WO2013148348A1 - Micro-organismes chimiotrophes manipulés fixant le co2 et produisant des produits carbonés, et procédés d'utilisation de ces micro-organismes - Google Patents

Micro-organismes chimiotrophes manipulés fixant le co2 et produisant des produits carbonés, et procédés d'utilisation de ces micro-organismes Download PDF

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WO2013148348A1
WO2013148348A1 PCT/US2013/032362 US2013032362W WO2013148348A1 WO 2013148348 A1 WO2013148348 A1 WO 2013148348A1 US 2013032362 W US2013032362 W US 2013032362W WO 2013148348 A1 WO2013148348 A1 WO 2013148348A1
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microorganism
nucleic acid
bacterial cell
exogenous nucleic
lipids
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PCT/US2013/032362
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English (en)
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Itzhak Kurek
John S. REED
Lisa Dyson
Michael Siani-Rose
Henrik Fyrst
Christer Jansson
David GALGOCZY
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Kiverdi, Inc.
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Priority claimed from US13/623,089 external-priority patent/US9879290B2/en
Application filed by Kiverdi, Inc. filed Critical Kiverdi, Inc.
Priority to US14/388,756 priority Critical patent/US20150017694A1/en
Publication of WO2013148348A1 publication Critical patent/WO2013148348A1/fr
Priority to US15/233,512 priority patent/US9957534B2/en
Priority to US15/936,440 priority patent/US20190040427A1/en

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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
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    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
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    • C12P5/023—Methane
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    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

  • This disclosure further relates to methods of fixing carbon from gas into useful organic molecules such as diacids, hydroxy acids, fatty acid alcohols, fatty acid aldehydes, fatty acids, unsaturated fatty acids, esters, lipids, alkanes, alkenes, and alkynes.
  • the bacteria of the invention can be genetically engineered for use in the methods or other aspects of the invention described herein.
  • the present invention further describes
  • Algal systems have been developed to create hydrocarbons through photo synthetic reactions, as well as heterotrophic reactions fed by sugar that indirectly depend upon photosynthesis, but insufficient yields limit the effectiveness, economic feasibility, practicality and commercial adoption.
  • Bacterial cells have been genetically engineered to process sugar feedstocks into useful hydrocarbons in heterotrophic fermentation systems, however, there are significant drawbacks for these systems.
  • Heterotrophic fermentations are vulnerable to contamination because heterotrophic microorganisms that can grow on fixed carbon nutrients are far more ubiquitous in the surface environment. Heterotrophic technologies also generally suffer limitations in terms of food versus fuel conflict and negative environmental impacts.
  • Gas-to-liquid (GTL) technologies have the benefit of allowing the utilization of waste carbon sources - including highly lignocellulosic waste through the conversion to synthesis gas (syngas) via gasification, as well as waste C0 2 through the provision of reduced hydrogen - in the production of liquid fuels and/or organic chemicals.
  • Syngas is a mix of gases that generally contains H 2 , CO, and C0 2 as major components, which can be generated through steam reforming of methane and/or liquid petroleum gas or through gasification of any organic material, including but not limited to biomass, waste organic matter, various polymers, and coal. Many gasification processes are available for the production of syngas.
  • a number of gasification processes subject the carbonaceous feedstock to partial oxidation at high temperatures (500-1500° C), with the oxygen supply restricted to prevent complete combustion, producing syngas with varying composition depending on feedstock and reaction conditions such that the ratio of H 2 :CO can range from 0.5: 1 to 3: 1.
  • the hydrogen component of syngas can be raised through the reaction of CO with steam in the water gas shift reaction with a concomitant increase in C0 2 in the syngas mix.
  • Some major technologies for syngas conversion to liquid fuels or chemicals include chemical catalytic processes such as the Fischer-Tropsch (F-T) as well as processes for the synthesis of methanol or other mixed alcohols, and biological gas fermentation processes.
  • F- T has been worked on for almost one hundred years and relies on metal-based, inorganic catalysts for the conversion of syngas into longer chain hydrocarbons. Difficulties with F-T include: a wide chain length distribution of products resulting in the need to reprocess short chain length products such as methane and LPG and/or the need to perform additional costly post-processing steps on long chain waxes and tars such as hydrocracking; high catalyst sensitivity to syngas impurities such as sulfur containing compounds, tars, and particulates, generally necessitating multiple costly gas clean up steps; relatively low flexibility in terms of accommodating various ratios of syngas constituents i.e.
  • biomass gasification has a long history going back to World War II where biomass gasification was used for running modified automobiles, boats, buses, and trucks.
  • biomass gasification technologies are at, or near commercialization (able to gasify 10,000 or more tons of biomass per year), and are generally used for the production of heat and/or electricity.
  • the synthesis of chemicals or fuels from syngas generated via biomass gasification is at an earlier stage of development, and is generally pre-commercial.
  • syngas and/or CO 2 and/or renewable H 2 in gas fermentation enables the utilization of cheaper and more flexible sources of energy and/or carbon for the biological synthesis of sustainable chemicals and fuels than is possible through heterotrophic or photo trophic synthesis.
  • syngas acts as both a carbon and energy source for the microbial culture.
  • a fermentation process based upon a gaseous feedstock such as syngas can allow for far lower negative environmental and food production impacts in the biological synthesis of liquid fuels and/or chemicals than the highly land and water intensive heterotrophic or photo trophic-based technologies.
  • gaseous feedstock such as syngas
  • current biological GTL technologies generally yield relatively short chain alcohols, or other short chain organic compounds, as products, which have relatively low energy density and infrastructure compatibility with current petrochemical and oleochemical processes.
  • syngas-growing microorganisms used in current biological GTL technologies are generally inappropriate for the synthesis of high energy density, infrastructure compatible fuels, or other longer carbon chain lipid-based chemicals.
  • Their short chain products are relatively low in value and they generally don't efficiently synthesize drop-in fuels such as diesel or jet fuel, or higher value lipid-based chemicals.
  • microorganisms that can grow in conventional and scalable contained reaction vessels and that produce commercially viable sets of organic carbon chains of at least eight carbon atoms long in a commercially feasible method.
  • microorganisms not limited metabolically by typically used carbon and energy inputs such as sugars, and a microorganism that can additionally utilize syngas, producer gas, as well as a wide array of abiotic sources of carbon and energy for the synthesis of drop-in fuels and chemicals, leading to a feedstock flexibility for the present technology that far exceeds comparable heterotrophic systems.
  • microorganisms that can utilize electron donors such as hydrogen, present in syngas, producer gas, as well as readily generated through a wide array of abiotic renewable energy technologies, for growth and carbon fixation.
  • microorganisms This has been accomplished through the use of thioesterases to change populations of fatty acids C8-C14 and the over-expression of thioesterases to increase shorter chain length fatty acids.
  • Examples in the prior art include C8-C14 thioesterase expression to produce shorter chain lengths in US 7,883,882 Renewable chemical production from novel fatty acid feedstocks, Franklin et al. Solazyme, p. 58.
  • Nutritionally important n-3 fatty acids include a-linolenic acid (ALA),
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • N-3 fatty acids that are important in human physiology are ⁇ -linolenic acid (18:3, n-3; ALA), eicosapentaenoic acid (20:5, n-3; EPA), and docosahexaenoic acid (22:6, n-3; DHA). These three polyunsaturates have either 3, 5, or 6 double bonds in a carbon chain of 18, 20, or 22 carbon atoms, respectively. As with most naturally produced fatty acids, all double bonds are in the cis-configuration.
  • a fatty acid desaturase is an enzyme that removes two hydrogen atoms from a fatty acid, creating a carbon/carbon double bond. These desaturases are classified as delta - indicating that the double bond is created at a fixed position from the carboxyl group of a fatty acid (for example, ⁇ 9 desaturase creates a double bond at the 9th position from the carboxyl end), omega (e.g. co3desaturase) - indicating the double bond is created between the third and fourth carbon from the methyl end of the fatty acid.
  • omega e.g. co3desaturase
  • an elongase alternates with different desaturases (for example, A6desaturase) repeatedly inserting an ethyl group, then forming a double bond.
  • Most polyunsaturated oils come from fish and there is a need for alternate, and particularly microbial sources of polyunsaturated fatty acids, given depleting fish stocks and increasing pollution in the oceans.
  • the present invention allows microorganisms to be genetically engineered to convert
  • C0 2 gas and/or syngas and/or producer gas to higher value and/or more infrastructure compatible products than current biologically based syngas and/or C0 2 conversion technologies.
  • the present technology allows the development of new genetically enhanced strains of microorganisms that can be used for gas fermentation within biological gas-to- liquid (GTL) processes to produce and/or secrete drop-in liquid fuels directly from C0 2 or from syngas, as well as various other relatively long chain organic compounds that are drop- in, and are currently only produced in bulk from petroleum or higher plants.
  • GTL biological gas-to- liquid
  • the present invention relates to the engineering of microorganisms, including but not limited to hydrogen oxidizing, carbon monoxide oxidizing, and knallgas microorganisms, with a natural capability to grow and synthesize biomass on gaseous carbon sources such as syngas and/or C0 2 , such that the engineered microorganisms synthesize targeted products, including chemicals and fuels, under gas fermentation.
  • the microorganisms and methods of the present invention enable low cost synthesis of chemicals and fuels, which can compete on price with petrochemicals and higher-plant derived oleochemicals, and which will generally have a substantially lower price than oleochemicals produced through heterotrophic or photo trophic synthesis.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids.
  • the composition comprises a microorganism, wherein the microorganism is a carbon monoxide-oxidizing microorganism.
  • the composition comprises a microorganism, wherein the microorganism is a knallgas microorganism.
  • the composition comprises a microorganism, wherein the microorganism is chosen from the genera Rhodococcus or Gordonia.
  • the composition comprises a microorganism, wherein the microorganism is Rhodococcus opacus. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Rhodococcus opacus (DSM 43205) or Rhodococcus sp (DSM 3346). In some embodiments, the composition comprises a microorganism, wherein the microorganism is chosen from the genera Ralstonia or Cupriavidus. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Cupriavidus necator.
  • the composition comprises a microorganism wherein the microorganism can naturally grow on H 2 /CO 2 and/or syngas, and wherein the microorganism can naturally accumulate lipid to 50% or more of the cell biomass by weight.
  • the microorganisms have a native ability to send a high flux of carbon down the fatty acid biosynthesis pathway.
  • the microorganism exhibiting these traits is Rhodococcus opacus (DSM 43205 or DSM 43206).
  • the composition comprises a microorganism that can naturally grow on H 2 /CO 2 and/or syngas, and wherein the microorganism can naturally accumulate polyhydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) to 50% or more of the cell biomass by weight.
  • PHB polyhydroxybutyrate
  • PHA polyhydroxyalkanoate
  • the microorganisms have a native ability to direct a high flux of carbon through the acetyl-CoA metabolic intermediate, which can lead into fatty acid biosynthesis, along with a number of other synthetic pathways including PHA and PHB synthesis.
  • a microorganism is considered to direct a high flux of carbon through acetyl-CoA if a product of a synthesis pathway going through the acetyl-CoA metabolic intermediate, including but not limited to polyhydroxybutyrate (PHB) or
  • polyhydroxyalkanoate can represent 50% or more of the cell biomass by weight.
  • the microorganism exhibiting these traits is Cupriavidus necator (DSM 531 or DSM 541).
  • the invention relates to a non-naturally occurring
  • microorganism capable of converting syngas or other gaseous carbon sources into targeted oleochemical and/or monomer products, where the wild-type microorganism is capable of growing on syngas or other gaseous carbon sources, but is either not capable of synthesizing said targeted oleochemical and/or monomer products, or is capable of synthesizing the targeted oleochemicals and/or monomers, but is not capable of synthesizing the targeted biochemical products at the concentration and/or efficiency of the non-natural
  • microorganisms In such microorganisms, one or more proteins or enzymes are expressed in the microorganism, thereby modifying, extending, diverting, enhancing, promoting, or otherwise altering the lipid biosynthesis pathway or its regulation for the synthesis and/or enhanced synthesis of a targeted lipid-based product, oleochemical, monomer, or
  • the invention relates to a non-naturally occurring
  • microorganism capable of converting syngas or other gaseous carbon sources into targeted oleochemical and monomer products, where the wild-type microorganism is capable of growing on syngas or other gaseous carbon sources and is capable of synthesizing said targeted oleochemical and monomer products, but the non-naturally occurring microorganism is capable of synthesizing the targeted biochemical products at a higher concentration and/or efficiency than the wild-type microorganism due to the overexpression and/or
  • the invention relates to compositions comprising one or more bacterial cells that consist of one, two, or three exogenous nucleic acid sequences where said bacteria can grow using syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas as a source of carbon and/or energy.
  • the invention relates to compositions comprising one or more bacterial cells of Rhodococcus opacus (DSM 43205) that consist of zero, one, two, or three exogenous nucleic acid sequences.
  • DSM 43205 Rhodococcus opacus
  • one, two, or three exogenous nucleic acid sequences encode one or more thioesterase proteins.
  • one, two, or three exogenous nucleic acid sequences encode one or more CYP52A proteins.
  • one, two, or three exogenous nucleic acid sequences encode a CYP709C1 and/or a CYP81B1 protein.
  • the source of thioesterase is inherent to the production organisms. In some embodiments the source of thioesterase is Rhodococcus opacus B4. In some embodiments the thioesterase is derived from bacteria or plants other than the host microorganism.
  • the invention relates to compositions comprising one or more bacterial cells that consist of two exogenous nucleic acid sequences that encode the following proteins: fatty acid acyl-ACP reductase, a fatty acid aldehyde decarbonylase, where said bacteria can grow using syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas as a source of carbon and/or energy.
  • the invention relates to compositions comprising one or more bacterial cells that consist of three exogenous nucleic acid sequences that encode the following proteins: fatty acid acyl-ACP reductase, a fatty acid aldehyde decarbonylase, and a thioesterase, where said bacteria can grow using syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas as a source of carbon and/or energy.
  • the bacterial cell produces and/or secretes one or more lipids in an amount that is greater than the amount of lipids produced and/or secreted by the same cell not comprising the exogenous nucleic acid sequence.
  • the bacterial cell produces and/or secretes one or more lipids having a given carbon chain length, where the amount of said lipid produced and/or secreted is greater than the amount produced and/or secreted by the same cell not comprising the exogenous nucleic acid sequence.
  • the bacterial cell produces and/or secretes one or more lipid molecules in an amount that is less than the amount of lipids produced and/or secreted by the same cell not comprising the exogenous nucleic acid sequence.
  • the bacterial cell produces and/or secretes one or more hydrocarbons in an amount that is greater than the amount of hydrocarbons produced and/or secreted by the same cell not comprising the exogenous nucleic acid sequence.
  • the bacterial cell produces and/or secretes one or more lipids or hydrocarbons in a ratio greater than the ratio of lipids or hydrocarbons produced and/or secreted by the same cell not comprising the one or more exogenous nucleic acid sequences. In some embodiments, the bacterial cell produces and/or secretes one or more lipids or hydrocarbons, wherein at least 50% of the one or more lipids or hydrocarbons have 8 to 18 carbon atoms. In some embodiments, the bacterial cell produces and/or secretes one or more lipids or hydrocarbons, wherein at least 60% of the one or more lipids or hydrocarbons have 8 to 18 carbon atoms. In some embodiments, the bacterial cell produces and/or secretes one or more lipids or hydrocarbons, wherein at least 70% of the one or more lipids or
  • the bacterial cell produces and/or secretes one or more lipids or hydrocarbons, wherein at least 75% of the one or more lipids or hydrocarbons have 8 to 18 carbon atoms. In some embodiments, the bacterial cell produces and/or secretes one or more lipids or hydrocarbons, wherein at least 80% of the one or more lipids or hydrocarbons have 8 to 18 carbon atoms.
  • the bacterial cell or compositions comprising the bacterial cell comprise at least one exogenous nucleic acid sequence that is integrated into the genome of the cell.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more hydrocarbons including unsaturated hydrocarbons, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase.
  • the microorganism is Rhodococcus opacus.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more hydrocarbons, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase, wherein the one or more hydrocarbons have a carbon chain length of at least 8 carbon atoms.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more hydrocarbons, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the one or more hydrocarbons comprise a mixture of hydrocarbon molecules having a carbon chain length from 8 carbon atoms to 18 carbon atoms.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids
  • the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the one or more lipids comprise a quantity of at least one alkane, alkene, alkyne, fatty alcohol, and/or fatty aldehyde at a level higher than the quantity of the alkane, alkene, alkyne, fatty alcohol, and or fatty aldehyde in the same microorganism not comprising the heterologous nucleic acid sequences.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein
  • microorganism produces and/or secretes at least 10% of one or more lipids by weight.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 20% of one or more lipids by weight.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 30% of one or more lipids by weight.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 40% of one or more lipids by weight.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 50% of one or more lipids by weight.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 60% of one or more lipids by weight.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein
  • microorganism produces and/or secretes at least 70% of one or more hydrocarbons by weight.
  • the invention relates to a composition
  • a composition comprising a
  • microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 75% of one or more lipids by weight.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein
  • microorganism produces and/or secretes at least 80% of one or more lipids by weight.
  • the invention relates to a composition
  • a composition comprising a
  • microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 85% of one or more lipids by weight.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more hydrocarbons, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein less than 10% by weight of the hydrocarbons produced is methane.
  • the invention relates to a composition
  • a composition comprising a microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more organic compounds, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein less than 10% by weight of the organic compounds produced are organic acids with carbon chain length of four carbons or less.
  • the invention relates to a composition
  • a composition comprising a
  • microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more lipids or hydrocarbons, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein at least one lipid produced is a component or a precursor of a component of jet fuel, diesel fuel, or biodiesel fuel.
  • the invention relates to a composition
  • a composition comprising a
  • microorganism that converts syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas into one or more hydrocarbons
  • the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the hydrocarbons produced comprise a mixture of at least two hydrocarbons having a carbon backbone from 8 to 18 carbon atoms.
  • the present invention also relates to a bacterial cell comprising at least two exogenous nucleic acid sequences, wherein the at least two exogenous nucleic acid sequences encode fatty acid acyl-ACP reductase and fatty acid aldehyde decarbonylase, and wherein the cell converts gaseous C0 2 and/or gaseous H 2 and/or syngas into lipids.
  • the invention relates to a bacterial cell comprising at least two exogenous nucleic acid sequences, wherein the at least two exogenous nucleic acid sequences encode fatty acid acyl- ACP reductase and fatty acid aldehyde decarbonylase, and wherein the cell converts gaseous C0 2 and/or gaseous H 2 and/or syngas into lipid; wherein the cell produces and/or secretes at least 75% of one or more hydrocarbons by weight.
  • the invention relates to a bacterial cell comprising at least two exogenous nucleic acid sequences, wherein the at least two exogenous nucleic acid sequences encode fatty acid acyl-ACP reductase and fatty acid aldehyde decarbonylase, and wherein the cell converts gaseous C0 2 and/or gaseous H 2 and/or syngas into lipid; wherein the cell produces and/or secretes at least 75% of one or more hydrocarbons by weight when cultured at least 42 degrees Celsius for at least 1 hour.
  • the bacterial cell is cultured without exposure to light.
  • the invention relates to a bacterial cell comprising at least two exogenous nucleic acid sequences, wherein the at least two exogenous nucleic acid sequences encode fatty acid acyl-ACP reductase and fatty acid aldehyde decarbonylase, and wherein the cell converts gaseous C0 2 and/or gaseous H 2 and/or syngas into a hydrocarbon or mixture of hydrocarbons, and/or other lipids; wherein the cell is a strain of Rhodococcus opacus.
  • the invention relates to a bacterial cell comprising at least two exogenous nucleic acid sequences, wherein the at least two exogenous nucleic acid sequences encode fatty acid aldehyde acyl-ACP and fatty acid aldehyde decarbonylase, and wherein the cell converts gaseous C0 2 and/or gaseous H 2 and/or syngas into a hydrocarbon or mixture of hydrocarbons, and/or other lipids; wherein the cell is a strain of Cupriavidus necator.
  • the invention relates to a bacterial cell comprising a first, a second, and a third exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase, the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase, and the third exogenous nucleic acid sequence encodes a thioesterase; and wherein the cell converts gaseous C0 2 and/or gaseous H 2 and/or syngas into a lipid or mixture of lipids.
  • the bacterial cell comprises no more than eight exogenous nucleic acids that encode a lipid pathway enzyme.
  • the bacterial cell comprises no more than seven exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than six exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than five exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than four exogenous nucleic acids that encode a lipid pathway enzyme. In some
  • the bacterial cell comprises no more than three exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than two exogenous nucleic acids that encode a lipid pathway enzyme. In some
  • the bacterial cell comprises no more than one exogenous nucleic acid that encodes a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than eight exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than seven exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than six exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than five exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than four exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than three exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than two exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than one exogenous nucleic acid that encodes a protein.
  • the invention relates to a method of producing a lipid or mixture of lipids in a microorganism population comprising the cell or the composition described herein, wherein the method comprises: culturing a population of microorganisms comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas.
  • the invention relates to a method of producing a lipid or mixture of lipids, wherein the method comprises: culturing a population of bacterial cells comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas.
  • the microorganism population comprises a bacterial strain of Rhodococcus opacus.
  • the microorganism population comprises a bacterial strain of Rhodococcus opacus (DSM 43205 or 43206).
  • the invention relates to a method of producing a lipid or mixture of lipids, wherein the method comprises: culturing a population of bacterial cells comprising the cell or the composition described herein in a feedstock comprising methanol, a common impurity of syngas, with or without the addition of syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas.
  • the microorganism comprises: culturing a population of bacterial cells comprising the cell or the composition described herein in a feedstock comprising methanol, a common impurity of syngas, with or without the addition of syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas.
  • the microorganism population comprises a bacterial strain of Rhodococcus opacus.
  • the microorganism population comprises a bacterial strain of Rhodococcus opacus (DSM 43205).
  • the invention relates to a method of producing a lipid or mixture of lipids, wherein the method comprises: culturing a population of bacterial cells comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas.
  • the microorganism population comprises a bacterial strain of Cupriavidus necator.
  • the molecule produced is one or more alkane, alkene, alkyne, fatty alcohol, and/or fatty aldehyde.
  • the method produces a lipid or mixture of lipids at a quantity higher than the quantity of lipid or mixture of lipids in the same bacterial cell population not comprising the exogenous nucleic acids described herein.
  • the one or more lipids comprise a quantity of at least one alkane, alkene, alkyne, fatty alcohol, and/or fatty aldehyde at a level higher than the quantity of the alkane, alkene, alkyne, fatty alcohol, and or fatty aldehyde in the same microorganism not comprising the exogenous nucleic acid sequences.
  • the method comprises a population of microorganisms or bacterial cell described herein that produces and/or secretes lipids of a weight equal to or greater than 10% of the total percentage of cellular dry matter.
  • the method comprises a population of
  • the method comprises a population of microorganisms or bacterial cell described herein that produces and/or secretes lipids of a weight equal to or greater than 20% of the total percentage of cellular dry matter.
  • the method comprises a population of microorganisms or bacterial cell described herein that produces and/or secretes lipids of a weight equal to or greater than 30% of the total percentage of cellular dry matter.
  • the method comprises a population of microorganisms or bacterial cell described herein that produces and/or secretes lipids of a weight equal to or greater than 40% of the total percentage of cellular dry matter.
  • the method comprises a population of microorganisms or bacterial cell described herein that produces and/or secretes lipids of a weight equal to or greater than 50% of the total percentage of cellular dry matter. In some embodiments, the method comprises a population of microorganisms or bacterial cells described herein that produces and/or secretes lipids of a weight equal to or greater than 60% of the total percentage of cellular dry matter. In some embodiments, the method comprises a population of microorganisms or bacterial cells described herein that produces and/or secretes lipids of a weight equal to or greater than 70% of the total percentage of cellular dry matter.
  • the method comprises a population of microorganisms or bacterial cell described herein that produces of secretes lipids of a weight equal to or greater than 75% of the total percentage of cellular dry matter. In some embodiment, the method comprises a population of microorganisms or bacterial cell described herein that produces of secretes lipids of a weight equal to or greater than 80% of the total percentage of cellular dry matter. In some embodiments, the method comprises a population of microorganisms or bacterial cell described herein that produces of secretes lipids of a weight equal to or greater than 85% of the total percentage of cellular dry matter.
  • the bacterial cell or composition comprising the bacterial cell produces and/or secretes at least 10% of the total percentage of the cellular dry matter or 10% by weight.
  • the method comprises a population of microorganisms comprising a bacterial cell described herein that produces or secretes lipids, wherein at least 5% of the lipids have carbon backbones from 8 to 18 carbon atoms in length.
  • the method comprises a population of microorganisms comprising a bacterial cell described herein that produces or secretes lipids, wherein at least 10% of the lipids have carbon backbones from 8 to 18 carbon atoms in length.
  • the method comprises a population of microorganisms comprising a bacterial cell described herein that produces or secretes lipids, wherein at least 15% of the lipids have carbon backbones from 8 to 18 carbon atoms in length. In some embodiments, the method comprises a population of microorganisms comprising a bacterial cell described herein that produces or secretes lipids, wherein at least 20% of the lipids have carbon backbones from 8 to 18 carbon atoms in length.
  • the molecule is chosen from one or more alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or dicarboxylic acid (diacid).
  • the one or more lipids comprise a quantity of at least one alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or diacid at a level higher than the quantity of the alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or diacid in the same microorganism not comprising the exogenous nucleic acid sequences.
  • the invention relates to a method of producing and/or secreting a lipid or mixture of lipids by culturing a population of microorganisms comprising a bacterial cell described herein, wherein the exogenous nucleic acid sequences are operably linked to a promoter that is inducible in response to a first stimulus, and wherein the method further comprises: culturing the population of bacterial cells for a first period of time in the presence of a first stimulus to produce one or more lipids chosen from an alkane, alkene, alkyne, fatty acid, unsaturated fatty acid, diacid, hydroxy acid, alcohol, and/or fatty acid aldehyde.
  • the invention relates to a method of fixing carbon from a gaseous feedstock containing carbonaceous molecules, wherein the method comprises the step of exposing a composition comprising exposing a bacterial cell to syngas and/or gaseous C0 2 and/or gaseous H 2 ; wherein the bacterial cell comprises at least one exogenous nucleic acid sequence.
  • the exogenous nucleic acid sequences are fatty acid acyl-ACP reductase or a fatty acid aldehyde decarbonylase.
  • the bacterial cell comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase.
  • the bacterial cell is Rhodococcus opacus or the population of microorganisms comprises a Rhodococcus cell.
  • the bacterial cell is Cupriavidus necator or the population of microorganisms comprises a Cupriavidus cell.
  • the bacterial cell comprises at least a first, a second, and a third exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase, the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase, and the third exogenous nucleic acid sequence encodes a thioesterase.
  • the bacterial cell comprises at least a first exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a thioesterase.
  • the bacterial cell comprises no more than five exogenous nucleic acid sequences that encode a lipid pathway enzyme.
  • the composition comprises a microorganism, wherein the microorganism is Rhodococcus opacus (DSM 43205 or 43206) or Rhodococcus sp (DSM 3346).
  • the composition comprises a microorganism, wherein the microorganism is chosen from the genera Ralstonia or Cupriavidus.
  • the composition comprises a microorganism, wherein the microorganism is Cupriavidus necator.
  • the microorganism is from the suborder corynebacterineae or the family burkholderiaceae.
  • the microorganism through its native machinery produces a complement of fatty acids described in the Fatty Acid Output section below.
  • the bacterial cell comprises at least a first and a second exogenous nucleic acid sequence but no more than five exogenous nucleic acid sequences, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase.
  • the invention relates to a method of producing one or more hydroxyacid, diacid, or unsaturated fatty acid, alcohols, fatty acid aldehydes, alkanes, alkenes, alkynes, or any combination thereof comprising exposing a bacterial cell to syngas and/or gaseous C0 2 or a mixture of gaseous C0 2 and gaseous H 2 ; wherein the bacterial cell is capable of fixing gaseous C0 2 into one or more fatty acid alcohols, alkanes, alkenes, or alkynes and wherein the microorganism comprises at least a first exogenous nucleic acid and a second exogenous nucleic acid, wherein the first exogenous nucleic acid encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid encodes fatty acid aldehyde decarbonylase.
  • the first and second exogenous nucleic acids are heterologous nucleic acid sequences.
  • the bacterial cell comprises at least a first, a second, and a third exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase, the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase, and the third exogenous nucleic acid sequence encodes a thioesterase.
  • the bacterial cell comprises at least a first exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a thioesterase.
  • the composition comprises a bacterial cell, wherein the bacteria is Rhodococcus opacus (DSM 43205 or 43206) or Rhodococcus sp (DSM 3346).
  • the bacterial cell is chosen from the genera Ralstonia or Cupriavidus.
  • the bacterial cell is Cupriavidus necator.
  • the bacterial cell is from the suborder corynebacterineae or the family burkholderiaceae.
  • the bacterial cell through its native machinery produces a complement of fatty acids described in the Fatty Acid Output section below.
  • the invention relates to a method of producing one or more unsaturated fatty acids, comprising exposing a bacterial cell to syngas and/or gaseous C02 or a mixture of gaseous C0 2 and gaseous H 2 ; wherein the bacterial cell is capable of fixing gaseous C0 2 into one or more unsaturated fatty acids and wherein the microorganism comprises at least a first exogenous nucleic acid, wherein the first exogenous nucleic acid encodes a desaturase that introduces double bonds to fatty acids.
  • the first exogenous nucleic acids is a heterologous nucleic acid sequence.
  • the bacterial cell comprises at least a first, and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a desaturase, the second exogenous nucleic acid sequence encodes a thioesterase.
  • the composition the bacterial cell comprises a microorganism, wherein the microorganism is Rhodococcus opacus (DSM 43205 or 43206) or Rhodococcus sp (DSM 3346).
  • the composition comprises a microorganism, wherein the microorganism is chosen from the genera Ralstonia or Cupriavidus.
  • the composition comprises a microorganism, wherein the microorganism is Cupriavidus necator.
  • the microorganism is from the suborder corynebacterineae or the family burkholderiaceae.
  • the microorganism through its native machinery produces a complement of fatty acids described in the Fatty Acid Output section below.
  • the invention relates to a method of producing one or more hydroxy fatty acids (hydroxy acids), comprising exposing a bacterial cell to syngas and/or gaseous C0 2 or a mixture of gaseous C0 2 and gaseous H 2 ; wherein the bacterial cell is capable of fixing gaseous C0 2 into one or more hydroxy acids and wherein the microorganism comprises at least a first exogenous nucleic acid, wherein the first exogenous nucleic acid encodes a P450-dependent fatty acid hydroxylase that introduces hydroxyl groups at positions along the fatty acid chain.
  • the first exogenous nucleic acids is a heterologous nucleic acid sequence.
  • the bacterial cell comprises at least a first, and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a P450-dependent fatty acid hydroxylase, the second exogenous nucleic acid sequence encodes a thioesterase.
  • the composition comprises a microorganism, wherein the microorganism is Rhodococcus opacus (DSM 43205 or 43206) or Rhodococcus sp (DSM 3346).
  • the composition comprises a microorganism, wherein the microorganism is chosen from the genera Ralstonia or Cupriavidus.
  • the composition comprises a microorganism, wherein the microorganism is Cupriavidus necator.
  • the microorganism is from the suborder corynebacterineae or the family burkholderiaceae.
  • the microorganism through its native machinery produces a complement of fatty acids described in the Fatty Acid Output section below.
  • the invention relates to a method of producing one or more hydroxyacid, diacid, or unsaturated fatty acid, alcohols, fatty acid aldehydes, alkanes, alkenes, alkynes, or any combination thereof comprising exposing a bacterial cell to syngas and/or gaseous C0 2 or a mixture of gaseous C0 2 and gaseous H 2 ; wherein the bacterial cell is capable of fixing gaseous C0 2 into one or more lipids; wherein the lipids are recovered from the bioreactor and fed to a second bioreactor wherein the lipids are postprocessed to generate hydroxyacid, diacid, and/or unsaturated fatty acids via a second microorganism such as but not limited to Candida tropicalis.
  • a method of producing one or more hydroxyacid, diacid, or unsaturated fatty acid, alcohols, fatty acid aldehydes, alkanes, alkenes, alkynes, or any combination thereof comprising
  • the invention relates to a method of manufacturing one or more lipids, comprising (a) culturing a cell described herein in a reaction vessel or bioreactor in the presence of syngas and/or gaseous C0 2 or a mixture of gaseous C0 2 and gaseous H 2 , wherein the cell produces and/or secretes one or more lipids in an quantity equal to or greater than at least 10% of the cell's total dry cellular mass; and (b) separating the one or more lipids from reaction vessel.
  • the method further comprises purifying the one or more lipids after separation from the reaction vessel or bioreactor.
  • the one or more lipids is a component of or a precursor to a component of jet fuel, diesel fuel, or biodiesel fuel.
  • the invention relates to a method of producing a alkene, fatty alcohol, alkyne, or alkane in a bacterial cell comprising at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase and the second exogenous nucleic acid encodes a fatty acid aldehyde decarbonylase.
  • the bacterial cell producing a alkene, fatty alcohol, alkyne, or alkane comprises at least a first, a second, and a third exogenous nucleic acid sequences, wherein the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase and the second exogenous nucleic acid encodes a fatty acid aldehyde decarbonylase, and the third exogenous nucleic acid encodes a thioesterase.
  • the invention relates to a method of producing cycloalkanes in a bacterial cell comprising at least a first exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a fatty acyl-CoA reductase.
  • the cycloalkane is cyclotetradecane.
  • the bacterial cell is Cupriavidus necator or the population of microorganisms comprises a Cupriavidus cell.
  • the nucleic acid sequence comprises or consists of SEQ ID NO:5 and/or SEQ ID NO: 6.
  • the nucleic acid sequence has at least 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide homology to one or more of SEQ ID NOs: 5 or 6.
  • the invention relates to a bioreactor comprising the composition or bacterial cells described herein.
  • the invention relates to a system for the production of one or more lipids or mixture of lipids, comprising a bioreactor, which comprises: (a) a microorganism population comprising a cell described herein; and (b) an inlet connected to a feedstock source allowing delivery of a feedstock comprising syngas and/or gaseous C0 2 or a mixture of gaseous C0 2 and gaseous H 2 .
  • the lipid or mixture of lipids comprise at least one component of or one precursor to a component of jet fuel, diesel fuel, or biodiesel fuel.
  • the invention relates to the population of fatty acids being modified to produce molecules of desired carbon chain length by incorporation of one or more thioesterases.
  • the invention relates to the population of fatty acids being modified to add additional carboxylic acid (-COOH) groups using exogenous enzymes.
  • the invention relates to the population of fatty acids being modified to add hydroxyl groups (-OH) using the exogenous enzymes (hydroxylases).
  • the invention relates to the population of fatty acids being modified to add desaturation through the incorporation of one or more double bonds, using the exogenous enzymes (desaturases). In some embodiments, the invention relates to a method for generating hydroxylated fatty acids in microbes through the transfer of enzymes that are known to hydroxylate fatty acids in plants or microbes into microorganisms where the enzyme is not native.
  • the invention relates to a microorganism comprising at least a first exogenous nucleic acid sequence wherein the microorganism converts gaseous C02 and/or gaseous H2 and/or syngas into one or more hydroxylated fatty acids.
  • the invention further provides a composition wherein the first exogenous nucleic acid sequence encodes a hydroxylating ezyme.
  • the invention further comprises a second exogenous nucleic acid sequence encoding a thioesterase enzyme.
  • the invention further provides a composition wherein the
  • microorganism is the genera Rhodococcus or Gordonia. In some embodiments, the invention further provides a composition wherein the microorganism is Rhodococcus opacus. In some embodiments, the invention further provides a composigion wherein the microorganism is Rhodococcus opacus (DSM 43205) or Rhodococcus opacus (DSM 43206) or Rhodococcus opacus (DSM 44193). In some embodiments, the invention further provides a composition wherein the microorganism is of the family Burkholderiaceae. In some embodiments, the invention further provides a composition wherein the microorganism is Cupriavidus necator. In some embodiments, the invention further provides a composition wherein the
  • the microorganism is Cupriavidus metallidurans .
  • the invention further provides a composition wherein the microorganism is a knallgas microorganism, also known as an oxyhydrogen microorganism.
  • the invention further provides a composition wherein the microorganism is a chemoautotrophic microbe.
  • the invention further provides a composition wherein the wild-type or mutant of the microorganism naturally has a capability for accumulating and/or synthesizing high quantities of triacylglycerol where a high quantity is considered to be 10% or more of the dry cell mass; 20% or more of the dry cell mass; 30% or more of the dry cell mass; 40% or more of the dry cell mass; 50% or more of the dry cell mass; 60% or more of the dry cell mass; 70% or more of the dry cell mass.
  • the invention further provides a composition wherein the microorganism is a hydrogen- oxidizing chemoautotroph.
  • the invention further provides a composition wherein the microorganism is capable of growing on syngas as the sole energy and carbon source.
  • the invention further provides a composition wherein the microorganism is capable of growing on untreated crude glycerol as the sole energy and carbon source.
  • the invention relates to a method for producing hydroxylated fatty acids wherein the method comprises culturing an engineered microorganism or a natural strain in a bioreactor or solution with a feedstock comprising syngas and/or gaseous C02 and/or a mixture of C02 gas and H2 gas.
  • the invention further provides a step of up-regulating an endogenous or exogenous thioesterase gene of the microorganism.
  • the invention further provides a step of down- regulating an endogenous or exogenous thioesterase gene of the microorganism. In some embodiments, the invention further provides a step of down-regulating an endogenous or exogenous acyl carrier protein gene of the microorganism.
  • the invention relates to a microorganism comprising at least a first exogenous nucleic acid sequence wherein the microorganism converts gaseous C0 2 and/or gaseous H 2 and/or syngas into one or more shorter-chain fatty acids.
  • the invention further provides a composition wherein the first exogenous nucleic acid sequence encodes a fatty acyl-CoA binding protein.
  • the invention further comprises a second exogenous nucleic acid sequence encoding a thioesterase enzyme.
  • the invention further provides a composition wherein the microorganism is the genera Rhodococcus or Gordonia.
  • the invention further provides a composigion wherein the microorganism is Rhodococcus opacus. In some embodiments, the invention further provides a composition wherein the microorganism is Rhodococcus opacus (DSM 43205) or Rhodococcus opacus (DSM 43206) or Rhodococcus opacus (DSM 44193). In some embodiments, the invention further provides a composition wherein the microorganism is of the family Burkholderiaceae. In some embodiments, the invention further provides a composition wherein the microorganism is Cupriavidus necator. In some embodiments, the invention further provides a composition wherein the microorganism is Cupriavidus metallidurans .
  • the invention further provides a composition wherein the microorganism is a knallgas microorganism, also known as an oxyhydrogen microorganism. In some embodiments, the invention further provides a composition wherein the microorganism is a chemoautotrophic microbe.
  • the invention further provides a composition wherein the wild-type or mutant of the microorganism naturally has a capability for accumulating and/or synthesizing high quantities of triacylglycerol where a high quantity is considered to be 10% or more of the dry cell mass; 20% or more of the dry cell mass; 30% or more of the dry cell mass; 40% or more of the dry cell mass; 50% or more of the dry cell mass; 60% or more of the dry cell mass; 70% or more of the dry cell mass.
  • the invention further provides a composition wherein the microorganism is a hydrogen-oxidizing chemoautotroph.
  • the invention further provides a composition wherein the microorganism is capable of growing on syngas as the sole energy and carbon source.
  • the invention further provides a composition wherein the microorganism is capable of growing on untreated crude glycerol as the sole energy and carbon source.
  • the invention relates to a method for producing shorter-chain fatty acids wherein the method comprises culturing an engineered microorganism or a natural strain in a bioreactor or solution with a feedstock comprising syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas.
  • the invention further provides a step of enhancing expression of enzymes through heat.
  • the invention further provides a step of up-regulating an endogenous or exogenous thioesterase gene of the microorganism.
  • the invention further provides a step of down- regulating an endogenous or exogenous thioesterase gene of the microorganism.
  • the invention further provides a step of down-regulating an endogenous or exogenous acyl carrier protein gene of the microorganism.
  • the instant invention provides a method of producing butanediol, or other biochemical precursors to butanediol by microbial fermentation under
  • microaerophilic or anaerobic conditions including: supplying an inorganic substrate as a primary source of metabolic energy, fermentation in a bioreactor containing a culture of microorganisms utilizing an inorganic substrate as a primary source of metabolic energy and carbon dioxide or other inorganic carbon as the primary source of carbon.
  • the invention further provides a method wherein the inorganic substrate comprises hydrogen (H2).
  • the invention further provides a method whereinthe butanediol product is 2,3 butanediol, 1,4 butanediol, and/or 1,3 butanediol.
  • the invention further provides a method wherein the level of hydrogen is supplied at such a level such that butanediol is produced. In some embodiments, the invention further provides a method wherein the level of C0 2 is supplied at a level such that butanediol is produced. In some embodiments, the invention further provides a method wherein the culture is propogated in the bioreactor in which oxygen is introduced at a certain flow rate, and the oxygen level is subsequently changed to a lower flow rate, and the oxygen level is subsequently changed to a lower flow rate such that butanediol is produced at enchanced levels.
  • the invention further provides a method wherein the electron donors include but are not limited to one or more of the following reducing agents: ammonia; ammonium; carbon monoxide; dithionite; elemental sulfur; hydrogen; metabisulfites; nitric oxide; nitrites; sulfates such as thiosulfates including but not limited to sodium thiosulfate (Na 2 S203) or calcium thiosulfate (CaS 2 03); sulfides such as hydrogen sulfide; sulfites; thionate; thionite.
  • the invention further provides a method wherein the primary fermentation microbe is of the genera Rhodococcus or
  • the invention further provides a method wherein the primary fermentation microbe is the species Rhodococcus sp. DSM 3346 or DSM364. In some embodiments, the invention further provides a method wherein the primary
  • the fermentation microbe is a Rhodococcus opacus.
  • the invention further provides a method wherein the primary fermentation microbe is a Rhodococcus opacus (DSM 43205) or a Rhodococcus opacus (DSM 43206) or a Rhodococcus opacus (DSM
  • the invention further provides a method wherein the primary fermentation microbe is of the family Burkholderiaceae. In some embodiments, the invention further provides a method wherein the primary fermentation microbe is Cupriavidus necator. In some embodiments, the invention further provides a method wherein the primary fermentation microbe is Cupriavidus metallidurans. In some embodiments, the invention further provides a method wherein the primary fermentation microbe is a knallgas microorganism, also known as an oxyhydrogen microorganism. In some embodiments, the invention further provides a method wherein the primary fermentation microbe is a chemoautotrophic microbe.
  • the invention further provides a method wherein the wild-type or mutant of the microorganism naturally has a capability for accumulating and/or synthesizing high quantities of triacylglycerol where a high quantity is considered to be 10% or more of the dry cell mass; 20% or more of the dry cell mass; 30% or more of the dry cell mass; 40% or more of the dry cell mass; 50% or more of the dry cell mass; 60% or more of the dry cell mass; 70% or more of the dry cell mass.
  • the invention further provides a method wherein the primary fermentation microbe is a hydrogen- oxidizing chemoautotroph.
  • the invention further provides a composition wherein the primary fermentation microbe is capable of growing on syngas as the sole energy and carbon source.
  • the invention further provides a composition wherein the primary fermentation microbe is capable of growing on untreated crude glycerol as the sole energy and carbon source. In some embodiments, the invention further provides a step of up-regulating an endogenous or exogenous gene regulating the pathway for the production of butanediol. In some
  • the invention further provides a step of down-regulating an endogenous or exogenous gene regulating the pathway for the production of butanediol.
  • a chemotroph capable of C0 2 fixation is engineered to produce a carbon-based product having a desired chemical structure to a level sufficient for commercial production.
  • the product generated may be native to the organism, but produced in non-optimal quantities in the absence of engineering, or completely lacking in the absence of engineering.
  • a host cell is genetically modified with an exogenous nucleic acid sequence encoding a single protein involved in a biosynthetic pathway generating a carbon- based product or intermediate.
  • a host cell is genetically modified with an exogenous nuceic acid sequence encoding multiple proteins involved in a biosynthetic pathway generating a carbon-based product or intermediate.
  • a host cell is genetically modified with multiple exogenous nucleic acid sequences encoding multiple proteins involved in a biosynthetic pathway generating a carbon-based product or intermediate, or multiple carbon-based products or intermediates.
  • a host cell is genetically modified with an exogenous nucleic acid sequence encoding a single protein affecting the generation of a carbon-based product or intermediate, but in a manner that does not directly add to or modify the biosynthetic pathway protein sequences.
  • a host cell is genetically modified with an exogenous nucleic acid sequence encoding multiple proteins affecting the generation of a carbon-based product or intermediate, but in a manner that does not directly add to or modify the biosynthetic pathway protein sequences.
  • a chemotroph capable of C0 2 fixation is engineered to produce two or more carbon-based products having desired chemical structures to a level sufficient for commercial production.
  • the products generated may be native to the organism, but produced in non-optimal quantities in the absence of engineering, or completely lacking in the absence of engineering.
  • such organisms produce at least 1 mg of carbon-based product of interest per liter of fermentation suspension.
  • the product is secreted by the organism into culture medium.
  • the product is retained in the organism in the course of fermentation.
  • the product may be recovered by lysing the cells and separating the product.
  • the product may have commercial value in the intact organism without significant preparation or purification of the product from the organism.
  • production of one of more other fermentation byproducts are attenuated or eliminated by downregulation of pathway genes that leads to its production by recombinant DNA methods, including gene knockouts, gene replacement, or partial or complete replacement of gene promoter sequences affecting genes in these pathways.
  • pathway genes that leads to its production by recombinant DNA methods, including gene knockouts, gene replacement, or partial or complete replacement of gene promoter sequences affecting genes in these pathways.
  • these include pathways leading to production of ethanol, acetate, lactate, succinate, butyrate, and butanol.
  • alcohols short or long chain, branched or straight- chain, saturated or unsaturated
  • production of alcohols is optimized by introduction of one or more exogenous nucleic acids encoding proteins in alcohol synthesis pathways.
  • Alcohols can be used as products or used to create products comprised of fatty acid esters, alkyl esters, isoprenyl esters, or other esters.
  • such organisms are modified such that they produce or upregulate production of polyhydroxybutyrate (PHB) or other products classified as polyhydroxyalkanoates (PHAs).
  • PHB polyhydroxybutyrate
  • PHAs polyhydroxyalkanoates
  • Organisms that already produce a specific PHA may be modified to produce more of the same or of a different PHA under cultivation conditions appropriate for chemoautotrophic cultivation.
  • organisms that do not produce PHAs may be modified to produce one or multiple types of PHAs.
  • pathway genes that enable production of PHAs include the following, for production of PHB: a beta- ketothiolase (which converts acetyl-CoA to acetoacetyl-CoA and CoA), Acetoacetyl-CoA reductase (which converts acetoacetyl-CoA and NADPH to 3-hydroxybutyryl-CoA), and PHA synthase (which converts 3-hydroxybutyryl-CoA to PHB and CoA).
  • An example of such a pathway, enabling production of PHB is encoded by the Ralstonia eutropha phaCAB operon.
  • specific modifications are made by recombinant methods to knockout or attenuate genes that degrade or prevent the accumulation of PHAs.
  • An example of such a gene is poly[(R)-3-hydroxybutanoate] hydrolase.
  • such organisms are modified such that they produce detectable levels of hydrocarbons or fatty acids of desired structure from inorganic energy and C0 2 .
  • desired structures or characteristics includes carbon chain length, branching, and saturation levels.
  • such organisms are modified such that they produce high yields of desired hydrocarbons.
  • hydrocarbons produced are secreted by passive transport proteins, active transport proteins or combinations thereof.
  • secretion is optimized for maximum yield of secreted hydrocarbons by introducing one or more exogenous nucleic acid sequences encoding transport proteins or gene regulatory sequences (e.g., promoters) that directly modify expression of transport proteins.
  • such organisms are optimized for maximum yield of secreted, desired hydrocarbons by introducing one or more exogenous nucleic acid sequences encoding proteins that regulate the expression of transport proteins or gene regulatory sequences (e.g., promoters) that directly modify expression of transport proteins.
  • such organisms are optimized for maximum yield of secreted hydrocarbons by introduction of one or more nucleic acid sequences that knock out or attenuate expression of certain endogenous transport proteins or proteins that regulate endogenous transport proteins.
  • the microorganisms are introduced with one or more exogenous nucleic acid sequences encoding acetyl-CoA carboxylase activity (accBCAD), aldehyde dehydrogenase activity (adhA, adhB), alcohol dehydrogenase activity (ADH I), alkane 1-monooxygenase activity (alkB), 3-hydroxyacyl-ACP dehydratase activity (fabA), 3-ketoacyl-ACP synthase activity (fabB), malonyl-CoA:ACP transacylase activity (fabD), 3-ketoacyl-ACP reductase activity (fabG), acetyl-CoA:ACP transacylase activity (fabH), enoyl-ACP reductase activity (fabl), acyl-ACP hydrolase activity (FASl), the Elp dehydrogense component of the pyruvate dehydrogenase complex, the E2p dihydrolipoamide acyltrans
  • such organisms are modified to secrete fatty acid chains by introduction of one or more exogenous nucleic acid sequences encoding an acyl- ACP-thioesterase, wherein the acyl-ACP-thioesterases liberate fatty acid chains from ACP- thioesters.
  • production of fatty acids of specific lengths, or enriched for specific lengths and structure can be produced by the introduction of one or more nucleic acid sequences encoding specific acyl-ACP- thioesterases showing a bias for producing fatty acid chains of a specific length and structure.
  • an organism may be modified by introduction of one or multiple exogenous nucleic acid sequences encoding multiple acyl-ACP-thioesterase proteins into the same organism such that the organism produces fatty acids of multiple specific lengths and structures, or enriched for multiple specific lengths and structures.
  • exogenous nucleic acid sequences encoding multiple acyl-ACP-thioesterase proteins
  • thioesterases are available in the art, published in the patent literature or in the open literature.
  • such organisms are modified by the introduction of one or more nucleic acid sequences to enable or enhance the ability of the organism to utilize inorganic energy, C02, and water to generate carbon-based products, including amino acids, acrylate, acrylic acid, adipic acid, alcohol, ascorbate, ascorbic acid, aspartate, aspartic acid, 1,3 - butadiene, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, butanol, caprolactam, carotenoid, citrate, citric acid, DHA, diesel, docetaxel, e-caprolactone, erythromycin 7- ADCA/cephalosporin, ethanol, ethyl ester, ethylene, fatty acid ester, fatty alcohols, fuel oxygenates, gamma butyrolactone, gasoline, glucose, fructose, carbohydrate, glutamate, glutamic acid, HPA, hydrocarbons, hydroxy
  • such organisms provided by the invention comprises a cell line selected from eukaryotic plants, algae, cyanobacteria, green-sulfur bacteria, green non- sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophiles, yeast, fungi, proteobacteria, engineered organisms thereof, and synthetic organisms.
  • such organisms are chemoautotrophic microorganisms that include, but are not limited to, one or more of the following: Acetoanaerobium sp.,
  • Acetobacterium sp. Acetogenium sp., Achromobacter sp., Acidianus sp., Acinetobacter sp., Actinomadura sp., Aeromonas sp., Alcaligenes sp., Alcaliqenes sp., Arcobacter sp.,
  • Aureobacterium sp. Bacillus sp., Beggiatoa sp., Butyribacterium sp., Carboxydothermus sp., Clostridium sp., Comamonas sp., Dehalobacter sp., Dehalococcoide sp., Dehalospirillum sp., Desulfobacterium sp., Desulfomonile sp., Desulfotomaculum sp., Desulfovibrio sp.,
  • Desulfurosarcina sp. Ectothiorhodospira sp., Enterobacter sp., Eubacterium sp.,
  • Ferroplasma sp. Halothibacillus sp., Hydro genobacter sp., Hydro genomonas sp.,
  • Lepto spirillum sp. Metallosphaera sp., Methanobacterium sp., Methanobrevibacter sp., Methanococcus sp., Methanosarcina sp., Micrococcus sp., Nitrobacter sp., Nitrosococcus sp., Nitrosolobus sp., Nitrosomonas sp., Nitrosospira sp., Nitrosovibrio sp., Nitrospina sp.,
  • chemoauto trophic microorganisms that are generally categorized as sulfur- oxidizers, hydrogen-oxidizers, iron-oxidizers, acetogens, and methanogens, as well as a consortiums of microorganisms that include chemoautotrophs.
  • Such organisms also include but are not limited to extremophiles that can withstand extremes in various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and chemicals. They include hyperthermophiies, such as Pyrol hus fumarii; tbermopbiles, such as Synechococcus lividis; mesophiles, and psychrophiles, such as Psychrobacter. Radiation tolerant organisms include Deinococcus radiod mm. Pressure tolerant organisms include piezophiles or barophiles. Dessicant tolerant and anhydrobiotic organisms include xerophiles such as A emia salimi microbes and fungi.
  • Salt tolerant organisms include halophiles, such as Halobacteriacea and Diinaliella salina.
  • pH tolerant organisms include alkaliphiles such as Na ronobacterium, Bacillus fir us OF4, Spimlina spp., and acidop les such as Cyanidium eaidariwn,
  • Ferroplasma sp Gas tolerant organisms, which tolerate pure C0 2 include Cyanidium caldarium and metal tolerant organisms include metalotolerants such as Ferroplasma acidarmanus, Ralstonia sp.
  • Such organisms also include algae and cyanobacteria, which include, but are not limited to the following genera: Acantiwceras, Acanthococcus, A aryochloris, A hnanth s, Achnanthidium, Aclinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Arnphikrikos, Amphiple ra, Amphiprora, Amphithrix, Amphora, Anabaena, Anahaenopsis, Aneumastus, Ankis rodesmus, Ankyra, Anomoeoneis, Apatococcus,
  • Aulacoseira Bacillaria, Balbiania, Batnbiisina, Bangia, B sichlamys, Bainichospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryo occus, Botryospkaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria,
  • Chlorolobion Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromuiina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroornonas, Ckroothece, Chrysamoeba, Chrysapsis, Chrysidiastnim, Chrysocapsa,
  • Chrysocapsella Chrysochaete, Chry so chromuiina, Chrysococcus, Chrysocrinus,
  • Chrysolepidomonas Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerelia, Chrysostephanosphaer , Ciodophora, Clasiidium, Closteriopsis,
  • Compsogonopsis Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladiurn, Crateriportuia, Craticuia, Cririalium, Crucigeni ,
  • Cyanonephron Cyanophor , Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cycloiella, Cylindrocapsa, Cylindrocysiis, Cylindwspermum,
  • Cylindrotheca Cymatopleura, Cymbella, Cymbellonitzschi , Cystodiniurn Dactylococcopsis, Debarya, Denticula, Derrnatochrysis, Dermocarpa, Dertnocarpella, Desniatractum,
  • Desmidium Desmococcus, Desmonema, Desmosiphon, Diacanihos, Diacronema, Diadesmis, Diaioma, Diaioniella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaeie,
  • Dictyochloris Dictyococcus. DictyospJu ium, Didymocystis. Didymogenes, Didymosphenia, Dilahifilum, Dirnorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis,
  • Entomoneis Entophysalis, Epichrysis, Epipyxi , Epithemia, Eremosphaera, Euaslmpsis, Euasirum, Eucapsis, Eucocconeis, Eudoriria, Euglena, Euglenophyta, Eu iolia,
  • Eustigmatophyta Eutreptia, Failacia, Fischereila, Fragiiaria, Fragilariforma, Franceia, Frustuiia.
  • Curcilla Geminella, Genicularia, Glaucocystis, Giaucophyta, Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeockrysis, Gloeococcus, Gloeocystis,
  • Gloeodendron Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golerikinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphone na, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyosiomum,
  • Granulochloris Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, H ' ammatoidea, Hannaea, Hanizschia,
  • Planctonema Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigtna, Pleurosira, Pleurotaenium,
  • Pocillomonas Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Poly taenia, Poly Soma, Polytomella, Porphyridium, Posteriochromonas, P rasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Prot siphon, Provasoliella, Piymnesium, Psammodictyon, Pfelnothidium, Pseudanabaena, Pseudenoclonium, Psuedocaneria, Pseudochate,
  • Pseudoncobyrsa Pseudoquadrigula, Pseudosphaerocystis, Pseudoslauraslrwn,
  • Rhodomonas Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossiihidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocy stop sis, Dimonsenia, Siphononema. Sirocladiuni,
  • Stephanodiscus, Steph noporos, Stephanospli era Slichococcus, Stichogloea, Stigeoclonumi, Stigonema, Slipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylo sphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochrornonas, Synura, Tabellaria, Tabularia, Molingia, Temnogamelum, Tetmemorus, Tetracnloreila. Tetracyclus, Tetradesmus.
  • Such organisms also include green non- sulfur bacteria, which include but are not limited to the following genera: Chlorofiexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicwbi m.
  • Such organisms also include green sulfur bacteria., which include but are not limited to the following genera: Chlorobium, Clathrochloris, and Prosthecochloris ,
  • Such organisms also include purple sulfur bacteria, which include but are not limited to the following genera: A llochromatium, Chromati m, Halochromalium, Isochromatium, Marichromatium, Rhodovulum, Th.ermochwmatium, Tniocapsa, Tniorhodococcus, and Thiocystis.
  • Such organisms also include purple non-sulfur bacteria, which include but are not limited to the following genera.: Phaeospirilh i, Rhodobaca, Rhodobacter, Rhodomicrobi m, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio, and
  • Such organisms also include aerobic chemolithotrophic bacteria, which include but are not limited to nitrifying bacteria such as Nitrobacteraceae sp,, Nitrobacter sp., Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosornonas sp., Nitrosococcus sp., Nhrosospira sp., Nitrosolobus sp., Nitrosovibrio sp.; colorless sulfur bacteria such as, Thiovulum sp..
  • Thiob cUlus sp. Thiormcrospira sp., Thiosphaera sp., Thermothrix sp.; obligateiy chemoliihoiropliic hydrogen bacteria such as Hydrogenobacter sp,, iron and manganese- oxidizing and/or depositing bacteria such as Siderococcus sp., and magnetotaetic bacteria such as Aquas pi ri Hum sp.
  • Such organisms also include arch aeobacteria, which include but are not limited to methanogenic archaeobacteria such as Methanohacteruinr sp., Methanohre vibacter sp., Median other nius sp., M ihanococcus sp., Methano icrobium sp., M ethanospirillum sp., Methanogenium sp., Methanosarcina sp., Methanolobus sp., Methanothrix sp.,
  • methanogenic archaeobacteria such as Methanohacteruinr sp., Methanohre vibacter sp., Median other nius sp., M ihanococcus sp., Methano icrobium sp., M ethanospirillum sp., Methanogenium sp., Methanosarcina sp.
  • Methanococcoides sp. Methanoplanus sp.
  • extremely thermophilic sulfur-metabolizers such as Thermoproteus sp., Pyrodictium sp., Sulfoiobus sp., Acidianus sp.
  • a oxyhydro en microorganism such as but not limited to Ralstonia eutropha, Alcaligenes eutrophus or Cup.riav.idus necator, is grown up to a high cell density in micro aerobic conditions using syngas components as a carbon source and energy, including, but not limited to 1.1 C02 and / or CO, and / or using methanol and / or using glycerol, including crude glycerol, which is a by-product of biodiesei or oleochemical manufacturing.
  • Exemplary oxyhydrogen microorganisms that can be used in one or more process steps of certain embodiments of the present invention include but are not limited to one or more of the following: purple non-sulfur pliotosynthetic bacteria including but not limited to Rhodopseudomonas palustris, Rhodopseudomonas capsuiala, Rhodopseudomonas viridis, Rhodopseudomonas sulfoviridis, Rhodopseudomonas hiastica, Rhodopseudomonas spheroides, Rhodopseudomonas acidophila and other Rhodopseudomonas sp.,
  • purple non-sulfur pliotosynthetic bacteria including but not limited to Rhodopseudomonas palustris, Rhodopseudomonas capsuiala, Rhodopseudomonas viridis, Rh
  • Rhodospirillum rubrum and other Rhodospirilium sp.
  • Rhodococcus opacus and other Rhodococcus sp Rhizobium japonicum and other Rhizobium sp
  • Pseudomonas hydrogenovora Pseudomonas
  • Hydrogenobacter thermophilus and other Hydrogenobacter sp. Hydrogenovibrio m rinus and ofti r. Hydrogenovibrio sp.; Helicobacter pylori and other Helicobacter sp.; Xanthobacter sp.; Hydrogenoph ga sp.; Brady rhizohi m japonic m and other Bradyrhizobium sp.;
  • One feature of certain embodiments of the present invention is the inclusion of one or more process steps within a chemical process for the conversion of CI carbon sources including but not limited to carbon monoxide, methane, methanol, formate, or formic acid, and/or mixtures containing CI chemicals including but not limited to various syngas compositions generated from various gasified, pyrolyzed, or steam- reformed fixed carbon feedstocks, that utilize oxyhydrogen microorganisms and/or enzymes from oxyhydrogen microorganisms as a biocatalyst for the conversion of CI chemicals into longer chain organic chemicals (i.e. C2 or longer and, in some embodiments, €5 or longer carbon chain molecules).
  • CI carbon sources including but not limited to carbon monoxide, methane, methanol, formate, or formic acid
  • mixtures containing CI chemicals including but not limited to various syngas compositions generated from various gasified, pyrolyzed, or steam- reformed fixed carbon feedstocks, that utilize oxyhydr
  • CI containing syngas, or process gas, or CI chemicals in a pure liquid form or dissolved in solution is pumped or otherwise added to a vessel or enclosure containing nutrient media and oxyhydrogen microorganisms, hi some such cases oxyhydrogen microorganisms perform biochemical synthesis to elongate CI chemicals into longer carbon chain organic chemicals using the chemical energy stored in the CI chemical, and/or molecular hydrogen and/or valence or conduction electrons in solid state electrode materials and/or one or more of the following list of electron donors pumped or otherwise provided to the nutrient media including but not limited to: ammonia; ammonium; carbon monoxide; dithionite; elemental sulfur; hydrocarbons; metabisulfites; nitric oxide; nitrites; sulfates such as thiosulfates including but not limited to sodium thiosulfate
  • Na.2S203 calcium thiosulfate
  • CaS203 calcium thiosulfate
  • sulfides such as hydrogen sulfide; sulfites; thionate; tliionite: transition metals or their sulfides, oxides, chaicogenides, halides, hydroxides, oxyhydroxides, sulfates, or carbonates, in soluble or solid phases.
  • the electron donors can be oxidized by electron acceptors in a chemosynthetic reaction.
  • Electron acceptors that may be used at this reaction step include oxygen and/or other electron acceptors including but not limited to one or more of the following: carbon dioxide, f erric iron or other transition metal ions, nitrates, nitrites, oxygen, or holes in solid state electrode materials.
  • C2 or longer and, in some embodiments, C5 or longer carbon chain molecules can be performed in aerobic, microaerobic, anoxic, anaerobic conditions, or facultative conditions.
  • a facultative environment is considered to be one having aerobic upper layers and anaerobic lower layers caused by stratification of the water column.
  • the present invention relates to the engineering of microorganisms, including but not limited to hydrogen oxidizing and / or carbon monoxide oxidizing knallgas microorganisms, with a natural capability to grow and synthesize biornass on gaseous carbon sources such as syngas and/or CO 2 , such that the natural or engineered microorganisms synthesize targeted products, including chemicals and fuels, under gas cultivation.
  • microorganisms including but not limited to hydrogen oxidizing and / or carbon monoxide oxidizing knallgas microorganisms, with a natural capability to grow and synthesize biornass on gaseous carbon sources such as syngas and/or CO 2 , such that the natural or engineered microorganisms synthesize targeted products, including chemicals and fuels, under gas cultivation.
  • the composition comprises a microorganism that can naturally grow on H2/C02 and/or syngas, and wherein the microorganism can naturally accumulate polyhyclroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) to 50% or more of the cell biornass by weight, in some embodiments the microorganisms have a native ability to direct a high flux of carbon through the acetyl -Co A metabolic intermediate, which can lead into fatty acid biosynthesis, along with a number of oilier synthetic pathways including PHA and PHB synthesis.
  • PHB polyhyclroxybutyrate
  • PHA polyhydroxyalkanoate
  • a microorganism is considered to direct a high flux of carbon through acetyl-CoA if a product of a synthesis pathway going through the acetyl-CoA metabolic intermediate, including but not limited to poly hydrox butyrate (PHB) or polyhydroxyalkanoate (PHA), can represent 50% or more of the cell biornass by weight, In some embodiments the
  • DSM 531. or DSM 541 Cupriavidus necator
  • aspects of the invention relate to a bacterial cell comprising at least a first exogenous nucleic acid sequence wherein the cell converts gaseous C0 2 and/or gaseous H 2 and/or syngas into one or more lipids or hydrocarbons.
  • the first exogenous nucleic acid sequence encodes a protein selected from the group consisting of a fatty acid acyl-ACP reductase and a fatty acid aldehyde decarbonylase. In some embodiments, the first exogenous nucleic acid sequence encodes a CYP52A protein. In certain embodiments, the first exogenous nucleic acid sequence encodes a protein selected from the group consisting of a CYP709C1 and
  • the first exogenous nucleic acid sequence encodes a thioesterase protein.
  • the cell further comprises a second exogenous nucleic acid sequence.
  • the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase.
  • the cell comprises a first and second exogenous nucleic acid wherein the second exogenous nucleic acid encodes a thioesterase protein or a fatty acyl-CoA ligase.
  • the cell further comprises a third exogenous nucleic acid sequence that encodes a thioesterase.
  • the bacterial cell is of the suborder corynebacterineae. In some embodiments, the bacterial cell is of the family burkholderiaceae. In some
  • the cell is of the genera Rhodococcus or Gordonia. In certain embodiments, the cell is a Rhodococcus opacus. In some embodiments, the bacterial cell is an oxyhydrogen microorganisms including oxyhydrogen microorganisms selected from one or more of the following genera: Rhodopseudomonas sp.; Rhodospirillum sp.; Rhodococcus sp.; Nocardia sp.; Mycobacterium sp.; Gordonia sp.; Tsukamurella sp.; Rhodobacter sp.; Rhizobium sp.; Thiocapsa sp.; Pseudomonas sp.; Hydro genomonas sp.; Hydro genobacter sp.;
  • the bacterial cell produces and/or secretes at least 10% of one or more lipids or hydrocarbons by weight. In some embodiments, the bacterial cell produces and/or secretes one or more lipids or hydrocarbons, wherein at least 50% of the one or more lipids or hydrocarbons have 6 to 30 carbon atoms. In some embodiments, less than 10% by weight of the lipids or hydrocarbons is methane. In some embodiments, less than 10% by weight of the lipids or hydrocarbons is organic acid.
  • the one or more lipids or hydrocarbons comprise at least one organic molecule having a carbon chain length of at least 8 carbon atoms and at least one carbon-carbon double bond. In some embodiments, the one or more lipids or hydrocarbons comprise at least one diacid acid molecule having a carbon chain length of at least 6 carbon atoms. In some embodiments, the one or more lipids or hydrocarbons comprise at least one desaturated hydrocarbon molecule having a carbon chain length of at least 6 carbon atoms.
  • the one or more lipids or hydrocarbons comprise at least one fatty acid molecule having a carbon chain length of at least 6 carbon atoms. In some embodiments, the one or more lipids or hydrocarbons comprise at least one unsaturated fatty acid molecule having a carbon chain length of at least 6 carbon atoms. In some
  • the one or more lipids or hydrocarbons comprise at least one hydroxyl acid molecule having a carbon chain length of at least 6 carbon atoms. In some embodiments, the one or more lipids or hydrocarbons comprise at least one dicarboxylic acid molecule having a carbon chain length of at least 6 carbon atoms.
  • the one or more lipids or hydrocarbons comprise at least one alkane, alkene, alkyne, fatty alcohol, and/or fatty aldehyde at a level higher than the quantity of the alkane, alkene, alkyne, fatty alcohol, and or fatty aldehyde in the same microorganism not comprising the exogenous nucleic acid sequences.
  • the one or more lipids or hydrocarbons comprise at least one component of or one precursor to a component of jet fuel, diesel fuel, or biodiesel fuel.
  • lipid or a hydrocarbon or a mixture of lipids or hydrocarbons including culturing a bacterial cell in a feedstock comprising syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas.
  • the H 2 is generated or recycled using renewable, alternative, or conventional sources of power that are low in greenhouse gas emissions, and wherein said sources of power are selected from at least one of photovoltaics, solar thermal, wind power, hydroelectric, nuclear, geothermal, enhanced geothermal, ocean thermal, ocean wave power, and tidal power.
  • the syngas is generated from lignocellulosic energy crops, crop residue, bagasse, saw dust, forestry residue, food waste, municipal solid waste, biogas, landfill gas, or stranded natural gas.
  • the lipid or hydrocarbon or mixture of lipids or hydrocarbons produced is one or more alkane, alkene, alkyne, fatty alcohol, and/or fatty aldehyde.
  • at least one exogenous nucleic acid sequences of the bacterial cell is operably linked to a promoter that is inducible in response to a first stimulus, and wherein the method further comprises culturing a population of the bacterial cell of claim 1 for a first period of time in the presence of a first stimulus to produce one or more lipids or hydrocarbons.
  • Further aspects of the invention relate to culturing of a bacterial cell in a feedstock comprising syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas in a reaction vessel or a bioreactor wherein the one or more lipids or hydrocarbons are separated from the reaction vessel or bioreactor.
  • the method further comprises purifying the one or more lipids or hydrocarbons or a mixture of lipids or hydrocarbons after separation from the reaction vessel or bioreactor.
  • a microorganism comprising at least a first exogenous nucleic acid sequence wherein the microorganism converts gaseous C0 2 and/or gaseous H 2 and/or syngas into one or more hydroxylated fatty acids.
  • the first exogenous nucleic acid sequence encodes a hydroxylating ezyme.
  • the cell further comprises a second exogenous nucleic acid sequence encoding a thioesterase enzyme.
  • the microorganism is the genera Rhodococcus or Gordonia.
  • the microorganism is the species Rhodococcus sp. DSM 3346 or DSM 364.
  • the microorganism is Rhodococcus opacus. In certain embodiments, the microorganism is Rhodococcus opacus (DSM 43205) or Rhodococcus opacus (DSM 43206) or Rhodococcus opacus (DSM 44193). In some embodiments, the microorganism is family Burkholderiaceae. In some embodiments, the microorganism is Cupriavidus necator. In some embodiments, the microorganism is
  • the microorganism is a knallgas microorganism, also known as an oxyhydrogen microorganism. In some embodiments, herein the microorganism is a chemoautotrophic microbe.
  • the wild-type or mutant of the microorganism naturally has a capability for accumulating and/or synthesizing high quantities of triacylglycerol where a high quantity is considered to be 10% or more of the dry cell mass.
  • the microorganism is a hydrogen-oxidizing chemoautotroph.
  • the microorganism is capable of growing on syngas as the sole energy and carbon source.
  • the microorganism is capable of growing on untreated crude glycerol as the sole energy and carbon source.
  • the method further comprises the step of up- regulating an endogenous or exogenous thioesterase gene of the microorganism. In some embodiments, the method further comprises the step of down-regulating production of an endogenous or exogenous thioesterase gene of the microorganism. In some embodiments, the method further comprises the step of down regulating an endogenous or exogenous acyl carrier protein gene of the microorganism.
  • aspects of the invention relate to a microorganism comprising at least a first exogenous nucleic acid sequence wherein the microorganism converts gaseous C0 2 and/or gaseous H 2 and/or syngas into one or more shorter-chain fatty acids.
  • the first exogenous nucleic acid sequence encodes a fatty acyl-CoA binding protein.
  • the microorganism further comprises a second exogenous nucleic acid sequence encoding a thioesterase enzyme.
  • the microorganism is of the genera Rhodococcus or Gordonia.
  • the microorganism is the species Rhodococcus sp. DSM 3346 or DSM 364.
  • the microorganism is a Rhodococcus opacus. In some embodiments, the microorganism is a Rhodococcus opacus (DSM 43205) or a Rhodococcus opacus (DSM 43206) or a Rhodococcus opacus (DSM 44193). In some embodiments, the microorganism is family burkholderiaceae. In some embodiments, the microorganism is Cupriavidus necator. In some embodiments, the microorganism is Cupriavidus metallidurans . In some embodiments, the microorganism is a knallgas microorganism, also known as an oxyhydrogen microorganism. In some
  • the microorganism is a chemoautotrophic microbe.
  • the wild-type or mutant of the microorganism naturally has a capability for accumulating and/or synthesizing high quantities of triacylglycerol where a high quantity is considered to be 10% or more of the dry cell mass.
  • the microorganism is a hydrogen-oxidizing chemoautotroph.
  • the microorganism is capable of growing on syngas as the sole energy and carbon source.
  • the microorganism is capable of growing on untreated crude glycerol as the sole energy and carbon source.
  • Further aspects of the invention relate to a method for producing shorter-chain fatty acids including in a bioreactor or solution, culturing an engineered microorganism as in claim 55 or a natural strain with a feedstock comprising syngas and/or gaseous C0 2 and/or a mixture of C0 2 gas and H 2 gas.
  • the method further comprises the step of enhancing expression of enzymes through heat.
  • the method further comprises the step of up-regulating an endogenous or exogenous thioesterase gene of the microorganism.
  • the method further comprise the step of down- regulating an endogenous or exogenous thioesterase gene of the microorganism.
  • the method further comprises the step of down regulating an endogenous or exogenous acyl carrier protein gene of the microorganism.
  • Further aspects of the invention relate to a method of producing butanediol, or other biochemical precursors to butanediol by microbial fermentation under rnicroaerophi lie or anaerobic conditions, including: supplying an inorganic substrate as a primary source of metabolic energy, whereby the substrate consists of one or more electron donors and one or more electron acceptors; and fermentation in a bioreactor containing a culture of
  • microorganisms utilizing an inorganic substrate as a primary source of metabolic energy and carbon dioxide or other inorganic carbon as the primary source of carbon.
  • the inorganic substrate comprises hydrogen (H2).
  • the butanediol product is 2,3-butanediol, 1,4 butanediol or 1,3 butanediol.
  • the level of hydrogen is supplied at a level such that butanediol is produced.
  • the level of C0 2 is supplied at a level such that butanediol is produced.
  • the culture is propagated in the bioreactor in which oxygen is introduced at a certain flow rate, and the oxygen level is subsequently changed to a lower flow rate such that butanediol is produced at enhanced levels.
  • the electron donors include but are not limited to one or more of the following reducing agents: ammonia; ammonium; carbon monoxide; dithionite; elemental sulfur; hydrogen; metabisulfites; nitric oxide; nitrites; sulfates such as thiosulfates including but not limited to sodium thiosulfate (Na 2 S 2 0 3 ) or calcium thiosulfate (CaS 2 0 3 ); sulfides such as hydrogen sulfide; sulfites; thionate; thionite and said electron acceptors include but are not limited to one or more of the following oxidizing agents: carbon dioxide, ferric iron or other transition metal ions, nitrates, nitrites, oxygen, or holes in solid state electrode materials.
  • the primary fermentation microbe is of the genera Rhodococcus or Gordonia. In some embodiments, the primary fermentation microbe is the species
  • the primary fermentation microbe is a Rhodococcus opacus. In some embodiments, the primary fermentation microbe is a Rhodococcus opacus (DSM 43205) or a Rhodococcus opacus (DSM 43206) or a
  • Rhodococcus opacus (DSM 44193).
  • the primary fermentation microbe is family burkholderiaceae.
  • the primary fermentation microbe is Cupriavidus necator.
  • the primary fermentation microbe is Cupriavidus metallidurans.
  • the primary fermentation microbe is a knallgas microorganism, also known as an oxyhydrogen microorganism.
  • the primary fermentation microbe is a chemoautotrophic microbe.
  • the wild-type or mutant of the primary fermentation microbe naturally has a capability for accumulating and/or synthesizing high quantities of triacylglycerol where a high quantity is considered to be 10% or more of the dry cell mass.
  • the primary fermentation microbe is a hydrogen- oxidizing
  • the primary fermentation microbe is capable of growing on syngas as the sole energy and carbon source. In some embodiments, the primary fermentation microbe is capable of growing on untreated crude glycerol as the sole energy and carbon source.
  • the method further comprises the step of up-regulating an endogenous or exogenous gene regulating the pathway for the production of butanediol. In some embodiments, the method further comprises the step of down-regulating an endogenous or exogenous gene regulating the pathway for the production of butanediol.
  • Figure 1 describes the taxonomic names afforded to the chemoautotrophic and oleaginous microorganisms used in selected embodiments of the invention.
  • Figure 2 shows the 16S rRNA gene based- rooted phylogenetic tree of gordoniaceae, mycobacteriaceae, nocardiaceae and burkholderiaceae.
  • Figure 3 shows the sequence similarity of Rhodococcus opacus (DSM 43205) 16S rRNA gene (NR_026186.1) to members of the family gordoniaceae, mycobacteriaceae, nocardiaceae and burkholderiaceae.
  • Genbank accession numbers, DNA length and % identity of analyzed genes are indicated.
  • Figure 4 describes the nucleotide sequence alignment of the 16S rRNA genes SEQ ID NOs: 20-49.
  • Figure 5 demonstrates the growth of chemotrophic and oleaginous microorganisms on different carbon sources. Bacterial growth was measured using optical density (OD) detection at 650 nm after the indicated days (in parentheses). Media and growth conditions described in the Examples section below. ND, not done.
  • Figure 6 describes the measured lipid content of microorganisms on heterotrophic and chemoautotrophic growth conditions as a percentage of total cellular dry matter (CDM). Cells were grown under conditions described in Figure 5, harvested after 72hr (unless otherwise indicated) and analyzed by gas chromatography. For CDM, total dry weight was determined gravimetrically.
  • CDM total dry weight was determined gravimetrically.
  • Figure 7 describes the fatty acid profile of R. opacus (DSM 44193) under
  • Cells were harvested after 72hr and analyzed by gas chromatography.
  • Figure 8 describes the fatty acid profile R. opacus (DSM43205) under heterotrophic (A) and chemoautotrophic (B) growth conditions. Cells were harvested after 72 hours of growth and analyzed by gas chromatography.
  • Figure 9 describes the fatty acid profile Rhodococcus sp. (DSM 3346) under heterotrophic (A) chemoautotrophic (B) growth conditions. Cells were harvested after 72hr and analyzed by gas chromatography.
  • Figure 10 describes shuttle vectors (A) and genetic elements (B) for transformation and gene expression of in chemoautotrophic and oleaginous microorganisms.
  • MCS multiple cloning site.
  • Figure 11 describes the map of the plasmids pSeqC01(A; SEQ ID: 01), pSeqC02 (B; SEQ ID: 02), pVerl(C; SEQ ID: 03) and pVer2 (D; SEQ ID: 04) described in Fig.10. The genetic elements are indicated.
  • Figure 12 describes the transformation of chemoautotrophic and oleaginous microorganisms with shuttle vectors described in Figure 10.
  • Figure 13 describes the growth of Cupriavidus necator (DSM531) transformed with the plasmid (Y) pSeqC02 (SEQ ID:2) and untransformed (N) on different kanamycin concentrations.
  • DSM531 Cupriavidus necator transformed with the plasmid (Y) pSeqC02 (SEQ ID:2) and untransformed (N) on different kanamycin concentrations.
  • the growth was measured using O.D650 after the indicated number of days.
  • Figure 14 describes the formation of fatty alcohols in oleaginous bacteria.
  • the role of the fatty acyl-CoA reductases (FAR) gene in the biosynthesis pathway is shown.
  • the Arabidopsis genes FAR1 (SEQ ID: 05), FAR2 (SEQ ID: 06) and FAR3 (SEQ ID: 07) were cloned into pSeqC02 plasmid using the indicated restriction sites to give pSeqC02::FARl, pSeqC02::FAR2, pSeqC02::FAR3.
  • Figure 15 describes the pathway for formation of fatty alcohols in burkholderiaceae using of the fatty acyl-CoA reductases (FAR) gene.
  • Figure 16 describes the cloning strategy of FAR gene into pSeqC02 plasmids.
  • the Arabidopsis genes FARl (SEQ ID: 05), FAR2 (SEQ ID: 06) and FAR3 (SEQ ID: 07) were cloned into pSeqC02 plasmid using the indicated restriction sites to give pSeqC02::FARl, pSeqC02::FAR2, pSeqC02::FAR3.
  • Figure 17 describes the effect of FAR genes expression on fatty acid synthesis in
  • C. necator cells were transformed with pSeqC02::FARl (Cn-Fl), pSeqC02::FAR2 (Cn-F2) and control pSEqC02 (Cn-P).
  • Cells were harvested (3,000 x g for 20min at 4°C) and fatty acids were analyzed by gas chromatography.
  • Figure 18 describes the pathway for formation of hydrocarbons in oleaginous bacteria using the enzymes fatty acid acyl-ACP reductase (FadDR) and fatty acid aldehyde decarbonylase by (FAD) genes.
  • FadDR fatty acid acyl-ACP reductase
  • FAD fatty acid aldehyde decarbonylase by
  • Figure 19 describes the pathway for formation of hydrocarbons in burkholderiaceae using the enzymes fatty acid acyl-ACP reductase (FadDR) and fatty acid aldehyde decarbonylase by (FAD) genes
  • Figure 20 describes the restriction map related to the cloning strategy of FadDR and Fad genes into pSeqC02 plasmid transformed for the experiment.
  • Genes from the cyanobacterium (Synechocystis sp. PCC 6803) used in the experiment were FadR (SEQ ID: 08) and FAD (SEQ ID: 09) driven by the Synechocystis sp. Rubisco large subunit promoter (SEQ ID: 10) were cloned into pSeqC02 plasmid using the indicated restriction sites to give pSeqC02::FUEL.
  • Figure 21 describes the production of Alkanes in Cupriavidus necator transformed with pSeqC02: :FUEL (Cn_FUEL2.1 ) and empty vector (Cn-P) .
  • GC chromatogram of hydrocarbon peaks indicated with label extracted from transformants grown in 50ml LB media under previously identified conditions.
  • Figure 22 describes the hydrocarbon specific products and distribution (percentage in parentheses) from Cupriavidus necator transformed with pSeqC02::FUEL (Cn_FUEL2.1 and Cn_FUEL2.2 ) and empty vector (Cn-P).
  • Figure 23 describes the effect of pSeqC02::FUEL (Cn_FUEL2.1 and 2.2 ) and empty vector (Cn-P) on the fatty acids distribution under the experimental conditions described previously.
  • Figure 24 describes the modification of the fatty acid chain length by the enzymatic action of thioesterase (TE) in oleaginous bacteria.
  • Figure 25 describes the modification of the fatty acid chain length by the enzymatic action of fatty acyl-ACP thioesterase (TE) in burkholderiaceae.
  • Figure 26 describes the similarity of Rhodococcus opacus (B4) thioesterases protein sequence (YP_002784058.1) to other organisms.
  • the Genbank accession numbers, amino acid length and % identity of analyzed proteins are indicated.
  • Figure 27 describes the fluorescence intensity of Rhodococcus Sp exposed to 0, 5, 10, and 20 seconds of (Fig. 27B, 27C, 27D and 27E respectively) of UV light and stained with Nile Red. FACS analysis of untreated cells (negative control; no Nile Red staining and no UV exposure) (Fig. 27F) and mutated population with increased lipid content (G; P3) are shown.
  • Figure 28 describes the chemoautotrophic growth of Cupriavidus necator transformed with pSeqC02::FUEL (Cn-FUEL2.1), empty vector (Cn-P) and untransformed (Cn).
  • Bacterial growth was measured at ⁇ . ⁇ 6 5 ⁇ after 12 days. Media and growth conditions described in Fig 7.
  • Figure 29 describes the affect of FAR genes expression on biosynthesis of cyclotetradecane in Cupriavidus necator. C. necator cells were transformed with
  • pSeqC02::FARl Cn-Fl
  • pSeqC02::FAR2 Cn-F2
  • control pSEqC02 Cn-P
  • Figure 30 shows a schematic block flow diagram of a process for utilizing a gaseous CI feedstock such as syngas to produce hydrocarbons using the microorganisms of the present invention.
  • Figure 31 shows a schematic block flow diagram of a process for utilizing a gaseous
  • CI feedstock such as syngas to produce lipids using the microorganisms of the present invention with additional post-processing steps converting the lipids to drop-in fuels such as jet fuel and/or diesel.
  • Figure 32 shows octadecanoic acid derivatives produced by at least one Kiverdi chemoautotrophic production strain.
  • Experimental runs for fatty acid percent yields (grams of product/100 grams total fatty acid) from organisms Rhodococcus opacus (DSM 44193), Rhodococcus opacus (DSM 43205), and Cupriavidus necator.
  • Figure 33 shows putative 12-hydroxylases culled by word searching Genbank.
  • Figure 34 shows genes related to Vicia sativa P450 omega hydroxylases.
  • Figure 35 shows a list of P450-dependent fatty acid omega hydroxylases.
  • Figure 36 shows a list fatty acid hydroxylases.
  • Figure 37 shows the percent fatty acid production for plasmid control (TK04-P), thioesterase expression (TK04-TE), and fatty acyl-CoA binding protein (TK04-ACoA-BP).
  • Figure 38 shows the percent fatty acid production for fatty acyl-CoA binding protein
  • Figure 39 shows (A) Fatty acid percentages (C12, C14, C16, and C18 chain lengths) for Cupriavidus necator (DSM531) organism with control plasmid pSeqC02 (CN-P), with expression of exogenous thioesterase (CN-TE), and expression of fatty acyl-CoA binding protein (CN-ACBP). (B) Fatty acid percentages (C12 and C14) with expression of exogenous thioesterase (CN-TE), and expression of fatty acyl-CoA binding protein (CN- ACBP) compared with control (CN-P).
  • Figure 41 shows the map of the plasmid pSeqC02::ACBP. The genetic elements are indicated.
  • Figure 42 shows growth (optical density) of Alcaligenes eutrophus on H2, C02 and 02 to a cell density of 35 g/1 (dry cell weight). Alcaligenes eutrophus was grown
  • microaerobically Several aspects involve growing Alcaligenes eutrophus or other oxyhydrogen microbes, either engineered or not engineered, to a high cell density microaerobically on syngas components (H2, C02 and / or CO) then switching to anaerobic bioprocessing for the production of 1,3 butandiol and other organic compounds, which are secreted.
  • syngas components H2, C02 and / or CO
  • Figure 43 shows 2.3 Butatadiol pathways.
  • Figure 44 shows the pathway of introducing BDO metabolic pathway to a organism.
  • amino acid refers to a molecule containing both an amine group and a carboxyl group that are bound to a carbon, which is designated the a-carbon.
  • Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes.
  • a single “amino acid” might have multiple sidechain moieties, as available per an extended aliphatic or aromatic backbone scaffold. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include amino acid analogs.
  • biodiesel refers to a biologically produced fatty acid alkyl ester suitable for use as a fuel in a diesel engine.
  • biomass refers to a material produced by growth and/or propagation of cells. Biomass may contain cells and/or intracellular contents as well as extracellular material, includes, but is not limited to, compounds secreted by a cell.
  • biomass refers to a closed or partially closed vessel in which cells are grown and maintained.
  • the cells may be, but are not necessarily held in liquid suspension.
  • cells may alternatively be growing and/or maintained in contact with, on, or within another non-liquid substrate including but not limited to a solid growth support material.
  • catalyst refers to a chemical actor, such as a molecule or macromolecular structure, which accelerates the speed at which a chemical reaction occurs where a reactant or reactants is converted into a product or products, while the catalyst is not turned into a product itself, or otherwise changed or consumed at the completion of the chemical reaction.
  • a catalyst After a catalyst participates in one chemical reaction, because it is unchanged, it may participate in further chemical reactions, acting on additional reactants to create additional products.
  • a catalyst decreases the activation energy barrier across the reaction path allowing it to occur at a colder temperature, or faster at a given temperature. In this way a more rapid approach of the system to chemical equilibrium may be achieved.
  • Catalysts subsume enzymes, which are protein catalysts.
  • cellulosic material refers to any material with a high amount of cellulose, which is a polysaccharide having the formula (C 6 H 10 O5) n , that generally consists of a linear chain of hundreds to thousands of ⁇ (1 ⁇ 4) linked D-glucose monomers.
  • Sources of cellulosic material include but are not limited to cardboard, cotton, corn stover, paper, lumber chips, sawdust, sugar beet pulp, sugar cane bagasses, and switchgrass.
  • CoA or "coenzyme A” refers to an organic cofactor for condensing enzymes involved in fatty acid synthesis and oxidation, pyruvate oxidation, acetyl or other acyl group transfer, and in other acetylation.
  • cofactor subsumes all molecules needed by an enzyme to perform its catalytic activity. In some embodiments, the cofactor is any molecule apart from the substrate.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C, H), nonpolar side chains (e.g., G, A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H).
  • basic side chains e.g., K, R, H
  • acidic side chains e.g., D, E
  • uncharged polar side chains e.g., G, N, Q, S, T, Y, C, H
  • nonpolar side chains e.g.
  • a predicted nonessential amino acid residue in an amino acid sequence encoded by an exogenous nucleic acid sequence is replaced with another amino acid residue from the same side chain family.
  • Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other biochemical properties (e.g. 2-thienylalanine for phenylalanine).
  • enzyme fragment is meant to refer to a fragment of an enzyme that includes the sequences sufficient to function substantially similar to the function of the wild- type enzyme upon which the fragment sequence is based. Fragments are generally 10 or more amino acids in length. Some preferred lengths of fatty acid reductase are at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210 at least 215, at least 220
  • Some preferred lengths of fatty acid reductase fragments are 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, 45 or fewer, 50 or fewer, 55 or fewer, 60 or fewer, 65 or fewer, 70 or fewer, 75 or fewer, 80 or fewer, 85 or fewer, 90 or fewer, 95 or fewer, 100 or fewer, 105 or fewer, 110 or fewer, 115 or fewer, 120 or fewer, 125 or fewer, 130 or fewer, 135 or fewer, 140 or fewer, 145 or fewer, 150 or fewer, 155 or fewer, 160 or fewer, 165 or fewer, 170 or fewer, 175 or fewer, 180 or fewer, 185 or fewer, 190 or fewer, 195 or fewer, 200 or fewer, 205 or fewer, 210 or fewer, 215 or fewer, 220 or fewer, 225 or fewer, 230 or fewer,
  • Some preferred lengths of fatty acid decarbonylase are at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210 at least 215, at least 220, at least 225, least 230 at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least
  • the lengths of the fatty acid decarbonylase fragments are 15 or fewer, amino acids, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, 45 or fewer, 50 or fewer, 55 or fewer, 60 or fewer, 65 or fewer, 70 or fewer, 75 or fewer, 80 or fewer, 85 or fewer, 90 or fewer, 95 or fewer, 100 or fewer, 105 or fewer, 110 or fewer, 115 or fewer, 120 or fewer, 125 or fewer, 130 or fewer, 135 or fewer, 140 or fewer, 145 or fewer, 150 or fewer, 155 or fewer, 160 or fewer, 165 or fewer, 170 or fewer, 175 or fewer, 180 or fewer, 185 or fewer, 190 or fewer, 195 or fewer, 200 or fewer, 205 or fewer, 210 or fewer, 215 or fewer, 220 or fewer, 225 or fewer,
  • Some preferred lengths of thioesterase fragments are at least 10 amino acids, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210 at least 215, at least 220, at least 225, least 230 at least 235, at least 240, at least 245, at least 250 or at least 255.
  • Some preferred lengths of thioesterase fragments are 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, 45 or fewer, 50 or fewer, 55 or fewer, 60 or fewer, 65 or fewer, 70 or fewer, 75 or fewer, 80 or fewer, 85 or fewer, 90 or fewer, 95 or fewer, 100 or fewer, 105 or fewer, 110 or fewer, 115 or fewer, 120 or fewer, 125 or fewer, 130 or fewer, 135 or fewer, 140 or fewer, 145 or fewer, 150 or fewer, 155 or fewer, 160 or fewer, 165 or fewer, 170 or fewer, 175 or fewer, 180 or fewer, 185 or fewer, 190 or fewer, 195 or fewer, 200 or fewer, 205 or fewer, 210 or fewer, 215 or fewer, 220 or fewer, 225 or fewer, 230 or fewer, 235 or
  • ranges may be any number set forth as an "at least” size to any number set forth as an "less than t" size in order to provide a range of sizes such as 20-400, 20-30, 40-100, etc.
  • exogenous gene or "exogenous nucleic acid” means a nucleic acid that has been recombinantly introduced into a cell, which encodes the synthesis of RNA and/or protein.
  • the exogenous gene is introduced by transformation.
  • the exogenous gene is introduced into the cell by electroporation.
  • a transformed cell may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced.
  • the exogenous gene put into the host species may be taken from a different species (this is called heterologous), or it may naturally occur within the same species (this is homologous as defined below).
  • exogenous genes subsume homologous genes that are integrated within or introduced to regions of the genome, episome, or plasmid that differ from the locations where the gene naturally occurs. Multiple copies of the exogenous gene may be introduced into the cell. An exogenous gene may be present in more than one copy within the host cell or transformed cell.
  • the microorganism comprises between and including 1 and 1,000 copies of the nucleic acid that encodes an exogenous protein. In some embodiments, the microorganism comprises between and including 1 and 10,000 copies of the nucleic acid that encodes an exogenous protein. In some embodiments, the microorganism comprises between and including 1 and 500 copies of the nucleic acid that encodes an exogenous protein.
  • the exogenous gene is maintained by a cell as an insertion into the genome or as an episomal molecule.
  • the microorganism comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 1000 copies of the one or more nucleic acids that encode one or more exogenous proteins.
  • the term "expressible form” refers to gene constructs that contain the necessary regulatory elements operably linked to a coding sequence that encodes an enzyme or fragment thereof capable of conferring enzymatic activity to a cell, such that when present in the cell, the coding sequence will be expressed.
  • the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than ten expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than nine expressible forms of exogenous nucleic acid sequences.
  • the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than eight expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than seven expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than six expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than five expressible forms of exogenous nucleic acid sequences.
  • the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than four expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than three expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than two expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than one expressible form of an exogenous nucleic acid sequences. In other embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprises more than ten expressible forms of exogenous nucleic acid sequences.
  • SEQ ID NO: l refers to Sequesco plasmid sequence 1.
  • SEQ ID NO:2 refers to Sequesco plasmid sequence 2.
  • SEQ ID NO: 3 refers to Sequesco plasmid Verl plasmid sequence.
  • SEQ ID NO:4 refers to Sequesco plasmid Ver2 plasmid sequence.
  • SEQ ID NO:5 refers to Arabidopsis gene FAR1.
  • SEQ ID NO: 6 refers to Arabidopsis gene FAR2.
  • SEQ ID NO: 7 refers to Arabidopsis gene FAR3.
  • SEQ ID NO: 8 refers to cyanobacterium FadR.
  • SEQ ID NO:9 refers to cyanobacterium FAD.
  • SEQ ID NO: 10 refers to cyanobacterium Rubisco large subunit promoter
  • SEQ ID NO: 11 refers to the 16S rRNA sequence from the genus Rhodococcus opacus DSM43205
  • SEQ ID NO: 12 refers to the 16S rRNA sequence from the genus Rhodococcus opacus
  • SEQ ID NO: 13 refers to the 16S rRNA sequence from the genus Ralstonia.
  • SEQ ID NO: 14 refers to Rhodococcus opacus TE
  • fatty acyl-ACP thioesterase mean an enzyme that catalyzes the cleavage of a fatty acid from an acyl carrier protein (ACP) during lipid synthesis.
  • fatty acyl-CoA reductase refers to an enzyme catalyzing the reaction that produces a fatty alcohol from an acyl-CoA molecule by reduction.
  • fatty acyl-ACP/acyl-CoA reductase refers to an enzyme catalyzing the reaction that produces a fatty aldehyde from an acyl-ACP or acyl-CoA molecule by reduction.
  • fatty aldehyde decarbonylase refers to an enzyme catalyzing the reaction that produces an alkane from a fatty aldehyde molecule by decarbonylization.
  • fatty aldehyde reductase refers to an enzyme catalyzing the reaction that produces a fatty alcohol from a fatty aldehyde molecule by reduction.
  • the term "functional fragment” is meant to refer to a fragment of any polypeptide or amino acid sequence that is encoded by an exogenous nucleic acid sequence of the present invention which retains its ability to function like the amino acid sequence to which the fragment is homologous.
  • Functional fragments of enzymes are at least about 5 amino acids in length derived from enzyme and may comprise non- wild-type amino acid sequences.
  • One having ordinary skill in the art can readily determine whether a protein or peptide is a functional fragment of a particular amino acid sequence by examining its sequence and testing its ability to function in a fashion similar to that function of the amino acid sequence upon which the fragment is based. Truncated versions of exogenous proteins may be prepared and tested using routine methods and readily available starting material.
  • the term "functional fragment” is also meant to refer to peptides, polypeptides, amino acid sequence linked by non-peptidal bonds, or proteins which comprise an amino acid sequence that is identical or substantially homologous to at least a portion of the exogenous amino acid sequence and which are capable of functioning in a similar function to the exogenous amino acid sequence to which the fragment is homologous.
  • substantially homologous refers to an amino acid sequence that has conservative substitutions.
  • One having ordinary skill in the art can produce functional fragments of the FAR, FadD, FAD, thioesterase, cytochrome P450 enzyme, desaturase, and hydroxylase amino acid sequences following the disclosure provided herein and well known techniques. The functional fragments thus identified may be used and formulated in place of full length FAR, FadD, FAD, thioesterase, cytochrome P450 enzyme, desaturase, and hydroxylase without undue experimentation.
  • gasification refers to a generally high temperature (>700°C) process that converts carbonaceous materials into a mixture of gases including hydrogen, carbon monoxide, and carbon dioxide called syngas or producer gas.
  • the process generally involves partial combustion and/or the application of externally generated heat along with the controlled addition of oxygen and/or steam.
  • homologous refers to the sequences homology between two nucleic acid sequences or two amino acid sequences. Two nucleic acid sequences or two amino acid sequences that are sufficiently homologous to retain immunogenic function are
  • homologues Sequence homology for nucleotides and amino acids may be determined using FASTA, BLAST and Gapped BLAST (Altschul et al., Nuc. Acids Res., 1997, 25, 3389, which is incorporated herein by reference in its entirety) and PAUP* 4.0M0 software (D. L. Swofford, Sinauer Associates, Massachusetts). "Percentage of similarity” is calculated using PAUP* 4.0bl0 software (D. L. Swofford, Sinauer Associates, Massachusetts). The average similarity of the enzymatic sequence or 16S rRNA sequence is calculated compared to all sequences in the phylogenic tree.
  • BLAST algorithm which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403410, which is incorporated herein by reference in its entirety).
  • Software for performing BLAST analyses is publicly available though the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • HSPs high scoring sequence pair
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or 3) the end of either sequence is reached.
  • the Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci.
  • a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • hydrocarbon refers to a molecule composed exclusively of carbon and hydrogen atoms with the carbons bonded covalently in a branched, cyclic, linear, or partially cyclic chain and with hydrogen atoms covalently bonded to the carbons such that the chemical octet rule for the carbons is generally satisfied. In some hydrocarbons there may occur some number of double or triple bonds between adjacent carbon atoms in the chain. Thus, the label hydrocarbon subsumes branched, cyclic, linear, branched, or partially cyclic alkanes (also called paraffins), alkenes (also called olefins), and alkynes.
  • hydrocarbon molecules range from the smallest, methane (CH 4 ), a primary component of natural gas, to high molecular weight complex molecules including asphaltenes present in bitumens crude oil, and petroleum.
  • CH 4 methane
  • Other examples include dodecane (CI 2), hexadecane (CI 6), or octadecane (CI 8) etc.
  • Hydrocarbons of the present invention may be in gaseous, liquid, or solid phases, either as singly or in multiply coexisting phases. In some
  • the hydrocarbons are selected from one or more of the following: linear, branched, cyclic, or partially cyclic alkanes, alkenes, alkynes, lipids, and paraffin.
  • the hydrocarbon are selected from one or more of the following: octane, squalene Spiro[4.5]decane, Bicyclo[10.8.0]eicosane, cis,cis-l,6-Dimethylspiro[4.5]decane, 1,19-Eicosadiene, Cyclooctacosane, Bicyclo[10.8.0]eicosane, 1-Pentadecyne, 1-Pentadecyne, Heptacosyl acetate, 5-Cyclohexyl-l-pentene, 1-Hexadecyne and Cyclodecacyclotetradecene, -eicosahydro.
  • hydrophobic fraction gives the fraction of matter that has low solubility in water and greater solubility in a hydrophobic phase than in an aqueous phase.
  • the hydrophobic fraction is non-polar.
  • the genetically modified bacterial cells described herein increase the hydrophobic fraction in a cell as compared to the same cell that is not genetically modified.
  • improve lipid yield refers to an increase in the lipid production of an organism through any means.
  • the increase is caused by raising the cell dry weight density of a microbial culture and/or raising the fraction of cell mass that is composed of lipid and/or reducing the cell doubling time and/or the biomass doubling time, resulting in an overall increase in the lipid production rate per unit volume.
  • jet fuel means a fuel useful for igniting in the engine of an aircraft comprising a mixture of kerosene (mixture of C9 - C16 alkanes of a certain percentage) combined with typical additives.
  • the jet fuel may comprise a mixture of ingredients specified by the Jet A-l, Jet A, Jet B, JP1, JP-2, JP-3, JP-4, JP-5, JP-6, JP-7, JP-8, or other similar compositions.
  • the jet fuels comprise at least one or more typical additive chosen from antioxidants (including phenolic antioxidants), static inhibitors, corrosion inhibitors, fuel system icing inhibitors, lubrication improvers, biocides, and thermal stability improvers (DOD 1992; IARC 1989; Pearson 1988). These additives are used only in specified amounts, as governed by military specifications (DOD 1992; IARC 1989).
  • Straight-run kerosene the basic component of the kerosene used for jet fuels, consists of hydrocarbons with carbon numbers mostly in the C9-C16 range. Like all jet fuels, straight- run kerosene consists of a complex mixture of aliphatic and aromatic hydrocarbons (LARC 1989).
  • Aliphatic alkanes paraffins
  • cycloalkanes naphthenes
  • the jet fuel comprises from between about 10% - 20 % aromatics and less than 1% of olefins.
  • the boiling range of the jet fuels is well above the boiling point of benzene.
  • the jet fuel comprises less than or equal to 0.02% of benzene and less than or equal to 0.01% of PAHs.
  • knallgas refers to the mixture of molecular hydrogen and oxygen gas.
  • a "knallgas microorganism” is a microbe that can use hydrogen as an electron donor and oxygen as an electron acceptor in the generation of intracellular energy carriers such as Adenosine-5'-triphosphate (ATP).
  • ATP Adenosine-5'-triphosphate
  • microorganism can be used synonymously with “knallgas” and “knallgas microorganism” respectively.
  • lignocellulosic material is any material composed of cellulose
  • Lignocellulosic materials subsume agricultural residues (including corn stover and sugarcane bagasse), most biomass energy crops, wood residues (including sawmill and paper mill discards), and a substantial fraction of municipal waste.
  • lipids refers to category of molecules that can be dissolved in nonpolar solvents (such as chloroform and/or ether) and which also have low or no solubility in water.
  • nonpolar solvents such as chloroform and/or ether
  • hydrophobic character of lipids molecules typically results from the presence of long chain hydrocarbon sections within the molecule.
  • Lipids subsume the following molecule types: hydrocarbons, fatty acids (saturated and unsaturated), fatty alcohols, fatty aldehydes, hydroxy acids, diacids, monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, sterols such as cholesterol and steroid hormones, fat-soluble vitamins (such as vitamins A, D, E and K), polyketides, terpenoids, and waxes.
  • hydrocarbons fatty acids (saturated and unsaturated), fatty alcohols, fatty aldehydes, hydroxy acids, diacids, monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, sterols such as cholesterol and steroid hormones, fat-soluble vitamins (such as vitamins A, D, E and K), polyketides, terpenoids, and waxes.
  • lipid modification enzyme corresponds to an enzyme that catalyzes a reaction changing a lipid's covalent bonds such as TE, FAR, FadR, FAD, fatty aldehyde reductase, lipase, cytochrome P450 enzyme, desaturase, or hydroxylase. Any enzyme that catalyzes a reaction step or steps in lipid synthesis, catabolism, or modification, including carrier proteins, is called a "lipid pathway enzyme”.
  • lysate refers to the liquid containing a mixture and/or a solution of cell contents that result from cell lysis.
  • the methods of the present invention comprise a purification of hydrocarbons or mixture of hydrocarbons in a cellular lysate.
  • the methods of the present invention comprise a purification of lipids and/or hydrocarbons and/or a mixture of hydrocarbons in a cellular lysate.
  • lysis refers to the rupture of the plasma membrane and if present the cell wall of a cell such that a significant amount of intracellular material escapes to the extracellular space. Lysis can be performed using electrochemical, mechanical, osmotic, thermal, or viral means.
  • the methods of the present invention comprise performing a lysis of cells or microorganisms described herein in order to separate a hydrocarbon or mixture of hydrocarbons from the contents of a bioreactor.
  • the methods of the present invention comprise performing a lysis of cells or microorganisms described herein in order to separate a lipid or hydrocarbon or mixture of lipids or hydrocarbons or a mixture of lipids and hydrocarbons from the contents of a bioreactor.
  • microorganism and “microbe” mean microscopic single celled life forms.
  • molecule means any distinct or distinguishable structural unit of matter comprising one or more atoms, and includes for example hydrocarbons, lipids, polypeptides and polynucleotides.
  • natural strain means any wild-type or mutant organism that has not had exogenous genes encoded in it.
  • oil refers to something that is rich in oil or produces oil in high quantities.
  • organic compound refers to any gaseous, liquid, or solid chemical compounds which contain carbon atoms with the following exceptions that are considered inorganic: carbides, carbonates, simple oxides of carbon, cyanides, and allotropes of pure carbon such as diamond and graphite.
  • jet fuel is a complex mixture of hydrocarbons that varies depending on crude source and manufacturing process. Consequently, it is impossible to define the exact composition of jet fuel. Specification of jet fuel has therefore evolved primarily as a performance specification rather than a compositional specification and the hydrocarbons typically range between 8 and 17 carbon atoms in hydrocarbon chain length.
  • a precursor to jet fuel may be composition comprising at least one hydrocarbon having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more carbon atoms and having the commonly known specifications for Jet A-l, Jet A, Jet B, JP1, JP-2, JP-3, JP- 4, JP-5, JP-6, JP-7, JP-8 fuel when in isolation or mixture with other hydrocarbons.
  • the precursor to jet fuel is a mixture of different carbon backbone lengths of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more carbon atoms with the commonly known specifications for Jet A-l, Jet A, Jet B, JP1, JP-2, JP-3, JP-4, JP-5, JP-6, JP-7, JP-8 fuel, or other jet fuels.
  • the precursor to jet fuel may be one or more hydrocarbons that, when exposed to cracking and/or deoxygention and/or isomerization, may be used as a component of Jet A-l, Jet A, Jet B, JP1, JP-2, JP-3, JP-4, JP-5, JP-6, JP-7, JP-8 fuel or other jet fuels.
  • producing includes both the production of compounds intracellularly and extracellularly, which is to include the secretion of compounds from the cell.
  • Promoter is a control DNA sequence that regulates transcription.
  • a promoter may includes nucleic acid sequences near the start site of transcription that are required for proper function of the promoter, as for example, a TATA element for a promoter of polymerase II type.
  • Promoters of the present invention can include distal enhancer or repressor elements that may lie in positions up to many thousands of base pairs away from the start site of transcription.
  • inducible promoter refers to an operable linkage between a promoter and a nucleic acid where the promoter's mediation of nucleic acid transcription is sensitive to a specific stimulus.
  • the inducible promoter requires a cofactor which can be added to the environment of the composition comprising the nucleic acid sequence that contains the inducible promoter.
  • An "operable linkage" refers to an operative connection between nucleic acid sequences, such as for example between a control sequence (e.g. a promoter) and another sequence that codes for a protein i.e. a coding sequence. If a promoter can regulate transcription of an exogenous gene then it is in operable linkage with the gene.
  • gas from synthetic gas or synthesis gas refers to a gas mixture that contains various proportions of hydrogen, carbon monoxide, and carbon dioxide, and which typically also includes a variety of impurities such as methane, hydrogen sulfide, condensable gases, and tars.
  • Producer gas is a related term that generally refers to gas mixes similar to syngas except for the presence of a large N 2 component that results from using air directly in the gasification process.
  • the invention relates to chemotrophic bacterial strains that comprise one or more exogenous nucleic acid sequences.
  • the present invention results from the discovery that chemotrophic bacteria and particular related microorganisms provide unforeseen advantages in the economic and large scale production of chemicals, oils, fuels, and other hydrocarbon or lipid substances from gaseous and waste carbon feedstocks, and also from the discovery of genetic techniques and systems for modifying these microorganisms for improved
  • the lipids and other biochemicals synthesized by the microorganisms of the present invention can be applied to uses including but not limited to transportation fuel, petrochemical substitutes, monomers, feedstock for the production of polymers, lubricants, as ingredients in animal feed, food, personal care, and cosmetic products.
  • triglycerides produced in the present invention can be converted by transesterification to long-chain fatty acid esters useful as biodiesel fuel.
  • enzymatic and chemical processes can be utilized to produce alkanes, alkenes, alkynes, hydroxy acids, fatty aldehydes, fatty alcohols, fatty acids, diacids, and unsaturated fatty acids.
  • Some embodiments enable the production of renewable jet fuel, diesel, or other hydrocarbons.
  • the present invention gives methods for culturing and/or modifying chemotrophic bacteria for improved lipid yield and/or lower production costs.
  • the genetically modified bacteria produce more of a certain type or types of lipid molecules as compared to the same bacteria that is not genetically modified.
  • the present invention relates to compositions comprising and methods of using genetically modified microorganisms to produce and/or secrete carbon-based products from conversion of gaseous carbon feedstocks including but not limited to syngas or producer gas.
  • the present invention relates to methods and mechanisms to confer production and/or secretion of carbon-based products of interest including but not limited to ethylene, chemicals, monomers, polymers, n-alkanes, branched alkanes, cycloalkanes, alkenes, alkynes, hydroxy acids, fatty alcohols, fatty acids, diacids, unsaturated fatty acids, aldehydes, hydrocarbons, isoprenoids, proteins, polysaccharides, nutraceutical or pharmaceutical products or intermediates thereof in obligate or facultative chemotrophic organisms such that these organisms convert carbon dioxide and/or other forms of inorganic carbon and/or syngas and/or other Ci compounds such as methanol and/or the liquid, gaseous, and solid products of pyro
  • hydrocarbons hydrocarbons, isoprenoids, proteins, polysaccharides, nutraceutical or pharmaceutical products or intermediates thereof.
  • Chemoautotrophs are capable of performing chemo synthetic reactions that fix C02, and/or other forms of inorganic carbon, to organic compounds, using the potential energy stored in inorganic chemicals to drive the reaction, rather than radiant energy from light as in microorganisms performing photosynthesis [Shively et al, 1998; Smith et al, 1967; Hugler et al, 2005; Hugker et al., 2005; Scott and Cavanaugh, 2007].
  • Carbon fixing biochemical pathways that occur in chemoautotrophs include the reductive tricarboxylic acid cycle, the Calvin-Benson-Bassham cycle [Jessup Shively, Geertje van Kaulen, Wim Meijer, , Annu. Rev. Microbiol., 1998, 191-230], and the Wood-Ljungdahl pathway [Ljungdahl, 1986;
  • the invention relates to compositions comprising and methods of using
  • chemoautotrophic metabolism to produce ATP for the support of ATP consuming synthetic reactions and cellular maintenance, without the co-production of methane or short chain organic acids such as acetic or butyric acid, by means of energy conserving reactions for the production of ATP using inorganic electron donors, including but not limited to the oxyhydrogen reaction.
  • hydrocarbons or other lipids with carbon chain lengths longer than C 4 is most commonly and efficiently accomplished biologically through fatty acid
  • acetyl-CoA acetyl- coenzyme A
  • the invention utilizes microorganisms with a naturally occurring pathway for the conversion of CO, C0 2 and/or H 2 to acetyl-CoA.
  • the invention utilizes microorganisms that can fix CO and/or C0 2 through the reductive tricarboxylic acid cycle, the Calvin-Benson-Bassham cycle, and/or the Wood- Ljungdahl pathway. In some embodiments the invention utilizes microorganisms that fix CI compounds through a methanotropic pathway.
  • the microorganisms naturally produce enzymes that catalyze the fixation of gaseous inorganic carbon to produce acetyl-CoA, utilizing gaseous electron donors such as are present in syngas as reducing agents, with such enzymatic proteins including but not limited to acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase, cobalamide corrinoid/iron- sulfur protein, carbon monoxide dehydrogenase, hydrogenase, and methyltransferase.
  • gaseous electron donors such as are present in syngas as reducing agents
  • enzymatic proteins including but not limited to acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase, cobalamide corrinoid/iron- sulfur protein, carbon monoxide dehydrogenase, hydrogenase, and methyltransferase.
  • Methanogenic archaea ecologically relevant differences in energy conservation. Nat Rev Microbiol 6, 579-591, doi:nrmicrol931 [pii] ] or acetic acid production, and one ATP per 10H 2 for butyric acid production [Papoutsakis, Biotechnology & Bioengineering (1984) 26, 174-187; Heise, Muller, Gottschalk, J. of Bacteriology (1989) 5473-5478; Lee, Park, Jang, Nielsen, Kim, Jung, Biotechnology & Bioengineering (2008) 101, 2, 209-228].
  • the invention relates to a microorganism or compositions comprising a microorganism, wherein the microorganism produces ATP from an inorganic electron donor such as but not limited to H2 without synthesis of methane or short chain organic acids.
  • Hydrogen-oxidizing microorganisms that use more electronegative electron acceptors in energy conserving reactions for ATP production, such as but not limited to
  • hydrogenotrophic oxyhydrogen or knallgas microbes that link the oxyhydrogen reaction, 2 H 2 + 0 2 -> 2 H 2 0, to ATP production, can produce more ATP per H 2 consumed than acetogens or methanogens.
  • knallgas microorganisms can produce up to two ATP per H 2 consumed [Bongers, J. Bacteriology, (Oct 1970) 145-151], which is eight times more ATP produced per H 2 consumed than what can be produced in microorganisms undergoing methanogenesis or acetogenesis.
  • microorganisms that can utilize more electronegative electron acceptors in the production of ATP, such as but not limited to knallgas microbes, in fatty acid biosynthesis from syngas or H 2 , can be more efficient for supporting fatty acid biosynthesis than using the acetogens or methanogens that are currently used in biological GTL technologies.
  • the invention relates to a microorganism or compositions comprising a microorganism, wherein the microorganism is a knallgas microbe and comprises at least one or more exogenous nucleic acid sequences that encodes one or more enzymes to enable fixation of a carbon-containing gas feedstock, including but not limited to syngas or producer gas, into useful carbon-based products of interest including but not limited to ethylene, chemicals, monomers, polymers, n- alkanes, branched alkanes, cycloalkanes, alkenes, alkynes, hydroxy acids,fatty alcohols, fatty acids, diacids, unsaturated fatty acids, fatty aldehydes, hydrocarbons, isoprenoids, polypeptides, polysaccharides, nutraceutical or pharmaceutical products.
  • the microorganism is a knallgas microbe and comprises at least one or more exogenous nucleic acid sequences that encodes one or more enzymes to enable fixation of
  • the microorganism or composition comprising the microorganism comprises at least one or more exogenous nucleic acid sequences that encodes one or more enzymes that allows the microorganism to convert a carbon-containing gas feedstock, including but not limited to syngas or producer gas, into jet fuel, diesel fuel, biodiesel fuel, or a component or precursor thereof.
  • the invention relates to a genetically modified microorganism and compositions comprising such a microorganism, wherein the microorganism comprises one or more exogenous genes and wherein the microorganism grows on carbon-containing gas or utilizes a gaseous feedstock selected from syngas, C0 2 , H 2 , CO, or mixtures of gas comprising one or more gases selected from syngas, C0 2 , H 2 , or CO.
  • a gaseous feedstock selected from syngas, C0 2 , H 2 , CO, or mixtures of gas comprising one or more gases selected from syngas, C0 2 , H 2 , or CO.
  • the invention relates to a cell and compositions comprising a cell of the class
  • Actinobacteria comprising at least one exogenous gene.
  • the invention also relates to cells and compositions comprising cells of the family of Nocardiaceae comprising at least one exogenous gene.
  • the invention relates to cells and compositions comprising cells of Corynebacterium, Gordonia, Rhodococcus, Mycobacterium and Tsukamurella comprising at least one exogenous gene.
  • the invention relate to cells of the family of Nocardiaceae comprising an exogenous gene, wherein the cell is not a cell of the genus Mycobacterium.
  • the invention provides a cell and compositions comprising a cell of the genus Rhodococcus comprising an exogenous gene, and in some embodiments the cell is a strain of the species Rhodococcus sp., Rhodococcus opacus, Rhodococcus aurantiacus; Rhodococcus baikonurensis; Rhodococcus boritolerans;
  • Rhodococcus rhodochrous Rhodococcus rhodnii; (synonym: Nocardia rhodnii); Rhodococcus ruber (synonym: Streptothrix rubra); Rhodococcus sp. RHA1; Rhodococcus triatomae;
  • the cell comprising one or more exogenous genes is strain Rhodococcus opacus DSM number 43205 or 43206. In some embodiments the cell comprising one or more exogenous genes is strain Rhodococcus sp. DSM number 3346.
  • the invention provides cells and compositions comprising a cell of the genus Rhodococcus comprising an exogenous gene, wherein the cell or composition comprising a cell of Rhodococcus is non-infectious to animals and/or plants. In some embodiments, the invention provides cells and compositions comprising a cell of the genus Rhodococcus comprising an exogenous gene, wherein the Rhodococcus cell or composition comprising a Rhodococcus cell is non-infectious to humans.
  • the invention provides cells and compositions comprising a cell of the genus Rhodococcus comprising an exogenous gene, wherein the Rhodococcus cell or composition comprising a Rhodococcus cell is non-infectious to plants.
  • the invention provides cells and compositions comprising cells of the genus Rhodococcus comprising an exogenous gene, wherein, if the cell is from Rhodococcus equi or Rhodococcus fascians species, the species is non-infectious to animals and/or plants.
  • the invention relates to a Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is not a species selected from Rhodococcus equi or Rhodococcus fascians.
  • the invention relates to a Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is incapable of producing any acrylic acid or acrylamide. In some embodiments, the invention relates to a Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell produces less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of its weight of total dry cellular matter in acrylamide or
  • the invention relates to a Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is not from the species Rhodococcus rhodochrous. In some embodiments, the invention relates to Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is incapable of producing 10-hydroxy-12-octadecenoic acid. In some embodiments, the invention relates to a
  • Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is unable to produce more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of its weight of total dry cellular matter in 10-hydroxy-12-octadecenoic acid.
  • the invention relates to
  • Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is incapable of producing optically- active 4-amino-3-hydroxybutyric acid.
  • the invention relates to a Rhodococcus cell or composition comprising a
  • Rhodococcus cell wherein the cell is unable to produce more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of its weight of total dry cellular matter in optically- active 4-amino-3-hydroxybutyric acid.
  • the cell or compositions comprising one of more cells is not E. coli. In some embodiments, the cell or compositions comprising one of more cells is from the genus Rhodococcus but is not for the species equi. In some embodiments, the cell of the present invention is not pathogenic to animals or plants. In some embodiments, the cell of the present invention is not pathogenic to humans. In some embodiments, the cell or compositions comprising one of more cells is from the genus Ralstonia. In some
  • the cell or compositions comprising one of more cells is from the species
  • the cell comprising one or more exogenous genes is strain Cupriavidus necator DSM number 531 or 541.
  • the cell or compositions comprising the one or more cells have a 16S rRNA sequence with at least 50, 60, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide homology to one or more of SEQ ID NOs: 11 or 12.
  • the cell or compositions comprising the one or more cells have a 16S rRNA sequence with at least 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide homology to one or more of SEQ ID NOs: 11.
  • the cell or compositions comprising the one or more cells have a 16S rRNA sequence with at least 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide homology to one or more of SEQ ID NOs: 12.
  • the cell or compositions comprising the one or more cells have a 16S rRNA sequence with at least 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide homology to one or more of SEQ ID NOs: 13.
  • the microorganism of the claimed invention is not dependent upon light to grow and/or metabolize and/or synthesize lipid molecules.
  • the microorganism of the claimed invention does not require any type of sugar to grow and/or metabolize and/or synthesize lipid molecules. In some embodiments, the microorganism of the claimed invention does not require any type of organic compound to grow and/or metabolize and/or synthesize lipid molecules. In some embodiments, the microorganism of the claimed invention does not require any type of fixed carbon to grow and/or metabolize and/or synthesize lipid molecules. In some embodiments, the
  • microorganism can grow and/or metabolize lipids in a slightly anaerobic or extremely anaerobic environment.
  • the microorganism of the claimed invention is a facultative microorganism
  • Microbial culturing in the present invention is performed both for the sake of implementing genetic modifications, and for production of organic compounds, and specifically lipids and/or hydrocarbons (e.g., alkenes, alkynes, alkanes, unsaturated fatty acids, fatty acids, fatty alcohols, fatty aldehydes, triacylglycerols, hydroxy acids, diacids).
  • Microbial culturing with the aim of genetic manipulation is generally performed at a small benchtop scale and often under conditions that select for genetically modified traits.
  • Microbial culturing aimed at the commercial production of organic compounds and specifically lipids and/or hydrocarbons is typically performed in bioreactors at much greater scale (e.g., 500 L, 1,000 L 5,000 L, 10,000 L, 50,000 L, 100,000 L, 1,000,000 L bioreactor volumes and higher).
  • the chemoautotrophs of the present invention are grown in a liquid media inside a bioreactor using the methods of the invention.
  • the bioreactor containing the microorganisms is constructed of opaque materials that keep the culture in darkness. Bioreactors constructed out of opaque materials such as steel or reinforced concrete can be designed to have extremely big working volumes.
  • steel fermenters 50,000 liter and greater in volume are utilized.
  • egg- shape or cylindrical digesters 3,000,000 liters and greater in volume are utilized.
  • the bioreactor comprising the microorganism does not allow light to penetrate its interior.
  • the bioreactor or fermentor is used to culture cells through the various phases of their physiological cycle.
  • a bioreactor is utilized for the cultivation of cells, which may be maintained at particular phases in their growth curve.
  • the use of bioreactors is advantageous in many ways for cultivating chemoautotrophic growth.
  • oleaginous cell mass which is used to produce fuel, is grown to high densities in liquid suspension.
  • control of growth conditions including control of dissolved carbon dioxide, oxygen, and other gases such as hydrogen, as well as other dissolved nutrients, trace elements, temperature and pH, is facilitated in a bioreactor.
  • Nutrient media as well as gases can be added to the bioreactor as either a batch addition, or periodically, or in response to a detected depletion or programmed set point, or continuously over the period the culture is grown and/or maintained.
  • the bioreactor at inoculation is filled with a starting batch of nutrient media and/or gases at the beginning of growth, and no additional nutrient media and/or gases are added after inoculation.
  • nutrient media and/or gases are added periodically after inoculation.
  • nutrient media and/or gas is added after inoculation in response to a detected depletion of nutrient and/or gas.
  • nutrient media and/or gas is added continuously after inoculation.
  • the bioreactors have mechanisms to enable mixing of the nutrient media that include but are not limited to spinning stir bars, blades, impellers, or turbines, spinning, rocking, or turning vessels, gas lifts and sparging.
  • the culture media may be mixed continuously or intermittently.
  • the ports that are standard in bioreactors may be utilized to deliver, or withdraw, gases, liquids, solids, and/or slurries, into the bioreactor vessel enclosing the microbes of the present invention.
  • Many bioreactors have multiple ports for different purposes (e.g. ports for media addition, gas addition, probes for pH and DO, sampling), and a given port may be used for various purposes during the course of a fermentation run.
  • a port might be used to add nutrient media to the bioreactor at one point in time and at another time might be used for sampling.
  • the multiple use of a sampling port can be performed without introducing contamination or invasive species into the growth environment.
  • a valve or other actuator enabling control of the sample flow or continuous sampling can be provided to a sampling port.
  • the bioreactors are equipped with at least one port suitable for culture inoculation that can additionally serve other uses including the addition of media or gas.
  • Bioreactors ports enable control of the gas composition and flow rate into the culture environment.
  • the ports can be used as gas inlets into the bioreactor through which gases are pumped.
  • gases that may be pumped into a bioreactor include syngas, producer gas, hydrogen gas, C02, air, air/C0 2 mixtures, ammonia, nitrogen, noble gases, such as argon, as well as other gases.
  • C0 2 may come from sources including but are not limited to: C0 2 from the gasification of organic matter; C0 2 from the calcination of limestone, CaC0 3 , to produce quicklime, CaO; C0 2 from methane steam reforming, such as the C0 2 byproduct from ammonia or hydrogen production; combustion; C0 2 byproduct of sugar fermentation; C0 2 byproduct from sodium phosphate production; geologically or geothermally produced C0 2 .
  • Raising the gas flow rate into a bioreactor can enhance mixing of the culture and produce turbulence if the gas inlet is positioned under the surface of the liquid media such that gas bubbles or sparges up through the media.
  • a bioreactor comprises gas outlet ports for gas escape and pressure release.
  • gas inlets and outlets are preferably equipped with check valves to prevent gas backflow.
  • the present invention relates to bioreactors that comprise a cell, which comprises at least one exogenous nucleic acid sequences that encodes a lipid pathway enzyme.
  • the present invention relates to a system of at least one bioreactor that comprise a cell, which comprises at least one exogenous nucleic acid sequences that encodes a lipid pathway enzyme.
  • the system comprises two or more, three or more, or four or more bioreactors, at least one of which comprise a cell, which comprises at least one exogenous nucleic acid sequences that encodes a lipid pathway enzyme.
  • the system of bioreactors comprises at least a first and second bioreactor, wherein the first bioreactor comprises a cell, which comprises at least one exogenous nucleic acid sequences that encodes a lipid pathway enzyme; and wherein the second bioreactor comprises a microorganism derived from a different species, wherein the microorganism from a different species comprises at least one exogenous nucleic acid sequence that encodes a lipid pathway enzyme.
  • the system of bioreactors comprises a first bioreactor that comprises the cell of the present invention and a second bioreactor comprising a microalgal, yeast, or bacterial cell.
  • the cells of the present invention are capable of producing desaturated alkanes between 8 and 18 carbon atoms long at greater than 18 grams per liter volume of culture per three day period. In some embodiments, the cells of the present invention are capable of producing desaturated alkanes between 8 and 18 carbon atoms long at greater than or equal to 18 grams per liter volume of culture per three day period, wherein the desatruated alkanes are desatuated at a carbon position other than carbon-9.
  • the present invention relates to methods of modifying a bacterial cell to express one or more exogenous nucleic acid sequences that encodes one or more enzymes to enable fixation of a carbon-containing gas feedstock into useful carbon-based products of interest in an amount greater than an amount of carbon-based products produced by the same bacterial cell that does not express the exogenous nucleic acid sequences.
  • Methods of selecting and manufacturing nucleic acid sequences for modification of bacterial cells are known and can be performed by transformation, electroporation, phage infection of bacteria, or other techniques for nucleic acid transfer generally known in the art. Standard recombinant DNA and molecular cloning techniques useful for the invention are well known in the art and are described by Sambrook, J., Fritsch, E. F.
  • the invention relates to genetic constructs comprising one or more exogenous genes that encode one or more amino acid sequences to enable fixation of a carbon-containing gas feedstock, including but not limited to syngas or producer gas, into useful carbon-based products of interest in an amount greater than an amount of carbon-based products produced by the same bacterial cell that does not express the exogenous nucleic acid sequence or sequences.
  • Another aspect of the present invention relates to compositions that comprise at least one bacterial cell, which comprises at least one nucleic acid sequence that encodes at least one exogenous amino acid sequence that functions as a fatty acid acyl-ACP reductase, a fatty acid aldehyde decarbonylase and/or a thioesterase.
  • the bacterial cell is transformed with one or more, two or more, three or more, four or more, or five or more exogenous nucleic acid sequences that encode one or more amino acid sequences to enable fixation of a carbon-containing gas feedstock, including but not limited to syngas or producer gas, into useful carbon-based products of interest in an amount greater than an amount of carbon-based products produced by the same bacterial cell that does not express the exogenous nucleic acid sequence or sequences.
  • genetic material that encodes the enzyme is delivered to a bacterial cell in an expressible form.
  • the genetic material, DNA or RNA is taken up by the cells of the invention and expressed.
  • the enzyme or enzymes that are thereby produced can biochemically modify lipid molecules to remove or add hydroxyl groups, remove or add carbonyl groups, remove or add carbon-carbon double bonds, remove or add carbon-carbon triple bonds, remove or add aldehyde groups, remove or add hydroxy groups, remove or add carboxylic acid groups, or remove or add ester groups to lipid molecules in lipid.
  • the genetic constructs of the present invention comprise DNA, RNA, or combinations of both DNA and RNA.
  • the genetic construct of the present invention is a plasmid.
  • the plasmid contains a variety of open reading frames (ORFs) encoding proteins of many diverse functions, including those enzymes that enable hydrocarbon or lipid modification, glutathione-S transferase (GST) activity, origins of replication, multiple cloning sites, promoters, and/or termination sequences. It is contemplated therefore that a host cell transformed with the plasmid will demonstrate the ability to modify a variety of lipids or hydrocarbons as well as maintain its copy number in the cytoplasm of the cell.
  • ORFs open reading frames
  • GSTs represent a large group of detoxification enzymes.
  • GSTs catalyze the conjugation of glutathione, homoglutathione and other glutathione-like analog via sulfhydryl group, to a large range of hydrophobic, electrophilic compounds. The conjugation can result in detoxification of these compounds.
  • GST genes are found in both prokaryotic (e.g., E. coli) and eukaryotic organisms (e.g., yeast, plant and human).
  • a host strain transformed with the plasmid will have the ability detoxify harmful compounds via conjugation of those compounds to glutathione.
  • the instant plasmid additionally encodes a variety of maintenance proteins, useful for maintaining, stabilizing and replicating the plasmid. It is contemplated that these genes may be used in conjunction with other bacterial plasmids deficient in these functions for the increased stabilization or robust maintenance of the plasmid.
  • the plasmid comprises maintenance proteins of particular interest including the REP origin of replication (encoded by ORF 38) the TRA proteins (TRAI, TRAJ and TRAK, encoded by ORF's 23, 24 and 25 respectively) and the VAG proteins (VAGD and VAGC, encoded by ORF's 33 and 34 respectively).
  • the tra gene family is known to be involved in plasmid conjugation, a process that promotes DNA transfer from a donor to a recipient cell mediated by physical contact (Firth et al, Escherichia coli and Salmonella: Cellular and Molecular Biology, ASM press (1996)).
  • Tral and TraK proteins are reported to be required for efficient plasmid site- specific recombination (Paterson et al. J. Bacteriol 181:2572-2583 (1999)).
  • Tral is required for conjugal DNA transfer.
  • TraJ has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA.
  • TraJ, traJ gene product regulates transcription originating at the tra operon promoter P.sub.traY. (Firth et al., Escherichia coli and Salmonella: Cellular and Molecular Biology, ASM press (1996)).
  • the stabilization proteins VAGC and VAGD encoded by vag and vagD are involved in maintaining the plasmid as an autonomous replicating unit.
  • Non- limiting examples of bacterial maintenance proteins of particular interest on the pSeq and pVer plasmids are represented by the following DNA and protein sequences:
  • GAAAAGCCCC TTTACACGCGCG CGGGCGCAAG GACGACCGCG TGGGCCTGTT TCTGGCGCTG GACTTCCCGC TGTTCCGTCA
  • GCAGCTTTTC GCCCACGGCC TTGATGATCG CGGCGGCCTT GGCCTGCATA TCCCGATTCA ACCCGGACAA AGACCGCGAC
  • GCGCCAGTAC ACGCCCGACC AAAGCAGTGC CACCTACGAC GCCGTCACGA TGCGATCCGC CCCGTACTTG TCCGCCAGCC
  • CCCATCGCTT CATCGGTGCT GGCCCGACCG GCAGTACTGC ATGAGCTGGC GGTTGTGTCG CAGGAACGCG GCGAAGAGAC
  • CTTTGCTTCG CAAAGTCGTG ACCGCCTACG GCGGCTGCGG CGCCCTACGG GCTTGCTCTC GGAGGCCGCC CGGTGAGTTA
  • CACGGCGGCA CCACCAACTA GTGCTGCTGG TCCTGCTTAG GCTGTTGGGG CATGGCGACC TGCGCCGCAT CCCGACCCTG
  • GCTATTTAAC GACCCTGCCC CCGGAGTCCG TAAACTCTTC GTGTGCCAGT GTGACGAAGG CCATCAGTTA TTTGGCCATT
  • AAAC GAAAG CTTAAAGACG GTAAGTAGGC GAATAATAGT GAATAAGTCC GCATCGTGGT CCGCAAATTC CCGTGGTTAT
  • AATTTAACGC GAATTTTAAC TGACGGAATT TTTTTAATGC GGGGCGGGAC GGTGAGTAGC GTCAGCCGGA TAACCAATTT
  • CACATACGCC CGGACTGCCT AGTTGCAAGG GTGGCTCGGT GTCGAGATGT TCATCTGGTC GGCGATCTGC CGGTACTTCA
  • GCCGCCACTT CCACGCCACC CAGGTCACCC GCTGACTAAA GCCGATCTGC TCGGCCTCGG CCCGACTCAT GGGGCCGATC
  • CACACAGTGA TCGACGGGTA GTTCTGTTTC CGGATCTCGC GGTAGGCCCA TTCCCGGGTG CGGTCGAACA GTTCGACGTT
  • GCCAGCTTGT CAAGCTGCAA GGCCGGGCAA AGCCAGGACT CCTGTGTCTT GCGGCCGTAG TCCGGTGGGG CGGGGAAACG
  • GCAACCGTCG AATCGATTTC GAGCAGAGCG AGCAGAGCAA GATATTCCAA AACTCCGGGG TTCCTCGGCG GCCTCCCCCG
  • GGAAGCCATC ACAAACGGCA TGATGAACCT GAATCGCCAG CGGCATCAGC ACCTTGTCGC CTTGCGTATA ATATTTGCCC
  • CTCCACCCAA GCGGCCGGAG AACCTGCGTG CAATCCATCT TGTTCAATCA TGCGAAACGA TCCTCATCCT GTCTCTTGAT
  • GCTTTTGTCA GCCGGTCGGC CACGGCTTCC GGCGTCTCAA CGCGCTTTGA GATTCCCAGC TTTTCGGCCA ATCCCTGCGG
  • AAAAGCCGGT TAGGGACGCC
  • ACGTATCCGC GCACCGAGCT CCGCTTGCGG GCTGATGGTG ACGTGGCCCA CTGGTGGCCG
  • AAAAC C AC AG GTTGGCCGAG CTGCCCCCGT CGCGTTCCGC CACGGAGGCC GCCCGGTGAG TTACGAACTC ATATGAGTGA
  • CACGGCGGCA CCACCAACTA GTGCTGCTGG TCCTGCTTAG GCTGTTGGGG CATGGCGACC TGCGCCGCAT CCCGACCCTG
  • GCTATTTAAC GACCCTGCCC CCGGAGTCCG TAAACTCTTC GTGTGCCAGT GTGACGAAGG CCATCAGTTA TTTGGCCATT
  • AAAC GAAAG CTTAAAGACG GTAAGTAGGC GAATAATAGT GAATAAGTCC GCATCGTGGT CCGCAAATTC CCGTGGTTAT
  • AATTTAACGC GAATTTTAAC TGACGGAATT TTTTTAATGC GGGGCGGGAC GGTGAGTAGC GTCAGCCGGA TAACCAATTT
  • CTTACACTTA TTTCCGGCCT ATTTTGAACA CGAATAAAAA CTTTACGGTC TTTAAAAAGG CCGTAATATC CAGCTGAACG
  • GTAAGGGCCC ACGCCAGCTT GTCAAGCTGC AAGGCCGGGC AAAGCCAGGA CTGGACACAG AACGCCGGCA TCAGGCCACC
  • GACTAGCCGC GGCTGGTGGC TCCGCTAGTC GGGGACCAAG TGGGCCAGCA TCTCGGCGTC GCCCGGGACA GCCCGACGGA
  • GACATGGCTA GCCCCCTCCG CGCGGCGTTT AATAAATTCT CAGAGCGATC GTTTGGTACA GTCCACAACG CCACCCAAGG
  • TTCAGCAAGA GGTGTGTCTG AACTTCGGTG TTTTTTTGGG GGGTGACTCC AGCGGGGTGG CTTAAAGCAG CGCGCTTTTG
  • CGGCCTCCCC CGTCTGTTTG CTCAACCGAG GGAGACCTGG CGGTCCCGCG TTTCCGGACG GCTCGTCTCG TTCTATAAGG
  • CTCGGGCCGT CTCTTGGGCT CTAGCGCCGC CGGAACCGGA CGTATAGGGC TAAGTTGCCG GGGTCCCGCA GGTCTTGCCC
  • GGAGCATCTT GCGGACTTAC GCGCACACTG CACGGAACGA CGGGAGCTAC GGGGCAACGT CGGGATCTAG CCGGTGTCGC
  • CAGCGGCACC ACGAACGCGG CGGCGTTTGC ACCAGACCAG CGCCCAGTAG ACGCGAAACA ACGGCTACTT GAGGAACCGG
  • GCGAAGTAGC CACGACGACC GGCGGGTCAC GAGCAAGAGA CCGCAGGACG ACCGCAGTCG CAACCCGCAG AGCGCGAGCG
  • CTACGGCGGC TGCGGCGCCC TACGGGCTTG CTCTCCGGGC TTCGCCCTGC GCGGTCGCTG CGCTCCCTTG CCAGCCCGTG
  • the cell comprises a plasmid that contains one or more exogenous nucleic acid sequences encoding enzymes or proteins that include but are not limited to one or more of the following: an acyl carrier protein, a TE, a FAR, a FadR, a FAD, a fatty aldehyde reductase, a cytochrome P450 enzyme, a NADH or NADPH cytochrome P450 reductase, a desaturase, a hydroxylase, and an antibiotic resistance enabling protein; wherein the plasmid is at least 20, 30, 40, 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
  • the exogenous nucleic acid sequence is incorporated into the genome of the cell.
  • the cell or composition comprising a cell comprises at least one exogenous nucleic acid that encodes a FAR or a functional fragment of a FAR derived from one of the following organisms: Arabidopsis thaliana, Arabidopsis lyrata, Vitis vinifera, Populus trichocarpa, Artermisia annua, Ricinus communis, Simmondsia chineis, Oryza sativa japonica, Hevea brasiliensis, Hordeum vulgare, Triticum aestivum, Sorghum bicolor, Zea mays, and Selaginella moelllendorff .
  • the exogenous gene encodes a FAR. In some cases, the FAR encoded by the exogenous gene catalyzes the reduction of a 20 to 30-carbon fatty acyl-CoA to a corresponding primary alcohol. In some cases, the FAR encoded by the exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding primary alcohol. In some cases, the FAR encoded by the exogenous gene catalyzes the reduction of a 10 to 14-carbon fatty acyl-CoA to a corresponding primary alcohol. In one embodiment, the FAR encoded by the exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol.
  • the exogenous gene encodes a FadR. In some cases, the reductase encoded by the exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding aldehyde. In one embodiment, the reductase encoded by the exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanal.
  • the invention relates to a bacterial cell or a compositions comprising at least one bacterial cell that comprises at least a first and a second exogenous nucleic acid sequence, wherein the first nucleic acid sequence encodes a FadR or a functional fragment of a FadR and the second exogenous nucleic acid sequence encodes a fatty acyl- CoA ligase or a functional fragment thereof.
  • the functional fragments of the enzymes encoded by the one or more exogenous nucleic acid sequences are at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the nucleic acid sequences that encode the full-length amino acid sequence upon which the functional fragment is based.
  • Any enzyme disclosed in this application and part of the invention may be replaced with a functional fragment or variant.
  • Any composition or cell disclosed in the application may be used in any disclosed method of this application.
  • the genetic constructs contain sequences directing transcription and translation of the relevant exogenous (either heterologous or homologous) gene, a selectable marker, and/or sequences allowing autonomous replication or chromosomal integration.
  • suitable vectors comprise a region 5' of the gene or DNA fragment which harbors transcriptional initiation controls and a region 3' of the gene or DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
  • the exogenous gene is coding sequence and is in operable linkage with a promoter, and in some embodiments the promoter is derived from a gene endogenous to a species of the genus Rhodococcus or Ralstonia.
  • Initiation control regions or promoters, which are useful to drive expression of the instant ORFs in the desired host cell are numerous and familiar to those skilled in the art.
  • Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO; and lac, ara, tet, trp, IP L , IP R , T7, tac, and trc as well as the amy, apr, npr promoters and various phage promoters useful for expression in the lipid-producing bacteria of the present invention.
  • the promoter is upregulated in response to reduction or elimination of a cofactor in the culture media of the cell, such as at least a 3-fold upregulation as determined by transcript abundance in a cell when the cell is exposed to extracellular environment changes from containing at least 10 mM or 5 mM cofactor to containing no cofactor.
  • Termination control regions may also be derived from various genes native to the preferred hosts.
  • the genetic constructs of the present invention do not comprise a termination control region.
  • the bacterial cell or the composition comprising the bacterial cell comprises at least one genetic construct, which comprises one or more coding sequences.
  • the invention relates to the bacterial cell or the composition comprising at least one bacterial cell wherein the at least one cell comprises two or more genetic constructs, three or more genetic constructs, or four or more genetic constructs, each comprising one or more coding sequences.
  • the coding sequences of the claimed invention encode at least one protein that modifies or accelerates lipid production in the host cell.
  • the coding sequence encodes at least one protein that alters the levels of individual lipids or hydrocarbons produced by the cell as compared to the same cell not modified by an exogenous nucleic acid sequence.
  • the coding sequence may encode at least one protein that alters the amount of one specific lipid or hydrocarbon molecule of the cell as compared to the same cell not modified by the nucleic acid.
  • the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes an increase in the ratio of C14:C16:C18 lipids or hydrocarbons produced or secreted by the cell as compared to the C14:C16:C18 lipids or hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the lipid pathway enzyme.
  • the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes a decrease in the ratio of C14:C16:C18 lipids or hydrocarbons produced or secreted by the cell as compared to the C14:C16:C18 lipids or hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the lipid pathway enzyme.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 5% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 5% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the bacterial cell produces and/or secretes one or more unsaturated lipids or hydrocarbons in a ratio greater than the ratio of unsaturated lipids or hydrocarbons produced and/or secreted by the same cell not cells comprising one or more exogenous nucleic acid sequences.
  • the bacterial cell produces and/or secretes at least 6% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 7% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 8% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 9% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 10% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 15% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 20% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 25% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 30% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 35% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 40% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 45% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 50% more C8 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 5% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 6% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 7% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 8% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 9% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 10% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 15% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 20% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 25% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 30% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 35% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 40% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 45% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 50% more C9 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 5% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 6% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 7% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 8% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 9% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 10% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 15% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 20% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 25% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 30% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 35% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 40% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 45% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 50% more CIO hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 5% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 6% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 7% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 8% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 9% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 10% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 15% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 20% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 25% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 30% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 35% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 40% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 45% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 50% more CI 1 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 5% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 6% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 7% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 8% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 9% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 10% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 15% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 20% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 25% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 30% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 35% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 40% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 45% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 50% more C12 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 5% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 6% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 7% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 8% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 9% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 10% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 15% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 20% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 25% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 30% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 35% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 40% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 45% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 50% more C13 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 5% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 6% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 7% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 8% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 9% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 10% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 15% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 20% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 25% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 30% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 35% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 40% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 45% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 50% more C14 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 5% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 6% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 7% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 8% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 9% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 10% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 15% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 20% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 25% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 30% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 35% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 40% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 45% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 50% more C15 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
  • the one or more cells comprising one or more exogenous nucleic acid sequences produces at least 5% more C16 hydrocarbon as compared to the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.

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Abstract

La présente invention concerne des micro-organismes contenant des séquences d'acides nucléiques exogènes ou hétérologues, lesdits micro-organismes étant capables de croître sur du dioxyde de carbone gazeux, de l'hydrogène gazeux, un gaz de synthèse, ou des combinaisons de ceux-ci. Selon certains modes de réalisation, les micro-organismes sont des bactéries chimiotrophes qui produisent ou sécrètent au moins 10 % de lipide en poids. L'invention concerne également des procédés de fixation de carbone gazeux dans des molécules carbonées organiques utiles pour des procédés industriels. L'invention concerne également des procédés de fabrication de produits chimiques ou de production de précurseurs de produits chimiques utiles dans le carburant aviation, le carburant diesel et le carburant biodiesel. Des exemples de produits chimiques ou de précurseurs de produits chimiques utiles dans la production de carburant sont les alcanes, alcènes, alcynes, alcools d'acides gras, aldéhydes d'acides gras, hydrocarbures désaturés, acides gras insaturés, hydroxyacides ou diacides contenant des chaînes carbonées de six à trente atomes de carbone de long. L'invention concerne également des micro-organismes et des procédés faisant appel aux micro-organismes de l'invention pour la production de butanediol et de ses précurseurs chimiques par une fermentation pauvre en oxygène ou anaérobie. L'invention concerne également des micro-organismes et des procédés faisant appel aux micro-organismes de l'invention pour la génération d'acides gras hydroxylés dans des microbes grâce au transfert d'enzymes qui sont connues pour hydroxyler des acides gras chez les plantes ou les microbes. L'invention concerne également des micro-organismes et des procédés faisant appel aux micro-organismes de l'invention pour la production d'acides gras à chaîne plus courte dans des microbes grâce à l'introduction de protéines de liaison à l'acyl-CoA exogènes.
PCT/US2013/032362 2008-11-06 2013-03-15 Micro-organismes chimiotrophes manipulés fixant le co2 et produisant des produits carbonés, et procédés d'utilisation de ces micro-organismes WO2013148348A1 (fr)

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US14/388,756 US20150017694A1 (en) 2008-11-06 2013-03-15 Engineered CO2-Fixing Chemotrophic Microorganisms Producing Carbon-Based Products and Methods of Using the Same
US15/233,512 US9957534B2 (en) 2008-11-06 2016-08-10 Engineered CO2-fixing chemotrophic microorganisms producing carbon-based products and methods of using the same
US15/936,440 US20190040427A1 (en) 2008-11-06 2018-03-27 Engineered CO2-Fixing Chemotrophic Microorganisms Producing Carbon-Based Products and Methods of Using the Same

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US201261616560P 2012-03-28 2012-03-28
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US201261635238P 2012-04-18 2012-04-18
US61/635,238 2012-04-18
US13/623,089 2012-09-19
US13/623,089 US9879290B2 (en) 2008-11-06 2012-09-19 Industrial fatty acid engineering general system for modifying fatty acids
US201261708057P 2012-10-01 2012-10-01
US61/708,057 2012-10-01

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US9957534B2 (en) 2008-11-06 2018-05-01 Kiverdi, Inc. Engineered CO2-fixing chemotrophic microorganisms producing carbon-based products and methods of using the same
US10376837B2 (en) 2013-03-14 2019-08-13 The University Of Wyoming Research Corporation Conversion of carbon dioxide utilizing chemoautotrophic microorganisms systems and methods
US10557155B2 (en) 2013-03-14 2020-02-11 The University Of Wyoming Research Corporation Methods and systems for biological coal-to-biofuels and bioproducts
US9556462B2 (en) 2013-03-15 2017-01-31 Kiverdi, Inc. Methods of using natural and engineered organisms to produce small molecules for industrial application
US10179920B2 (en) 2013-08-22 2019-01-15 Kiverdi, Inc. Microorganisms for biosynthesis of limonene on gaseous substrates
US9506086B2 (en) 2013-08-22 2016-11-29 Kiverdi, Inc. Microorganisms for biosynthesis of limonene on gaseous substrates
CN113667659A (zh) * 2014-10-21 2021-11-19 卡比欧斯公司 具有聚酯降解活性的多肽及其用途
US10544436B2 (en) 2015-05-06 2020-01-28 Trelys, Inc. Compositions and methods for biological production of methionine
EP3199620A1 (fr) * 2016-01-29 2017-08-02 Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives Utilisation d'oxyde nitrique ou d'un donneur d'oxyde nitrique pour induire la production de triacylglycérols dans des micro-algues
US10975399B2 (en) 2016-01-29 2021-04-13 Commissariat A L'energie Atomique Et Aux Energies Alternatives Use of nitric oxide or nitric oxide donor for inducing the production of triacylglycerols in microalgae
US11725290B2 (en) 2016-03-19 2023-08-15 Kiverdi, Inc. Microorganisms and artificial ecosystems for the production of protein, food, and useful co-products from C1 substrates
US11203738B2 (en) 2017-02-03 2021-12-21 Kiverdi, Inc. Microbial conversion of CO2 and other C1 substrates to protein and meat substitute products
US11466246B2 (en) 2017-02-03 2022-10-11 Kiverdi, Inc. Microbial conversion of CO2 and other C1 substrates to vegan nutrients, fertilizers, biostimulants, and systems for accelerated soil carbon sequestration
CN113646437A (zh) * 2019-03-28 2021-11-12 帝斯曼知识产权资产管理有限公司 温室气体改进发酵
CN114599779A (zh) * 2019-10-29 2022-06-07 太阳食物有限公司 用于单细胞蛋白质或生物质生产的菌株和方法
CN114599779B (zh) * 2019-10-29 2024-05-24 太阳食物有限公司 用于单细胞蛋白质或生物质生产的菌株和方法
CN111057570B (zh) * 2019-11-26 2022-05-27 大连立和盈海科技有限公司 一种用于制备生物燃料的系统及方法
CN111057570A (zh) * 2019-11-26 2020-04-24 大连立和盈海科技有限公司 一种用于制备生物燃料的系统及方法
GB202306850D0 (en) 2023-05-09 2023-06-21 Farmless Holding B V Fermentation process

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