WO2014198560A2 - Verfahren zur herstellung von organischen verbindungen aus knallgas und co2 über acetoacetyl-coa als zwischenprodukt - Google Patents
Verfahren zur herstellung von organischen verbindungen aus knallgas und co2 über acetoacetyl-coa als zwischenprodukt Download PDFInfo
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- WO2014198560A2 WO2014198560A2 PCT/EP2014/061245 EP2014061245W WO2014198560A2 WO 2014198560 A2 WO2014198560 A2 WO 2014198560A2 EP 2014061245 W EP2014061245 W EP 2014061245W WO 2014198560 A2 WO2014198560 A2 WO 2014198560A2
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- 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
- C12P7/52—Propionic acid; Butyric acids
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
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- 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
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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- 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
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
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- 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
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
- C12P7/28—Acetone-containing products
- C12P7/30—Acetone-containing products produced from substrate containing inorganic compounds other than water
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- 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)
- C12Y203/01009—Acetyl-CoA C-acetyltransferase (2.3.1.9)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Definitions
- the present invention is a process for the preparation of organic compounds comprising the process steps
- Fuel value can be converted.
- carbohydrates such as glucose, dextrose or glycerol, are subject to strong
- Carbohydrates such as glucose, dextrose or glycerin, are not available in sufficient quantities as raw materials in all regions.
- Fermentation raw materials are in competition with the use as food.
- Biocatalysts and with H 2 and C0 2 as raw materials under energetically favorable, aerobic Conditions are not yet available today, but would make it possible, in terms of raw materials and regional highly flexible, these chemicals from low-cost raw materials or waste streams (natural gas, municipal waste, biomass, converter gas, etc.).
- Another object of the invention is the use of the disclosed in the context of the invention oxyhydrogen bacteria for the production of organic compounds.
- the present invention thus provides a process for the preparation of an organic compound comprising the process steps
- explosive gas bacterium a bacterium capable of growing chemolithoautotrophically and of H 2 and C0 2 in the presence of oxygen
- the oxyhydrogen bacteria used in the process according to the invention are preferably those which already represent oxyhydrogen bacteria as wild-type.
- Oxy-gas bacteria preferably used according to the invention are selected from the genera Achromobacter, Acidithiobacillus, Acidovorax, Alcaligenes, Anabena, Aquifex, Arthrobacter, Azospirillum, Bacillus, Bradyrhizobium, Cupriavidus, Derxia, Helicobacter, Herbaspirillum, Hydrogenobacter, Hydrogenobaculum, Hydrogenophaga, Hydrogenophilus,
- Methanobrevibacter Myobacterium, Nocardia, Oligotropha, Paracoccus, Pelomonas,
- Rhodopseudomonas Rhodospirillum, Streptomyces, Thiocapsa, Treponema, Variovorax, Xanthobacter, Wautersia, cupriavidus being particularly preferred
- Cupriavidus necator also known as Ralstonia eutropha, Wautersia eutropha, Alcaligenes eutrophus, Hydrogenomonas eutropha
- Achromobacter ruhlandii Acidithiobacillus ferrooxidans, Acidovorax facilis
- Alcaligenes hydrogenophilus Alcaligenes latus
- Anabena cylindrica Anabena oscillaroides, Anabena sp., Anabena spiroides, Aquifex aeolicus, Aquifex pyrophilus
- Arthrobacter strain 11X Bacillus schlegelii, Bradyrhizobium japonicum, Cupriavidus necator, Derxia gummosa, Escherichia coli, Heliobacter pylori, Herbaspirillum autotrophicum, Hydrogenobacter hydrogenophilus, Hydrogenobacter thermophilus, Hydro
- Treponema monia, Variovorax paradoxus, Xanthobacter autrophicus, Xanthobacter flavus in particular from the strains Cupriavidus necator H 16, Cupriavidus necator H1 or Cupriavidus necator Z-1.
- the explosive gas bacterium of the method according to the invention has an increased compared to its wild type activity of an enzyme E- ⁇ .
- wild-type of a cell is meant herein a cell whose genome is in a state as naturally evolved by evolution. The term is used for both the entire cell and for individual genes therefore in particular not such cells or genes whose gene sequences have been at least partially modified by humans by means of recombinant methods.
- enhanced activity of an enzyme is preferably to be understood as meaning that the wild type has been genetically engineered in such a way that the corresponding increase in activity occurs to understand increased intracellular activity.
- an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence or gene sequences which code for the enzyme, using a strong promoter, changing the codon usage of the gene, in various ways the half-life of the mRNA or of the enzyme that increases
- Microorganisms are generated, for example, by transformation, transduction, conjugation, or a combination of these methods with a vector containing the desired gene, an allele of this gene or parts thereof, and a promoter enabling 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.
- the quantification of the increase in enzyme activity can be determined in a simple manner by comparing the 1- or 2-dimensional protein separations between wild-type and genetically modified cell.
- a common method for preparing the protein gels in bacteria and for identifying the proteins is that of Hermann et al.
- Protein concentration can also be assessed by Western blot hybridization with an antibody specific for the protein to be detected (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989) and subsequent optical evaluation with appropriate software for concentration determination (Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 1 1 1: 2630- 2647).
- This method is also always suitable when possible products of the catalyzed by the enzyme activity to be determined reaction in the microorganism quickly
- the enzyme E-i whose activity in the oxyhydrogen bacterium is increased compared to its wild-type, is preferably selected from acetyl-CoA: acetyl-CoA C-acetyltransferases from the enzyme class EC 2.3.1 .9.
- accession numbers cited in connection with the present invention correspond to the ProteinBank database entries of NCBI dated 26.06.2012; As a rule, the version number of the entry is indicated by "number” such as "1".
- Acetyl-CoA preferred according to the invention acetyl-CoA C-acetyltransferases are selected from the list
- AAC26023.1, ABR35750.1 and ABR25255.1 such as
- Biocatalyst is understood without the presence of the reference protein, wherein the activity in this context, the reaction of two acetyl-CoA to acetoacetyl-CoA and CoA is understood.
- the explosive gas bacterium is provided in process step A) in an aqueous medium.
- the aqueous medium used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington D.C, USA, 1981).
- the medium may contain nitrogen sources, nitrogen source may be organic nitrogenous compounds such as peptones, yeast extract, meat extract, malt extract,
- Corn steep liquor, soybean meal and urea or inorganic compounds such as
- Ammonium nitrate, ammonia, ammonium hydroxide or ammonia water can be used.
- the nitrogen sources can be used singly or as a mixture.
- the medium may contain phosphorus sources, phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts may be used as the phosphorus source.
- the medium may also contain sources of carbon, but should be limited in so far as that the oxyhydrogen bacterium is dependent substantially on the utilization of carbon-containing gases contained in the gas containing H 2, C0 2 and 0. 2
- the medium must continue to contain salts of metals such. As magnesium sulfate or iron sulfate, which are necessary for growth.
- essential growth factors such as amino acids and vitamins can be used in addition to the above-mentioned substances.
- suitable precursors may be added to the medium.
- Feedstocks may be added to the culture as a one-time batch or fed in a suitable manner during the cultivation.
- 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.
- process step B) the aqueous medium is brought into contact with a gas containing H 2, C0 2 and 0. 2
- Carbon source provided.
- the oxyhydrogen bacterium synthesizes at least partially from this carbon source acetoacetyl-CoA, which is metabolized to other organic compounds.
- process step B) of the invention is Unlike in an acetogenic process process, which takes place under strictly anaerobic conditions, process step B) of the invention
- the partial pressure of hydrogen in the H is 2, C0 2 and 0 2 gas containing 0.1 to 100 bar, preferably from 0.2 to 10 bar, particularly preferably 0.5 to 4 bar.
- the partial pressure of carbon dioxide in the H 2, C0 2 and 0 2 -containing gas is preferably 0.03 to 100 bar, particularly preferably 0.05 to 1 bar, in particular 0.05 to 0.3 bar
- the partial pressure of oxygen in the H 2, C0 2 and gas containing 0 2 is preferably 0.001 to 100 bar, particularly preferably 0.04 to 1 bar, in particular 0.04 to 0.5 bar.
- the gas in process step B) contains synthesis gas.
- Syngas can, for. B. be provided from the by-product of the coal gasification. The oxyhydrogen bacterium thus converts a substance that is a waste product into a valuable raw material.
- synthesis gas may be provided by the gasification of widely available, inexpensive agricultural raw materials for the process of the present invention.
- raw materials that can be converted into synthesis gas, since almost all forms of vegetation can be used for this purpose.
- Preferred raw materials are selected from the group comprising perennial grasses such as miscanthus,
- Grain residues processing waste such as sawdust.
- synthesis gas is recovered in a gasification apparatus from dried biomass, mainly by pyrolysis, partial oxidation and steam reforming, the primary products being CO, H 2 and CO 2 .
- the carbon contained in the synthesis gas in
- Process step B) at least 50 wt .-%, preferably at least 70 wt .-%, particularly preferably at least 90 wt .-% of the carbon of all carbon sources, the
- Knallgasbakterium in process step B) are available, makes up, wherein the weight percent of carbon refer to the carbon atoms. The remaining
- Carbon sources may, for example, be present in the form of carbohydrates in the aqueous medium or may also be C0 2 from a source other than synthesis gas.
- Other C0 2 sources include exhaust gases such as flue gas, petroleum refinery emissions, gases resulting from yeast fermentation or clostridial fermentation, gasification gases, cellulosic materials or coal gasification.
- the organic substance to be produced by the process according to the invention is preferably selected from substances comprising three or four carbon atoms, in particular butanol, butene, propene, butyric acid, acetone, 2-hydroxyisobutyric acid and 2-propanol and 2-hydroxyisobutyric acid.
- Butanol A first embodiment of the process according to the invention is characterized in that the organic substance is butanol, preferably E-1 is selected from acetyl-CoA: acetyl-CoA C-acetyltransferases and preferably the oxyhydrogen bacterium an increased activity of an enzyme E 2 compared to its wild-type which is the implementation
- the enzyme E 2 is preferably selected from 3-hydroxybutyryl-CoA dehydrogenases from the enzyme class EC: 1.1.1 .157.
- Preferred 3-hydroxybutyryl-CoA dehydrogenases according to the invention are selected from the list
- a preferred first embodiment of the method according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and optionally E 2 , the oxyhydrogen bacterium has an increased activity of an enzyme E 3 compared to its wild-type, which is the reaction
- the enzyme E 3 is preferably selected from 3-hydroxybutyryl-CoA dehydratases from the enzyme class EC: 4.2.1.55.
- Preferred 3-hydroxybutyryl-CoA dehydratases according to the invention are selected from the list
- Amino acid residues are altered by deletion, insertion, substitution or a combination thereof with respect to the abovementioned reference sequences and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein is the increase in the activity of the cells used as biocatalyst, ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of Biocatalyst is understood without the presence of the reference protein, wherein under the
- Activity in this context and in connection with the determination of the activity of the enzyme E 3 is generally understood in particular the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA and water.
- a further preferred first embodiment of the method according to the invention is characterized in that the oxyhydrogen gas bacterium in addition to the increased activity of the enzyme egg and optionally E 2 and / or E 3 increased compared to its wild-type activity an enzyme E 4 , which is the reaction
- the enzyme E 4 is preferably selected from butyryl-CoA dehydrogenases from the
- Enzyme class EC 1.3.99.2
- Butyryl-CoA dehydrogenases preferred according to the invention are selected from the list NP_349317.1, YP_001307466.1 and CAQ53135.1
- a further preferred first embodiment of the process according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and optionally E 2 , E 3 and / or E 4 , the oxyhydrogen bacterium has an activity of an enzyme E 5 which is increased compared to its wild-type
- butyryl-CoA and NADH or NADPH to butyraldehyde NAD + or NADP + and HS-CoA and of butyraldehyde and NADH or NADPH to catalyze n-butanol and NAD + or NADPH +.
- the enzyme E 5 is selected from bifunctional aldehyde / alcohol dehydrogenases from the enzyme class EC: 1 .2.1 .10 or EC: 1 .1.1.1 or from butyraldehyde dehydrogenases from the enzyme class EC: 1 .2.1.10.
- the former enzymes catalyze both of the aforementioned reactions, while butyraldehyde dehydrogenases react only butyryl-CoA.
- Preferred bifunctional aldehyde / alcohol dehydrogenases according to the invention are selected from the list
- Amino acid residues are altered by deletion, insertion, substitution or a combination thereof with respect to the abovementioned reference sequences and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein is the increase in the activity of the cells used as biocatalyst, ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of Biocatalyst is understood without the presence of the reference protein, wherein under the
- Butyraldehyde dehydrogenases preferred according to the invention are selected from the list YP_001310903.1 and CAQ57983.1
- a further preferred first embodiment of the inventive method is characterized in that the explosive gas bacterium in addition to the increased activity of the enzyme Ei and optionally E 2 , E 3 , E 4 and / or E 5 increased compared to its wild-type activity of an enzyme E 6 , which the implementation
- the enzyme E 6 is selected from butanol dehydrogenases from the enzyme class EC: 1.1 .1 .-.
- Butanol dehydrogenases preferred according to the invention are selected from the list
- Polypeptide sequence in which up to 60%, preferably up to 25%, more preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues relative to the aforementioned reference sequences are altered by deletion, insertion, substitution or a combination thereof and which are still at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90% of the activity of the protein with the corresponding
- the oxyhydrogen gas bacterium has an increased activity E 6 when it has an increased activity of an enzyme E 5 , which requires only the reaction of butyryl-CoA and NADH or NADPH to butyraldehyde, NAD + or NADP + and HS-CoA catalyze and thus is preferably a butyraldehyde dehydrogenase.
- a further preferred first embodiment of the method according to the invention is characterized in that the oxyhydrogen gas bacterium, in addition to the increased activity of the enzyme Ei and optionally E 2 , E 3 , E 4 , E 5 and / or E 6, has an increased activity of an enzyme E compared to its wild-type 7 selected from electron transfer flavoproteins from the
- Enzyme class EC 2.8.3.9.
- an enzyme E 7 which is a heterodimeric enzyme, composed of two subunits, in which the
- alpha subunit is selected from NP_349315.1, YP_001307468.1 and CAQ53137.1 and the
- beta subunit is selected from NP_349316.1, YP_001307467.1 and CAQ53136.1 and proteins having a polypeptide sequence in which up to 60%, preferably up to 25%, more preferably up to 15%, in particular up to 10, 9, 8 , 7, 6, 5, 4, 3, 2, 1% of
- Amino acid residues with respect to the aforementioned reference sequences by deletion, insertion, substitution or a combination thereof are changed and still at least 50%, preferably 65%, more preferably 80%, in particular more than 90% of the activity of the protein having the corresponding above-mentioned reference sequence.
- alpha and beta subunits from the same organism are combined.
- the explosive gas bacterium used has an increased activity E 7 if an increased activity of E 4 already exists.
- the oxyhydrogen bacterium has increased activities in the combinations
- E1 E2E 3 egg ESE 4 , E1 E 5 E 6 , E
- a second embodiment of the method according to the invention is characterized in that the organic substance is butene, preferably E-1 is selected from acetyl-CoA: acetyl-CoA C-acetyltransferases and preferably the oxyhydrogen bacterium an increased activity of an enzyme E 2 compared to its wild type.
- the enzyme E 2 is preferably selected from 3-hydroxybutyryl-CoA dehydrogenases from the enzyme class EC: 1.1.1 .157.
- Preferred 3-hydroxybutyryl-CoA dehydrogenases according to the invention are selected from the list
- Amino acid residues are altered by deletion, insertion, substitution or a combination thereof with respect to the abovementioned reference sequences and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein is the increase in the activity of the cells used as biocatalyst, ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of Biocatalyst is understood without the presence of the reference protein, wherein the activity in this context and in connection with the determination of the activity the enzyme E 2 is generally understood in particular the reaction of acetoacetyl-CoA and NADH to 3-hydroxybutyryl-CoA and NAD +.
- a preferred second embodiment of the erfindungewelen method is characterized in that the explosive gas bacterium in addition to the increased activity of the enzyme Ei and optionally E 2 increased compared to its wild-type activity of an enzyme E 3 , which is the reaction
- the enzyme E 3 is preferably selected from 3-hydroxybutyryl-CoA dehydratases from the enzyme class EC: 4.2.1.55.
- Preferred 3-hydroxybutyryl-CoA dehydratases according to the invention are selected from the list
- Amino acid residues are altered by deletion, insertion, substitution or a combination thereof with respect to the abovementioned reference sequences and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein, the increase in the activity of the biocatalyst used
- Cells ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of the biocatalyst without the presence of the reference protein, wherein the activity in this context and in connection with the determination of the activity of the enzyme E 3 is generally understood in particular the reaction of 3-hydroxybutyryl-CoA to crotonyl-CoA and water.
- a further preferred second embodiment of the process according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and optionally E 2 and / or E 3 , the oxyhydrogen gas bacterium has an increased activity of an enzyme E 4 compared to its wild-type
- the enzyme E 4 is preferably selected from butyryl-CoA dehydrogenases from the enzyme class EC: 1.3.99.2
- Butyryl-CoA dehydrogenases preferred according to the invention are selected from the list NP_349317.1, YP_001307466.1 and CAQ53135.1
- Amino acid residues are altered by deletion, insertion, substitution or a combination thereof with respect to the abovementioned reference sequences and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein is the increase in the activity of the cells used as biocatalyst, ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of Biocatalyst is understood without the presence of the reference protein, wherein the activity in this context and in connection with the determination of the activity of the enzyme E 4 is generally understood in particular the implementation of crotonyl-CoA and NADH to butyryl-CoA and NAD +.
- a further preferred second embodiment of the process according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and optionally E 2 , E 3 and / or E 4 , the oxyhydrogen bacterium has an increased activity of an enzyme E 5 compared to its wild-type
- the enzyme E 5 is selected from bifunctional aldehyde / alcohol dehydrogenases from the enzyme class EC: 1 .2.1 .10 or EC: 1 .1.1 .1 or from butyraldehyde dehydrogenases from the enzyme class EC: 1 .2.1.10.
- bifunctional aldehyde / alcohol dehydrogenases are selected from the list
- Amino acid residues are altered by deletion, insertion, substitution or a combination thereof with respect to the abovementioned reference sequences and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein, the increase in the activity of the biocatalyst used
- Cells ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of the biocatalyst without the presence of the reference protein, wherein the activity in this context and in connection with the determination of the activity of the enzyme E 5 in the case of a bifunctional aldehyde / alcohol dehydrogenase, in particular the reaction of at least one of the two reactions
- Butyraldehyde dehydrogenases preferred according to the invention are selected from the list YP_001310903.1 and CAQ57983.1
- Amino acid residues are altered by deletion, insertion, substitution or a combination thereof with respect to the abovementioned reference sequences and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein is the increase in the activity of the cells used as biocatalyst, ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of Biocatalyst is understood without the presence of the reference protein, wherein the activity in this context and in connection with the determination of the activity of the enzyme E 5 in the case of a butyraldehyde dehydrogenase in particular the reaction of butyryl-CoA and NADH to butyraldehyde, NAD + and HS-CoA is understood.
- a further preferred second embodiment of the erfindungeingen method is characterized in that the explosive gas bacterium in addition to the increased activity of the enzyme Ei and optionally E 2 , E 3 , E 4 and / or E 5 increased compared to its wild type activity of an enzyme E 6 , which the implementation
- the enzyme E 6 is selected from butanol dehydrogenases from the enzyme class EC: 1.1 .1 .-.
- Butanol dehydrogenases preferred according to the invention are selected from the list
- Cell dry weight [U / g CDW]) is understood in comparison to the activity of the biocatalyst without the presence of the reference protein, wherein the activity in this context and in connection with the determination of the activity of the enzyme E 6 in general in particular the implementation of Butyraldehyde and NAD (P) H to n-butanol and NAD (P) + is understood.
- the oxyhydrogen gas bacterium has an increased activity E 6 when it has an increased activity of an enzyme E 5 , which requires only the reaction of butyryl-CoA and NADH or NADPH to butyraldehyde, NAD + or NADP + and HS-CoA catalyze and thus is preferably a butyraldehyde dehydrogenase.
- a further preferred second embodiment of the method according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and optionally E 2 , E 3 , E 4 , E 5 and / or E 6 , the oxyhydrogen bacterium is enhanced compared to its wild type Activity of an enzyme E 7 selected from electron transfer flavoproteins from the enzyme class EC: 2.8.3.9.
- an enzyme E 7 which is a heterodimeric enzyme, composed of two subunits, in which the
- alpha subunit is selected from NP_349315.1, YP_001307468.1 and CAQ53137.1 and the
- beta subunit is selected from NP_349316.1, YP_001307467.1 and CAQ53136.1 and proteins having a polypeptide sequence in which up to 60%, preferably up to 25%, more preferably up to 15%, in particular up to 10, 9, 8 , 7, 6, 5, 4, 3, 2, 1% of
- Amino acid residues with respect to the aforementioned reference sequences by deletion, insertion, substitution or a combination thereof are changed and still at least 50%, preferably 65%, more preferably 80%, in particular more than 90% of the activity of the protein having the corresponding above-mentioned reference sequence.
- alpha and beta subunits from the same organism are combined.
- a further preferred second embodiment of the method according to the invention is characterized in that the oxyhydrogen gas bacterium in addition to the increased activity of the enzyme Ei and optionally E 2 , E 3 , E 4 , E 5 , E 6 and / or E 7 increased compared to its wild type activity an enzyme E 8 , which is the reaction
- the enzyme E 8 is preferably selected from oleate hydratases from the enzyme class
- Preferred oleate hydratases according to the invention are selected from the list
- AAA87627.1 and for both classes of enzymes proteins having a polypeptide sequence in which up to 60%, preferably up to 25%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3 , 2, 1% of the amino acid residues with respect to the aforementioned reference sequences by deletion, insertion, substitution or a combination thereof are changed and at least 50%, preferably 65%, more preferably 80%, in particular more than 90% of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein, the increase of the activity as a biocatalyst used cells, ie the amount of substance reacted per unit time based on the amount of cell used (units per gram cell dry weight [U / g CDW]) compared to the activity of the biocatalyst is understood without the presence of the reference protein, wherein the activity in this In connection with the determination of the activity of the enzyme E 8 is generally understood in particular the reaction of n-butanol to 1-butene
- the oxyhydrogen bacterium has increased activities in the combinations E-
- E 1 E 2 E 7 6 E3E4E5E E8, E 1 E 3 E 7 6 E4E5E E8, E 1 E 3 E4E5E 7 E8, E 1 E 2 E3E4E5E 7 E8 is particularly preferred.
- a third embodiment of the process according to the invention is characterized in that the organic substance is propene or butyric acid, preferably E-1 is selected from acetyl-CoA: acetyl-CoA C-acetyltransferases and preferably the oxyhydrogen bacterium increased compared to its wild-type Activity of an enzyme E 2 , which is the reaction of acetoacetyl-CoA and NADH or NADPH to 3-hydroxybutyryl-CoA and NAD + or NADP +
- the enzyme E 2 is preferably selected from 3-hydroxybutyryl-CoA dehydrogenases from the enzyme class EC: 1.1.1 .157.
- Preferred 3-hydroxybutyryl-CoA dehydrogenases according to the invention are selected from the list
- a preferred third embodiment of the erfindunbeen method is characterized in that the explosive gas bacterium in addition to the increased activity of the enzyme Ei and optionally E 2 increased compared to its wild-type activity of an enzyme E 3 , which is the reaction
- the enzyme E 3 is preferably selected from 3-hydroxybutyryl-CoA dehydratases from the enzyme class EC: 4.2.1.55.
- Preferred 3-hydroxybutyryl-CoA dehydratases according to the invention are selected from the list
- Amino acid residues are altered by deletion, insertion, substitution or a combination thereof with respect to the abovementioned reference sequences and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein is the increase in the activity of the cells used as biocatalyst, ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of Biocatalyst is understood without the presence of the reference protein, wherein the activity in this context and in connection with the determination of the activity the enzyme E 3 is generally understood in particular the reaction of 3-hydroxybutyryl-CoA to crotonyl-CoA and water.
- a further preferred third embodiment of the method according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and optionally E 2 and / or E 3 , the oxyhydrogen bacterium has an increased activity of an enzyme E 4 compared to its wild-type, which is the reaction
- the enzyme E 4 is preferably selected from butyryl-CoA dehydrogenases from the
- Butyryl-CoA dehydrogenases preferred according to the invention are selected from the list NP_349317.1, YP_001307466.1 and CAQ53135.1
- a further preferred third embodiment of the process according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and optionally E 2 , E 3 and / or E 4 , the oxyhydrogen bacterium has an activity of an enzyme E 9 which is increased compared to its wild type and which is the reaction
- P in this context, stands for an inorganic phosphate.
- the enzyme E 9 is preferably selected from phosphate butyryltransferases from the enzyme class EC: 2.3.1.19.
- Preferred phosphate butyryltransferases according to the invention are selected from the list ABR32393.1 and ZP_05394269.1
- a further preferred third embodiment of the method according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and optionally E 2 , E 3 , E 4 and / or E 9 , the oxyhydrogen bacterium is enhanced compared to its wild-type
- the enzyme E 10 is preferably selected from butyrate kinases from the enzyme class EC: 2.7.2.7.
- Butyrate kinases preferred according to the invention are selected from the list
- Amino acid residues with respect to the aforementioned reference sequences are altered by deletion, insertion, substitution or a combination thereof and which is at least 50%, preferably 65%, more preferably 80%, in particular more than 90% of the activity of Have protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein, the increase in the activity of the cells used as a biocatalyst, ie the amount of substance converted per unit time based on the amount of cells used (units per gram cell dry weight [U / g CDW]) compared to the activity of the biocatalyst without the presence of the reference protein, wherein the activity in this context and in connection with the determination of the activity of the enzyme E 10 in general in particular the reaction of butyryl phosphate and ADP to butyrate and ATP is understood.
- a further preferred third embodiment of the method according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and optionally E 2 , E 3 , E 4 , E 9 and / or E 10 , the oxyhydrogen bacterium has an increased activity of an enzyme En which is the implementation
- the enzyme En is selected from cytochrome P450 of the CYP152 family.
- cytochrome P450 of the CYP152 family according to the invention are selected from the list
- the oxyhydrogen bacterium increased activity in the combinations E 1 E2E 3 E 4 E 9, E 1 E 2 E 3 E 4 E 10, egg E2EsE 4 Eii, E "
- E 1 E 2 E 3 E 4 E 9 E 1 OE 11 E 1 E 3 E 4 E 9 E 10 E 11 is particularly preferred.
- the combination of the enzymes E 9 E 10 also in combination with the abovementioned other enzymes as described above, which in total catalyzes the reaction of n-butyryl-CoA to butyrate, replaced by at least one enzyme in which the n-butyryl-CoA is converted to butyrate by a thioesterase or acyl-CoA synthetase or acyl-CoA acylate: CoA transferase.
- a fourth embodiment of the process according to the invention is characterized in that the organic substance is acetone, preferably E-1 is selected from acetyl-CoA: acetyl-CoA C-acetyltransferases, and the oxyhydrogen bacterium an increased activity of an enzyme E 12 compared to its wild type.
- the enzyme E 12 is selected from acetoacetyl-CoA: acetate / acyl: CoA transferases from the enzyme class EC: 3.1.2.1 1, from butyrate-acetoacetate-CoA transferases from
- the alpha subunit is selected from NP_149326.1, YP_001310904.1 and CAQ57984.1 and the
- beta subunit is selected from NP_149327.1, YP_001310905.1 and CAQ57985.1 and proteins having a polypeptide sequence in which up to 60%, preferably up to 25%, more preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues are modified with respect to the abovementioned reference sequences by deletion, insertion, substitution or a combination thereof and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90% of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein, the increase in the activity of the cells used as biocatalyst, ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of the biocatalyst without the presence of the reference protein, whereby under the activity in this connection and in connection with the determination of the activity of the enzyme
- Butyrate acetoacetate-CoA transferases preferred according to the invention are selected from ctfA and ctfB from Clostridium acetobutylicum and atoD and atoA from Escherichia coli as well as proteins having a polypeptide sequence in which up to 60%, preferably up to 25%, particularly preferably up to 15% in particular, up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues are altered from the abovementioned reference sequences by deletion, insertion, substitution or a combination thereof and which is at least 50%, preferably 65% %, more preferably 80%, in particular more than 90% of the activity of the protein having the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein, the increase in the activity of the cells used as biocatalyst, ie the amount converted per unit time based on the used cell volume (units per gram of cell dry weight [U / g CDW]) compared to the activity of the biocataly
- Activity in this context and in connection with the determination of the activity of the enzyme E 12 is generally understood in particular the reaction of acetoacetyl-CoA to acetoacetate and CoA.
- Acyl-CoA hydrolases which are preferred according to the invention are selected from
- a preferred fourth embodiment of the method according to the invention is characterized in that the oxyhydrogen gas bacterium in addition to the increased activity of the enzyme Ei and optionally E 12 a compared to its wild type increased activity of an enzyme E 13 , which is the reaction
- the enzyme E 13 is preferably selected from acetoacetate decarboxylases from the
- Enzyme class EC 4.1.1.4 or from acetone: C02 Ligases from the enzyme class EC 6.4.1 .6.
- Acetoacetate decarboxylases preferred according to the invention are selected from the list NPJ 49328.1, YP_001310906.1 and CAQ57986.1
- Amino acid residues are altered by deletion, insertion, substitution or a combination thereof with respect to the abovementioned reference sequences and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein, the increase in the activity of the biocatalyst used
- Cells ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of the biocatalyst without the presence of the reference protein is understood, wherein under Activity in this context and in connection with the determination of the activity of the enzyme E 13 is generally understood in particular the reaction of acetoacetate to acetone and C0 2 .
- Acetone preferred according to the invention is C02 ligases are selected from the list of oligomeric proteins with sequences.
- C02 ligase A method for determining the activity of acetone: C02 ligase is described by Miriam K. Sluis et al in Proc. Natl. Acad Sci U S A. 1997 Aug. 5; 94 (16): 8456-8461, acetoacetate, AMP and orthophosphate being used here as substrates.
- the oxyhydrogen bacterium has increased activities in the combinations E "
- a fifth embodiment of the inventive method is characterized in that the organic substance is propan-2-ol, preferably E- ⁇ is selected from acetyl-CoA: acetyl-CoA C-acetyltransferases, and the oxyhydrogen bacterium compared one increased to its wild-type activity of an enzyme E 12 , which is the reaction
- the enzyme E 12 is preferably selected from acetoacetyl-CoA: acetate / acyl: CoA transferases from the enzyme class EC: 3.1.2.1 1, from butyrate-acetoacetate-CoA transferases from the enzyme class EC 2.8.3.9 or from acyl-CoA-hydrolases from the enzyme class EC 3.1 .2.20.
- Acetoacetyl-CoA preferred according to the invention: acetate / acyl: CoA transferases are selected from the list
- the alpha subunit is selected from NP_149326.1, YP_001310904.1 and CAQ57984.1 and the
- beta subunit is selected from NP_149327.1, YP_001310905.1 and CAQ57985.1 and proteins having a polypeptide sequence in which up to 60%, preferably up to 25%, more preferably up to 15%, in particular up to 10, 9, 8 , 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the aforementioned reference sequences by deletion, insertion, substitution or a combination thereof are changed and still at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90% of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein, the increase in the activity of the cells used as biocatalyst, ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry matter ( cell dry weight) [U / g CDW]) in comparison to the activity of the biocatalyst without the presence of the reference protein, whereby under the activity in this Zusa mmenhang and in connection
- Butyrate acetoacetate-CoA transferases preferred according to the invention are selected from ctfA and ctfB from Clostridium acetobutylicum and atoD and atoA from Escherichia coli as well as proteins having a polypeptide sequence in which up to 60%, preferably up to 25%, particularly preferably up to 15% in particular, up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues are altered from the abovementioned reference sequences by deletion, insertion, substitution or a combination thereof and which is at least 50%, preferably 65% %, more preferably 80%, in particular more than 90% of the activity of the protein having the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein, the increase in the activity of the cells used as biocatalyst, ie the amount converted per unit time based on the used cell volume (units per gram of cell dry weight [U / g CDW]) compared to the activity of the biocataly
- Acyl-CoA hydrolases which are preferred according to the invention are selected from
- a preferred fifth embodiment of the process according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and, where appropriate, E 12 , the oxyhydrogen bacterium has an increased activity of an enzyme compared to its wild-type
- the enzyme E 13 is preferably selected from acetoacetate decarboxylases from the
- Enzyme class EC 4.1.1 .4 or from acetone: C02 Ligases from the enzyme class EC 6.4.1 .6.
- Acetoacetate decarboxylases preferred according to the invention are selected from the list
- Amino acid residues are altered by deletion, insertion, substitution or a combination thereof with respect to the abovementioned reference sequences and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein, the increase in the activity of the biocatalyst used
- Cells ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of the biocatalyst without the presence of the reference protein is understood, wherein under Activity in this context and in connection with the determination of the activity of the enzyme E 13 is generally understood in particular the reaction of acetoacetate to acetone and C0 2 .
- a method for determining the activity of acetone: C02 ligase is described by Miriam K. Sluis et al in Proc Natl. Acad. See USA 1997 August 5; 94 (16): 8456-8461, acetoacetate, AMP and orthophosphate being used here as substrates.
- a further preferred fifth embodiment of the method according to the invention is characterized in that, in addition to the increased activity of the enzyme Ei and optionally E 12 , and / or E 13, an increased activity of an enzyme E 14 , which enhances the conversion compared to its wild-type
- the enzyme E 14 is preferably selected from propan-2-ol: NADP + oxidoreductase from the enzyme class EC: 1 .1.1.80.
- the oxyhydrogen bacterium has increased activities in the combinations E-
- the propan-2-ol in process step C) is isolated by distillation from the aqueous solution as an azeotrope.
- Methods for the isolation of propan-2-ol are described, inter alia, in Lei Zhigang, Zhang Jinchang, Chen Biaohua Separation of aqueous isopropanol by reactive extractive distillation in Journal of Chemical Technology and Biotechnology Volume 77, Issue 1 1 pages 1251-1254, November 2002, Lloyd Berg et al., Separation of the propylene alcohols from water by azeotropic or extractive distillation US5085739 and Berg, Lloyd Separation of ethanol, isopropanol and water mixtures by azeotropic distillation US5762765 described.
- a particularly preferred fifth embodiment of the method according to the invention comprises a method step D).
- the isolated propan-2-ol in process step D) by chemical
- a sixth embodiment of the process according to the invention is characterized in that the organic substance is 2-hydroxyisobutyric acid or a salt of 2-hydroxyisobutyric acid, preferably E-1 is selected from acetyl-CoA: acetyl-CoA C-acetyltransferases, the oxyhydrogen bacterium optionally an increased compared to its wild-type activity of an enzyme E 2 , which is the reaction
- Enzymes E 2 preferred in this context are those given as preferred in the first embodiment.
- the enzyme E 15 is preferably a hydroxyl isobutyryl-CoA mutase, an isobutyryl-CoA mutase (EC 5.4.99.13) or a methylmalonyl-CoA mutase (EC 5.4.99.2), in each case preferably a coenzyme B12 dependent mutase.
- the enzyme E 15 is preferably those enzymes which are derived from the
- the enzyme E 15 is a heterodimeric enzyme comprising sequences selected from SEQ ID NO. 78 and 80
- Substitution or a combination thereof are modified and which still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90% of the activity of the protein with the corresponding above-mentioned reference sequence, wherein below 100% activity of the reference protein increases the Activity of used as a biocatalyst
- Cells ie the amount of substance reacted per unit time based on the amount of cells used (units per gram of cell dry weight [U / g CDW]) compared to the activity of the biocatalyst without the presence of the reference protein, wherein the activity in this context and in connection with the determination of the activity of the enzyme E 15 is generally understood in particular the reaction of 3-hydroxybutyryl-coenzyme A to 2-hydroxyisobutyryl-coenzyme A.
- the oxyhydrogen bacterium has increased activities in the combinations on.
- the explosive gas bacterium increased compared to its wild-type amount of MeaB protein, in particular one with Seq ID no. 82, has.
- the MeaB protein is preferably selected from Sequence ID No. 82, YP_001023545.1 ⁇ Methylibium petroleiphilum PM1), YP_001409454.1 ⁇ Xanthobacter autotrophicus Py2), YP_001045518.1 ⁇ Rhodobacter sphaeroides ATCC 17029),
- enzymes E 15 and the MeaB protein can be expressed as fusion proteins, as disclosed, for example, in PCT / EP2010 / 065151 and which are used with particular preference.
- Figure 1 Exemplary acetone and isopropanol production C. necator H 16 pBBR- EcatoDAB
- Figure 2 Exemplary butanol production with C. necator H 16 pBBR-RephaABJ-CaadhE2
- necator (nRBS-EcatoD-nRBS-EcatoA-nRBS-EcatoB-T-EcatoB; Seq ID No. 16) to the C. necator phaA gene encoding the thiolase PhaA (SEQ ID NO: 17), including the phaA ribosome binding site, and the C.
- acetobutylicum ctfAB operon encoding the a and ⁇ -subunit acetyl / butyryl-CoA-acetoacetate: CoA transferase CtfA (SEQ ID NO: 18) and CtfB (SEQ ID NO: 19), including the ctfA and cff ⁇ ribosome binding sites and the ctfAB terminator, where the CtfA and CtfB coding regions are in C.
- necator codon optimized (nRBS-RephaA-nRBS-CactfA-nRBS-CactfB-T-CactfAB; SEQ ID NO: 20) of the ribosome binding site of the C. necator groEL gene (SEQ ID NO: 1), C.
- acetobutylicum thIA gene encoding the thiolase ThIA (SEQ ID NO: 21) and the C. acetobutylicum ctfAB operon encoding the ⁇ and ⁇ subunits of acetyl butyryl CoA acetoacetate: CoA transferase CtfA (Seq ID No. 18) and CtfB (SEQ ID NO: 19), inclusive of the native ctfA and cifss ribosome binding sites and the native cfAAss terminator, respectively, with the ThIA, CtfA and CtfB coding regions codon optimized for translation into C.
- acetobutylicum adc gene encoding the acetoacetate decarboxylase Ade (SEQ ID NO: 24), including the ac / c terminator, where the ThIA, YbgC and Ade coding regions were codon optimized for translation into C. necator (RBS-RegroEL-CathlA-RBS-RegroEL-HiybgC-RBS-RegroEL-Caadc-T-Caadc; Seq ID No. 25) to the E. coli / acZ promoter (Seq ID No. 26), the Ribosome binding site of the C. necator groEL gene (Seq ID No. 1) and the C.
- acetobutylicum adc gene coding for the acetoacetate Decarboxylase Ade (SEQ ID NO: 24), including the ac / c terminator, where the Ade coding region was codon optimized for translation into C. necator (Plac-RBS-RegroEL-Caadc-T-Caadc; Seq ID No.
- EcatoDAB EcatoDAB, pBBR-RephaA-CactfAB, pBBR-CathlA-ctfAB and pBBR-CathlA-HiybgC-Caadc, and corresponding to Seq ID Nos. 28, 29, 30 and 31.
- EcatoDAB caadc, pBBR-RephaA-CactfAB
- adc refer to and correspond to Seq ID Nos. 32, 33 and 34.
- CoA Acetoacetyl-CoA transferase AtoD (SEQ ID NO: 13), the ⁇ -subunit of acetyl-CoA: acetoacetyl-CoA transferase AtoA (SEQ ID NO: 14) and the thiolase AtoB (Seq-ID No. 15), including the atoD, atoA and aio ⁇ ribosome binding sites and the atoB terminator, whereby the regions coding for AtoD, AtoA and AtoB were codon optimized in C. necator (nRBS-EcatoD-nRBS- EcatoA-N RBS EcatoB-T
- Subunit acetyl- / butyryl-CoA-acetoacetate CoA transferase CtfA (SEQ ID NO: 18) and CtfB (SEQ ID NO: 19), including the ctfA and ctfB ribosome binding sites and the cf ⁇ 4ß- Terminator, wherein the CtfA and CtfB coding regions were codon optimized in C. necator (nRBS-RephaA-nRBS-CactfA-nRBS-CactfB-T-CactfAB; Seq ID No. 20) of the ribosome binding site of C. necator groEL gene (SEQ ID NO: 1), C.
- acetobutylicum thIA gene encoding the thiolase ThIA (SEQ ID NO: 21) and the C. acetobutylicum ctfAB operon, encoding the a- and ⁇ -subunit acetyl / butyryl-CoA-acetoacetate: CoA transferase CtfA (SEQ ID NO: 18) and CtfB (SEQ ID NO: 19), including the native ctfA and cff ⁇ ribosome binding sites, respectively, and the native ctfAB terminator, where the regions encoding ThIA, CtfA and CtfB codon optimized for translation into C.
- influenzae ybgC gene encoding the thioesterase YbgC (SEQ ID NO: 23) and finally the ribosome binding site of the C. necator groEL gene (SEQ ID NO: 1), followed by the Cupriavidus necator JMP134 acss gene for the acetone carboxylase beta subunit AcbB (Seq ID No. 9), the ribosome binding site of the C. necator groEL gene (Seq ID No. 1), followed by the Cupriavidus necator JMP134 acbA gene encoding the acetone carboxylase alpha subunit AcbA (Seq ID No. 8) and the
- Cupriavidus necator JMP134 acbC gene encoding the acetone carboxylase gamma subunit AcbC (SEQ ID NO: 86), including the ac / c terminator, where the ThIA and YbgC coding regions were codon optimized for translation into C. necator (RBS-RegroEL-CathlA-RBS-RegroEL-HiybgC-RBS-RegroEL-AcbB-RBS-RegroEL-AcbA-RBS-RegroEL-AcBC-T-Caadc; Seq ID No. 7) 5. the £. coli / acZ promoter (SEQ ID NO: 26), the ribosome binding site of the C.
- necator groEL gene (SEQ ID NO: 1) followed by the Cupriavidus necator JMP134 acbB gene encoding the acetone carboxylase beta subunit AcbB (SEQ ID NO: 9), the ribosome binding site of the C. necator groEL gene (SEQ ID NO: 1), followed by
- Subunit AcbA (SEQ ID NO: 8) and the ribosome binding site of the C. necator groEL gene (SEQ ID NO: 1), followed by the Cupriavidus necator JMP134 acbC gene encoding the acetone carboxylase gamma subunit AcbC (Seq ID No. 86) incl.
- the adc terminator (Plac RBS RegroEL AcbB RBS RegroEL AcbA RBS RegroEL AcBC T Caadc; Seq ID No. 6)
- the resulting expression plasmids were named pBBR-EcatoDAB, pBBR-RephaA-CactfAB, pBBR-CathlA-ctfAB and pBBR-CathlA-HiybgC-ReacbBAC and correspond to Seq ID Nos. 28, 29, 30 and 5.
- Components consist of: the C. necator phaA gene encoding the thiolase PhaA (SEQ ID NO: 17), including the phaA ribosome binding site, the C. necator phaBl gene, encoding the (R) -3-hydroxybutyryl-CoA Dehydrogenase PhaB1 (Seq ID No. 35), including the pftassi ribosome binding site, the Aeromonascaviae phaJ gene, encoding the (R) -3-hydroxybutyryl-CoA deydratase PhaJ (Seq ID No. 36) , including the phaJ ribosome binding site, the ribosome binding site of the C.
- necator groEL gene (SEQ ID NO: 1), the C. acetobutylicum adhE2 gene, encoding the bifunctional butyryl-CoA / butyraldehyde dehydrogenase adhE2 (Seq ID No. 37), and the A. caviae phaJ terminator (SEQ ID NO: 38), where the translation coding regions for PhaJ and AdhE2 were codon optimized in C. necator (nRBS-RephaA-nRBS- RephaB1-nRBS-AcphaJ-RBS-RegroEL-CaadhE2-T-AcphaJ; Seq ID No. 39) to the C.
- necator phaA gene encoding the thiolase PhaA (SEQ ID NO: 17), incl phaA ribosome binding site, the C. necator phaBl 'gene, encoding f for the (R) -3-hydroxybutyryl-CoA dehydrogenase PhaB1 (SEQ ID NO. 35), including the phaB 7-ribosome binding site and the Aeromonascaviae phaJ gene, encoding the (R) -3-hydroxybutyryl-CoA deydratase PhaJ (Seq ID No.
- acetobutylicum hbd gene encoding the (S) -3-hydroxybutyryl-CoA dehydrogenase Hbd (Seq ID No. 41), the ribosome binding site of the C. necator groEL gene (Seq ID No. 1) of the C. acetobutylicum adhE2 gene encoding the bifunctional butyryl-CoA / butyraldehyde dehydrogenase AdhE2 (SEQ ID NO: 37) and the C. acetobutylicum ctfAB terminator (SEQ ID NO: 42); coding for ThIA, Hbd and AdhE2 regions for translation in C. necator (RBS-RegroEL-CathlA-RBS-RegroEL-Cahbd-RegroEL-CaadhE2-T-CactfAB; Seq ID No. 43)
- acetobutylicum hbd gene encoding the (S) -3-hydroxybutyryl-CoA dehydrogenase Hbd (SEQ ID NO: 41) and the C. acetobutylicum cfAA ⁇ terminator (SEQ ID NO: 42), which are for ThIA and Hbd coding regions were optimized for translation into C. necator (RBS-RegroEL-CathlA-RBS-RegroEL-Cahbd-T-CactfAB; Seq ID No. 44).
- E. coli / acZ promoter (SEQ ID NO: 26), the C. necator etfBA bcd operon, encoding the ⁇ subunit of the electron transfer protein EtfB (Seq ID No. 45) , the a-subunit of the electron transfer protein EtfA (Seq ID No. 46) and the butyryl-CoA dehydrogenase Bcd (SEQ ID NO: 46), incl. the eifss, etfA and bcd ribosome binding sites and the C.
- E. coli / acZ promoter (Seq ID No. 26), the C. acetobutylicum c / f gene, coding for crotonase Crt (Seq ID No. 50), incl. f-ribosome binding site, the C.
- acetobutylicum etfBA-bcd operon encoding the ⁇ -subunit of the electron transfer protein EtfB (SEQ ID NO: 51), the ⁇ -subunit of the electron transfer protein EtfA (SEQ ID NO: 52) and the butyryl CoA dehydrogenase Bcd (SEQ ID NO: 53), including the etfB, etfA and bcd ribosome binding sites, and the C.
- Expression vector pBBR1 MCS-2 (SEQ ID NO: 10) is cloned so that the expression of the genes is under the control of the E. coli / acZ promoter.
- the resulting expression plasmids were named pBBR-RephaABJ-CaadhE2, pBBR-RephaABJ, pBBR-CathlA-hbd-adhE2-ctfAB and pBBR-CathlA-hbd-ctfAB, and correspond to Seq ID Nos. 56, 57, 58 and 59.
- the expression cassette Plac-nRBS-ReetfB-nRBS-ReetfA-nRBS-Rebcd-nT was then transcribed via Hind ⁇ / Bam ⁇ into the vectors pBBR-RephaABJ-CaadhE2 and pBBR-RephaABJ (Seq-ID Nos. 56 and 57 ) cloned.
- the resulting expression plasmids were named pBBR-RephaABJ-CaadhE2
- the resulting expression plasmids were identified as pBBR-RephaABJ-CaadhE2
- the expression cassette Plac-nRBS-CaetfB-nRBS-CaetfA-nRB-Cabcd-T-Rebcd was then transcribed via Hind ⁇ / Bam ⁇ into the vectors pBBR-RephaABJ-CaadhE2 and pBBR-RephaABJ (SEQ ID Nos. 56 and 57).
- the resulting expression plasmids were named pBBR-RephaABJ-CaadhE2
- a synthetic expression cassette was synthesized consisting of the following components: 1 . the C. necator groEL promoter (Seq ID No. 68), the ribosome binding site of the C. necator groEL gene (Seq ID No. 1), followed by Jeotgalicoccus sp. ATCC 8456 o / eT gene encoding the terminal olefin-forming fatty acid decarboxylase OleT (SEQ ID NO: 69), the ribosome binding site of the C. necator gro £ Z gene (SEQ ID NO: 1) and finally the C.
- Cabuk-T-Captb-buk was then transfected via BamH ⁇ / Sac ⁇ into the vectors pBBR-RephaABJ
- the resulting expression plasmids were identified as pBBR-RephaABJ
- Embodiment 5 Construction of plasmids for the preparation of 2-propanol with Cupriavidus necator
- Codon-optimized region for translation in C. necator PRegroESL-RBS-Cbadh-T-Rebcd; SEQ ID NO: 12).
- the resulting expression plasmids were designated as pBBR-EcatoDAB
- Cbadh refer to and correspond to Seq ID Nos. 83, 84 and 85.
- Embodiment 6 Generation of vector pBBR 1 MCS-2 :: HCM-phaA and pBBR 1 MCS-2 :: meaBhcmA-hcmB-phaA
- the vector pBBR1 MCS-2 :: HCM-phaA was generated starting from the plasmid pBBR1 MCS-2 :: HCM (generation and properties described in patent application EP12173010).
- a synthetic expression cassette was synthesized, consisting of the following components:
- the plasmid pBBR1 MCS-2 :: HCM was linearized with the Restricktionsendonuklease Xbal and then with the also restricted by XbaI and so prepared
- the expression of the genes takes place after successful cloning under control of the E. coli lacZ promoter.
- the resulting expression plasmid is designated pBBR1 MCS-2 :: HCM-phaA and corresponds to SEQ ID NO. 88th
- the vector pBBR1 MCS-2 :: meaBhcmA-hcmB-phaA was generated starting from the plasmid pBBR1 MCS-2 :: meaBhcmA-hcmB (generation and properties described in patent application WO201 1/057871).
- For the construction was a synthetic
- Synthesized expression cassette consisting of the following components: 1 . the C. necator phaA gene encoding the thiolase PhaA (SEQ ID NO: 17), incl. the p /? a4 ribosome binding site; up and downstream of the sequence will be Xbal
- the plasmid pBBR1 MCS-2 :: meaBhcmA-hcmB was linearized with the Restricktionsendonuklease Sacl and then ligated with the also restricted by Sacl and thus prepared expression cassette nRBS-RephaA. All molecular biological work is carried out in a manner known to the person skilled in the art.
- the expression of the genes takes place after successful cloning under the control of the E. coli lacZ promoter.
- the resulting expression plasmid is designated pBBR1 MCS-2 :: meaBhcmA-hcmB-phaA and corresponds to SEQ ID NO. 89th
- Exemplary embodiment 7 Introduction of plasmids for the production of acetone, 2-propanol, 1-butanol, butyrate, 2-hydroxyisobutyric acid and 1-propene in Cupriavidus necator
- the plasmids are transferred into competent E. coli S 17-1 cells, one strain , with which the conjugative transfer of plasmids and others in Cupriavidus necaior strains is possible.
- a spotmating conjugation as described in FRIEDRICH et al., 1981, Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus J Bacteriol 147: 198-205) with the respective plasmids carrying £.
- transconjugants which carry the respective plasmids and denote the corresponding strains as follows:
- Exemplary embodiment 8 Quantification of acetone, 2-propanol, 1-butanol,
- the quantification of butanol and butyrate from a fermentation broth is carried out by means of HPLC / RID and DAD.
- Mobile phase Mobile phase A1: H 2 O (Millipore) + 5 mM aqueous H 2 S0 4
- Measuring range For all analytes approx. 0.100 g / L - 12.0 g / L
- Quantification standard is trimethylpropionic acid.
- the determination of the analytes acetone, isopropanol and 1-propene in the aqueous phase is carried out by means of headspace GC / FID measurement and standard addition. The most important
- Exemplary embodiment 9 Preparation of acetone, 2-propanol, 1-butanol, butyrate and 1-propene with recombinant Ralstonia eutropha cells
- the medium consists of (NH 4 ) 2 HP0 4 2.0 g / l; KH 2 P0 4 2.1 g / l; MgS0 4 * 7H 2 0 0.2 g / l; FeCl 3 * 6H 2 0 6 mg / l; CaCl 2 x 2 H 2 O 10 mg; Trace element solution (Pfennig and Lippert, 1966) 0.1 ml.
- the trace element solution consists of Titriplex III 10 g / l FeS0 x 7 H 2 0 4 g / l ZnS0 4 x 7 H 2 0 0.2 g / 1, MnCl 2 ⁇ 4 H 2 O 60 mg / l, H 3 B0 3 0.6 g / l, CoCl 2 ⁇ 6 H 2 O 0.4 g / l, CuCl 2 ⁇ 2 H 2 O 20 mg / l , NiCl 2 x 6 H 2 0 40 mg / l, Na 2 Mo 4 x 2 H 2 0 60 mg / l together.
- the medium of the preculture was additionally supplemented with fructose 5 g / l, kanamycin 300 ⁇ g / ml.
- the flasks were inoculated 1% (v / v) from a cryoculture.
- the cultures are incubated on a shaker at 30 ° C and 150 rpm for 24 h. Thereafter, the cultures are combined and determined an OD 6 oo of about 3.5.
- the main culture is chemolithoautotrophic in a 21 stainless steel reactor, Biostat B from Sartorius, filled with 1 -2 l medium of composition (NH 4 ) 2 HP0 2.0 g / l, KH 2 P0 2.1 g / l, MgS0 4 x 7 H 2 0 3 g / l, FeCl 3 * 6 H 2 0 6 mg / l, CaCl 2 x 2 H 2 0 10 mg, biotin 1 mg / l, thiamine HCl 1 mg / l, Ca-pantothenate 1 mg / l, nicotinic acid 20 mg / l, trace element solution 0.1 ml and polypropylene glycol (PPG 1000 diluted 1: 5 with water).
- PPG 1000 diluted 1: 5 with water polypropylene glycol
- the cultivation is carried out at 30 ° C, 500 - 1500 rpm, pH 7.
- the pH is unilaterally controlled with 1 M NaOH. Fumigation is carried out with a gas mixture of H 2 90%, C0 2 6%, 0 2 4% with an overpressure of 0 - 2 bar with a gassing rate of 0.19 vvm.
- the reactor is inoculated with 0.1% of the preculture. For this purpose, the required volume of preculture in 50 ml Falcontubes 10 min, centrifuged at 20 ° C and 4500 rpm and resuspended in 10 ml phosphate buffered saline. The washing is repeated 3 times, the last resuspension is carried out in 10 ml of main culture medium.
- the cultivation period is 76 - 150 h.
- 10 ml of sample are withdrawn via a septum using a syringe with a sterile cannula. Determined is OD 6 oo, BTM and the expected product according to strain according to example 7.
- Production phase used by a plasmid-bearing Cupriavidus necator In this approach, the bacterium absorbs H 2 and C0 2 from the gaseous phase and forms 2HIB.
- pressure-resistant glass bottles which can be hermetically sealed with a butyl rubber stopper, were used.
- plasmid-carrying C. necator strains the strains C. necator pBBR1 MCS2 :: HCM and C necator pBBR1 MCS2 :: HCM-phaA were used.
- Hydroxyisobutyric acid first streaked on LB-R agar plates with antibiotic and incubated for 3 days at 30 ° C.
- the strains were cultured in 200 ml H16 mineral medium (modified according to Schlegel et al., 1961) in pressure-resistant 500 ml glass bottles.
- the medium consisted of Na 2 HPO 4 ⁇ 12 H 2 O 9.0 g / L; KH 2 P0 4 1, 5g / l; NH 4 Cl 1, 0 g / l; MgSO 4 ⁇ 7H 2 O 0.2 g / l; FeCl 3 * 6H 2 0 10 mg / l; CaCl 2 x 2 H 2 O 0.02 g / l; Trace element solution SL-6 (Pfennig, 1974) 1 ml / l.
- the trace element solution was composed of ZnS0 4 x 7 H 2 0 100 mg / l, MnCl 2 x 4 H 2 0 30 mg / l, H 3 BO 3 300 mg / l, CoCI 2 x 6 H 2 0 200 mg / l, CuCl 2 ⁇ 2 H 2 O 10 mg / l, NiCl 2 ⁇ 6 H 2 O 20 mg / l, Na 2 Mo 4 ⁇ 2 H 2 O 30 mg / l together.
- the pH of the medium was adjusted to 6.8 by addition of 1 M NaOH.
- the flasks were inoculated with a single colony from the incubated agar plates and cultivated chemolithoautotrophically on a N 2 / H 2 / O 2 / C0 2 mixture (ratio
- the cultures were incubated in an open water bath shaker at 28 ° C, 150 rpm and a fumigation of 1 l / h for 137 h to an OD> 1, 0. Gas was introduced into the medium through a gassing frit having a pore size of 10 ⁇ m, which was attached to a gassing tube in the middle of the reactor. The cells were then centrifuged off, washed with 10 ml of washing buffer (0.769 g / L NaOH, at least 1 h at 28 ° C. and 150 rpm with a gas containing 6% C0 2 ) and again centrifuged off.
- washing buffer 0.69 g / L NaOH, at least 1 h at 28 ° C. and 150 rpm with a gas containing 6% C0 2
- T (M) SP Sodium trimethylsilylpropionate
- Production phase used by a plasmid-bearing Cupriavidus necator In this approach, the bacterium absorbs H 2 and C0 2 from the gaseous phase and forms 2HIB.
- pressure-resistant glass bottles which can be hermetically sealed with a butyl rubber stopper, are used.
- plasmid-carrying C. necator strains the strains C. necator pBBR1 MCS2 :: meaBhcmA-hcmB and C. necator pBBR1 MCS2 :: meaBhcmA-hcmB-phaA are used.
- the plasmid-bearing C. necaior strains are used to study the formation of 2-
- Hydroxyisobutyric acid first streaked on LB-R agar plates with antibiotic and incubated for 3 days at 30 ° C.
- the strains are cultured in 200 ml H16 mineral medium (modified according to Schlegel et al., 1961) in pressure-resistant 500 ml glass bottles.
- the medium consists of Na 2 HPO 4 x 12 H 2 0 9.0 g / l; KH 2 P0 4 1, 5g / l; NH 4 Cl 1, 0 g / l; MgS0 4 * 7H 2 0 0.2 g / l; FeCl 3 * 6H 2 0 10 mg / l;
- the trace element solution consists of ZnS0 4 ⁇ 7 H 2 O 100 mg / l, MnCl 2 ⁇ 4 H 2 O 30 mg / l, H 3 O 3 300 mg / l, CoCl 2 ⁇ 6 H 2 O 200 mg / l, CuCl 2 ⁇ 2 H 2 0 10 mg / l, NiCl 2 x 6 H 2 0 20 mg / l, Na 2 Mo 4 x 2 H 2 0 30 mg / l together.
- the pH of the medium is adjusted to 6.8 by addition of 1 M NaOH. All media used are supplied with 300 ⁇ g ml kanamycin and 76 nM CoB 12 .
- the flasks are inoculated with an individual colony from the agar plates incubated and the cultivation takes place chemolithoautotrophically on a N 2 / H 2/0 2 / C0 2 mixture (ratio
- the cultures are incubated in an open water bath shaker at 28 ° C, 150 rpm and a fumigation of 1 l / h for 137 h to an OD> 1, 0.
- the gas is introduced into the medium through a gassing frit with a pore size of 10 ⁇ m, which is attached to a gassing tube in the middle of the reactor.
- the cells are then centrifuged off, washed with 10 ml of washing buffer (0.769 g / L NaOH, at least 1 h at 28 ° C and 150 rpm with a gas with 6% C0 2 effetgast) and again centrifuged.
- the gas is introduced into the medium through a gassing frit with a pore size of 10 ⁇ m, which is attached to a gassing tube in the middle of the reactors.
- a gassing frit with a pore size of 10 ⁇ m, which is attached to a gassing tube in the middle of the reactors.
- the product concentration is determined by means of semi-quantitative 1 H NMR spectroscopy.
- the internal quantification standard is sodium trimethylsilylpropionate (T (M) SP).
- necator pBBR1 MCS2 meaBhcmA-hcmB up to 1 13 mg / l 2HIB, while in the strain C.
- necator pBBR1 MCS2 meaBhcmA-hcmB-phaA form up to 156 mg / l 2HIB.
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US14/898,417 US20160138058A1 (en) | 2013-06-14 | 2014-05-30 | Method for producing organic compositions from oxyhydrogen and co2 via acetoacetyl-coa as intermediate product |
CA2915524A CA2915524A1 (en) | 2013-06-14 | 2014-05-30 | Method for producing organic compositions from oxyhydrogen and co2 via acetoacetyl-coa as intermediate product |
CN201480043976.5A CN105431538A (zh) | 2013-06-14 | 2014-05-30 | 经由作为中间产物的乙酰乙酰辅酶a由氢氧气和co2制备有机化合物的方法 |
KR1020167000831A KR20160019940A (ko) | 2013-06-14 | 2014-05-30 | 산수소 및 CO2로부터 중간체 생성물로서의 아세토아세틸-CoA를 통해 유기 조성물을 제조하는 방법 |
BR112015031211A BR112015031211A2 (pt) | 2013-06-14 | 2014-05-30 | Método para a produção de composições orgânicas de oxihidrogênico e co2 através de acetoacetil-coa-redutase como produto intermediário |
EP14727495.5A EP3008194A2 (de) | 2013-06-14 | 2014-05-30 | Erfahren zur herstellung von organischen verbindungen aus knallgas und co2 über acetoacetyl-coa als zwischenprodukt |
MX2015016967A MX2015016967A (es) | 2013-06-14 | 2014-05-30 | Metodo para producir composiciones organicas de oxihidrogeno y co2 via acetoacetil-coa como producto intermedio. |
RU2016100993A RU2016100993A (ru) | 2013-06-14 | 2014-05-30 | СПОСОБ ПОЛУЧЕНИЯ ОРГАНИЧЕСКИХ КОМПОЗИЦИЙ ИЗ ГРЕМУЧЕГО ГАЗА И CO2 ЧЕРЕЗ АЦЕТОАЦЕТИЛ-КоА В КАЧЕСТВЕ ПРОМЕЖУТОЧНОГО ПРОДУКТА |
ZA2016/00119A ZA201600119B (en) | 2013-06-14 | 2016-01-07 | Method for producing organic compositions from oxyhydrogen and co2 via acetoacetyl-coa as intermediate product |
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WO2016097293A1 (en) * | 2014-12-19 | 2016-06-23 | Global Bioenergies | Process for the enzymatic production of 1-butene from 2-pentenoyl-coa |
EP3301181A1 (de) | 2016-09-30 | 2018-04-04 | B.R.A.I.N. Biotechnology Research And Information Network AG | Autotrophe mikrobielle elektrosynthese |
CN108884467A (zh) * | 2015-10-13 | 2018-11-23 | 朗泽科技新西兰有限公司 | 包含产能发酵途径的基因工程菌 |
US10239898B2 (en) | 2016-12-22 | 2019-03-26 | Evonik Degussa Gmbh | Compounds based on adducts with isocyanates for coating compositions |
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EP2944697A1 (de) | 2014-05-13 | 2015-11-18 | Evonik Degussa GmbH | Verfahren zur Herstellung von Nylon |
EP3390622B1 (de) | 2015-12-17 | 2020-05-13 | Evonik Operations GmbH | Genetisch modifizierte acetogene zelle |
CN109790106A (zh) | 2016-07-27 | 2019-05-21 | 赢创德固赛有限公司 | N-乙酰基高丝氨酸 |
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2014
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- 2014-05-30 KR KR1020167000831A patent/KR20160019940A/ko not_active Application Discontinuation
- 2014-05-30 CN CN201480043976.5A patent/CN105431538A/zh active Pending
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US20160138058A1 (en) | 2016-05-19 |
RU2016100993A (ru) | 2017-07-17 |
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KR20160019940A (ko) | 2016-02-22 |
CA2915524A1 (en) | 2014-12-18 |
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ZA201600119B (en) | 2018-11-28 |
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