DE102007015583A1 (de) Ein Enzym zur Herstellung von Methylmalonyl-Coenzym A oder Ethylmalonyl-Coenzym A sowie dessen Verwendung
WO2007141208A2 (fr) Fabrication microbiologique d'acide 3-hydroxyisobutyrique
EP2354235B1 (fr) Méthode de production d'acides aminés L par fermentation au moyen de bactéries corynéformes
US8435768B2 (en) Method for producing succinic acid using a yeast belonging to the genus Yarrowia
WO2008148640A1 (fr) Production microbiologique d'aldéhydes, notamment de 3-hydroxy propionaldéhyde
DE102012201360A1 (de) Zellen und Verfahren zur Herstellung von Rhamnolipiden
KR102430878B1 (ko) 당 포스포트랜스퍼라제 시스템 (pts)을 코딩하는 유전자를 포함하는 미생물에 의한 관심 분자의 발효적 생산을 위한 방법
EP3824078A1 (fr) Fabrication biotechnologique de cannabinoïdes
CN112204146A (zh) 具有受抑制的乙醇产生途径的耐酸酵母及使用其生产乳酸的方法
US20160326554A1 (en) Recombinant microorganisms for producing organic acids
US20160348082A1 (en) Microorganisms for Succinic Acid Production
DE102007059248A1 (de) Zelle, welche in der Lage ist, CO2 zu fixieren
JP5651989B2 (ja) コハク酸の製造方法
WO2009053489A1 (fr) Production par fermentation d'acide alpha-cétoglutarique
Peters-Wendisch et al. Use of Corynebacterium glutamicum for the Production of High-Value Chemicals from New Carbon Sources

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11724632

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11724632

Country of ref document: EP

Kind code of ref document: A1