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WO2012015949A2 - Procédés et compositions pour amélioration des rendements de produits réduits de microorganismes photosynthétiques - Google Patents

Procédés et compositions pour amélioration des rendements de produits réduits de microorganismes photosynthétiques Download PDF

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Publication number
WO2012015949A2
WO2012015949A2 PCT/US2011/045572 US2011045572W WO2012015949A2 WO 2012015949 A2 WO2012015949 A2 WO 2012015949A2 US 2011045572 W US2011045572 W US 2011045572W WO 2012015949 A2 WO2012015949 A2 WO 2012015949A2
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Prior art keywords
host cell
atp
recombinant protein
gene
group
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WO2012015949A3 (fr
Inventor
Frank Anthony Skraly
Nikos Basil Reppas
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Joule Unlimited Technologies Inc
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Joule Unlimited Technologies Inc
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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
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • 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
    • C12N1/12—Unicellular algae; Culture media therefor
    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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

Definitions

  • the present invention relates to methods and compositions for increasing a host cell's production of a carbon-based product of interest, including a reduced product of interest.
  • the invention provides, in certain embodiments, an engineered host cell, comprising one or more recombinant protein activities selected from at least one futile cycle pathway, wherein the recombinant protein activities facilitate dissipation of excess energy stored in the engineered host cell.
  • dissipation of excess energy stored in a host cell is implemented exogenously, e.g., through addition of an energy-dissipating factor to the host cell growth medium.
  • the host cell is a gram-negative or gram-positive bacterium.
  • the host cell is a photosynthetic microbe.
  • the host cell is a cyanobacterium.
  • the present invention relates to methods and compositions which dissipate excess energy in a host cell.
  • the excess energy is stored in an ATP molecule.
  • the excess energy is stored as a proton gradient.
  • the proton gradient stores energy as a proton concentration gradient across membrane-bound compartments. It can be used to generate proton motive force and drive ATP synthesis.
  • the host cell is engineered to dissipate excess ATP.
  • the host cell is engineered to dissipate excess proton gradient.
  • excess energy dissipation increases the host cell's production of a reduced product of interest.
  • the present invention also provides for dissipation of excess energy stored in ATP, wherein the excess energy is defined by an amount of ATP in a host cell modified to produce a carbon-based product of interest that is greater than the amount of ATP at the metabolic equilibrium of a background host cell, wherein the background host cell lacks such modifications.
  • the excess energy is defined by the energy charge in a host cell modified to produce a carbon based product of interest, wherein said cell is at metabolic equilibrium, and the energy charge is higher than a corresponding characteristic energy charge of a background host cell lacking such modifications.
  • the characteristic energy charge in a background host cell is proportional to the mole fraction of ATP plus half the mole fraction of ADP in relationship to the mole fraction of ATP, ADP, and AMP combined.
  • the energy charge is decreased by the compositions and methods provided herein.
  • the engineered host cell has excess energy stored in ATP, wherein the amount of ATP in the host cell is greater than the amount of ATP at metabolic
  • the amount of ATP at metabolic equilibrium can be determined by the characteristic energy charge of the background host cell.
  • the present invention provides an engineered host cell, comprising one or more recombinant protein activities selected from at least one futile cycle pathway, wherein the recombinant protein activities facilitate dissipation of an excess energy stored in an engineered host cell, and wherein the host cell produces a carbon-based product of interest.
  • the excess energy is produced during the production of a carbon-based product of interest.
  • the carbon-based product o fmterest is a reduced product.
  • the carbon-based product of interest is a hydrocarbon.
  • the carbon-based product of interest is an alcohol.
  • a method is provided to increase production of a reduced product in a host cell, comprising the step of manipulating the host cell to dissipate excess energy stored in the host cell, and culturing the host cell in a host cell growth medium.
  • an exogenous ATP depletion agent is added to the host cell growth medium.
  • the reduced product is ethanol.
  • the host cell comprises a recombinant NADPH-dependent adh gene.
  • the exogenous ATP depletion agent is administered in an amount effective to mitigate ATP synthesis in said host cell, wherein the yield of the reduced product by the host cell is increased by at least 5% relative to the yield of the reduced product by the host cell cultured in an otherwise identical host cell growth medium lacking said exogenous ATP depletion agent.
  • exogenous ATP depletion agent is selected from the group consisting of: ⁇ , ⁇ '-dicyclohexylcarbodiimide, 3,3',4',5-tetrachlorosalicylanilide, 2,4-dinitrophenol, and carbonyl cyanide-/?-trifluoromethoxyhydrazone.
  • the exogenous ATP depletion agent is ⁇ , ⁇ '-dicyclohexylcarbodiimide and is present in the host cell growth medium at a concentration of 0.25 ⁇ to ⁇ .
  • the presence of ⁇ , ⁇ '-dicyclohexylcarbodiimide in the culture medium results in a 5 to 30% increase in ethanol productivity of the host cell relative to ethanol productivity of the host cell cultured in an otherwise identical culture medium lacking said 3,3',4',5- tetrachlorosalicylanilide.
  • the exogenous ATP depletion agent is 3,3',4',5-tetrachlorosalicylanilide and is present in the host cell growth medium at a concentration of ⁇ . ⁇ to 0.25 ⁇ .
  • the presence of 3,3',4',5- tetrachlorosalicylanilide in the culture medium results in a 5 to 30% increase in ethanol productivity of the host cell relative to ethanol productivity of the host cell cultured in an otherwise identical culture medium lacking said 3,3',4',5-tetrachlorosalicylanilide.
  • the present invention provides for a method to increase production of a reduced product in a host cell, comprising the step of manipulating the host cell to dissipate excess energy stored in the host cell, and culturing the host cell in a host cell growth medium.
  • the host cell is genetically modified to dissipate the excess energy.
  • the genetically modified host cell comprises one or more recombinant protein activities selected from a futile cycle pathway.
  • an exogenous ATP synthase inhibitor is added to the host cell growth medium.
  • the exogenous ATP synthase inhibitor is selected from the group consisting of: ⁇ , ⁇ '- dicyclohexylcarbodiimide, 3,3',4',5-tetrachlorosalicylanilide, 2,4-dinitrophenol, and carbonyl cyanide -p-trifluoromethoxyhydrazone.
  • the invention provides for a method for increasing the rate of production of a carbon-based product of interest by an engineered host cell, comprising the step of introducing one or more recombinant protein activities from a futile cycle pathway into a host cell, and culturing the engineered host cell under conditions that promote production of the carbon-based product of interest.
  • the one or more recombinant protein activities are under the control of an inducible promoter.
  • the inducible promoter is P(mV07) (SEQ ID NO: l).
  • the one or more recombinant protein activities are selected from Table 7.
  • the one or more recombinant protein activities are comprised of a gene product of a gene selected from the group consisting of pntAB and udhA.
  • the one or more recombinant protein activities are comprised of a gene product of a gene selected from the group consisting of pntAB and udhA.
  • the one or more recombinant protein activities are comprised
  • the recombinant protein activities are selected from the group consisting of a membrane-bound proton-translocating transhydrogenase and a soluble pyridine nucleotide transhydrogenase.
  • the one or more recombinant protein activities comprise a gene product of a gene selected from the group consisting of glgC, glgA, and glgP.
  • the one or more recombinant activities are selected from the group consisting of glucose- 1 -phosphate adenylyltransferase, glucose pyrophosphorylase, and glycogen phosphorylase.
  • the one or more recombinant protein activities comprise a gene product of a gene selected from the group consisting of pfkA and fbp. In yet another embodiment, the one or more recombinant protein activities are selected from the group consisting of phosphofructokinase and fructose-bisphosphatase. In another
  • the one or more recombinant protein activities comprise a gene product of a gene selected from the group consisting of pta, ackA, and acs.
  • the one or more recombinant protein activities are selected from the group consisting of phosphotransacetylase, acetate kinase, and acetyl coenzyme synthetase.
  • the one or more recombinant protein activities comprise a gene product of a gene selected from the group consisting of pykF and ppsA.
  • the one or more recombinant protein activities are selected from the group consisting of pyruvate kinase and phosphoenolpyruvate synthase.
  • the one or more recombinant protein activities comprise a gene product of a gene selected from the group consisting of pyc,pck, and pykF.
  • the one or more recombinant protein activities are selected from the group consisting of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase.
  • the one or more recombinant protein activities comprise a gene product of a gene selected from the group consisting of mdh, sfcA, and pyc. In one embodiment, the one or more recombinant protein activities are selected from the group consisting of malate dehydrogenase and pyruvate carboxylase. In a further aspect,
  • the one or more recombinant protein activities comprise a gene product of a gene selected from the group consisting of mdh, ppc, sucCD, maeB, sad, and sucD.
  • the one or more recombinant protein activities are selected from the group consisting of malate dehydrogenase, phosphoenolpyruvate carboxylase, succinyl-CoA synthetase, and succinate semialdehyde dehydrogenase.
  • the one or more recombinant protein activities comprise a gene product of a gene selected from the group consisting of mdh, mdhP, gap2, pgk, did, mgsA, gloA, and gloB.
  • the one or more recombinant protein activities are selected from the group consisting of NAD-specific malate dehydrogenase, NADP-specific malate dehydrogenase, glyceraldehyde-3 -phosphate dehydrogenase, phosphoglycerate kinase, D-lactate
  • the one or more recombinant protein activities comprise a gene product of a gene selected from the group consisting of zwf, pgl, and gnd. In one aspect, the one or more recombinant protein activities are selected from the group consisting of glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, and gluconate-P dehydrogenase.
  • activity of the futile cycle is increased without overexpression of the genes of the futile cycle pathway.
  • the activity of at least part of a futile cycle pathway is increased by overexpression of another gene.
  • the gene is outside the futile cycle pathway.
  • the gene is endogenous.
  • the gene is recombinant.
  • the overexpression of the group 2 sigma factor sigE increases the activity of a futile cycle pathway.
  • the futile cycle pathway is an oxidative pentose phosphate pathway comprising an enzyme expressed by a gene selected from the group consisting of zwf (glucoses- phosphate dehydrogenase), pgl (6-phosphogluconolactonase), and gnd (gluconate-P dehydrogenase).
  • the present invention also relates to methods for mitigating ATP accumulation in an engineered host cell.
  • the method provides for addition of salt to the host cell medium.
  • the method provides for acidification or alkalinization of the host cell medium.
  • the temperature of the host cell medium is adjusted.
  • a toxic compound is introduced to the medium.
  • the toxic compound induces an efflux reaction in the host cell.
  • one embodiment of the present invention provides a method for production of a host cell engineered to produce a carbon-based product of interest, wherein said host cell dissipates accumulated excess ATP, comprising the steps of: (i) performing random mutagenesis on the host cell, and (ii) screening the host cell for dissipation of excess ATP.
  • a composition for producing ethanol comprising a host cell engineered to produce ethanol, and a medium comprising an ATP depletion agent, is provided in one aspect of the invention.
  • the ATP depletion agent is selected from the group consisting of: ⁇ , ⁇ '-dicyclohexylcarbodiimide, 3,3',4',5-tetrachlorosalicylanilide, 2,4- dinitrophenol, and carbonyl cyanide-p-trifluoromethoxyhydrazone.
  • the ATP depletion agent is ⁇ , ⁇ '-dicyclohexylcarbodiimide at a concentration of 0.25 to 0.75 ⁇ .
  • the ATP depletion agent is 3,3',4',5-tetrachlorosalicylanilide at a concentration of 0.1 to 0.25 ⁇ .
  • the host cell is a photosynthetic microbe. In another embodiment, the host cell is cyanobacterium. In still another
  • the host cell comprises a recombinant NADPH-dependent adh gene.
  • the invention provides a method to increase the production of ethanol in a photosynthetic microbe comprising a recombinant NADPH- dependent adh gene, comprising the step of culturing said photosynthetic microbe in a medium comprising ⁇ , ⁇ '-dicyclohexylcarbodiimide at a concentration of 0.75 ⁇ , wherein said ⁇ , ⁇ '-dicyclohexylcarbodiimide increases the ethanol productivity of the photosynthetic microbe.
  • the invention provides a method to increase the production of ethanol in a photosynthetic microbe comprising a recombinant NADPH- dependent adh gene, comprising the step of culturing said photosynthetic microbe in a medium comprising 3,3',4',5-tetrachlorosalicylanilide at a concentration of 0.1 ⁇ , wherein said 3,3',4',5-tetrachlorosalicylanilide increases the ethanol productivity of the photosynthetic microbe.
  • the photosynthetic microbe is cyanobacterium
  • a composition for producing ethanol comprising a photosynthetic microbe having a recombinant NADPH-dependent adh gene, and a medium comprising ⁇ , ⁇ '-dicyclohexylcarbodiimide at a concentration of 0.75 ⁇ .
  • a composition is provided for producing ethanol, comprising a photosynthetic microbe having a recombinant NADPH-dependent adh gene, and a medium comprising 3,3',4',5-tetrachlorosalicylanilide at a concentration of ⁇ . ⁇ .
  • the photosynthetic microbe is cyanobacterium.
  • Figure 1 Effect of ⁇ , ⁇ '-dicyclohexylcarbodiimide on growth curves of JCC1581 as measured by optical density at 730 nm over a 264 hr time course.
  • Figure 2 Effect of ⁇ , ⁇ '-dicyclohexylcarbodiimide on cumulative ethanol production by JCC1581 as measured over a 264-hr time course.
  • Figure 3 Effect of ⁇ , ⁇ '-dicyclohexylcarbodiimide on acetaldehyde levels produced by JCC1581 as measured over a 264-hr time course.
  • nucleic acid comprising SEQ ID NO: l refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO: 1 , or (ii) a sequence complementary to SEQ ID NO: 1.
  • the choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.
  • the term "recombinant” refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature.
  • the term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids.
  • an endogenous nucleic acid sequence in the genome of an organism is deemed "recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered.
  • a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof).
  • a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become
  • a nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome.
  • an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention.
  • a "recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.
  • vector as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid generally refers to a circular, double-stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme.
  • PCR polymerase chain reaction
  • Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC).
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as
  • expression control sequence refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and/or translation of nucleic acid sequences.
  • Expression control sequences include, e.g., appropriate transcription initiation, termination, promoter and enhancer sequences; efficient R A processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • the nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • recombinant host cell (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant nucleic acid sequence has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • a recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.
  • background host cell as used herein, is intended to refer to a cell that lacks one or more recombinant nucleic acid sequences that are to be introduced to create a recombinant host cell or host cell.
  • carbon-based products of interest includes alcohols such as ethanol, propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, wax esters; hydrocarbons and alkanes such as propane, octane, diesel, Jet Propellant 8 (JP8); polymers such as terephthalate, 1,3 -propanediol, 1 ,4-butanediol, polyols, Polyhydroxyalkanoates (PHA), poly-beta-hydroxybutyrate (PHB), acrylate, adipic acid, ⁇ -caprolactone, isoprene, caprolactam, rubber; commodity chemicals such as lactate, Docosahexaenoic acid (DHA), 3-hydroxypropionate, ⁇ -valerolactone, lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbic acid, is
  • Biofuel refers to any fuel that derives from a biological source.
  • Biofuel can refer to one or more hydrocarbons, one or more alcohols, one or more fatty esters or a mixture thereof.
  • hydrocarbon generally refers to a chemical compound that consists of the elements carbon (C), hydrogen (H) and optionally oxygen (O).
  • C carbon
  • H hydrogen
  • O optionally oxygen
  • hydrocarbons e.g., aromatic hydrocarbons, saturated hydrocarbons and unsaturated hydrocarbons such as alkenes, alkynes, and dienes.
  • the term also includes fuels, biofuels, plastics, waxes, solvents and oils.
  • Hydrocarbons encompass biofuels, as well as plastics, waxes, solvents and oils.
  • the term "futile cycle” as used herein refers to two metabolic pathways running simultaneously in opposite directions whose primary purpose is to dissipate energy. This can include consumption of ATP or reduction of a proton motive force in a host cell by rebalancing a proton gradient.
  • the simultaneous carrying out of glycolysis and gluconeogenesis is an example of a futile cycle, where glucose would be converted to pyruvate by glycolysis and then converted back to glucose by gluconeogenesis, resulting in an overall consumption of ATP.
  • One or more genes encoding enzymes in pathways related through a futile cycle can be engineered into a host cell, where their expression results in an overall dissipation of excess energy.
  • reduced product refers to a product that has been synthesized by reducing a reactant via an endogenous or exogenous pathway in a host cell.
  • the reduced product such as, e.g., ethanol, is a carbon-based product of interest.
  • proton gradient refers to an imbalance in the concentration of protons across one or more intracellular compartments separated by a membrane.
  • the "proton gradient” can also be described as a charge separation providing electrical potential energy. It can be generated by a variety of phenomena, including the operation of an electron transport chain, and the hydrolysis of ATP by ATP synthase. Excess energy created by metabolic process in the host cell can be stored as a proton gradient.
  • proton motive force refers to energy that is generated by the transfer of protons or electrons across an energy-transducing membrane and can be used for chemical, osmotic, or mechanical work.
  • the proton motive force can be described as the work performed by the proton or voltage gradient across a membrane.
  • Metabolic equilibrium refers to a state within a host cell that is actively regulated by several complex biochemical interactions and reactions. Metabolic equilibrium in the present application refers to the metabolic equilibrium state of an unmodified (i.e., background) host cell at homeostasis.
  • a genetically modified or engineered host cell may have an internal metabolic environment which is altered from the metabolic equilibrium of the unmodified host cell which affects metabolic reaction rates, for example, e.g. addition of a pathway to produce reduced products may affect the normal energetic flow of a host cell, affecting the energy charge of the modified host cell in a way which inhibits production of reduced products.
  • the term "energy charge” as used herein refers to an index of the energy status of a host cell, which is proportional to the mole fraction of ATP plus half the mole fraction of ADP, given that ATP contains two anhydride bonds whereas ADP contains one, and where the mole fraction is defined as the fraction of the species over the total amount of ATP, ADP, and AMP, combined. Metabolic pathway reactions are affected by the energy charge of the host cell. A 'characteristic energy charge' is an inherent property of an unmodified (i.e., background) host cell at metabolic equilibrium or homeostasis.
  • Excess energy refers to an energy level in a host cell that is outside of metabolic equilibrium or homeostasis, or to an energy level that inhibits production of a reduced product in the host cell. Excess energy can also refer to an energy charge which is outside of the normal value for the host cell, e.g., energy charge that significantly exceeds the value in a corresponding unmodified (i.e., background) host cell. Excess energy in the host cell can be stored or can physically manifest itself, for example, as a proton gradient, capable of generating a proton motive force, or as excess ATP.
  • Excess ATP is defined as an amount of ATP that gives a higher than normal energy charge for a host cell, or an amount that inhibits synthesis of a reduced product. Dissipation of an excess energy refers to a mechanism to reduce the physical manifestation of an excess energy.
  • the methods of the invention are based on principles of metabolic engineering, and uses, e.g., engineered pathways as described in, e.g., WO 2007/136762 and WO
  • 2007/139925 (each of which is incorporated by reference in its entirety for all purposes) to make products from energy captured by photoautotrophic organisms.
  • carbon-based products of interest are produced by expressing a gene or a set of genes in a photoautotrophic microorganism, e.g., cyanobacteria, as described herein.
  • Plasmids are constructed to express various proteins that are useful in production of carbon-based products, as described in the Examples herein, e.g., Example 1.
  • the constructs can be synthetically made or made using standard molecular biology methods and all the cloned genes are put under the control of constitutive promoters or inducible promoters. Plasmids containing the genes of interest are transformed into the host and corresponding transformants are selected in LB plate supplemented with antibiotics such as spectinomycin, carbenicillin, etc.
  • cells in which a nucleic acid molecule has been introduced are transformed to express or over-express desired genes while other nucleic acid molecules are attenuated or functionally deleted.
  • Transformation techniques by which a nucleic acid molecule can be introduced into such a cell including, but not limited to, transfection with viral vectors, conjugation, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • Transformants are inoculated into a suitable medium. The samples containing the transformants are grown at suitable temperatures in a shaker until they reach at certain OD. The cells are then spun down at and the cell pellets are suspended. Separation techniques allows for the sample to be subjected to GC/MS analysis. Total yield is determined.
  • Microorganism Includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista.
  • microbial cells and “microbes” are used interchangeably with the term microorganism.
  • Photoautotrophic organisms include eukaryotic plants and algae, as well as prokaryotic cyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria.
  • the host cell can be a Gram-negative bacterial cell or a Gram-positive bacterial cell.
  • a Gram-negative host cell of the invention can be, e.g., Gluconobacter, Rhizobium,
  • Bradyrhizobium Alcaligenes, Rhodobacter, Rhodococcus. Azospirillum, Rhodospirillum,
  • Sphingomonas Burkholderia, Desuifomonas, Geospirillum, Succinomonas, Aeromonas,
  • a Gram-positive host cell of the invention can be, e.g., Fibrobacter,
  • Acidobacter Bacteroides, Sphingobacterium, Actinomyces, Corynebacterium, Nocardia,
  • Rhodococcus Propionibacterium, Bifidobacterium, Bacillus, Geobacillus, Paenibacillus,
  • Extremophiles are also contemplated as suitable organisms. Such organisms withstand various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and chemicals. They include
  • hyperthermophiles which grow at or above 80°C such as Pyrolobus fumarii; thermophiles, which grow between 60-80°C such as Synechococcus lividis; mesophiles, which grow between 15-60°C and psychrophiles, which grow at or below 15°C such as Psychrobacter and some insects.
  • Radiation-tolerant organisms include Deinococcus radiodurans.
  • Pressure- tolerant organisms include piezophiles or barophiles, which tolerate pressure of 130 MPa.
  • Hypergravity- (e.g., >lg) hypogravity- (e.g., ⁇ lg) tolerant organisms are also contemplated.
  • Vacuum-tolerant organisms include tardigrades, insects, microbes and seeds.
  • Dessicant- tolerant and anhydrobiotic organisms include xerophiles such as Artemia salina; nematodes, microbes, fungi and lichens.
  • Salt-tolerant organisms include halophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina.
  • pH-tolerant organisms include alkaliphiles such as Natronobacterium, Bacillus firmus OF4, Spirulina spp. (e.g., pH > 9) and acidophiles such as Cyanidium caldarium, Ferroplasma sp. (e.g., low pH).
  • Anaerobes which cannot tolerate 0 2 such as Methanococcus jannaschii; microaerophils, which tolerate some 0 2 such as
  • Clostridium and aerobes, which require 0 2 are also contemplated.
  • Gas-tolerant organisms, which tolerate pure C0 2 include Cyanidium caldarium and metal-tolerant organisms include metalotolerants such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn), Ralstonia sp. CH34 (e.g., Zn, Co, Cd, Hg, Pb). Gross, Michael. Life on the Edge: Amazing Creatures Thriving in Extreme Environments. New York: Plenum (1998) and Seckbach, J.
  • Plants include but are not limited to the following genera: Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea.
  • Algae and cyanobacteria include but are not limited to the following genera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira,
  • Chrysonebula Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella,
  • Chrysostephanosphaera Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,
  • Coenocystis Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,
  • Cyanothece Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella,
  • Cymbellonitzschia Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,
  • Distrionella Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis,
  • Entophysalis Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium,
  • Gloeocapsa Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron,
  • Gloeomonas Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,
  • Gonatozygon Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,
  • Granulochloris Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia,
  • Hapalosiphon Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitonia, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon,
  • Microglena Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,
  • Myochloris Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium,
  • Pocillomonas Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Poly goniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate,
  • Pseudoncobyrsa Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum,
  • Rhabdoderma Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium,
  • Sirogonium Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum,
  • Stauerodesmus Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Molingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis,
  • Tetraspora Tetrastrum
  • Thalassiosira Thamniochaete
  • Thorakochloris Thorea
  • Tolypella Tolypothrix
  • Trachelomonas Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella,
  • Green non-sulfur bacteria include but are not limited to the following genera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicrobium .
  • Green sulfur bacteria include but are not limited to the following genera:
  • Purple sulfur bacteria include but are not limited to the following genera:
  • Rhodovulum Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis.
  • Purple non-sulfur bacteria include but are not limited to the following genera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila,
  • Rhodopseudomonas Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira.
  • Aerobic chemolithotrophic bacteria include but are not limited to nitrifying bacteria such as Nitrobacteraceae sp., Nitrobacter sp., Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp., Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibrio sp.; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp.,
  • Archaeobacteria include but are not limited to methanogenic archaeobacteria such as Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus sp., Methanomicrobium sp., Methanospirillum sp., Methanogenium sp., Methanosarcina sp., Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanus sp.; extremely thermophilic sulfur-metabolizers such as Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., Acidianus sp.
  • methanogenic archaeobacteria such as Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus sp.,
  • microorganisms such as, Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp., Mycobacteria sp., and oleaginous yeast.
  • Hyperphotosynthetic conversion requires extensive genetic modification; thus, in some embodiments the parental photoautotrophic organism can be transformed with exogenous DNA.
  • Organisms for hyperphotosynthetic conversion include: Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, and Zea mays (plants), Botryococcus braunii, Chlamydomonas reinhardtii and Dunaliela salina (algae), Synechococcus sp. PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp.
  • PCC 6803 and Thermosynechococcus elongatus BP-1 (cyanobacteria), Chlorobium tepidum (green sulfur bacteria), Chloroflexus auranticus (green non-sulfur bacteria), Chromatium tepidum, and Chromatium vinosum (purple sulfur bacteria), Rhodospirillum rubrum, Rhodobacter capsulatus, and
  • Rhodopseudomonas palusris purple non-sulfur bacteria.
  • suitable organisms include synthetic cells or cells produced by synthetic genomes as described in Venter et al. US Pat. Pub. No. 2007/0264688, and cell-like systems or synthetic cells as described in Glass et al. US Pat. Pub. No. 2007/0269862.
  • microorganisms that can be engineered to fix carbon dioxide bacteria such as Escherichia coli, Acetobacter aceti, Bacillus subtilis, yeast and fungi such as Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis.
  • carbon dioxide bacteria such as Escherichia coli, Acetobacter aceti, Bacillus subtilis, yeast and fungi such as Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis.
  • a common theme in selecting or engineering a suitable organism is autotrophic fixation of C0 2 to products. This covers photosynthesis and methanogenesis. Acetogenesis, encompassing the three types of C0 2 fixation; Calvin cycle, acetyl-CoA pathway and reductive TCA pathway is also covered. The capability to use carbon dioxide as the sole source of cell carbon (autotrophy) is found in almost all major groups ofprokaryotes. The C0 2 fixation pathways differ between groups, and there is no clear distribution pattern of the four presently-known autotrophic pathways. Fuchs, G. 1989. Alternative pathways of autotrophic C0 2 fixation, p. 365-382. In H. G. Schlegel, and B. Bowien (ed.), Autotrophic bacteria. Springer- Verlag, Berlin, Germany. The reductive pentose phosphate cycle
  • Light is delivered through a variety of mechanisms, including natural illumination (sunlight), standard incandescent, fluorescent, or halogen bulbs, or via propagation in specially-designed illuminated growth chambers (for example Model LI 15 Illuminated Growth Chamber (Sheldon Manufacturing, Inc. Cornelius, OR). For experiments requiring specific wavelengths and/or intensities, light is distributed via light emitting diodes (LEDs), in which wavelength spectra and intensity can be carefully controlled (Philips).
  • LEDs light emitting diodes
  • Carbon dioxide is supplied via inclusion of solid media supplements (i.e., sodium bicarbonate) or as a gas via its distribution into the growth incubator or media.
  • solid media supplements i.e., sodium bicarbonate
  • Most experiments are performed using concentrated carbon dioxide gas, at concentrations between 1 and 30%, which is directly bubbled into the growth media at velocities sufficient to provide mixing for the organisms.
  • concentrated carbon dioxide gas the gas originates in pure form from commercially-available cylinders, or preferentially from concentrated sources including off-gas or flue gas from coal plants, refineries, cement production facilities, natural gas facilities, breweries, and the like.
  • Synechococcus sp. PCC 7002 cells are transformed according to the optimized protocol previously described [Essich ES, Stevens Jr E, Porter RD "Chromosomal
  • KH2P04 1 g/L Trizma base pH 8.2, 4 ⁇ g/L Vitamin B12, 3.89 mg/L FeC13. 6 H20, 34.3 mg/L H3B03, 4.3 mg/L MnC12. 4 H20, 315 ⁇ g/L ZnC12, 30 ⁇ g/L Mo03, 3 ⁇ g/L CuS04. 5 H20, 12.2 ⁇ g/L CoC12. 6 H20) [Stevens SE, Patterson COP, and Myers J. "The production of hydrogen peroxide by green algae: a survey.” J. Phycology (1973). 9:427-430] plus 5g/L of NaN03 to approximately 108 cells/ mL.
  • Transformants are picked 3-4 days later. Selections are typically performed using 200 ⁇ g/ml kanamycin, 8 ⁇ g/ml chloramphenicol, 10 ⁇ g/ml spectinomycin on solid media, whereas 150 ⁇ g/ml kanamycin, 7 ⁇ g/ml chloramphenicol, and 5 ⁇ g/ml spectinomycin are employed in liquid media. As described in Example 1 , selection is performed using A+ containing 3 mM urea, 50 ⁇ g/ml kanamycin, and a 25 ⁇ g/ml spectinomycin underlay.
  • Non-genetic ATP-mitigating factors ⁇ e.g. inhibitors of ATP synthesis
  • ATP synthase inhibitors were evaluated in the ethanol-producing strains JCC1581 and JCC1510, described herein.
  • Table 1 Examples of chemical compounds used to deplete the cellular ATP pool.
  • PMF proton motive force.
  • the JCC 1581 strain was constructed by standard homologous recombination techniques.
  • the starting material was wild-type Synechococcus sp. PCC7002 (JCC138), which was obtained from the Pasteur Collection or ATCC.
  • Gene, promoter, terminator, and marker constructs made synthetically were obtained from DNA2.0 or by PCR,
  • DCCD ⁇ , ⁇ '- dicyclohexylcarbodiimide
  • TCS 3,3',4',5-tetrachlorosalicylanilide
  • 2,4-DNP 2,4- dinitrophenol
  • CCCP carbonyl cyanide-/?-trifluoromethoxyhydrazone
  • a pAQ7 Aldh targeting plasmid (see Genbank # CP000957) was constructed containing the Moorella alcohol dehydrogenase gene (adhAM) under the control of the lambda cl promoter.
  • This plasmid (pJB594) was naturally transformed into JCC 138 (see Table 2) using a standard cyanobacterial transformation protocol, yielding strain JCC1034. Briefly, JCC138 culture was grown to an OD 73 o of approximately 1.0, after which 5-10 ⁇ g of plasmid DNA was added to 1 ml of neat JCC138 culture. The cell-DNA mixture was incubated at 37 °C for 4 hours in the dark with gentle mixing, plated onto A+ plates, and incubated in a
  • the growth medium for liquid culture was A+ with 50 ⁇ g/ml kanamycin.
  • JCC 1034 was then transformed with pJB 1156 which introduced a two-gene operon, driven by P(nir07) (SEQ ID NO: 1), to an ectopic location on pAQ3.
  • This operon contained adhAMas well as the pyruvate decarboxylase gene from Zymomonas mobilis (pdcZm).
  • the resulting strain, JCC 1581 therefore had two independent adhAM transgenes and one pdcZm transgene.
  • the protocol used to transform pJBl 156 into JCC 1034 generating JCC 1581 was the same with the exception that the selection media is A+ containing 3 mM urea, 50 ⁇ g/ml kanamycin, and a 25 ⁇ g/ml spectinomycin underlay.
  • Table 2 Transformation of host cell with integrative plasmid generated JCC 1034 and JCC1518 strains from wild-type Synechococcus sp. PCC7002 (JCC138).
  • P(w/V07) promoter is a synthetic construct based on the nirA promoter from Synechococcus sp. PCC 7942. (Shin-Ichi Maeda et al. (1998). cist-Acting Sequences Required for NtcB-Dependent, Nitrite-Responsive Positive Regulation of the Nitrate Assimilation Operon in the Cyanobacterium Synechococcus sp. Strain PCC 7942. J. Bacteriol. 180:4080- 4088. The ATG in bold represents the start codon of an expressed gene.
  • transformation was inoculated into 5 ml A + broth with 5 mM urea, 50 ⁇ g/ml kanamycin, and 100 ⁇ g/ml spectinomycin in a 16mM x 150mM plastic-capped culture tube and incubated in the Infors incubator (37C, 150rpm, 2%C02) at a ⁇ 60° angle.
  • the JCC 1510 strain was constructed by standard homologous recombination techniques from a cyanobacterial strain.
  • the engineered strain comprises a pyruvate decarboxylase gene under the control of a p(nir07) promoter, and an alcohol dehydrogenase gene under the control of a lambda cl promoter (Table 4).
  • Genetic modification of a host cell producing a carbon-based product of interest to mitigate excess energy accumulation is performed to enhance production of the carbon-based product of interest.
  • Genetic strategies for mitigating excess energy accumulation include but are not restricted to the sets of futile-cycle genes given in Table 7. Futile cycle pathways are inserted into modified host cells according to standard homologous recombination
  • sigE is overexpressed in a host cell, e.g., Synechocystis sp., resulting an increased activity of at least part of a futile cycle pathway, e.g. , the oxidative pentose phosphate pathway during ethanol production in light.
  • Table 7 Futile cycle sets of genes for dissipating excess ATP or proton motive force
  • Genes selected from a futile cycle are cloned or synthesized and transformed into production hosts, where they are expressed. Expression of the genes in the production host is inducible under certain conditions. These conditions include, for example, absence of ammonia, absence of copper, presence of nickel, or presence of a gratuitous inducer, such as IPTG.
  • Example 1 describes the engineering of an ethanologen comprising, in part, a urea-repressible, nitrate-inducible nirA-typc promoter, P(/»H)7) (SEQ ID NO: 1) which controls expression of an operon.
  • the ⁇ ( » ⁇ )7) promoter is a versatile inducible promoter and can be used to control expression of other genes and operons, e.g., genes encoding AAR and ADM enzymes as described in U.S. Patent Application 12/833821, filed July 9, 2010.

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Abstract

L'invention concerne des procédés et des compositions qui permettent l'amélioration de la biosynthèse d'éthanol dans une cellule hôte. L'invention concerne des gènes codant pour des enzymes de voies de cycle futile pour augmenter la production d'éthanol, des procédés d'optimisation de l'expression de ces enzymes dans des cellules hôtes et des procédés de production d'éthanol par ces cellules hôtes. L'invention concerne également des composés exogènes pour augmenter la productivité de l'éthanol par les cellules hôtes.
PCT/US2011/045572 2010-07-29 2011-07-27 Procédés et compositions pour amélioration des rendements de produits réduits de microorganismes photosynthétiques Ceased WO2012015949A2 (fr)

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WO2014003555A1 (fr) 2012-06-27 2014-01-03 Rijksuniversiteit Groningen Production améliorée de pénicilline
WO2014018902A3 (fr) * 2012-07-26 2014-04-03 Joule Unlimited Technologies, Inc. Procédés et compositions pour l'augmentation de la formation de pyruvate et d'acétyl-coa
US9309541B2 (en) 2011-07-27 2016-04-12 Alliance For Sustainable Energy, Llc Biological production of organic compounds
US9914947B2 (en) 2011-07-27 2018-03-13 Alliance For Sustainable Energy, Llc Biological production of organic compounds
EP2909305B1 (fr) * 2012-10-16 2018-12-26 DSM IP Assets B.V. Cellules à conversion de pentose améliorée
US10604773B2 (en) 2012-10-16 2020-03-31 Dsm Ip Assets B.V. Cells with improved pentose conversion
CN114426933A (zh) * 2020-10-29 2022-05-03 中国石油化工股份有限公司 一种提高亚硝酸细菌细胞产率的方法

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IL123986A (en) * 1997-04-24 2011-10-31 Organon Nv Medicinal compounds
AU2003903453A0 (en) * 2003-07-07 2003-07-17 The University Of Queensland Production of hydrogen
EP1650563A4 (fr) * 2003-08-04 2007-09-19 Kikkoman Corp Procede pour evaluer la fatigue
US7973214B2 (en) * 2006-09-25 2011-07-05 Ut-Battelle, Llc Designer organisms for photosynthetic production of ethanol from carbon dioxide and water
US20090155873A1 (en) * 2007-10-04 2009-06-18 Bio Architecture Lab, Inc. Biofuel production
EP2231857A2 (fr) * 2007-12-17 2010-09-29 Universiteit van Amsterdam Réduction de co2 générée par la lumière sur des composés organiques destinés à servir de combustibles ou de semi-produits industriels au moyen d'un organisme autotrophe contenant une cassette génétique fermentative
AU2009211551B2 (en) * 2008-02-08 2014-07-24 Algenol Biofuels Inc. Genetically modified photoautotrophic ethanol producing host cells, method for producing the host cells, constructs for the transformation of the host cells, method for testing a photoautotrophic strain for a desired growth property and method of producing ethanol using the host cells
EP2346998B1 (fr) * 2008-10-31 2016-01-27 GEVO, Inc. Micro-organismes manipulés capables de produire des composés cibles en conditions anaérobies

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US9309541B2 (en) 2011-07-27 2016-04-12 Alliance For Sustainable Energy, Llc Biological production of organic compounds
US9914947B2 (en) 2011-07-27 2018-03-13 Alliance For Sustainable Energy, Llc Biological production of organic compounds
WO2014003555A1 (fr) 2012-06-27 2014-01-03 Rijksuniversiteit Groningen Production améliorée de pénicilline
WO2014018902A3 (fr) * 2012-07-26 2014-04-03 Joule Unlimited Technologies, Inc. Procédés et compositions pour l'augmentation de la formation de pyruvate et d'acétyl-coa
EP2909305B1 (fr) * 2012-10-16 2018-12-26 DSM IP Assets B.V. Cellules à conversion de pentose améliorée
US10273507B2 (en) 2012-10-16 2019-04-30 Dsm Ip Assets B.V. Cells with improved pentose conversion
EP3492579A1 (fr) * 2012-10-16 2019-06-05 DSM IP Assets B.V. Cellules présentant une meilleure conversion de pentose
US10604773B2 (en) 2012-10-16 2020-03-31 Dsm Ip Assets B.V. Cells with improved pentose conversion
CN114426933A (zh) * 2020-10-29 2022-05-03 中国石油化工股份有限公司 一种提高亚硝酸细菌细胞产率的方法
CN114426933B (zh) * 2020-10-29 2023-07-04 中国石油化工股份有限公司 一种提高亚硝酸细菌细胞产率的方法

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