WO2009046929A2 - Fixation biotechnologique du dioxyde de carbone - Google Patents

Fixation biotechnologique du dioxyde de carbone Download PDF

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WO2009046929A2
WO2009046929A2 PCT/EP2008/008351 EP2008008351W WO2009046929A2 WO 2009046929 A2 WO2009046929 A2 WO 2009046929A2 EP 2008008351 W EP2008008351 W EP 2008008351W WO 2009046929 A2 WO2009046929 A2 WO 2009046929A2
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activity
sequence seq
ncbi
seq
nucleic acid
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PCT/EP2008/008351
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WO2009046929A8 (fr
WO2009046929A3 (fr
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Joachim W. Schmid
Klaus Mauch
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Insilico Biotechnology Ag
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Publication of WO2009046929A8 publication Critical patent/WO2009046929A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12P7/56—Lactic acid

Definitions

  • the invention relates to the biotechnological use of anorgani- see electron donors, especially of gaseous hydrogen for fixing carbon dioxide as a carbon source for the synthesis of organic compounds as energy sources and value products by fermentation in microorganisms.
  • Ralstonia metallidurans Ralstonia metallidurans (Ralstonia eutropha, Alcaligenes eutrophus), optionally autotrophic pseudomonads such as P. aeruginosa, P. saccharophila, P. facilis, P. hydrogenovora, P. hydrogenothermophila, P. carboxydi- hydrogena, P Compransoris, P. carboxydovarans, P. gasotropha and P. stanieri; Rhodopseudomonas palustris, R.
  • Clostridia capable of accepting hydrogen and homoacetate fermentation such as C. aceticum, C. magnum, C. thermoaceticum, C. scatologenes, C. deformicoaceticum and C. thermoautotrophicum; Acetobacterium woodii; Acetogenium kivui; Species of Rhodospirillales (C source carbon monoxide) and Rhodocyclus gelatinosus.
  • the invention is based on the technical problem of providing an improved process for the biotechnological production of organic compounds, especially in the form of C2 to C6 bodies, mainly from carbon dioxide as a carbon source with hydrogen as an "energy source” and means for carrying out this process
  • a technical problem also exists in making such a method usable, in particular, in known microorganisms or cell lines established in biotechnological / fermentative processes, which can be transfected with little effort using conventional recombination technologies to allow a stable synthesis of organic compounds such as carboxylic acids, short-chain fatty acids and alcohol mainly from CO2 as a carbon source and with hydrogen as an "energy source” with high yield, both in anaerobic as well may also occur in aerobic processes.
  • transgenic biological cell in particular a recombinant microorganism or parts thereof, which contains an enzyme equipment which is selected from:
  • cytoplasmic preferably NAD-reducing hydrogenase activity, and / or
  • Enzyme activity suitable for realizing a CO2-fixing metabolic pathway, selected from:
  • RTCC reductive tricarboxylic acid cycle
  • Citrate lyase activity Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
  • a cell according to the invention especially in the genome, especially in an expression cassette and operably linked to a promoter, contains at least one nucleic acid molecule with a nucleotide sequence which codes for at least one of the aforementioned enzyme activities.
  • the invention also relates to an expression cassette and a vector containing this expression cassette, which are suitable for mediating the expression of the enzyme activities provided according to the invention in the host cell.
  • the expression cassette for transformation of a host cell contains:
  • At least one heterologous nucleic acid molecule encoding enzyme activity selected from: Formyl-tetrahydrofolate ligase activity;
  • Citrate lyase activity Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
  • the invention preferably further provides that the cell additionally contains:
  • the Calvin-Benson-Bassham cycle can additionally be realized for realizing the CO2 fixation, specifically by expressing at least one heterologous enzyme activity selected from:
  • transgenic cell containing at least one recombinant nucleic acid molecule having a nucleotide sequence encoding at least one enzyme activity selected from:
  • Hydrogenase activity preferably cytoplasmic and / or membrane-bound hydrogenase activity
  • Enzyme activity suitable for realizing a CO2-fixing metabolic pathway, selected from:
  • RTCC Reductive tricarboxylic acid cycle
  • an expression cassette according to the invention for transforming a host cell additionally contains: phosphoribulosis kinase activity and ribulose bisphosphate carboxylase activity, nucleotide sequences being present in the expression cassette, in a transformation vector containing the expression cassette and in the host cell to be transformed coding for enzyme activity selected from fructose bisphosphatase activity and fructose bisphosphate aldolase activity, absent or absent or inhibiting this enzyme activity.
  • SC serine cycle serine cycle
  • RTCC reductive tricarboxylic acid cycle
  • the invention also provides a process for the biotechnological production of C2-C6 bodies as product of carbon dioxide as substrate and assimilation of hydrogen, use of transgenic biological cell for the biotechnological production of lactate or lactic acid and alcohol from carbon dioxide.
  • the invention makes use of the surprising finding that in a suitable transgenic or recombinant biological cell, in particular by recombinant expression of the abovementioned enzymes or enzyme activities, the fixation of carbon dioxide for the production of organic carbon compounds using an electron donor, especially hydrogen, with high Yield is made possible.
  • the invention thus provides means for carrying out biotechnological processes for a novel, highly effective conversion of, in particular, hydrogen and CO.sub.2 into readily transportable and usable fuels, for example alcohols, as well as basic chemicals and valuable substances, for example carboxylic acids and short-chain fatty acids
  • CO2 as a carbon source thus not only enables the realization of CO2-neutral processes, but also allows an independent choice of location due to the ubiquitous availability of CO2.
  • the present invention Above all, the use of hydrogen as an electron donor as an energy source for microbial processes is exploited.
  • the hydrogen is usually prepared from electrochemical processes from water or from the fermentation of organic waste.
  • the cell expresses at least one of the enzyme activities defined above.
  • the cell expresses two, more, and most preferably all of the enzyme activities defined above.
  • the at least one nucleic acid molecule is operatively linked to an expression system, that is to say with a promoter or promoter system. It is understood that preferably a constitutive promoter is used.
  • the wild type is preferably transformed with a suitable expression vector containing the expression cassette.
  • the transformation of the cells according to the invention and the incorporation of the isolated nucleic acid molecules takes place in a manner known per se, preferably by suitably suitable expression vectors or in conjunction with appropriate expression cassettes, preferably including appropriate expression vectors.
  • the de novo synthesis of nucleic acid molecules takes place with the desired nucleotide sequences, optionally the complete expression cassette in a suitable synthesis system.
  • Transgenic biological cell containing, preferably in the genome and in particular functionally linked to a promoter: (1) at least one nucleic acid molecule encoding hydrogenase activity; and
  • Citrate lyase activity Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
  • the transgenic biological cell contains, preferably in the genome and in particular functionally linked to a promoter, optionally in addition: phosphoribulose kinase activity and ribulose bisphosphate carboxylase activity, wherein enzyme activity selected from fructose bisphosphatase activity and fructose bisphosphate- Aldolase activity, not expressed or inhibited.
  • this cell contains, in particular for increasing the synthesis potential, additionally at least one heterologous nucleic acid molecule coding for enzyme activity, selected from:
  • the serine cycle is realized in the cell, preferably in place of the CBB.
  • the cell contains:
  • the cell further has the enzyme activity of a glycine cleavage system.
  • this cell additionally preferably contains at least one heterologous or homologous nucleic acid molecule coding for this enzyme activity.
  • the cell has the enzyme activity selected from:
  • this cell additionally contains formate dehydrogenase activity for realization using the serine cycle.
  • this cell additionally preferably contains at least one heterologous or homologous nucleic acid molecule coding for this enzyme activity.
  • the reductive tricarboxylic acid cycle is realized. The cell contains:
  • nucleic acid molecules encoding all of these activities are present, wherein at least one nucleic acid molecule encodes a heterologous enzyme activity.
  • the cell contains: at least one nucleic acid molecule encoding hydrogenase activity selected from:
  • membrane-bound hydrogenase activity preferably from the microorganism E. coli, selected from hydrogenase hvaABC and hydrogenase hybOCAB;
  • cytoplasmic NAD-reducing hydrogenase activity preferably from the microorganism Ralstonia eutropha with at least the structural genes hoxFUYH.
  • the invention basically envisages in all variants that only those genes are recombined heterologously which encode such enzyme activities of a metabolic pathway identified according to the invention. which do not belong to the enzyme equipment of the wild type of the recombined cell.
  • the homologous expression is preferably modified for homologous overexpression.
  • at least one of the enzyme activities involved is expressed both homologously and heterologously.
  • this cell expresses or has a lactate dehydrogenase activity.
  • this cell additionally preferably contains at least one heterologous or homologous nucleic acid molecule coding for this enzyme activity.
  • the invention preferably provides that in the cell according to the invention the intermediates formed during the CO2 assimilation can be converted predominantly or exclusively via the activity of a lactate dehydrogenase to lactate.
  • the expression of at least one lactate transporter is additionally realized in the cell according to the invention.
  • the invention provides that the intermediates formed in the CO2 assimilation in the cell according to the invention can be converted predominantly or exclusively via the activity of a lactate oxidoreductase to lactate.
  • the invention provides that the intermediates formed in the course of CO2 assimilation in the cell according to the invention can be converted predominantly or exclusively via the methylglyoxal route to lactate.
  • the invention provides that the intermediates formed in the CO2 assimilation in the cell according to the invention can be converted predominantly or exclusively via the activity of an aldehyde dehydrogenase to lactate.
  • an aldehyde dehydrogenase for enantiomerically pure lactate, the expression of activities of an enantiomer-selective enzyme is realized in the cell according to the invention.
  • the activity of optionally present homologous enzymes with lacking or reverse stereospecificity and, if present, lactate racemase activity is preferably inhibited in the cell according to the invention or preferably eliminated by de-ling of the genes involved.
  • the invention preferably includes those transgenic or recombinant cells selected or derived from bacteria, cyanobacteria, fungi and yeasts.
  • the cell is preferably selected from bacteria of the genera Escherichia, Corynebacterium, Ralstonia, Clostridium, Pseudomonas, Bacillus, Lactobacillus and Lactococcus.
  • the cell is particularly preferably selected from bacteria of the species Escherichia coli, Corynebacterium glutamicum, Ralstonia eutropha, Pseudomonas putida, Lactobacillus plantarum and Clostridium acetobutylicum.
  • the invention also includes those biological cells which are or derived from a synthetic microorganism.
  • a cell is also understood to mean membrane vesicles, membrane particles and parts and fragments thereof.
  • the enzyme activities are preferably located in the "inner” or cytosol or cytoplasm of the cell.
  • at least one of the enzyme activities is associated with a membrane, especially on a cell membrane.
  • the invention accordingly also comprises or parts or fragments of a transgenic invention Microorganism, which are preferred and in a conventional manner and while maintaining the enzyme activity associated or bound to carrier structures.
  • the invention also includes as a "cell" a biocatalyst in which the enzyme activities are realized by isolated or synthesized enzyme proteins having the amino acid sequences given herein, preferably by bypassing cellular expression and translation systems by incorporation of the enzyme proteins into the cell or biocatalyst.
  • Recombinant cells particularly recombinant microorganisms, which contain and express the genes according to the invention are produced in a manner known per se by customary recombination technologies. It is understood that the cells or microorganisms selected for recombination have sufficient resistance to the desired metabolic end products. Preference is given to microorganisms for which standardized cultivation conditions have been established in biotechnological production, so that elaborate adaptation of established biotechnological process management can be minimized or largely avoided.
  • the scope of the invention also covers those cells or microorganisms and cell lines which, even in the non-recombined / transgenic form, contain and express at least one or more of the genes coding for one of the enzyme activities provided according to the invention. It is understood that, depending on the activity of the homologous gene present, it may not be necessary to recombine with the analogous heterologous genes.
  • measures known per se for ensuring the homologous expression or overexpression of the homologous gene are made in order to completely eliminate the metabolic pathway according to the invention. Depending on the enzyme equipment of the starting organism, it may additionally be necessary to eliminate or suppress any existing competing metabolic pathways. This can be done by measures known per se.
  • a preferred embodiment of the invention is a transgenic or recombinant E. coli cell, for example derived from the cell lines K12.
  • An alternative preferred embodiment of the invention is a transgenic or recombinant Ralstonia eutropha cell.
  • nucleic acid molecules can be isolated therefrom and amplified by conventional methods.
  • Figure 1 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by the reactions of the Calvin-Benson-Bassham cycle.
  • Figure 2 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by parts of the Calvin-Benson-Bassham cycle.
  • Figures 3 and 4 show the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended to formyl tetrahydrofolate ligase.
  • Figures 5 and 6 show the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly expanded by a formyl tetrahydrofolate ligase and the key enzymes of the serine cycle.
  • FIGS. 7 and 8 show the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli, recombinantly expanded by the key enzymes of the reductive tricarboxylic acid cycle (citrate lyase, oxoglutarate oxidoreductase and fumarate reductase).
  • FIG. 9 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 1. All rivers are hydrogen (100%) based on the intake flow.
  • FIG. 10 shows a flux distribution which shows the synthesis potential for lactate from CO 2 and hydrogen with the reactivity shown in FIG. optimally exploited. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 11 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 3. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 12 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 4. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 13 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 5. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 14 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 6. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 15 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 7. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 16 shows a flux distribution which shows the synthesis potential for lactate from CO 2 and hydrogen with the reactivity shown in FIG. 8. optimally exploited. All rivers are based on the uptake of hydrogen (100%).
  • Hydrogenase activity is understood herein to mean the ability to assimilate elemental hydrogen to form reduction equivalents.
  • the invention thus preferably provides that the host cell is equipped with at least one heterologous hydrogenase operon.
  • at least one homologous hydrogenase activity existing in the WiId type of the cell is additionally expressed.
  • the introduction of the pHG1 megaplasmid from Ralstonia eutropha is preferably provided.
  • the nucleotide sequence of the pHG1 megaplasmid is described in Schwartz et al. 2003 (Schwartz E., Hen A., Cramm R, Eitinger T., Friedrich B., Gottschalk G. (2003).) Complete Nucleotide Sequence of PHG1: A Ralstonia Eutropha H16 Megaplasmid Encoding Key Enzymes of H2-based Lithoautotrophy and Anaerobiosis. J Mol Biol 332, 369-383.) And under Genbank Accession no.
  • the megaplasmid is 452 kbp in size and carries 429 potential genes. Including at least 41 genes for hydrogenase activity. In a preferred variant, it is therefore provided that a shortened or modified megaplasmide is incorporated into the cell according to the invention, which contains at least one hydrogenase operon of the gapase.
  • the invention preferably provides for the recombinant expression of the cytoplasmic NAD-reducing hydrogenase activity from Ralstonia eutropha.
  • the expression of the structural genes hoxFUYH preferably together with the genes involved in the maturation of the enzyme, hypC1, hypD1, hypE1 and hypABF is preferably realized in the cell according to the invention.
  • the expression of the H2 sensor system hoxA, hoxBC, hoxJ is preferred.
  • the heterologous hydrogenase activity preferably originates from the microorganism Ralstonia eutropha, in particular the enzyme NAD hydrogenase (EC 1.12.7.2), or is derived therefrom.
  • the invention makes use of the finding that, above all, the recombinant hydrogenase from Ralstonia eutropha, advantageously comparatively aerotolerant and already uses even the universally usable electron acceptor NAD.
  • the NAD hydrogenase (EC 1.12.7.2) from Ralstonia eutropha is a heterotetramer of 4 subunits.
  • Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 1 for hoxF subunit, SEQ ID NO: 3 for hoxU subunit, SEQ ID NO: 5 for hoxY subunit, and SEQ ID NO: 7 for hoxH subunit.
  • the enzyme protein thus preferably has the amino acid sequences SEQ ID NO: 2, 4, 6, and 8.
  • the invention thus preferably relates to a cell which contains at least one, preferably at least two, at least three or preferably all heterologous nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 1, 3, 5, 7;
  • nucleic acid molecules coding for amino acid molecules which contain or consist of the sequences SEQ ID NO: 2, 4, 6, 8;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably encode a hydrogenase activity.
  • the invention preferably further provides for the expression of the hydrogenase activity: the expression of at least one protein involved in the maturation of the hydrogenase activity.
  • the structural genes are selected from: nucleotide sequences encoding SEQ ID NO: 9 for hypC1.
  • the structural genes are selected from: nucleotide sequences encoding SEQ ID NO: 9 for hypC1.
  • the proteins hypA, hypB and hvpF preferably the coding nucleotide sequences SEQ ID NO: 15 for hypA1, SEQ ID NO: 17 for hvpB1 and SEQ ID NO: 19 for hypF1 provided.
  • SEQ ID NO: 21 for hypA2 SEQ ID NO: 23 for hypB2, and SEQ ID NO: 25 for hypF2.
  • the proteins relevant for the maturation thus preferably have the corresponding amino acid sequences SEQ ID NO: 10, 12, 14, as well as 16, 18, 20 and / or 22, 24, 26.
  • the invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, three, four, five, six, seven, eight or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25;
  • nucleic acid molecules which are suitable for amino acid molecules which have the sequences SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24,
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably encode a maturation protein for hydrogenase activity.
  • the invention preferably further provides in connection with the expression of the hydrogenase activity: the expression of at least one of the proteins involved in the H2 sensor system.
  • the H2 sensor protein is selected from: coding nucleotide sequences SEQ ID NO: 27 for hoxA, SEQ ID NO: 29 for hoxB subunit and SEQ ID NO: 31 for hoxC subunit of hoxBC and SEQ ID NO: 33 for hoxJ ,
  • the H2 sensor proteins therefore preferably have the corresponding amino acid sequences SEQ ID NO: 28, 30, 32 and 34.
  • the H2 sensor system (hoxA, hoxBC, hoxJ) is preferably only realized if a Ralstonia own transcription system is used.
  • the invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, at least three or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 27, 29, 31, 33;
  • nucleic acid molecules coding for amino acid molecules containing or consisting of the sequences SEQ ID NO: 28, 30, 32, 34;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an H 2 sensor Encode protein.
  • the invention provides alternatively or preferably additionally the expression of the activity of a membrane-bound hydrogenase from E. coli.
  • This is especially the enzyme hydrogenase hyaABC and the enzyme hydrogenase hvbOCAB.
  • the invention provides that, alternatively or preferably in addition, the activity of a membrane-bound hydrogenase is expressed.
  • the hydrogenase activity is derived from the microorganism E. coli, especially the enzyme hydrogenase hyaABC or hydrogenase hvbOCAB, or it is derived therefrom.
  • the expression of the membrane-bound hydrogenase hvaABC is realized.
  • the hydrogenase hvaABC from E. coli is a heteromer of 3 subunits. These subunit-encoding nucleotide sequences are SEQ ID NO: 137 for hvaA subunit, SEQ ID NO: 139 for hyaB subunit, and SEQ ID NO: 141 for hyaC subunit.
  • the enzyme protein hydrogenase hvaABC thus, it preferably has the amino acid sequences SEQ ID NO: 138, 140 and 142.
  • the invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, or preferably all, nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 137, 139, 141;
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 138, 140, 142;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b) and preferably encode a hydrogenase activity.
  • the expression of the membrane-bound hydrogenase hybOCAB is realized.
  • the hydrogenase hybOCAB from E. coli is a heteromer of 4 subunits. Nucleotide sequences encoding these subunits are SEQ ID NO: 149 for subunit hybO, SEQ ID NO: 155 for subunit hybC, and SEQ ID NO: 151 for subunit hybA and SEQ ID NO: 153 for subunit hybB.
  • the enzyme protein hydrogenase hybOCAB thus preferably has the amino acid sequences SEQ ID NO: 150, 156, 152 and 154.
  • the invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, at least three or preferably all nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which have the sequences SEQ ID NO:
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 150, 152, 154, 156;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b) and preferably encode a hydrogenase activity.
  • the expression of the associated chaperones hyaD, hyaE and hyaF or hybD, hybE, hybF and hybG is also at least partially realized.
  • the nucleotide sequences encoding the chaperones are SEQ ID NO: 143 for hyaD, SEQ ID NO: 145 for hyaE, and SEQ ID NO: 147 for hyaF.
  • the chaperones thus preferably have the amino acid sequences SEQ ID NO: 144, 146 and 148.
  • the chaperone genes hyaE and hyaF are optional and need not be expressed.
  • chaperones hybD, hybE. HybF and hybG coding nucleotide sequences are SEQ ID NO: 157 for hybP, SEQ IP NO: 159 ITy 1 b_E, SEQ IP NO: 161 for hybF and SEQ IP NO: 163 for hybG.
  • chaperones preferably have the amino acid sequences SEQ ID NO: 158, 160, 162 and 164.
  • the invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, at least three or preferably all nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 143, 145, 147, 157, 159, 161, 163;
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 144, 146, 148, 158, 160, 162, 164;
  • nucleic acid molecules which have at least 50%, preferably at least 50%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably code for hydrogenase chaperones.
  • enzymatic activity of an enzyme involved in CBB herein is meant the ability to react an intermediate of the CBB, Calvin cycle or reductive pentose phosphate pathway as a substrate into another intermediate product of the cycle as a product of the enzyme reaction.
  • 11 enzymes are involved in CBB.
  • the CBB can be divided into three sections: actual carbon fixation (carboxylation) tion), reduction phase (a C3 body is released as product per three cycles) and regeneration phase of the carbon acceptor ribulose-1, 5-bisphosphate.
  • the activities of a phosphoribulose kinase (phosphoribulokinase) and a ribulose bisphosphate carboxylase are considered as key enzyme activities of CBB.
  • the invention provides for the realization of the CBB in the cell according to the invention: the optionally heterologous expression of at least one phosphoribulose kinase activity and one ribulose bisphosphate carboxylase activity.
  • Figure 1 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate in Escherichia coli recombinantly extended by the reactions of the Calvin-Benson-Bassham cycle.
  • the invention provides, above all, that in the cell according to the invention the expression of fructose bisphosphatase activity and / or of fructose bisphosphate aldolase activity by deletion of these Activity-encoding nucleotide sequences is switched off.
  • the invention provides, above all, for predominantly or exclusively the Calvin-Benson-Bassham cycle (CBB) to be implemented for CO2 assimilation, wherein at least one enzyme activity involved in CBB is sufficiently expressed in the cell according to the invention. It is preferably provided that the expression of activities of enzymes selected from: Phosphoribulose kinase activity (phosphoribulokinase) and ribulose bisphosphate carboxylase activity
  • the invention further provides that the Calvin-Benson-Bassham cycle (CBB), while avoiding, inhibiting or deleting at least one, preferably both, enzyme activity optionally present in the wild type of the cell, is selected from:
  • the invention provides that the CBB is realized by inhibiting or deleting a fructose-bisphosphatase activity. This is preferably done according to the invention by inhibiting the expression and / or deletion of at least one nucleotide sequence coding for this enzyme activity or at least one of the genes. In connection with the use of a recombinant E. coli cell, the invention preferably provides for this:
  • the inhibition of the activity of the fructose bisphosphatase I fbp_ preferably by deletion of at least the gene for fbj), represented by the SEQ ID NO: 77 or coding for the amide Noklaresequenz SEQ ID NO: 78, and alternatively or preferably in addition
  • the inhibition of the activity of the fructose bisphosphatase II glpX preferably by deletion of at least the gene for glpX, represented by the SEQ ID NO: 79 or coding for the
  • the invention provides that the CBB is realized by inhibiting or deleting a fructose bisphosphate aldolase activity. This is done according to the invention preferably by inhibiting the expression and / or deletion of at least one nucleotide sequence encoding this enzyme activity or at least one of the genes.
  • the invention preferably additionally or alternatively additionally provides for this:
  • fructose bisphosphate aldolase II activity fbaA by deleting at least the gene for fbaA represented by SEQ ID NO: 81 or coding for the amino acid sequence SEQ ID NO: 82, and alternatively or preferably additionally
  • fructose bisphosphate aldolase I activity fbaB by deletion of at least the gene for fbaB represented by SEQ ID NO: 83 or coding for the amino acid sequence SEQ ID NO: 84.
  • the phosphoribulose kinase activity is from the microorganism Ralstonia eutropha, especially the enzyme Phosphoribulose kinase cbbPp, or is derived from it.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 35.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 36.
  • the ribulose bisphosphate carboxylase activity is derived from the microorganism Ralstonia eutropha, especially the enzyme ribulose bisphosphate carboxylase cbbSp / cbbLp, or is derived therefrom.
  • the ribulose bisphosphate carboxylase is composed of 2 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 37 for subunit cbbLp and SEQ ID NO: 39 for subunit cbbSp.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 40 and 42.
  • the invention preferably provides for the realization of the CBB in the cell according to the invention in the cell, the pHG1 megaplasmid from Ralstonia eutropha is present and there, in addition to the hydrogenase operon especially the phosphoribulose kinase operon and / or the Ribulose bisphosphate carboxylase operon are expressed. It is provided according to the invention to use a truncated or modified pHG1 megaplasmid, wherein the operons coding for fructose bisphosphatase activity and fructose bisphosphate aldolase activity are deleted or suppressed.
  • the invention preferably relates to a cell which contains at least one, preferably at least two or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 35, 37, 39;
  • nucleic acid molecules coding for amino acid molecules which contain or consist of the sequences SEQ ID NO: 36, 38, 40;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably a phosphoribulose kinase activity and / or encode a ribulose bisphosphate carboxylase activity.
  • the expression of a fructose-6-phosphate aldolase activity is realized.
  • the fructose-6-phosphate aldolase activity is derived from the microorganism E. coli, especially the enzyme fructose-6-phosphate aldolase fsaA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 85.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 86.
  • the enzyme is fructose 6-phosphate aldolase fsaB, or it is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 87.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 88.
  • the expression of a glycerol-3-phosphate dehydrogenase activity is realized.
  • the glycerol-3-phosphate dehydrogenase Activity from the microorganism E. coli, especially the enzyme glycerol-3-phosphate dehydrogenase gpsA, or is derived from it.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 89.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 90.
  • the enzyme glycerol-3-phosphate dehydrogenase qlpABC is preferred, or it is derived therefrom. According to the current state of knowledge, this glycerol-3-phosphate dehydrogenase is composed of 3 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 91 for subunit qlpA, SEQ ID NO: 93 for subunit qlpB and SEQ ID NO: 95 for subunit qlpC.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 92, 94 and 96.
  • the enzyme glycerol-3-phosphate dehydrogenase qlpD is preferred, or it is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 97.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 98.
  • the expression of a glycerol kinase activity is realized.
  • the glycerol kinase activity is derived from the microorganism E. coli, especially the enzyme glycerol kinase glpK, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 99.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 100.
  • the expression of a glycerol dehydrogenase activity is realized.
  • the glycerol dehydrogenase activity comes from the microorganism E. coli, especially the enzyme glycerol dehydrogenase gldA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 101.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 102.
  • transaldolase activity preferably originates from the microorganism E. coli, in particular the enzyme is the transaldolase talA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 103.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 104.
  • the enzyme is the transaldolase talB, or it is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 105.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 106.
  • the invention thus preferably relates to a cell which contains at least one, preferably at least two, three, four, five, six, seven, eight, nine, ten or preferably all nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105;
  • nucleic acid molecules which are suitable for amino acid molecules having the sequences SEQ ID NO: 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 include or consist of encode;
  • nucleic acid molecules which have at least 50%, preferably at least 50%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an enzyme activity selected from:
  • Fructose-6-phosphate aldolase activity Fructose-6-phosphate aldolase activity, glycerol-3-phosphate dehydrogenase activity, glycerol kinase activity,
  • Glycerol dehydrogenase activity and transaldolase activity are Glycerol dehydrogenase activity and transaldolase activity.
  • Figure 2 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by parts of the Calvin-Benson-Bassham cycle; Fructose bisphosphate aldolase and fructose bisphosphatase have been replaced by glycerol-3-phosphate dehydrogenase, glycerol kinase, glycerol dehydrogenase, fructose-6-phosphate aldolase and transaldolase to increase their synthetic potential.
  • the serine cycle (SC) is also available according to the invention. Due to the high
  • the energy requirement of fixing CO2 in CBB is a production of organic substances with this path of CO2 fixation (eg lactate) only possible if, in addition to CO2, another electron acceptor, for example oxygen, is available.
  • CO2 fixation eg lactate
  • the ribulose bisphosphate carboxylase preferably used in CBB has a comparatively low activity.
  • SC there is advantageously the possibility in principle to completely dispense with an additional electron acceptor besides CO2.
  • the SC is not dependent on the fixation of CO2 in the reaction of ribulose bisphosphate carboxylase.
  • this enzyme also catalyzes an undesirable side reaction with O 2 as a substrate, which reduces the efficiency of the metabolic pathway.
  • the invention preferably provides in this alternative variant for predominantly or exclusively the serine cycle (SC) to be implemented for CO2 assimilation.
  • SC serine cycle
  • the realization of this metabolic pathway according to the invention primarily provides for the expression of a formyl-tetrahydrofolate ligase activity.
  • Fig. 3 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by a formyl tetrahydrofolate ligase; For the assimilation of hydrogen and for the synthesis of glycine, homologous enzymes are used.
  • Fig. 4 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended to formyl tetrahydrofolate ligase; for the synthesis of glycine, homologous enzymes are used; the assimilation of hydrogen is by a recombinant hydrogenase.
  • the realization according to the invention of a formyl-tetrahydrofolate ligase activity ensures, above all, the introduction of formate into the transfer of C 1 -substances, in particular the transfer of C 1 -substances to glycine in connection with a serine hydroxymethyltransferase activity. This process is particularly relevant in connection with the use of E. coli as a host cell.
  • the homologous or heterologous expression of a, preferably cytosolic, formate dehydrogenase activity and / or a comparable activity is realized.
  • At least one formyl-tetrahydrofolate ligase activity is expressed according to the invention.
  • the formyl tetrahydrofolate ligase activity preferably originates from the microorganism Methylobacterium extorquens, especially the enzyme formyl tetrahydrofolate ligase ftfL, or is derived from it.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 107.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 108.
  • the invention thus preferably relates to a cell which contains at least one nucleic acid molecule, which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequence SEQ ID NO: 107;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an activity of formyl tetrahydrofolate ligase encode.
  • the SC is realized by including a glycine cleavage system activity present in the host cell.
  • a glycine cleavage system activity is expressed.
  • the glycine cleavage system activity qcvPHT and Issd is from E. coli or derived therefrom.
  • the glycine cleavage system of E. coli is composed of 4 subunits.
  • Preferred, subunit-encoding nucleotide sequences are SEQ ID NO: 189 for subunit qcvP.
  • the enzyme system thus preferably has the amino acid sequences SEQ ID NO: 190, 192, 194 and 64.
  • the invention thus preferably relates to a cell which contains at least one nucleic acid molecule, which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 189, 191, 193, 63;
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 190, 192, 194, 64;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an activity of the glycine cleavage Encode system.
  • the recombinant expression of activities of key enzymes of the SC selected from:
  • an enzyme activity of an enzyme involved in the SC herein is meant the ability to react an intermediate of the SC as a substrate into another intermediate of the cycle as a product of the enzyme reaction.
  • key enzymes of the SC are understood as meaning serine-glyoxylate aminotransferase, hydroxypyruvate reductase, malate thiokinase, glycerol dehydrogenase MaIyI-CoA lyase and isocitrate lyase.
  • all of the aforementioned enzyme activities are expressed, preferably all enzyme activities involved in SC are expressed to a sufficient extent.
  • at least one, more preferably at least two of these enzyme activities are heterologously expressed.
  • this allows circumvention of the glycine cleavage system activity present in connection with the use of E. coli as the host cell. It also uses the amino group from serine to synthesize glycine from glyoxylate.
  • the realization of a hydroxypyruvate reductase activity which is preferred according to the invention permits the conversion of the metabolic product of the serine-glyoxylate aminotransferase together with the glycerate kinase in the Embden-Meyerhof-Pamas route, which is preferably realized in the cell according to the invention.
  • the regeneration of glyoxylate is optional, alternatively or preferably in addition, via a homologous phosphoenolpyrovate carboxylase activity, tricarboxylic acid and glyoxylate pathway; This is especially relevant in connection with the use of E. coli as the host cell.
  • the preferred realization of the isocitrate-lyase activity according to the invention is preferably also active as part of the glyoxylate pathway at the glyoxylate regeneration in the serine pathway. This is particularly relevant in connection with the use of E. coli as a host cell.
  • the serine-glyoxylate aminotransferase activity derives from the microorganism Methylobacterium extorquens, especially the enzyme serine-glyoxylate aminotransferase sgaA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 113.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 114.
  • the hydroxypyruvate reductase activity preferably originates from the microorganism Methylobacterium extorquens, in particular the enzyme hydroxypyruvate reductase is hprA or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 1 15.
  • the enzyme protein therefore preferably has the amino acid sequence SEQ ID NO: 116.
  • the serine malate thiokinase activity preferably originates from the microorganism Methylobacterium extorquens, in particular the enzyme malate thiokinase is mtkAB, or is derived therefrom.
  • the malate thiokinase is composed of 2 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 117 for mtkA subunit and SEQ ID NO: 119 for mtkB subunit.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 118 and 120.
  • the glycerol dehydrogenase malyl CoA lyase activity is derived from the microorganism Methylobacterium extorquens, especially the enzyme glycerol dehydrogenase malyl CoA lyase mcIA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 121.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 122.
  • the isocitrate lyase activity preferably originates from E. coli, in particular the enzyme isocitrate lyase aceA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 11.1.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 1 12.
  • expression of isocitrate lyase activity is realized by homologous expression, preferably by homologous overexpression.
  • the invention thus preferably relates to a cell which contains at least one, preferably at least two, three, four, five or preferably all nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 1 1 1, 1 13, 1 15, 1 17, 1 19;
  • nucleic acid molecules coding for amino acid molecules containing or consisting of the sequences SEQ ID NO: 1 12, 1 14, 1 16, 1 18, 120;
  • nucleic acid molecules which, with the nucleic acid molecules described under a) and b), are at least 50%, preferably at least 60%, 70%, 80%, particularly preferably have at least 90% or more homology and prefer the activity of key enzymes of the SC selected from: serine-glyoxylate aminotransferase activity, hydroxypyruvate reductase activity, malate-thiokinase activity, glycerol dehydrogenase malyl-CoA-lyase-
  • the pHG1 megaplasmid from Ralstonia eutropha is introduced into the cell according to the invention for the purpose of realizing a recombinant hydrogenase activity according to the invention, at least one deletion of genes for key enzymes of CBB present on the complete pHG1 megaplasmide must have occurred there to suppress CBB ,
  • the genes for phosphoribulose kinase cbbPp SEQ ID NO: 35
  • ribulose bisphosphate carboxylase cbbLp SEQ ID NO: 37
  • cbbSp SEQ ID NO: 39.
  • the invention also provides the reductive tricarboxylic acid cycle (RTCC).
  • RTCC reductive tricarboxylic acid cycle
  • enzyme activity of an enzyme involved in the RTCC herein is meant the ability to react an intermediate of the RTCC as a substrate into another intermediate of the cycle as a product of the enzyme reaction.
  • Key enzymes of the RTCC are understood to mean citrate lyase, oxoglutarate oxidoreductase and fumarate reductase.
  • the invention preferably provides for predominantly or exclusively the reductive tricarboxylic acid cycle (RTCC) to be carried out for CO2 assimilation, wherein all the enzyme activities involved in the RTCC are sufficiently expressed in the cell according to the invention.
  • RTCC reductive tricarboxylic acid cycle
  • the expression of activities of key enzymes of the RTCC selected from:
  • Citrate lyase activity oxoglutarate oxidoreductase activity
  • Fumarate reductase activity and / or comparable activities are realized.
  • all of the abovementioned enzyme activities are expressed, preferably all enzyme activities involved in the RTCC are expressed to a sufficient extent.
  • at least one, more preferably at least two, of these enzyme activities are heterologously expressed.
  • the citrate-lyase activity preferably originates from the microorganism Chlorobium tepidum, especially the enzyme citrate-lyase ac-IAB, or is derived therefrom. According to the current state of knowledge, this citrate lyase is composed of 2 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 123 for subunit acIA, and SEQ ID NO: 125 for subunit acIB. The enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 124 and 126.
  • the citrate lyase activity is from E. coli, especially the enzyme citrate lyase citFED, or is derived therefrom. According to the current state of knowledge, this citrate lyase is composed of 3 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 127 for subunit citF, SEQ ID NO: 129 for subunit citE, and SEQ ID NO: 131 for subunit citD.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 128, 130 and 132.
  • the expression of an active citrate lyase activity is realized by homologous expression, preferably by homologous overexpression of, possibly by spontaneous mutation, mutant allele of the originally inactive citrate lyase of E. coli .
  • the oxoglutarate oxidoreductase activity preferably originates from the microorganism Hydrogenobacter thermophilus, in particular the enzyme oxoglutarate oxidoreductase korAB, or is derived therefrom. According to the current state of knowledge, this oxoglutarate oxidoreductase is composed of 2 subunits.
  • Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 133 for subunit korA, and SEQ ID NO: 135 for subunit korB.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 134 and 136.
  • the fumarate reductase activity is from E. coli, especially the enzyme fumarate reductase is frdDCBA or derived therefrom.
  • the fumarate reductase is composed of 4 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 57 for subunit frdD, SEQ ID NO: 55 for subunit frdC, SEQ ID NO: 53 for subunit frdB and SEQ ID NO: 51 for subunit frdA.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 58, 56, 54 and 52.
  • the expression of fumarate reductase activity is realized by homologous expression, preferably by homologous overexpression.
  • the invention thus preferably relates to a cell which contains at least one, preferably at least two, three, four, five, six, seven, eight, nine, ten or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain the sequences SEQ ID NO: 51, 53, 57, 123, 125, 127, 129, 131, 133, 135 or consist thereof;
  • nucleic acid molecules which are suitable for amino acid molecules having the sequences SEQ ID NO: 52, 54, 56, 58, 124, 126, 128,
  • nucleic acid molecules which have at least 50%, preferably at least 50%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b) and preferably an activity of key enzymes of the RTCC selected from citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
  • the homologous and preferably the heterologous expression of a, preferably reversible, pyruvate: ferredoxin oxidoreductase activity or pyruvate synthase activity and / or an activity comparable therewith is realized.
  • a, preferably reversible, pyruvate: ferredoxin oxidoreductase activity or pyruvate synthase activity and / or an activity comparable therewith is realized.
  • E. coli or Pseudomonas putida as the host cell is provided in an alternative variant, that preferably by the homologous or heterologous expression of enzyme activity of the malate pathway, preferably by an NAD-dependent malate enzyme activity, the NAD Dependent malate enzyme from E. coli, the intermediate oxaloacetate is irreversibly converted into pyruvate by decarboxylation.
  • the intermediate oxaloacetate by homologous or optionally heterologous expression of oxaloacetate decarboxylase activity, preferably the E. coli oxaloacetate decarboxylase.
  • oxaloacetate decarboxylase activity preferably the E. coli oxaloacetate decarboxylase.
  • the homologous overexpression of at least one of the aforementioned enzyme activities is provided to advantageously prevent byproducts from the tricarboxylic acid cycle from being produced instead of lactate.
  • this measure is preferably provided in order to realize the irreversible outflow from the tricarboxylic acid cycle.
  • this is usually associated with a pyruvate: ferredoxin oxidoreductase activity.
  • Figure 8 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly expanded by the key enzymes of the reductive tricarboxylic acid cycle (citrate lyase, oxoglutarate oxidoreductase and fumarate reductase).
  • oxygen is used as the terminal electron acceptor.
  • a recombi- nant hydrogenase is used for the assimilation of hydrogen.
  • the pHG1 megaplasmid from Ralstonia eutropha is introduced into the cell according to the invention for the purpose of realizing a recombinant hydrogenase activity according to the invention, at least one deletion of genes for key enzymes of CBB present on the complete pHG1 megaplasmide must have occurred there to suppress CBB ,
  • the genes for phosphoribulose kinase cbbPp and / or ribulose bisphosphate carboxylase cbbLp and cbbSp are preferably deleted on the megaplasmid.
  • the invention preferably further contemplates suppressing the potential degradation of lactate precursors and directing the material flow toward lactate. This is preferably realized by inhibiting any enzyme activities of competing metabolic pathways and / or comparable enzyme activities present in the cell according to the invention, preferably by inhibiting the expression and / or deletion of the nucleotide sequences or genes coding for these enzyme activities.
  • the production of lactic acid further provided to suppress acetate-converting pathways to suppress the formation of acetate as an undesired by-product.
  • the CBB is preferably at least one enzyme, preferably all enzymes selected from:
  • Acetate Kinase A / Propionate Kinase 2 Activity Phosphate Acetyl Transferase Activity, Phosphoenolpyruvate Carboxylase Activity, Pyruvate Formate Lyase I Activity, Acetaldehyde CoA Dehydrogenase Activity,
  • Fumarate reductase activity pyruvate dehydrogenase activity, succinate dehydrogenase activity
  • At least one enzyme preferably all enzymes, selected from:
  • the invention preferably provides for this: the inhibition of the phosphate acetyltransferase activity preferably by deletion of at least one of the genes for p_ta, represented by the SEQ ID NO: 41 or coding for the amino acid sequence SEQ ID NO: 42, and alternatively or preferably in addition
  • CBB optionally (in CBB) the inhibition of the phosphoenolpyruvate carboxylase activity, preferably by deletion of at least one of the genes for ppc, represented by SEQ ID NO: 43 or coding for the amino acid sequence SEQ ID NO: 44, wherein the inhibition of phosphoenolpyruvate
  • Carboxylase activity ppc is less preferred in this context.
  • the invention provides alternatively or preferably additionally:
  • the inhibition of pyruvate formate lyase I activity is preferred by deleting at least one of the genes for pflB, represented by SEQ ID NO: 45 or coding for the amino acid sequence SEQ ID NO: 46, and alternatively or preferably additionally
  • the inhibition of the acetate kinase A / propionate kinase 2 activity is preferred by deleting at least one of the genes for ackA represented by SEQ ID NO: 47 or coding for the amino acid sequence SEQ ID NO: 48, and alternatively or preferably additionally the inhibition of acetaldehyde CoA dehydrogenase activity preferably by deletion of at least one of the genes for ad ⁇ hE represented by SEQ ID NO: 49 or coding for the amino acid sequence SEQ ID NO: 50, and alternatively or preferably additionally
  • CBB optionally (in CBB) the inhibition of anaerobic fumarate reductase activity, preferably by deletion of at least one of the genes for frdABCD, represented by SEQ ID NOS: 51, 53, 55 and 57 or coding for the amino acid sequences SEQ ID NO : 52, 54, 56 and 58, more preferably frdA and / or frdB.
  • the invention alternatively provides for this:
  • the inhibition of pyruvate dehydrogenase activity is preferred by deleting at least one of the aceEF / lpdA genes represented by SEQ ID NOs: 59 and 61, 63 or coding for the amino acid sequences SEQ ID NOs: 60 and 62, and alternatively or preferably additionally
  • Activity is preferred by deleting at least one of the genes for ackA and alternatively or preferably additionally
  • the inhibition of acetaldehyde-CoA dehydrogenase activity is preferred by deleting at least one of the genes for ad; ITE and alternatively or preferably in addition the inhibition of succinate dehydrogenase activity, preferably by deletion of at least one of the genes for sdhABCD represented by SEQ ID NO: 65, 67, 69 and 71 or coding for the amino acid sequence SEQ ID NO: 66, 68, 70 and 72, especially preferably from sdhA and / or sdhB.
  • the invention preferably provides that the expression of a D-lactate dehydrogenase activity and / or a comparable activity is realized.
  • the D-lactate dehydrogenase activity originates from E. coli, in particular it is the enzyme lactate dehydrogenase IdhA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 73.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 74.
  • the D-lactate dehydrogenase activity originates from Lactobacillus plantarum, in particular it is the enzyme lactate dehydrogenase IdhD, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 185.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 186.
  • the expression of D-lactate dehydrogenase activity is realized by homologous expression, preferably by homologous overexpression.
  • homologous expression In the context of an aerobic process, heterologous expression or homologous overexpression of lactate dehydrogenase activity is mandatory.
  • the D-lactate transporter activity preferably originates from E. coli, in particular it is the transporter HdP or IctP, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 165.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 166.
  • the invention thus preferably relates to a cell which contains at least one, or preferably all, nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 73, 165, 185;
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 74, 166, 186;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably a D-lactate dehydrogenase activity and / or D-lactate transporter activity.
  • the invention additionally provides: the inhibition of homologous L-lactate dehydrogenase activity, preferably by deletion of the genes for HdD represented by SEQ ID NO: 75 or coding for the amino acid sequence SEQ ID NO: 76.
  • the invention additionally provides:
  • the inhibition of the homologous L-lactate dehydrogenase activity preferably by deletion of the gene for IdhLI, represented by SEQ ID NO: 167 or coding for the amino acid sequence SEQ ID NO: 168, and alternatively and preferably additionally by deletion of the gene for ldhL2 represented by SEQ ID NO: 169 or coding for the amino acid sequence SEQ ID NO: 170, and alternatively or preferably additionally
  • the inhibition of the homologous lactate racemase activity preferably by deletion of at least one of the genes for Ia 1 rABC1 C2E qlpFL represented by SEQ ID NO: 173, 175, 177, 179 and 181 or coding for the amino acid sequence SEQ ID NO: 174, 176, 178, 180 and 182.
  • the invention preferably provides that the expression of an L-lactate dehydrogenase activity and / or a comparable activity is realized.
  • the L-lactate dehydrogenase activity preferably originates from E. coli, in particular it is the enzyme L-lactate: quinone oxidoreductase (L-lactate dehydrogenase) IctD or HdD, or is derived therefrom.
  • this activity co- nucleotide sequence is SEQ ID NO: 75.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 76.
  • the homologous expression of L-lactate dehydrogenase activity is preferably realized by homologous overexpression of L-lactate-quinone oxidoreductase (L-lactate dehydrogenase) IctD or HdD.
  • the L-lactate dehydrogenase activity originates from the microorganism Lactobacillus plantarum. It is preferably the enzyme lactate dehydrogenase ldhL.1 or it is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 167.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 168.
  • it is the enzyme lactate dehydrogenase ldhL2 or it is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 169.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 170.
  • the invention thus preferably relates to a cell which contains at least one, preferably at least two, or preferably all, nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which have the sequences SEQ ID NO:
  • 167, 171, 75 contain or consist of;
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 168, 172, 76;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an L-lactate dehydrogenase activity and / or encode an L-lactate transporter activity.
  • the invention additionally provides:
  • the inhibition of the homologous fermentative D-lactate dehydrogenase activity preferably by deletion of the genes for IdhA, represented by SEQ ID NO: 73 or coding for the amino acid sequence SEQ ID NO: 74.
  • the invention additionally provides: the inhibition of the homologous D-lactate dehydrogenase activity, preferably by deletion of the genes for d] d, represented by SEQ ID NO: 187 or coding for the amino acid sequence SEQ ID NO: 188.
  • the invention additionally provides:
  • the inhibition of the homologous D-lactate dehydrogenase activity preferably by deletion of the gene for IdhD, represented by SEQ ID NO: 185 or coding for the amino acid sequence SEQ ID NO: 186, and alternatively or preferably additionally
  • the above-characterized recombinant microorganisms or transgenic biological cells according to the invention are particularly suitable for the biotechnological synthesis of lactate / lactic acid and other carboxylic acids or short-chain fatty acids. These are according to the invention C2 to C6 body, preferably C3 to C6 body.
  • Preferred metabolic end products which can be produced by means of the cells according to the invention are short-chain carboxylic acids, especially monocarboxylic and dicarboxylic acids, preferably D-lactate and L-lactate, acetate / acetic acid, formic acid / formic acid and succinate / succinic acid, and also mono- and dicarboxylic acids polyhydric alcohols, preferably ethanol. They serve as starting materials for further organic synthesis, for example for the production of plastics (eg polylactates).
  • the products which can be prepared according to the invention can also be used as energy carriers, especially as fuels / fuels and for their production.
  • the invention also provides a process for the biotechnological production of organic carbon compounds, especially C2 to C6 bodies, as a product of CO2 as substrate and an inorganic electron donor such as hydrogen, wherein in step (a) a cell according to the invention is provided, in step (b) the cell is contacted with the substrate and the electron donor and in step (c) the cell is cultured under conditions whereby the substrates are reacted and the cell forms the organic carbon bonds.
  • the product is isolated from the culture medium and / or the cell and optionally purified. Usually, the intracellularly formed product is released from the cell into the extracellular medium.
  • the cultivation preferably takes place in preferably liquid culture medium. It is preferably provided to cultivate the cell under anaerobic conditions. Depending on the host organism, it is also provided in an alternative variant to cultivate the cell under aerobic conditions. On the basis of the above description of the invention, the person skilled in the art can choose the respectively favorable enzyme equipment.
  • the electron donor is preferably elementary hydrogen, preferably gaseous hydrogen. Preferred alternative electron donors are selected from hydrogen sulfide, elemental sulfur, sulfite, thiosulfate, ammonium, nitrite and metals in reduced form.
  • At least one compound selected from O 2 is used as an electron acceptor.
  • nitrate, nitrite, sulfate, sulfite, thiosulfate and fumarate is used as an electron acceptor.
  • HypD1 involved in metallocene formation of hydrogenases
  • HypA1 involved in metallocene formation of hydrogenases [Ralstonia eutropha H 16] EC number
  • HoxC Designation HoxC component of the hydrogen sensing and signal transduction system
  • HoxJ histidine protein kinase component of the hydrogen senso ⁇ ng and signal transduction system Abbreviation hoxJ Designation HoxJ histidine protein kinase component of the hydrogen senso ⁇ ng and signal transduction system
  • NCBI GenelD 946778 DNA sequence SEQ ID NO 41 amino acid sequence SEQ ID NO 42 Abbreviation ppc
  • DNA sequence SEQ ID NO 43 amino acid sequence SEQ ID NO 44 Abbreviation pflb Name pyruvate formate lyase I [Escherichia coli
  • E3 component is part of three enzyme complexes, eg of pyruvate dehydrogenase [Escherichia coli K12]
  • NCBI-GenelD 946632 DNA sequence SEQ ID NO 83 amino acid sequence SEQ ID NO 84 Abbreviation fsaA
  • fructose-6-phosphate aldolase 1 [Escherichia coli K12]
  • fructose-6-phosphate aldolase 2 [Escherichia coli K12]
  • DNA sequence SEQ ID NO 91 amino acid sequence SEQ ID NO 92 Abbreviation glpB Name sn-glycerol-3-phosphate dehydrogenase
  • transaldolase A Esscherichia coli K12
  • transaldolase B Esscherichia coli K12
  • NCBI-GI 439605 (detail) NCBI-GenelD DNA sequence SEQ ID NO 113 amino acid sequence SEQ ID NO 114
  • mtkB Methylobacte ⁇ um extorquens malate thiokinase (alpha subunit)
  • citrate lyase, subunit 2 [Chlorobium tepi dum TLS]
  • citrate lyase, subunit 1 [Chlorobium tepidum TLS]
  • citrate lyase citrate-ACP transferase (alpha) subunit [Escherichia coli K12]
  • citrate lyase citryl-ACP lyase (beta) subunit [Escherichia coli K12]
  • citrate lyase acyl car ⁇ er (gamma) subunit [Escherichia coli K12]
  • HyaA and HyaB proteins [Escherichia coli K12]
  • DNA sequence SEQ ID NO 153 amino acid sequence SEQ ID NO 154 Abbreviation hybC Name hydrogenase 2, large subunit [Escherichia coli K12]
  • NCBI GenelD 945182 DNA sequence SEQ ID NO 155 amino acid sequence SEQ ID NO 156 Abbreviation hybD
  • IctP Name L-lactate transport protein [Lactobacillus plantarum WC FS 1]
  • gcvT Name aminomethyltransferase, tetrahydrofolate-dependent, subunit (T protein) of glycine cleavage complex [Escherichia coli K12]
  • Exemplary embodiment Synthesis of lactic acid from CO2 and H2 in a recombinant E. coli strain
  • the starting point for the transformation is E. coli strain K12.
  • expression cassettes were used for: NAL-reducing cytosolic hydrogenase activity from Ralstonia, structural hoxFUYH and maturation genes hvpC1, hypD1, hypE1 and hypABF, as well as formyl-tetrahydrofolate ligase activity from mammothacterium, especially containing nucleic acid molecules having the sequences SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 107, in expression vectors pUC or pPCU18 or the like cloned / ligated.
  • the ligation of the plasmid DNA is carried out in a conventional manner.
  • the vector is opened by hydrolysis with restriction endonuclease and the corresponding DNA sequences are inserted. Ligation takes place, for example, by cassette mutagenesis.
  • transformation-competent E. coli cells and the production of transformation-competent E. coli cells is preferably carried out according to the protocol of Hanahan, 1985 (Hanahan, D. in Glover, DM (ed.)) DNA Cloning: "A Practical Approach” IRL Press Oxford 109-135).
  • E. coli K12 strains for example TH1, TH2, TH5, TH5 ⁇ , C600, Top 10, XL1-Blue and their derivatives are used.
  • a fresh cell colony is suspended in 5 ml SOB medium and cultured with shaking at 37 ° C to a cell density of 1 to 2 x 10 8 / ml. Subsequently, 1: 1 with 40% glycerol / 60% SOB medium (1% yeast extract, 2% Bacto-trypton-mmol / l NaCl, 2.5 mmol / l KCl, after autoclaving to 10 mmol / l MgCl 2 and MgSO 4 ) diluted and cooled on ice. From the pre-culture, a cell smear is applied to an LM plate and incubated overnight at 37 ° C.
  • the cell suspension may, for example in 1, 5 ml reaction vessels after slow deep-freezing are stored at -70 0 C for several months.
  • the ligation is carried out in a molar ratio of DNA to vector DNA of 1: 1 to 1: 3.
  • the ligation mixture 4 .mu.l of sterile water, 2 .mu.l of 5 ⁇ Ligasebuffer (250 mmol / l Tris-HCl, pH 7.6, 50 mmol / l MgCl 2 , 5 mmol / l ATP, 5 mmol / l DTT, 25% PEE ( w / v)), 1 ml of amplified DNA, 2 ⁇ l of vector DNA (with 3 'T overhang in 1 ⁇ l T4).
  • Ligase is placed in a 1, 5 ml reaction vessel and incubated after mixing overnight at 14 ° C. In each case 1-5 ⁇ l of the mixture are used to transform transformation-competent E. coli cells; where appropriate, the ligation mixture may be stored at -20 0 C.
  • selection plates are used, in each case 100 .mu.l of a mixture of 10 .mu.l 100 mmol / l IPTG, 40 .mu.l 3% X-GaI and 50 .mu.l SOC medium are plated on an LB-AMP agar plate and this for about Incubated for 1 hour at 37 ° C. After incubation, in each case 200 ⁇ l of the cell suspension are plated out on the selection plate. On the basis of the ⁇ -complementation test, the colonies which do not contain a recombinant plasmid can easily be distinguished from the recombinant clones which have no coloration due to the blue coloration. The stable heterologous expression in the so available Recombinant E. coli cells are verified in a conventional manner.
  • the resulting recombinant E. coli cells are prepared after inoculation in a 50 L fermenter with culture medium and cultured at 25 to 40 0 C.
  • the culture is cultured with introduction of gaseous CO2 and H2 in the reactor.

Abstract

L'invention concerne l'utilisation biotechnologique de donneurs d'électrons inorganiques, principalement d'hydrogène gazeux pour la fixation du dioxyde de carbone comme source de carbone pour la synthèse de composés organiques comme vecteurs d'énergie et produits de valeur par fermentation dans des micro-organismes.
PCT/EP2008/008351 2007-10-02 2008-10-02 Fixation biotechnologique du dioxyde de carbone WO2009046929A2 (fr)

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DE102007047206.6A DE102007047206B4 (de) 2007-10-02 2007-10-02 Biotechnologische Fixierung von Kohlenstoffdioxid
DE102007047206.6 2007-10-02

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US10786064B2 (en) 2010-02-11 2020-09-29 Cj Cheiljedang Corporation Process for producing a monomer component from a genetically modified polyhydroxyalkanoate biomass
TWI573869B (zh) * 2011-07-29 2017-03-11 三井化學股份有限公司 導入有二氧化碳固定循環之微生物
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RU2639507C2 (ru) * 2012-04-24 2017-12-21 СиДжей ЧеилДжеданг Корпорейшн Штамм, продуцирующий d-молочную кислоту, и его применение
TWI652342B (zh) 2012-12-21 2019-03-01 國立中興大學 用於對糖發酵形成發酵產物之微生物
US10294505B2 (en) 2013-01-24 2019-05-21 Mitsui Chemcials, Inc. Microorganism for production of chemicals derived from acetyl-CoA
KR101714943B1 (ko) 2013-01-24 2017-03-09 미쓰이 가가쿠 가부시키가이샤 이산화탄소 고정 회로를 도입한 미생물
US9828618B2 (en) 2013-01-24 2017-11-28 Mitsui Chemicals, Inc. Microorganism having carbon dioxide fixation cycle introduced thereinto
KR20150041802A (ko) * 2013-01-24 2015-04-17 미쓰이 가가쿠 가부시키가이샤 이산화탄소 고정 회로를 도입한 미생물
WO2014115815A1 (fr) 2013-01-24 2014-07-31 三井化学株式会社 Micro-organisme dans lequel a été introduit un cycle de fixation du dioxyde de carbone
EP3013971A4 (fr) * 2013-06-29 2016-11-30 Univ California Plantes recombinantes et micro-organismes recombinants à voie inversée du glyoxylate
CN105518148A (zh) * 2013-06-29 2016-04-20 加利福尼亚大学董事会 具有逆向乙醛酸支路的重组植物和微生物
WO2015005406A1 (fr) 2013-07-09 2015-01-15 味の素株式会社 Procédé de fabrication de substance utile
EP3521433A1 (fr) 2013-07-09 2019-08-07 Ajinomoto Co., Inc. Procédé de production d'acide l-glutamique
WO2020225346A1 (fr) * 2019-05-09 2020-11-12 Enobraq Bactérie génétiquement modifiée pour produire du lactate à partir de co2
FR3095817A1 (fr) * 2019-05-09 2020-11-13 Enobraq Bactérie génétiquement modifiée pour une production améliorée de lactate à partir de CO2

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