Microorganisms and methods for continuous production of ethylene from C1 substrates

Abstract

Methods and microorganisms have been genetically engineered to continuously produce ethylene by microbial fermentation, particularly microbial fermentation of gaseous substrates. The microorganisms are C1-fixing. Furthermore, the gaseous substrates contain CO2 and an energy source. Ethylene production can be improved by various promoters or nutrient-limiting methods.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 366,758, filed June 21, 2022, U.S. Provisional Patent Application No. 63 / 506,350, filed June 5, 2023, and U.S. Provisional Patent Application No. 63 / 506,351, filed June 5, 2023, the entire contents of which are incorporated herein by reference.

[0002] Incorporation by reference of sequence listing This application contains a Sequence Listing that has been submitted electronically in ST.26 Sequence Listing XML format, which is incorporated herein by reference in its entirety. The ST.26 Sequence Listing XML, created on May 26, 2023, is named LT245WO1-Sequences.xml and is 24,583 bytes in size.

[0003] The present disclosure relates to genetically engineered microorganisms and methods for the continuous production of ethylene by microbial fermentation, particularly by microbial fermentation of gaseous substrates. [Background technology]

[0004] It has long been recognized that catalytic processes, such as the Fischer-Tropsch process, can be used to convert gases containing carbon dioxide (CO), carbon monoxide (CO), and / or hydrogen (H), such as industrial waste gases or syngas, into various fuels and chemicals. However, gas fermentation has recently emerged as an alternative platform for the biological fixation of such gases. Specifically, C1-fixing microorganisms have been shown to convert gases containing CO, CO, and / or H into products such as ethanol and 2,3-butanediol. Ethylene is the most widely produced organic compound in the world, useful in a wide range of industries, including plastics, solvents, and textiles. Ethylene is currently produced by steam cracking fossil fuels or dehydrogenating ethane. However, millions of tons of ethylene are produced each year, and these processes produce enough carbon dioxide to contribute significantly to global carbon emissions. Therefore, producing ethylene through regenerative methods could help meet the large demand from the energy and chemical industries while also protecting the environment. However, efficient production of such chemical products can be limited by slow microbial growth, limited gas uptake, sensitivity to toxins, or diversion of the carbon substrate to undesirable by-products. Thus, there is a continuing and unmet need to develop efficient production of ethylene by microbial fermentation of gaseous substrates that can be easily produced from renewable resources and offer a wide range of useful applications. Summary of the Invention

[0005] It is against this background that the present disclosure offers certain advantages and advancements over the prior art.

[0006] Although the disclosure disclosed herein is not limited to a particular benefit or functionality, the disclosure provides methods and genetically engineered microorganisms capable of producing ethylene from gaseous substrates, wherein the microorganism comprises a heterologous nucleic acid encoding an ethylene-forming enzyme (EFE).

[0007] In some embodiments of the methods disclosed herein, the microorganism is a recombinant C1-fixing microorganism capable of producing ethylene from a gaseous substrate comprising a nucleic acid encoding a group of exogenous enzymes including ethylene-forming enzymes (EFEs).

[0008] In some embodiments of the microorganisms disclosed herein, the microorganisms are recombinant C1-fixing microorganisms capable of switching cell load in the production of ethylene, wherein the microorganisms comprise nucleic acids encoding a group of exogenous enzymes including an ethylene-forming enzyme (EFE) and one or more inducible promoters.

[0009] The microorganism of an embodiment, further comprising a nucleic acid encoding a group of exogenous enzymes including alpha-ketoglutarate permease (AKGP), wherein the nucleic acid is operably linked to a promoter.

[0010] A microorganism of an embodiment, wherein the microorganism is selected from the group consisting of Cupriavidus necator and Ralstonia eutropha.

[0011] A microorganism of an embodiment, wherein the microorganism is Cupriavidus necator.

[0012] The microorganism of an embodiment, further comprising a nucleic acid encoding alpha-ketoglutarate, wherein the nucleic acid is codon optimized for expression in the microorganism.

[0013] The microorganism of an embodiment, wherein the one or more inducible promoters are selected from an H2-inducible promoter, a phosphate-limited inducible promoter, a nitrogen-limited inducible promoter, a CO2-inducible promoter, or any combination thereof.

[0014] A microorganism of an embodiment, wherein the CO2 inducible promoter is CBB.

[0015] 2. The microorganism of embodiment 1, wherein the EFE is codon-optimized for expression in the microorganism.

[0016] The microorganism of the embodiment, further comprising a disruptive mutation in one or more genes.

[0017]

[0023] The microorganism of an embodiment, wherein ethylene is converted into a derivative material selected from polyethylene (PE), polyethylene terephthalate (PET), polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), sustainable aviation fuel (SAF), or any combination thereof.

[0018] The microorganism of an embodiment, wherein the gaseous substrate comprises CO2 and an energy source.

[0019] The microorganism of an embodiment, wherein the gaseous substrate comprises CO2, and H2, O2, or both. One embodiment is directed to a method for the continuous production of ethylene, the process comprising passing a gaseous substrate through a bioreactor containing a culture of the recombinant C1-fixing microorganism of claim 1 in a culture medium such that the microorganism converts the gaseous substrate to ethylene, and recovering ethylene from the bioreactor.

[0020] In one embodiment, the present invention is directed to a method of culturing a microorganism according to claim 1, comprising growing the microorganism in a medium comprising a gaseous substrate, wherein the gaseous substrate comprises CO2.

[0021] The method of an embodiment, wherein the gaseous substrate comprises industrial waste or off-gas.

[0022] The method of an embodiment, further comprising an energy source.

[0023] The method of an embodiment, wherein the energy source is provided intermittently.

[0024] The method of the embodiment, wherein the energy source is H2.

[0025] One embodiment is directed to a method comprising growing a microorganism in a medium comprising a gaseous substrate, the gaseous substrate comprising CO2 and an energy source.

[0026] The method of the embodiment further comprises co-producing ethylene and microbial biomass.

[0027] The method of the embodiment, wherein switching the cellular load comprises limiting intracellular oxygen concentration.

[0028] The method of the embodiment, wherein the microbial biomass is suitable as animal feed.

[0029] The method of an embodiment, wherein the gaseous substrate further comprises H2, O2, or both.

[0030] In some embodiments of the microorganisms disclosed herein, the microorganisms produce commodity chemicals, microbial biomass, single cell proteins (SCPs), one or more intermediates, or any combination thereof.

[0031] In some aspects of the microorganisms disclosed herein, the microorganism is derived from a parent bacterium selected from the group consisting of Cupriavidus necator.

[0032] In some embodiments of the microorganisms disclosed herein, the product is 1-butanol, butyrate, butene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, terpenes, isoprene, fatty acids, fatty alcohols, 2-butanol, 1,2-propanediol, 1-propanol, 1-hexanol, 1-octanol, chorismic acid derived products, 3-hydroxybutyric acid, 1,3-butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, isoamyl alcohol, or monoethylene glycol.

[0033] The present disclosure further provides a genetically engineered C1-fixing microorganism, further comprising a microbial biomass and at least one excipient.

[0034] The present disclosure further provides genetically engineered C1-fixing microorganisms, wherein the animal feed may be suitable for feeding one or more of beef cattle, dairy cattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, banteng, gayal, yaks, chickens, turkeys, ducks, geese, quail, guinea pigs, squabs / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents.

[0035] The present disclosure further provides a genetically engineered C1-fixing microorganism, wherein the microorganism is suitable as a single cell protein (SCP).

[0036] The present disclosure further provides genetically engineered C1-fixing microorganisms, which are suitable as cell-free protein synthesis (CFPS) platforms.

[0037] The present disclosure further provides a genetically engineered C1-fixing microorganism, wherein the product is native to the microorganism.

[0038] In some embodiments of the methods disclosed herein, the substrate comprises one or more of CO, CO2, and H2.

[0039] In some embodiments, both anaerobic and aerobic gases can be used to feed separate cultures (e.g., an anaerobic culture and an aerobic culture) in two or more different bioreactors that are both incorporated into the same process stream.

[0040] In some embodiments, the present disclosure provides a method for storing energy in the form of a biopolymer, the method comprising: intermittently processing at least a portion of electrical energy generated from a renewable and / or non-renewable energy source in an electrolysis process to produce at least H, O, or CO; intermittently passing at least one of the H, O, or CO from the electrolysis process to a bioreactor containing a culture comprising a liquid nutrient medium and a microorganism capable of producing the biopolymer; and fermenting the culture.

[0041] In some embodiments, the present disclosure also provides a system for storing energy in the form of a biopolymer, the system comprising: an electrolysis process in intermittent fluid communication with a renewable and / or non-renewable energy source for producing at least one of H, O, or CO; an industrial plant for producing at least a C feedstock; and a bioreactor in intermittent fluid communication with the electrolysis process and / or in continuous fluid communication with the industrial plant, the bioreactor comprising a reaction vessel suitable for intermittently growing, fermenting, and / or culturing and housing a microorganism capable of producing the biopolymer.

[0042] In some embodiments, the disclosure provides a method for improving the performance and / or economics of a fermentation process, the fermentation process defining a bioreactor containing a bacterial culture in a liquid nutrient medium, the method including: passing a C1 feedstock comprising one or both of CO and CO2 from an industrial process to the bioreactor (the C1 feedstock having a per-unit cost); intermittently passing at least one of H2, O2, or CO2 from an electrolysis process to the bioreactor (the electrolysis process having a per-unit cost); and fermenting the culture to produce one or more fermentation products (each of the one or more fermentation products having a per-unit value). In certain instances, multiple electrolysis processes are utilized to provide one or all of CO2, CO2, and H2 to the bioreactor.

[0043] In another embodiment, the local grid supplies electricity delivered intermittently as electric energy generated by the grid based on the availability of electricity or the availability of electricity below a threshold price, where the price of electricity decreases as demand decreases or is set by the local grid.

[0044] In embodiments, the present disclosure can be operated intermittently by storing energy in the form of biopolymers, and product conversion can be intermittent during periods when the power grid is oversupplied with electricity, or can be on standby when there is a power shortage or demand. By storing energy in the form of biopolymers, the present disclosure provides a process that can be fine-tuned to assist in balancing the power grid system.

[0045] In one embodiment, the autotrophic microorganisms intermittently consume energy provided, in part or in whole, by the availability of electrical power.

[0046] In one embodiment, a system disclosed herein is associated with generating microbubbles and includes a vessel containing a liquid; a plate disposed above the vessel and including a plurality of orifices configured to accelerate at least a portion of the liquid within the vessel; and at least one sparger disposed within the vessel, the surface of the sparger being disposed about 50 mm to about 300 mm, 500 mm, or 1000 mm from the bottom of the plate. The sparger can be configured to inject gas bubbles into the liquid. In some examples, the sparger can be disposed within the vessel to form a first region for gas bubbles to rise within the vessel, and the accelerated liquid can break the gas bubbles into microbubbles, forming a second region for fluid to flow through the vessel. The fluid can comprise the liquid and the accelerated portion of the microbubbles. In yet another example, the superficial velocity of the gas phase within the vessel can be at least 30 mm / s. The sparger can be a sintered sparger or an orifice sparger. The thickness of the plate can be about 1 mm to about 25 mm. The accelerated liquid may have a velocity of about 8,000 mm / s to about 17,000 mm / s. In other examples, the accelerated liquid may have a velocity of about 12,000 mm / s to about 17,000 mm / s. In some examples, the gas bubbles injected into the liquid from the sparger may have a diameter of about 2 mm to about 20 mm. In other examples, the gas bubbles injected into the liquid from the sparger may have a diameter of about 5 mm to about 15 mm, or about 7 mm to about 13 mm. The fine gas bubbles may have a diameter of about 0.1 mm to about 5 mm, or about 0.2 mm to about 1.5 mm. The multiple orifices may also be configured to accelerate at least 90% of the liquid in the vessel.

[0047] In another embodiment, a method disclosed herein relates to generating microbubbles, which can include sparging a gas into a vessel containing a liquid through at least one sparger disposed within the vessel and configured to inject gas bubbles into the liquid; and accelerating a portion of the liquid in the vessel through a perforated plate disposed above the vessel, wherein the liquid is accelerated from the plate to break up the gas bubbles into microbubbles. In some examples, the superficial velocity of the gas phase within the vessel can be at least 30 mm / s. In other examples, the superficial velocity of the gas phase within the vessel can be between about 30 mm / s and about 80 mm / s. The sparger can be a sintered sparger or an orifice sparger. The liquid can be accelerated from the perforated plate at a velocity between about 8,000 mm / s and about 17,000 mm / s. In some examples, the liquid can be accelerated from the perforated plate at a velocity between about 12,000 mm / s and about 17,000 mm / s. The gas bubbles injected into the liquid through the sparger may have a diameter of about 2 mm to about 20 mm, or greater than 5 mm to about 15 mm, or about 7 mm to about 13 mm. In many cases, the gas bubbles injected into the liquid through the sparger are not spherical. The injected gas bubbles are sometimes called coarse bubbles. In contrast, fine gas bubbles may have a diameter of about 0.1 mm to about 5 mm, or about 0.2 mm to about 1.5 mm. Fine gas bubbles are usually spherical. The liquid flow may be introduced at a position close to the plate. The sparger can be positioned perpendicular or parallel to the plate, and the top or side of the sparger can be positioned about 50 mm to about 300 mm, 500 mm, or 1000 mm from the bottom of the plate.

[0048] In yet another embodiment, the system disclosed herein relates to a bioreactor comprising: a vessel containing a liquid growth medium; a plate disposed at an upper portion of the vessel, the plate may comprise a plurality of orifices configured to accelerate at least a portion of the liquid growth medium within the vessel; a substrate comprising at least one C1 carbon source; and at least one sparger disposed within the vessel, the surface of the sparger being disposed about 50 mm to about 300 mm, 500 mm, or 1000 mm from the bottom of the plate, the sparger being configured to inject gas bubbles of the substrate into the liquid growth medium. The sparger disposed within the vessel can form a first region for the substrate bubbles to rise within the vessel and a second region for the accelerated liquid growth medium to break down the substrate gas bubbles into microbubbles of the substrate and allow fluid to flow through the vessel. The fluid can comprise the accelerated portion of the liquid growth medium, the microbubbles of the substrate, and a culture of at least one microorganism in the liquid growth medium. The at least one microorganism culture can anaerobically ferment the substrate to produce at least one fermentation product.

[0049] In yet another embodiment, the method disclosed herein relates to generating microbubbles of a substrate in a bioreactor, and includes sparging bubbles of at least one C1 carbon source substrate into a vessel containing a liquid growth medium through at least one sparger disposed within the vessel, and accelerating a portion of the liquid growth medium in the vessel through a perforated plate disposed above the vessel. The accelerated liquid growth medium from the plate can break down the substrate bubbles into microbubbles of the substrate. The superficial velocity of the gas phase within the vessel can be at least 30 mm / s. A culture of at least one microorganism can be contained in the liquid growth medium and can anaerobically ferment the substrate to produce at least one fermentation product.

[0050] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description taken in conjunction with the appended claims. It should be noted that the scope of the claims is defined by the description therein, and not by the specific discussion of the features and advantages set forth herein. [Brief explanation of the drawings]

[0051] These and other aspects of the present disclosure, which is to be considered in all its novel aspects, will become apparent from the following description, given by way of example only, with reference to the accompanying drawings.

[0052] [Figure 1] FIG. 1 shows a schematic diagram illustrating the pathways of CO2 fixation, central carbon metabolism, and the TCA cycle in Cupriavidus necator with heterologous expression of ethylene-forming enzymes for ethylene production. [Figure 2] FIG. 2 shows ethylene production by Cupriavidus necator strains with ethylene-forming enzyme expression (pBBR1-Efe) and blank vector control (pBBR1) when grown on formate as the sole carbon and energy source. [Figure 3] FIG. 3 shows continuous ethylene production from CO2 as the sole carbon source in a CSTR over 11 days by Cupriavidus necator strains carrying ethylene-forming enzyme expression (pBBR1-Efe). [Figure 4] Figure 4 shows a schematic flux balance analysis predicted gene knockout strategy (red arrows) to eliminate unwanted by-products during ethylene production from CO and H in Cupriavidus necator. Gene annotations are presented below each enzyme name (see Table 1 for details). [Figure 5] FIG. 5 illustrates a schematic diagram of a system for generating gas bubbles in a container in accordance with the systems and methods disclosed herein. [Figure 6]FIG. 6 shows ethylene production by Cupriavidus necator strains carrying the blank vector control (pBBR1) when grown in phosphate-limiting minimal medium together with the ethylene-forming enzyme (Efe) expressed via a constitutive or phosphate-limited inducible promoter. [Figure 7] Figure 7 shows ethylene production by Cupriavidus necator strains carrying ethylene-forming enzyme variants from various organisms expressed via a chemically inducible promoter (rhamnose). Accession numbers for the EFE variants are: Pseudomonas syringae (AAD16440.1), Microcoleus asticus (NQE34890), Myxococcus stipitatus (WP_015351455.1), Nostoc sp. ATCC 43529 (RCJ18531), Ralstonia solanacearum (WP_014618742.1), and Scytonema sp. NIES-4073 (WP_096562523.1). [Figure 8] Figure 8 shows continuous ethylene production from CO as the sole carbon source in a CSTR over a period of 5.5 days by a Cupriavidus necator strain with the ethylene-forming enzyme expressed through a phosphate-limited inducible promoter. The CSTR was operated under phosphate-limited conditions starting at approximately day 14.7. [Figure 9] FIG. 9 shows continuous ethylene production from CO 2 as the sole carbon source in a CSTR over a 14-day period by a Cupriavidus necator strain with the ethylene-forming enzyme expressed via a synthetic CbbL promoter. [Figure 10] FIG. 10 shows ethylene production from CO as the sole carbon source during CSTR start-up by a Cupriavidus necator strain with the ethylene-forming enzyme expressed via a synthetic soluble hydrogenase promoter. [Figure 11] FIG. 11 shows that increasing FeSO4x7H2O concentrations lead to increased ethylene production in cells grown on fructose under PO4-limited conditions. DETAILED DESCRIPTION OF THE INVENTION

[0053] The following description of the embodiments is presented in general terms, and the present disclosure will be further elucidated from the disclosure set forth herein below under the heading "Examples," which provides supporting experimental data for the disclosure, specific examples of various aspects of the disclosure, and means for carrying out the disclosure.

[0054] To their surprise, the inventors were able to engineer a C1-fixing microorganism to produce ethylene continuously.

[0055] Unless otherwise specified, the following terms used throughout this specification are defined as follows:

[0056] The present disclosure provides microorganisms for the biological co-production of proteins, chemicals, and microbial biomass. A "microorganism" is a microscopic organism, particularly a bacterium, archaea, virus, or fungus. In an embodiment, the microorganism of the present disclosure is a bacterium.

[0057] The term "non-naturally occurring," when used with respect to a microorganism, is intended to mean that the microorganism has at least one genetic modification that is not found in naturally occurring strains of the referenced species, including wild-type strains of the referenced species. Non-naturally occurring microorganisms are typically developed in a laboratory or research facility. The microorganisms of the present disclosure are non-naturally occurring.

[0058] The terms "genetic modification," "genetic alteration," or "genetic engineering" broadly refer to the manipulation of the genome or nucleic acid of a microorganism by man. Similarly, the terms "genetically modified," "genetically altered," or "genetically engineered" refer to a microorganism that includes such a genetic modification, genetic alteration, or genetic engineering. These terms may be used to distinguish between laboratory-produced and naturally occurring microorganisms. Methods of genetic modification include, for example, heterologous gene expression, gene or promoter insertion or deletion, nucleic acid mutation, altered gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis, gene shuffling, and codon optimization. The microorganisms of the present disclosure are genetically engineered.

[0059] "Recombinant" indicates that a nucleic acid, protein, or microorganism is the product of genetic modification, manipulation, or recombination. Generally, the term "recombinant" refers to a nucleic acid, protein, or microorganism that contains or is encoded by genetic material derived from multiple sources, such as two or more different strains or species of a microorganism. The microorganisms of the present disclosure are generally recombinant.

[0060] "Wild-type" refers to the typical form of an organism, strain, gene, or trait that occurs in nature, as distinguished from mutant or variant forms.

[0061] "Endogenous" refers to a nucleic acid or protein present or expressed in the wild-type or parental microorganism from which the microorganism of the present disclosure is derived. For example, an endogenous gene is a gene that naturally occurs in the wild-type or parental microorganism from which the microorganism of the present disclosure is derived. In one embodiment, expression of an endogenous gene can be controlled by an exogenous regulatory element, such as an exogenous promoter.

[0062] "Exogenous" refers to a nucleic acid or protein that originates outside the microbial organism of the present disclosure. For example, an exogenous gene or enzyme can be artificially or recombinantly created and introduced into or expressed in a microbial organism of the present disclosure. An exogenous gene or enzyme can also be isolated from a heterologous microbial organism and introduced into or expressed in a microbial organism of the present disclosure. An exogenous nucleic acid can be adapted to integrate into the genome of a microbial organism of the present disclosure or to remain extrachromosomally in a microbial organism of the present disclosure, for example, a plasmid.

[0063] "Heterologous" refers to a nucleic acid or protein that is not present in the wild-type or parental microorganism from which the microorganism of the present disclosure is derived. For example, a heterologous gene or enzyme can be from a different strain or species and introduced into or expressed in a microorganism of the present disclosure. A heterologous gene or enzyme can be introduced into or expressed in a microorganism of the present disclosure in a form that occurs in the different strain or species. Alternatively, a heterologous gene or enzyme can be modified in several ways, for example, by codon-optimizing it for expression in a microorganism of the present disclosure, or by engineering it to change its function, for example, to reverse the direction of enzymatic activity or change its substrate specificity.

[0064] In order to provide a slightly less expensive range of products Bacillus Clostridium Cupriavi dus、Escherichia、Gluconobacter、Hyphomicrobium、Lysinibacillus、Pa enibacillus, Pseudomonas, Sedimenticola, Sporosarcina, Streptomyces, Thermithiobacillus, Thermotoga, Zea, Klebsiella, Mycobacterium, Sa lmonella、Mycobacteroides、Staphylococcus、Burkholderia、Listeria、Acinetobacter、Shigella、Neisseria、Bordetella、Streptococcus、Enter obacter, Vibrio, Legionella, Xanthomonas, Serratia, Cronobacter, Cupriavidus, Helicobacter, Yersinia, Cutibacterium, Francisella, Pectob acterium、Arcobacter、Lactobacillus、Shewanella、Erwinia、Sulfurospirillum、Peptococcaceae、Thermococcus、Saccharomyces、Pyrococcus、Gl ycine, Homo, Ralstonia, Brevibacterium, Methylobacterium, Geobacillus, bos, gallus, Anaerococcus, Xenopus, Amblyrhynchus, rattus, mouse, sus 、Rhodococcus、Rhizobium、Megasphaera、Mesorhizobium、Peptococcus、Agrobacterium、Campylobacter、Acetobacterium、Alkalibaculum、Blautia Butyribacterium Eubacterium Moorella Oxobacter Sporomusa Thermoanaerobacter Schizosaccharomyces Paenibacillus FictibacillusIt may be derived from Lysinibacillus, Ornithinibacillus, Halobacillus, Kurthia, Lentibacillus, Anoxybacillus, Solibacillus, Virgibacillus, Alicyclobacillus, Sporosarcina, Salimicrobium, Sporosarcina, Planococcus, Corynebacterium, Thermaerobacter, Sulfobacillus, or Symbiobacterium. ,

[0065] The terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can contain one or more modified nucleotides, such as methylated nucleotides or nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0066] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product."

[0067] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may be linear or branched, may comprise modified amino acids, or may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine, both D- and L-optical isomers, and amino acid analogs and peptidomimetics.

[0068] The term "copolymer" refers to a composition of the present disclosure that includes two or more types of monomers linked in the same polymer chain.

[0069] "Enzyme activity" or simply "activity" broadly refers to enzymatic activity, including, but not limited to, the activity of an enzyme, the amount of an enzyme, or the availability of an enzyme to catalyze a reaction. Thus, "increasing" enzyme activity includes increasing the activity of an enzyme, increasing the amount of an enzyme, or increasing the availability of an enzyme to catalyze a reaction. Similarly, "reducing" enzyme activity includes decreasing the activity of an enzyme, decreasing the amount of an enzyme, or decreasing the availability of an enzyme to catalyze a reaction.

[0070] "Mutated" refers to a nucleic acid or protein that is modified in a microorganism of the present disclosure compared to the wild-type or parent microorganism from which the microorganism of the present disclosure is derived. In one embodiment, the mutation may be a deletion, insertion, or substitution in a gene encoding an enzyme. In another embodiment, the mutation may be a deletion, insertion, or substitution of one or more amino acids in an enzyme.

[0071] A "disrupted gene" refers to a gene that has been modified in some way to reduce or eliminate gene expression, regulatory activity of the gene, or activity of the encoded protein or enzyme. A disruption can partially inactivate, completely inactivate, or delete a gene or enzyme. A disruption can be a knockout (KO) mutation that completely eliminates the expression or activity of a gene, protein, or enzyme. A disruption can also be a knockdown that reduces but does not completely eliminate the expression or activity of a gene, protein, or enzyme. A disruption can reduce, prevent, or block the biosynthesis of a product produced by the enzyme. A disruption can include, for example, a mutation in a gene encoding a protein or enzyme, a mutation in a genetic regulatory element involved in the expression of a gene encoding an enzyme, the introduction of a protein-producing nucleic acid that reduces or inhibits the activity of the enzyme, or the introduction of a nucleic acid (e.g., antisense RNA, RNAi, TALEN, siRNA, CRISPR, CRISPRi) or protein that inhibits the expression of a protein or enzyme. The disruption can be introduced using any method known in the art. For purposes of this disclosure, a disruption is created in a laboratory and not naturally occurring.

[0072] A "parental microorganism" is a microorganism used to generate a microorganism of the present disclosure. A parental microorganism can be a naturally occurring microorganism (i.e., a wild-type microorganism) or a microorganism that has been previously modified (i.e., a mutant or recombinant microorganism). A microorganism of the present disclosure can be modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parental microorganism. Similarly, a microorganism of the present disclosure can be modified to include one or more genes that were not included by the parental microorganism. A microorganism of the present disclosure can also be modified to not express or to express less of one or more enzymes than were expressed in the parental microorganism.

[0073] The microorganisms of the present disclosure can be derived from essentially any parent microorganism.

[0074] The term "derived from" indicates that a nucleic acid, protein, or microorganism is modified or adapted from a different (e.g., parental or wild-type) nucleic acid, protein, or microorganism to produce a new nucleic acid, protein, or microorganism. Such modifications or adaptations typically include insertions, deletions, mutations, or substitutions of nucleic acids or genes.

[0075] The microorganisms of the present disclosure can be further classified based on their organoleptic properties. For example, the microorganisms of the present disclosure can be or be derived from C1-fixing microorganisms, aerobes, anaerobes, acetogens, ethanologens, carboxydotrophs, autotrophs, and / or methanotrophs. The microorganisms of the present disclosure can be selected from chemoautotrophs, hydrogenotrophs, hydrogenotrophs, methanotrophs, or any combination thereof. In some embodiments, the microorganisms can be hydrogen-oxidizing, carbon monoxide-oxidizing, hydrogenotrophs, or any combination thereof, capable of growing and synthesizing biomass on a gaseous carbon source such as syngas and / or CO2, such that the producing microorganisms synthesize targeted chemical products under gaseous cultivation. The microorganisms and methods of the present disclosure can enable low-cost synthesis of biochemical agents, which can be price-competitive with petrochemical agents and higher-quality plant-derived amino acids, proteins, and other biological nutrients. In certain embodiments, these amino acids, proteins, and other biological nutrients may have a substantially lower price than amino acids, proteins, and other biological nutrients produced through heterotrophic or microbial phototrophic synthesis. Hydrogenobacterial microorganisms, hydrogenotrophs, carboxydotrophs, and chemoautotrophs can capture CO or CO as their sole carbon source to support biological growth. In some embodiments, this growth includes amino acid and protein biosynthesis. Hydrogenobacterial microorganisms and other hydrogenotrophs can use H as a source of reduced electrons for respiration and biochemical synthesis. In some embodiments of the present invention, hydrogenobacterial microorganisms and / or hydrogenotrophs and / or carboxydotrophs and / or other chemoautotrophic microorganisms grow on a gas stream including, but not limited to, one or more of the following: CO, CO, H, and inorganic minerals dissolved in an aqueous solution. In some embodiments, hydrogenobacterial microorganisms and / or hydrogenotrophs and / or carboxydotrophs and / or other chemoautotrophs and / or methanotrophic microorganisms convert greenhouse gases into biomolecules, including amino acids and proteins.

[0076] "C1" refers to a one-carbon molecule, e.g., CO, CO2, CH4, or CH3OH. "C1 oxygenate" refers to a one-carbon molecule that also contains at least one oxygen atom, e.g., CO, CO2, or CH3OH. "C1 carbon source" refers to a single carbon molecule that serves as a partial or sole carbon source for the microorganisms of the present disclosure. For example, a C1 carbon source can include one or more of CO, CO2, CH4, CH3OH, or CH2O2. Preferably, a C1 carbon source includes one or both of CO and CO2. "C1-fixing microorganism" is a microorganism that has the ability to produce one or more products from a C1 carbon source. Often, the microorganisms of the present disclosure are C1-fixing bacteria. In a preferred embodiment, the microorganisms of the present disclosure are derived from C1-fixing microorganisms.

[0077] An "anaerobe" is a microorganism that does not require oxygen for growth. Anaerobes may react negatively or die if oxygen is present above a certain threshold. However, some anaerobes can tolerate low levels of oxygen (e.g., 0.000001-5% oxygen), sometimes referred to as "micro-oxygen conditions." Often, the microorganisms of the present disclosure are anaerobes. In a preferred embodiment, the microorganisms of the present disclosure are derived from anaerobes.

[0078] "Acetogens" are obligate anaerobic bacteria that use the Wood-Ljungdahl pathway as their primary mechanism for energy conservation and synthesis of acetyl-CoA and acetyl-CoA-derived products, such as acetate (Ragsdale, Biochim Biophys Acta, 1784:1873-1898, 2008). Specifically, acetogens use the Wood-Ljungdahl pathway as (1) a mechanism for the reductive synthesis of acetyl-CoA from CO2, (2) a terminal electron acceptor, an energy-saving process, and (3) a mechanism for the fixation (assimilation) of CO2 in the synthesis of cellular carbon (Drake, Acetogenic Prokaryotes, In: The Prokaryotes, 3 rd(Eds., 1999, p. 354, New York, NY, 2006). All naturally occurring acetogens are C1-fixing, anaerobic, autotrophic, and non-methanotrophic. Often, the microorganisms of the present disclosure are acetogens. In a preferred embodiment, the microorganisms of the present disclosure are derived from acetogens.

[0079] An "ethanologen" is a microorganism that produces or is capable of producing ethanol. Often, the microorganisms of the present disclosure are ethanologens. In preferred embodiments, the microorganisms of the present disclosure are derived from ethanologens.

[0080] An "autotroph" is a microorganism that can grow in the absence of organic carbon. Instead, autotrophs use inorganic carbon sources, such as CO and / or CO. Often, the microorganisms of the present disclosure are autotrophs. In preferred embodiments, the microorganisms of the present disclosure are derived from autotrophs.

[0081] A "carboxydotroph" is a microorganism capable of utilizing CO as a sole source of carbon and energy. Often, the microorganisms of the present disclosure are carboxydotrophs. In preferred embodiments, the microorganisms of the present disclosure are derived from carboxydotrophs.

[0082] A "methanotroph" is a microorganism that is capable of utilizing methane as its sole source of carbon and energy. In certain embodiments, the microorganisms of the present disclosure are methanotrophs or are derived from methanotrophs. In other embodiments, the microorganisms of the present disclosure are not methanotrophs or are not derived from methanotrophs.

[0083] The term "hydrogen bacteria" refers to a mixture of molecular hydrogen and oxygen gas. A "hydrogen bacteria microorganism" is a microorganism that can use hydrogen as an electron donor and oxygen as an electron acceptor in respiration for the production of intracellular energy carriers such as adenosine-5'-triphosphate (ATP).

[0084] The terms "acid hydrogen" and "acid hydrogen microorganism" can be used synonymously with "hydrogen bacteria" and "hydrogen bacterial microorganism," respectively. Hydrogen bacterial microorganisms generally use molecular hydrogen through hydrogenases, and some of the electrons donated by H2 are converted to NAD + (and / or other intracellular reducing equivalents), and some of the electrons from H are used in aerobic respiration. Hydrogen-producing microorganisms generally fix CO autotrophically through pathways including, but not limited to, the Calvin cycle or the reverse citric acid cycle.

[0085] However, as noted above, the microorganisms of the present disclosure can also be derived from essentially any parental microorganism, for example, a parental microorganism selected from the group consisting of Escherichia coli and Saccharomyces cerevisiae.

[0086] In other embodiments, the microorganisms of the present disclosure are aerobic bacteria. In one embodiment, the microorganisms of the present disclosure include aerobic hydrogen bacteria. In one embodiment, the aerobic bacteria comprise at least one disrupted gene.

[0087] Numerous aerobic bacteria are known to be capable of carrying out fermentation for the methods and systems of the present disclosure. Examples of such bacteria suitable for use in the present invention include Cupriavidus and Ralstonia bacteria. In some embodiments, the aerobic bacterium is Cupriavidus necator or Ralstonia eutropha. In some embodiments, the aerobic bacterium is Cupriavidus alkaliphilus. In some embodiments, the aerobic bacterium is Cupriavidus basilensis. In some embodiments, the aerobic bacterium is Cupriavidus campinensis. In some embodiments, the aerobic bacterium is Cupriavidus gilardii. In some embodiments, the aerobic bacterium is Cupriavidus laharis. In some embodiments, the aerobic bacterium is Cupriavidus metallidurans. In some embodiments, the aerobic bacterium is Cupriavidus nantongensis. In some embodiments, the aerobic bacterium is Cupriavidus numazuensis. In some embodiments, the aerobic bacterium is Cupriavidus oxalaticus. In some embodiments, the aerobic bacterium is Cupriavidus pampae. In some embodiments, the aerobic bacterium is Cupriavidus pauculus. In some embodiments, the aerobic bacterium is Cupriavidus pinatubonensis. In some embodiments, the aerobic bacterium is Cupriavidus plantarum. In some embodiments, the aerobic bacterium is Cupriavidus respiraculi. In some embodiments, the aerobic bacterium is Cupriavidus taiwanensis. In some embodiments, the aerobic bacterium is Cupriavidus yeoncheonensis.

[0088] In some embodiments, the microorganism is Cupriavidus necator DSM248 or DSM541.

[0089] In some embodiments, the aerobic bacterium comprises one or more exogenous nucleic acid molecules encoding naturally occurring polypeptides, wherein the polypeptides are ribolose bisphosphate carboxylase, acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, butanol dehydrogenase, electron transfer flavoprotein large subunit, 3-hydroxybutyryl-CoA dehydrogenase, bifunctional acetaldehyde-CoA / alcohol dehydrogenase, acetaldehyde dehydrogenase, acetyl-CoA acetyltransfer ... hydrogenase, aldehyde decarbonylase, acyl-ACP reductase, L-1,2-propanediol oxidoreductase, acyltransferase, 3-oxoacyl-ACP synthase, 3-hydroxybutyryl-CoA epimerase / delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase / enoyl-CoA hydratase / 3-hydroxyacyl-CoA dehydrogenase, short-chain dehydrogenase, trans-2-enoyl-CoA reductase, or any combination thereof.

[0090] In the microorganisms of the present disclosure, carbon flux is strategically diverted from non-essential or undesired products to products of interest. In certain embodiments, these disrupted genes divert carbon flux from non-essential or undesired metabolic nodes through targeted metabolic nodes to improve production of products downstream of those targeted metabolic nodes. In one embodiment, limitations selected from nutrients, dissolved oxygen, or any combination thereof divert carbon flux to desired products.

[0091] In one embodiment, the fermentation broth comprises a feed stream in combination with aerobic microorganisms in a bioreactor. In some embodiments, feed streams, such as a carbon source feed stream, a combustible gas-containing stream, and an oxygen-containing gas feed stream, react with the microorganisms in the bioreactor to at least partially form a fermentation broth (which may also contain other products, by-products, and other media fed to the bioreactor). Unreacted oxygen, or oxygen not consumed by the microorganisms, exists as both dissolved oxygen and gaseous oxygen in the dispersed gas phase within the fermentation broth. The same is true for other gases that are soluble. The dispersed gas phase containing unreacted components, such as oxygen, nitrogen, hydrogen, carbon dioxide, and / or water vapor, rises to the headspace of the bioreactor.

[0092] In some embodiments, an oxygen-containing gas, e.g., air, can be fed directly to the fermentation broth. In one embodiment, the oxygen-containing gas can be an oxygen-enriched source, e.g., oxygen-enriched air or pure oxygen. In one embodiment, the oxygen-containing gas may contain more than 6.0% oxygen by volume, e.g., more than 10.0%, more than 20.0%, more than 40.0%, more than 60.0%, more than 80.0%, or more than 90.0% oxygen by volume. In some embodiments, the oxygen-containing gas may be pure oxygen.

[0093] In one embodiment, the microorganism of the present disclosure is capable of producing ethylene. One embodiment is directed to a recombinant C1-fixing microorganism capable of producing ethylene from a carbon source comprising a nucleic acid encoding a group of exogenous enzymes including at least one ethylene-forming enzyme (EFE). In some embodiments, the EEF is derived from Pseudomonas syringae. In one embodiment, the EEF has EC number 1.13.12.19. The microorganism of the embodiment comprises at least one EFE having EC number 1.13.12.19. The microorganism of the embodiment further comprises a nucleic acid encoding a group of exogenous enzymes including at least one alpha-ketoglutarate permease (AKGP).

[0094]

[0013] The microorganism of an embodiment, wherein the nucleic acid encoding the group of exogenous enzymes comprises at least one EFE, at least one AKGP, or any combination thereof. The microorganism of an embodiment, wherein the nucleic acid encoding the group of exogenous enzymes comprises at least one EFE and at least one AKGP. The microorganism of an embodiment, wherein the nucleotides encoding the group of exogenous enzymes are inserted into a bacterial vector plasmid, a high copy number bacterial vector plasmid, a bacterial vector plasmid with an inducible promoter, a nucleotide guide for a homologous recombination system, a CRISPR-Cas system, or any combination thereof. In one embodiment, the promoter is a phosphate-limited inducible promoter. In some embodiments, the promoter is a nitrogen-limited promoter. In some embodiments, the promoter is an NtrC-P activated promoter. In some embodiments, the promoter is an H2-inducible promoter. In one embodiment, the microorganism comprises intracellular oxygen concentration limitation. In another embodiment, the method limits intracellular oxygen concentration. In one embodiment, the method comprises controlling dissolved oxygen. In one embodiment, the method comprises decreasing ethylene production with a decrease in dissolved oxygen concentration. In some embodiments, the microorganism comprises a molecular switch. In some embodiments, the microorganism comprises the ability to switch cellular load under variable conditions.

[0095] In some embodiments, the microorganism is a natural or engineered microorganism capable of converting a gaseous substrate as a carbon and / or energy source. In one embodiment, the gaseous substrate comprises CO as a carbon source. In some embodiments, the gaseous substrate comprises H and / or O as an energy source. In one embodiment, the gaseous substrate comprises a mixture of gases including H and / or CO and / or CO.

[0096] In some embodiments, the gas fermentation product is selected from an alcohol, an acid, a diacid, an alkene, a terpene, an isoprene, and an alkyne. In some embodiments, the methods and microorganisms disclosed herein are for the improved production of ethylene. In one embodiment, the methods and microorganisms disclosed herein are for the improved production of gas fermentation products.

[0097] In one embodiment, the aerobic bacteria are capable of producing a product such as acetone, isopropanol, 3-hydroxyisovaleryl-CoA, 3-hydroxyisovalerate, isobutylene, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, isoprene, farnesene, 3-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxybutyrate, 3-hydroxybutyrylaldehyde, 1,3-butanediol, 2-hydroxyisobutyryl-CoA, 2-hydroxyisobutyrate, butyryl-CoA, butyrate, butanol, caproate, hexanol, octanoate, octanol, 1,3-hexanediol, 2-buten-1-ol, isovaleryl-CoA, isovalerate, isoamyl alcohol, methacrolein, methyl methacrylate, or any combination thereof.

[0098] In other embodiments, the bacteria of the present disclosure are capable of producing ethylene, ethanol, propane, acetate, 1-butanol, butyrate, 2,3-butanediol, lactate, butene, butadiene, methyl ethyl ketone (2-butanone), acetone, isopropanol, lipids, 3-hydroxypropionate (3-HP), terpenes, isoprene, fatty acids, 2-butanol, 1,2-propanediol, 1 propanol, 1 hexanol, 1 octanol, chorismic acid-derived products, 3-hydroxybutyric acid, 1,3 butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipic acid, 1,3 hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, isoamyl alcohol, monoethylene glycol, or a combination thereof.

[0099] The present disclosure provides a microorganism capable of producing ethylene, comprising culturing the microorganism of the present disclosure in the presence of a substrate, whereby the microorganism produces ethylene.

[0100] The enzymes of the present disclosure may be codon optimized for expression in the microorganisms of the present disclosure. "Codon optimization" refers to the mutation of a nucleic acid, such as a gene, for optimizing or improving translation of the nucleic acid in a particular strain or species. Codon optimization can result in a faster translation rate or higher translation accuracy. In a preferred embodiment, the genes of the present disclosure are codon optimized for expression in the microorganisms of the present disclosure. Although codon optimization refers to the underlying gene sequence, codon optimization often results in improved translation and therefore improved enzyme expression. Therefore, the enzymes of the present disclosure may also be described as being codon optimized.

[0101] One or more of the enzymes of the present disclosure may be overexpressed. "Overexpressed" refers to increased expression of a nucleic acid or protein in a microorganism of the present disclosure compared to the wild-type or parent microorganism from which the microorganism of the present disclosure is derived. Overexpression can be achieved by any means known in the art, including altering gene copy number, gene transcription rate, gene translation rate, or enzyme degradation rate.

[0102] Enzymes of the present disclosure may include disruptive mutations. A "disruptive mutation" is a mutation that reduces or eliminates (i.e., "disrupts") the expression or activity of a gene or enzyme. A disruptive mutation may partially inactivate, completely inactivate, or delete a gene or enzyme. A disruptive mutation may be a knockout (KO) mutation. A disruptive mutation may be any mutation that reduces, prevents, or inhibits the biosynthesis of a product produced by the enzyme. A disruptive mutation may include, for example, a mutation in a gene encoding the enzyme, a mutation in a genetic regulatory element involved in the expression of a gene encoding the enzyme, the introduction of a nucleic acid that produces a protein that reduces or inhibits the activity of the enzyme, or the introduction of a nucleic acid (e.g., antisense RNA, siRNA, CRISPR) or protein that inhibits the expression of the enzyme. A disruptive mutation may be introduced using any method known in the art.

[0103] Introduction of a disruptive mutation results in a microorganism of the present disclosure that does not produce the target product, or produces substantially no product, or a reduced amount of the target product, compared to the parent microorganism from which the microorganism of the present disclosure is derived. For example, a microorganism of the present disclosure may not produce the target product, or may produce at least about 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% less target product than the parent microorganism. For example, a microorganism of the present disclosure may produce less than about 0.001, 0.01, 0.10, 0.30, 0.50, or 1.0 g / L of product.

[0104] While exemplary sequences and sources of enzymes are provided herein, the present disclosure is in no way limited to these sequences and sources and encompasses variants. The term "variant" includes nucleic acids and proteins whose sequences vary from those of reference nucleic acids and proteins, such as those disclosed in the prior art or exemplified herein. The present disclosure can be practiced using variant nucleic acids or proteins that perform substantially the same function as the reference nucleic acid or protein. For example, the variant protein may perform substantially the same function or catalyze substantially the same reaction as the reference protein. A variant gene may encode the same or substantially the same protein as the reference gene. A variant promoter may have substantially the same ability to promote expression of one or more genes as the reference promoter.

[0105] Such nucleic acids or proteins may be referred to herein as "functionally equivalent variants." By way of example, functionally equivalent variants of nucleic acids may include allelic variants, gene fragments, mutated genes, polymorphisms, and the like. Homologous genes from other microorganisms are also examples of functionally equivalent variants. These include homologous genes from species such as Clostridium acetobutylicum, Clostridium beijerinckii, or Clostridium ljungdahlii, details of which are publicly available on websites such as Genbank or NCBI. Functionally equivalent variants also include nucleic acids whose sequences have been altered as a result of codon optimization for a particular microorganism. Functionally equivalent variants of nucleic acids preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98%, or more nucleic acid sequence identity (percent homology) with the reference nucleic acid. Functionally equivalent variants of a protein preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98%, or more amino acid identity (percent homology) with the reference protein. Functional equivalence of a variant nucleic acid or protein can be assessed using any method known in the art.

[0106] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by conventional Watson-Crick or other non-conventional methods. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10, which are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0107] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding can occur via Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR or the cleavage of a polynucleotide by an enzyme. A sequence that can hybridize to a given sequence is referred to as the "complement" of the given sequence.

[0108] Nucleic acids can be delivered to the microorganisms of the present disclosure using any method known in the art. For example, nucleic acids can be delivered as naked nucleic acids or can be formulated with one or more agents, such as liposomes. Nucleic acids can be DNA, RNA, cDNA, or a combination thereof, as needed. In certain embodiments, restriction inhibitors can be used. Additional vectors can include plasmids, viruses, bacteriophages, cosmids, and artificial chromosomes. In preferred embodiments, nucleic acids are delivered to the microorganisms of the present disclosure using plasmids. By way of example, transformation (including transduction or transfection) can be achieved by electroporation, sonication, polyethylene glycol-mediated transformation, chemical or natural competence, protoplast transformation, prophage induction, or conjugation. In certain embodiments with active restriction enzyme systems, it may be necessary to methylate the nucleic acids before introducing them into the microorganism.

[0109] Additionally, nucleic acids may be designed to include regulatory elements, such as promoters, to increase or otherwise control expression of a particular nucleic acid. The promoter may be a constitutive promoter or an inducible promoter. The promoter may be a Wood-Ljungdahl pathway promoter, a ferredoxin promoter, a pyruvate ferredoxin oxidoreductase promoter, an Rnf complex operon promoter, an ATP synthase operon promoter, or a phosphotransacetylase / acetate kinase operon promoter.

[0110] It should be understood that the present disclosure can be practiced using nucleic acids whose sequences vary from those specifically exemplified herein, provided that the sequences perform substantially the same function. For nucleic acid sequences encoding proteins or peptides, this means that the encoded protein or peptide has substantially the same function. For nucleic acid sequences representing promoter sequences, the variant sequence has the ability to promote expression of one or more genes. Such nucleic acids may be referred to herein as "functionally equivalent variants." By way of example, functionally equivalent variants of nucleic acids include allelic variants, fragments of genes, genes containing mutations (deletions, insertions, nucleotide substitutions, etc.), and / or polymorphisms. Homologous genes from other microorganisms may also be considered examples of functionally equivalent variants of the sequences specifically exemplified herein.

[0111] The phrase "functionally equivalent variants" should also be taken to include nucleic acids whose sequences are altered as a result of codon optimization for a particular microorganism. A "functionally equivalent variant" of a nucleic acid herein will preferably have at least about 70%, preferably about 80%, more preferably about 85%, preferably about 90%, preferably about 95% or more nucleic acid sequence identity with the specified nucleic acid.

[0112] It should also be understood that the present disclosure can be practiced using polypeptides whose sequences vary from the amino acid sequences specifically exemplified herein. These variants may be referred to herein as "functionally equivalent variants." Functionally equivalent variants of proteins or peptides include those proteins or peptides that share at least 40%, preferably 50%, preferably 60%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95%, or more amino acid identity with the identified protein or peptide and have substantially the same function as the peptide or protein of interest. Such variants include fragments of proteins or peptides within that range, where fragments include truncated forms of the polypeptide, and deletions may be 1 to 5, 10, 15, 20, or 25 amino acids, or may extend from residues 1 to 25 at either end of the polypeptide, or may be of any length within the region or at an internal location. Functionally equivalent variants of particular polypeptides herein should also be taken to include polypeptides expressed by homologous genes in other species of bacteria, for example, as exemplified in the previous paragraph.

[0113] The microorganisms of the present disclosure can be prepared from a parental microorganism and one or more exogenous nucleic acids using any number of techniques known in the art for producing recombinant microorganisms. For example, transformation (including transduction or transfection) can be achieved by electroporation, sonication, polyethylene glycol-mediated transformation, chemical or natural competence, or conjugation. Suitable transformation techniques are described, for example, in Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, 1989.

[0114] In certain embodiments, the nucleic acid introduced into the microorganism must be methylated by a restriction system that is active in the microorganism to be transformed. This can be done using a variety of techniques, including those described below and further exemplified in the Examples section later herein.

[0115] By way of example, in one embodiment, a recombinant microorganism of the present disclosure is produced by a method comprising the following steps: introducing (i) an expression construct / vector described herein and (ii) a methylation construct / vector comprising a methyltransferase gene into a shuttle microorganism; expressing the methyltransferase gene; isolating one or more constructs / vectors from the shuttle microorganism; and introducing one or more constructs / vectors into a destination microorganism.

[0116] In one embodiment, the methyltransferase gene of step B is constitutively expressed. In another embodiment, expression of the methyltransferase gene of step B is inducible.

[0117] The shuttle microorganism is a microorganism, preferably a restriction-negative microorganism, that promotes methylation of the nucleic acid sequences that make up the expression construct / vector. In particular embodiments, the shuttle microorganism is a restriction-negative E. coli, Bacillus subtilis, or Lactococcus lactis.

[0118] The methylation construct / vector comprises a nucleic acid sequence encoding a methyltransferase.

[0119] Upon introduction of the expression construct / vector and the methylation construct / vector into the shuttle microorganism, the methyltransferase gene present on the methylation construct / vector is induced. Induction may be by any suitable promoter system, but in one particular embodiment of the present disclosure, the methylation construct / vector contains an inducible lac promoter and is induced by the addition of lactose or an analog thereof, more preferably isopropyl-β-D-thiogalactoside (IPTG). Other suitable promoters include the ara, tet, or T7 systems. In a further embodiment of the present disclosure, the methylation construct / vector promoter is a constitutive promoter.

[0120] In certain embodiments, the methylation construct / vector has an origin of replication specific to the identity of the shuttle microorganism, such that any genes present on the methylation construct / vector are expressed in the shuttle microorganism. Preferably, the expression construct / vector has an origin of replication specific to the identity of the destination microorganism, such that any genes present on the expression construct / vector are expressed in the destination microorganism.

[0121] Expression of the methyltransferase enzyme results in methylation of the genes present on the expression construct / vector. The expression construct / vector may then be isolated from the shuttle microorganism according to any one of several known methods. By way of example only, the expression construct / vector may be isolated using the methodology described in the Examples section below.

[0122] In one particular embodiment, both constructs / vectors are isolated simultaneously.

[0123] The expression construct / vector may be introduced into the destination microorganism using any number of known methods. However, by way of example, the methodology described in the Examples section below may be used. Because the expression construct / vector is methylated, the nucleic acid sequences present on the expression construct / vector can be incorporated into and successfully expressed in the destination microorganism.

[0124] It is envisioned that the methyltransferase gene can be introduced into the shuttle microorganism and overexpressed. Thus, in one embodiment, the resulting methyltransferase enzyme can be harvested using known methods and used in vitro to methylate an expression plasmid. The expression construct / vector can then be introduced into a destination microorganism for expression. In another embodiment, the methyltransferase gene is introduced into the genome of the shuttle microorganism, followed by introducing the expression construct / vector into the shuttle microorganism, isolating one or more constructs / vectors from the shuttle microorganism, and then introducing the expression construct / vector into the destination microorganism.

[0125] It is envisioned that the expression constructs / vectors and methylation constructs / vectors defined above may be combined to provide compositions that have particular utility in circumventing restriction barrier mechanisms for generating recombinant microorganisms of the present disclosure.

[0126] In one particular embodiment, the expression construct / vector and / or the methylation construct / vector is a plasmid.

[0127] Those skilled in the art will recognize the use of several suitable methyltransferases in generating the microorganisms of the present disclosure. However, by way of example, the Bacillus subtilis phage ΦT1 methyltransferase and methyltransferases described in the Examples below may be used. Nucleic acids encoding suitable methyltransferases will be readily recognized given the sequence and genetic code of the desired methyltransferase.

[0128] Any number of constructs / vectors adapted to allow expression of a methyltransferase gene may be used to generate the methylation construct / vector.

[0129] In other embodiments, the substrate comprises CO and an energy source. In some embodiments, the substrate comprises CO and an energy source. In another embodiment, the substrate comprises CO, H, and O. In some embodiments, the substrate comprises CO and any suitable energy source. In one embodiment, the substrate comprises CO. In one embodiment, the substrate comprises CO and CO. In another embodiment, the substrate comprises CO and H. In another embodiment, the substrate comprises CO, CO, and H.

[0130] "Substrate" refers to a carbon and / or energy source for the microorganisms of the present disclosure. Often, the substrate is gaseous and includes a C1 carbon source, such as CO, CO, and / or CH. Preferably, the substrate includes a C1 carbon source of CO or CO + CO. The substrate may further include other non-carbon components, such as H, N, or electrons. However, in other embodiments, the substrate may be a carbohydrate, such as a sugar, starch, fiber, lignin, cellulose, or hemicellulose, or a combination thereof. For example, the carbohydrate may be fructose, galactose, glucose, lactose, maltose, sucrose, xylose, or some combination thereof. In some embodiments, the substrate does not include (D)-xylose (Alkim, Microb Cell Fact, 14:127, 2015). In some embodiments, the substrate does not include a pentose, such as xylose (Pereira, Metab Eng, 34: 80-87, 2016). In some embodiments, the substrate may include both gaseous and carbohydrate substrates. (Mixotrophic fermentation) The substrate may further include other non-carbon components such as H, N, or electrons.

[0131] In some embodiments, the gaseous substrate generally contains at least some amount of CO, such as about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mol% CO. The gaseous substrate can contain a range of CO, such as about 20-80, 30-70, or 40-60 mol% CO. Preferably, the gaseous substrate contains about 40-70 mol% CO (e.g., steel mill or blast furnace gas), about 20-30 mol% CO (e.g., basic oxygen furnace gas), or about 15-45 mol% CO (e.g., synthesis gas). In some embodiments, the gaseous substrate can contain relatively low amounts of CO, such as about 1-10 or 1-20 mol% CO. Microorganisms of the present disclosure typically convert at least a portion of the CO in the gaseous substrate to products. In some embodiments, the gaseous substrate is free or substantially free (<1 mol%) of CO.

[0132] The gaseous substrate may contain some amount of H. For example, the gaseous substrate may contain about 1, 2, 5, 10, 15, 20, or 30 mol% H. In some embodiments, the gaseous substrate may contain relatively large amounts of H, such as about 60, 70, 80, or 90 mol% H. In further embodiments, the gaseous substrate is free or substantially free (<1 mol%) of H.

[0133] The gaseous substrate may contain some amount of CO. For example, the gaseous substrate may contain about 1-80 or 1-30 mol% CO. In some embodiments, the gaseous substrate may contain less than about 20, 15, 10, or 5 mol% CO. In other embodiments, the gaseous substrate is free or substantially free (<1 mol%) of CO.

[0134] The gaseous substrate may also be provided in alternative forms, for example, the gaseous substrate may be dissolved in a liquid or adsorbed onto a solid support.

[0135] The gaseous substrate and / or C1 carbon source may be obtained as a by-product of an industrial process, such as from automobile exhaust or biomass gasification, or may be a waste gas or off-gas from some other source. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal product production, such as steel mill production, non-ferrous metal product production, petroleum refining, coal gasification, power generation, carbon black production, ammonia production, methanol production, and coke production. In these embodiments, the gaseous substrate and / or C1 carbon source may be captured from the industrial process before it is released into the atmosphere using any convenient method.

[0136] The gaseous substrate and / or C1 carbon source may be a synthesis gas, such as synthesis gas obtained by gasification of coal or refinery residues, gasification of biomass or lignocellulosic material, or reforming of natural gas. In another embodiment, the synthesis gas may be obtained from the gasification of municipal or industrial solid waste.

[0137] The terms "feedstock" or "gas fermentation feedstock" when used in the context of a stream flowing to a gas fermentation bioreactor (i.e., gas fermentor) should be understood to encompass any material (solid, liquid, or gas) or stream that can provide a substrate and / or a C1 carbon source to a gas fermentor or bioreactor, either directly or after processing.

[0138] The terms "waste gas" or "waste gas stream" may be used to refer to any gas stream that is either directly radiated, flared without additional value capture, or combusted for the purposes of energy recovery.

[0139] The term synthesis gas or syngas refers to a gaseous mixture containing at least one carbon source, such as carbon monoxide (CO), carbon dioxide (CO), or any combination thereof, and optionally hydrogen (H), which can be used as a feedstock for the disclosed gas fermentation processes and can be produced from a wide range of carbonaceous materials, both solid and liquid.

[0140] The substrate and / or C1 carbon source may be waste gas obtained as a by-product of an industrial process, or waste gas from another source, such as from automobile exhaust, biogas, landfill gas, direct air capture, or waste gas from electrolysis. The substrate and / or C1 carbon source may be syngas produced by pyrolysis, torrefaction, or gasification. In other words, carbon in waste may be recycled by pyrolysis, torrefaction, or gasification to produce syngas for use as the substrate and / or C1 carbon source. The substrate and / or C1 carbon source may be a gas containing methane.

[0141] In certain embodiments, the industrial process is selected from ferrous metals manufacturing, such as steel production, non-ferrous manufacturing, petroleum refining, power production, carbon black production, paper and pulp manufacturing, ammonia production, methanol production, coke production, petrochemical production, carbohydrate fermentation, cement manufacturing, aerobic digestion, anaerobic digestion, catalytic processes, natural gas extraction, cellulose fermentation, oil extraction, geological reservoirs, natural gas, gas from fossil sources such as coal and petroleum, or any combination thereof. Examples of specific processing steps within industrial processes include catalyst regeneration, fluid catalytic cracking, and catalyst regeneration. Air separation and direct air recovery are other suitable industrial processes. Specific examples in steel and ferroalloy production include blast furnace gas, basic oxygen furnace gas, coke oven gas, direct reduction of iron furnace top gas, and residual gas from iron smelting. In these embodiments, the substrate and / or C1 carbon source may be captured from the industrial process before it is released into the atmosphere using any known method.

[0142] The substrate and / or C1 carbon source may be synthesis gas, also known as syngas, which may be obtained from a reforming, partial oxidation, or gasification process. Examples of gasification processes include coal gasification, refinery residue gasification, petroleum coke gasification, biomass gasification, lignocellulosic material gasification, waste wood gasification, black liquor gasification, municipal solid waste gasification, municipal liquid waste gasification, industrial solid waste gasification, industrial liquid waste gasification, waste fuel gasification, sewage gasification, sewage sludge gasification, wastewater treatment sludge gasification, and biogas gasification. Examples of reforming processes include steam methane reforming, steam naphtha reforming, natural gas reforming, biogas reforming, landfill gas reforming, naphtha reforming, and dry methane reforming. Examples of partial oxidation processes include thermal and catalytic partial oxidation processes, catalytic partial oxidation of natural gas, and partial oxidation of hydrocarbons. Examples of municipal solid waste include tires, plastics, shoes, apparel, and textile fibers. Municipal solid waste may simply be landfill-type waste. Municipal solid waste may be sorted or unsorted. Examples of biomass may include lignocellulosic material and may include microbial biomass. Lignocellulosic material may include agricultural waste and forestry waste.

[0143] The substrate and / or C1 carbon source may be a gas stream containing methane. Such methane-containing gas may be obtained from fossil methane emissions, such as during fracking, wastewater treatment, livestock, agriculture, and municipal solid waste landfills. It is also contemplated that methane may be combusted to generate electricity or heat, and the C1 by-product may be used as a substrate or carbon source.

[0144] The composition of the gaseous substrate can significantly affect the efficiency and / or cost of the reaction. For example, the presence of oxygen (O2) can reduce the efficiency of an anaerobic fermentation process. Depending on the composition of the substrate, it may be desirable to treat, scrub, or filter the substrate to remove any undesirable impurities, such as toxins, undesirable components, or dust particles, and / or to increase the concentration of desired components.

[0145] Regardless of the source or exact content of gas used as a feedstock, the feedstock may be metered into the bioreactor to maintain control of the follow-up rate and amount of carbon provided to the culture (e.g., for carbon credit calculations or mass balancing of sustainable carbon and overall products). Similarly, the output of the bioreactor may be metered (e.g., for carbon credit calculations or mass balancing of sustainable carbon and overall products) or may be equipped with valve connections that can control the flow of the output and products (e.g., ethylene, ethanol, acetate, 1-butanol, etc.) produced via fermentation. Such valves or metering mechanisms may be useful for a variety of purposes, including, but not limited to, measuring the amount of output from a given bioreactor, as well as slugs of product through connected pipelines, so that if the product is mixed with other gases or liquids, the resulting mixture can later be mass balanced to determine the proportion of product produced from the bioreactor.

[0146] In certain embodiments, the fermentation is carried out in the absence of a carbohydrate substrate, such as sugar, starch, fiber, lignin, cellulose, or hemicellulose.

[0147] In addition to ethylene, the microorganisms of the present disclosure may be cultured to produce one or more co-products. For example, the microorganisms of the present disclosure may produce ethanol (WO 2007 / 117157), acetate (WO 2007 / 117157), 1-butanol (WO 2008 / 115080, WO 2012 / 053905, and WO 2017 / 066498), butyrate (WO 2008 / 115080), 2,3-butanediol (WO 2009 / 151342 and WO 2016 / 094334), milk, in addition to 2-phenylethanol. acid salts (WO 2011 / 112103), butenes (WO 2012 / 024522), butadiene (WO 2012 / 024522), methyl ethyl ketone (2-butanone) (WO 2012 / 024522 and WO 2013 / 185123), ethylene (WO 2012 / 026833), acetone (WO 2012 / 115527), isopropanol (WO 2012 / 115527), lipids (WO 2013 / 03 6147), 3-hydroxypropionate (3-HP) (WO 2013 / 180581), terpenes including isoprene (WO 2013 / 180584), fatty acids (WO 2013 / 191567), 2-butanol (WO 2013 / 185123), 1,2-propanediol (WO 2014 / 036152), 1-propanol (WO 2017 / 066498), and 1-hexanol (WO 2017 / 066498). , 1-octanol (WO 2017 / 066498), chorismic acid-derived products (WO 2016 / 191625), 3-hydroxybutyric acid (WO 2017 / 066498), 1,3-butanediol (WO 2017 / 066498), 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid (WO 2017 / 066498), isobutylene (WO 2017 / 066498), adipic acid (WO 2017 / 066498), 1,The microbial biomass may be capable of, or may be engineered to, produce 3-hexanediol (WO 2017 / 066498), 3-methyl-2-butanol (WO 2017 / 066498), 2-buten-1-ol (WO 2017 / 066498), isovalerate (WO 2017 / 066498), isoamyl alcohol (WO 2017 / 066498), and / or monoethylene glycol (WO 2019 / 126400). In certain embodiments, the microbial biomass itself may be considered a product. These products may be further converted to produce at least one component of diesel, jet fuel, sustainable aviation fuel (SAF), and / or gasoline. In certain embodiments, ethylene may be catalytically converted to another product, commodity, or any combination thereof. Additionally, the microbial biomass can be further processed to produce single-cell protein (SCP) by any method or combination of methods known in the art. In addition to one or more target chemical products, the microorganisms of the present disclosure can also produce ethanol, acetate, and / or 2,3-butanediol. In another embodiment, the microorganisms and methods of the present disclosure improve the production of products, proteins, microbial biomass, or any combination thereof.

[0148] A "natural product" is a product produced by a non-genetically modified microorganism. For example, ethanol, acetate, and 2,3-butanediol are natural products of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei. A "non-natural product" is a product produced by a genetically modified microorganism but not by the non-genetically modified microorganism from which the genetically modified microorganism is derived. Ethylene is not known to be produced by any naturally occurring microorganism, and is therefore a non-natural product of all microorganisms.

[0149] "Selectivity" refers to the ratio of target product production to total fermentation products produced by a microorganism. The microorganisms of the present disclosure can be engineered to produce products with a particular selectivity or with minimal selectivity. In one embodiment, the target product, such as ethylene glycol, accounts for at least about 5%, 10%, 15%, 20%, 30%, 50%, or 75% of the total fermentation products produced by the microorganisms of the present disclosure. In one embodiment, ethylene accounts for at least 10% of the total fermentation products produced by the microorganisms of the present disclosure, such that the microorganisms of the present disclosure have a selectivity for at least 10% ethylene glycol. In another embodiment, ethylene accounts for at least 30% of the total fermentation products produced by the microorganisms of the present disclosure, such that the microorganisms of the present disclosure have a selectivity for at least 30% ethylene.

[0150] At least one of the one or more fermentation products may be biomass produced by the culture. At least a portion of the microbial biomass may be converted into single-cell protein (SCP). At least a portion of the single-cell protein may be utilized as an ingredient in animal feed.

[0151] In one embodiment, the present disclosure provides an animal feed comprising a microbial biomass and at least one excipient, wherein the microbial biomass comprises microorganisms grown on a gaseous substrate comprising one or more of CO, CO2, and H2.

[0152] "Single-cell protein" (SCP) refers to microbial biomass that can be used in protein-rich human and / or animal feeds, often replacing traditional protein supplement sources such as soybean or fish meal. To produce single-cell protein or other products, the process may include additional separation, processing, or treatment steps. For example, the method may include sterilizing the microbial biomass, centrifuging the microbial biomass, and / or drying the microbial biomass. In certain embodiments, the microbial biomass is dried using spray drying or paddle drying. Because ingestion of a diet high in nucleic acid content can lead to the accumulation of nucleic acid degradation products and / or gastrointestinal disorders, the method may also include reducing the nucleic acid content of the microbial biomass using any method known in the art. The single-cell protein may be suitable for feeding to animals, such as livestock or pets. Specifically, the animal feed may be suitable for feeding one or more of beef cattle, dairy cattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, banteng, gayal, yaks, chickens, turkeys, ducks, geese, quails, guinea fowl, pigeons / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents. The composition of the animal feed may be tailored to meet the nutritional requirements of different animals. Additionally, the process may include mixing or combining the microbial biomass with one or more excipients.

[0153] "Microbial biomass" refers to biological material containing microbial cells. For example, microbial biomass can comprise or consist of pure or substantially pure cultures of bacteria, archaea, viruses, or fungi. When initially separated from a fermentation broth, microbial biomass generally contains large amounts of water. This water can be removed or reduced by drying or processing the microbial biomass.

[0154] "Excipient" may refer to any substance that can be added to microbial biomass to enhance or modify the form, characteristics, or nutritional content of animal feed. For example, an excipient may include one or more of carbohydrates, fiber, fat, protein, vitamins, minerals, water, flavorings, sweeteners, antioxidants, enzymes, preservatives, probiotics, or antibiotics. In some embodiments, an excipient may be hay, straw, silage, grain, oil or fat, or other plant material. An excipient may be any feed ingredient identified in Chiba, Section 18: Diet Formulation and Common Feed Ingredients, Animal Nutrition Handbook, 3rd revision, pages 575-633, 2014.

[0155] "Biopolymer" refers to a natural polymer produced by cells of living organisms. In certain embodiments, the biopolymer is a PHA. In certain embodiments, the biopolymer is a PHB.

[0156] "Bioplastic" refers to plastic materials produced from renewable biomass sources. Bioplastics can be produced from renewable sources such as vegetable fats and oils, corn starch, straw, wood chips, sawdust, or recycled food waste. Any reference herein to an acid (eg, acetic acid or 2-hydroxyisobutyric acid) should be interpreted as also including the corresponding salt (eg, acetate or 2-hydroxyisobutyrate).

[0157] Typically, culturing is carried out in a bioreactor. The term "bioreactor" includes a culturing / fermentation device consisting of one or more vessels, columns, or piping arrangements, such as a continuous stirred tank reactor (CSTR), immobilized cell reactor (ICR), trickle bed reactor (TBR), bubble column, gas lift fermenter, static mixer, or other vessel or device suitable for gas-liquid contact. In some embodiments, a bioreactor can include a first growth reactor and a second culture / fermentation reactor. Substrate may be provided to one or both of these reactors. As used herein, the terms "culturing" and "fermentation" are used interchangeably. These terms encompass both the growth phase and the product biosynthesis phase of the culturing / fermentation process.

[0158] Cultures are generally maintained in an aqueous medium containing sufficient nutrients, vitamins, and / or minerals to allow microbial growth. Preferably, the aqueous culture medium is an anaerobic microbial medium, such as a minimal anaerobic microbial growth medium. Suitable media are known in the art.

[0159] The culture / fermentation should desirably be carried out under conditions suitable for the production of ethylene glycol. If necessary, the culture / fermentation is carried out under anaerobic conditions. Reaction conditions to consider include pressure (or partial pressure), temperature, gas flow rate, liquid flow rate, medium pH, medium redox potential, agitation rate (if a continuous stirred tank reactor is used), inoculum level, maximum gas substrate concentration to ensure that the gas in the liquid phase does not become limiting, and maximum product concentration to avoid product inhibition. Specifically, the rate of substrate introduction may be controlled to ensure that the gas concentration in the liquid phase does not become limiting.

[0160] Operating a bioreactor at elevated pressure allows for an increased rate of gas mass transfer from the gas phase to the liquid phase. Therefore, it is generally preferable to conduct the culture / fermentation at a pressure higher than atmospheric pressure. Furthermore, because a given gas conversion rate is in part a function of substrate retention time, and retention time dictates the required volume of the bioreactor, the use of a pressurized system can significantly reduce the required bioreactor volume and, consequently, the capital costs of the culture / fermentation equipment. This means that the retention time, defined as the liquid volume in the bioreactor divided by the input gas flow rate, can be reduced when the bioreactor is maintained at a pressure higher than atmospheric pressure. Optimal reaction conditions depend in part on the specific microorganism used. However, it is generally preferable to conduct the fermentation at a pressure higher than atmospheric pressure. Furthermore, because a given gas conversion rate is in part a function of substrate retention time, and achieving a desired retention time further dictates the required volume of the bioreactor, the use of a pressurized system can significantly reduce the required bioreactor volume and, consequently, the capital costs of the fermentation equipment.

[0161] A "sparger" may comprise a device that introduces gas, injected as bubbles, into a liquid to agitate it or dissolve the gas in the liquid. Examples of spargers include orifice spargers, sintered spargers, and drilled pipe spargers. In certain configurations, perforated pipe spargers may be mounted horizontally. In other examples, spargers may be mounted vertically or horizontally. In some examples, spargers may be perforated plates or rings, sintered glass, sintered steel, porous rubber pipe, porous metal pipe, porous ceramic or stainless steel, perforated pipe, stainless steel perforated pipe, polymeric perforated pipe, etc. Spargers may be of various grades (porosities) and may have orifices of specific sizes to generate bubbles of specific sizes or ranges of bubble sizes.

[0162] A "vessel," "reactor," or "column" may refer to a vessel or container into which one or more gas and liquid streams or flows are introduced for bubble generation and / or microbubble generation, and subsequent gas-liquid contact, gas absorption, biological or chemical reactions, or surfactant adsorption. Within a reactor, gas and liquid phases may flow vertically. Within a reactor, large gas bubbles from a sparger may rise upward, having a buoyancy greater than the drag imposed by the liquid. As illustrated by the systems and methods disclosed herein, smaller microbubbles, having a buoyancy equal to or less than the drag imposed by the liquid, may flow downward with the liquid. A column or reactor may not be limited to any particular aspect ratio (height to diameter). A column or reactor is also not limited to any particular material and can be constructed of any material suitable for the process, such as stainless steel, PVC, carbon steel, or polymeric materials. A column or reactor may include internal components, such as one or more static mixers common in biological and chemical engineering processes. The reaction vessel may also be configured with external or internal heating or cooling elements, such as a water jacket, heat exchanger, or cooling coil. The reaction vessel may also be in fluid communication with one or more pumps to circulate the liquid, gas bubbles, microbubbles, and / or one or more fluids in the system.

[0163] A "perforated plate" or "plate" may comprise a plate or similar structure designed to facilitate the introduction of a liquid or additional liquid into a vessel, which may be in the form of multiple liquid jets (i.e., accelerated liquid streams). The perforated plate may have multiple pores or orifices, uniformly or non-uniformly distributed throughout the plate, that allow liquid to flow from the top of the plate to the bottom of the plate. In some examples, the orifices may be spherical, rectangular, hexagonal, conical, pentagonal, cylindrical, frustoconical, or circular. In other examples, the plate may comprise one or more nozzles configured to generate liquid jets that flow into the column. The plate may also comprise channels in any distribution or arrangement, configured to receive the liquid and facilitate its flow into the reaction vessel. The plate may be made of stainless steel with a predetermined number of pores or orifices sintered, machined, or drilled with a laser. The specific orifice size may depend on the required microbubble size and the required liquid, microbubble, and / or fluid velocity. A particular orifice shape may be required to achieve adequate liquid acceleration and velocity from the plate to break up or shear the sparger bubbles into the desired fine bubble size, and to generate sufficient overall fluid downward flow to carry the fine bubbles and liquid downward in the reactor vessel. The orifice shape may also affect ease of manufacture and associated costs. According to one embodiment, a straight orifice may be optimal for ease of manufacture.

[0164] The systems and methods disclosed herein use multiple liquid jets or portions of an accelerated liquid stream generated using a perforated plate within a vessel to accelerate the liquid and break down the gas bubbles into small, microscopic bubbles with a larger surface area than the original bubbles. The original bubbles are first generated by injecting gas using a sparger positioned entirely within the reaction vessel. In one example, the original gas bubbles injected into the liquid from the sparger may have a diameter of about 2 mm to about 20 mm. In another example, the original gas bubbles injected into the liquid from the sparger may have a diameter of about 5 mm to about 15 mm. In another example, the original gas bubbles injected into the liquid from the sparger may have a diameter of about 7 mm to about 13 mm. Upon injection, the original gas bubbles then travel upward through the liquid and encounter multiple liquid jets or portions of the accelerated liquid stream, which break down the original gas bubbles into microscopic bubbles. The resulting microscopic bubbles and liquid then flow down the reaction vessel in a downward fluid flow. The microbubbles in the substrate provide a carbon source, and optionally an energy source, for the microorganisms, which then produce one or more desired products. A sparger is positioned within the vessel to form a first region for the original bubbles to rise within the vessel, and a second region for the accelerated liquid to break the original bubbles into microbubbles and for the fluid to flow through the vessel, where the fluid is comprised of the accelerated portion of the liquid and the microbubbles.

[0165] Due to the nature of multiphase systems, one way to maximize product production is to increase gas-to-liquid mass transfer. The more gas substrate transferred to the reaction liquid, the more desired product is produced. The smaller microbubbles disclosed herein increase surface area, resulting in increased gas-to-liquid mass transfer rates and overcoming known solubility issues. Furthermore, the downflow reactor system disclosed herein is effective at increasing the residence time of the microbubbles. Increasing the residence time of the microbubbles in the reaction liquid generally increases the amount of reaction product produced and also increases the surface area in contact with microorganisms. Therefore, the systems and methods disclosed herein improve over conventional systems by generating microbubbles that maximize the gas-to-liquid surface area, resulting in high gas-to-liquid mass transfer rates. Furthermore, the systems and methods disclosed herein provide gas and liquid superficial velocities not achieved by conventional systems and methods, resulting in the efficient production of chemical and biological reaction products through the generation of microbubbles with long gas-phase residence times.

[0166] In certain embodiments, fermentation is carried out in the absence of light or in the presence of an amount of light insufficient to meet the energy requirements of the photosynthetic microorganisms, hi certain embodiments, the microorganisms of the present disclosure are non-photosynthetic microorganisms.

[0167] The target product can be separated or purified from the fermentation broth using any method or combination of methods known in the art, including, for example, fractional distillation, evaporation, pervaporation, gas stripping, phase separation, and extractive fermentation, including, for example, liquid-liquid extraction. In certain embodiments, the target product is recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating the microbial cells from the broth (conveniently by filtration), and recovering one or more target products from the broth. Alcohol and / or acetone may be recovered, for example, by distillation. Acids may be recovered, for example, by adsorption on activated carbon. The separated microbial cells are preferably returned to the bioreactor. The cell-free permeate remaining after the target product is removed is also preferably returned to the bioreactor. Additional nutrients (such as vitamin B) may be added to the cell-free permeate to replenish the medium before being returned to the bioreactor. Purification techniques can include affinity tag purification (e.g., His, Twin-Strep, and FLAG), bead-based systems, chip-based approaches, and FPLC systems for larger-scale automated purification. Purification methods that do not rely on affinity tags (e.g., salting out, ion exchange, and size exclusion) have also been described.

[0168] In some embodiments, the produced chemicals may be isolated and concentrated, including purified, using any suitable separation and / or purification technique known in the art. In one embodiment, the produced chemicals are gaseous. In one embodiment, the chemicals are liquid. In one embodiment, the gaseous chemicals may be passed through a filter, a gas separation membrane, a gas purifier, or any combination thereof. In one embodiment, the chemicals are separated using an absorption column. In another embodiment, the chemicals are stored in one or more cylinders after separation. In one embodiment, the chemicals are integrated into the infrastructure or process of an oil, gas, refinery, petrochemical operation, or any combination thereof. The infrastructure or process may be existing or new. In one embodiment, the gas fermentation product is integrated into an oil and gas production, transportation and refining, and / or chemical complex. In another embodiment, the source of the feedstock is from an oil, gas, refinery, petrochemical operation, or any combination thereof. In one embodiment, the gas fermentation product is integrated into the infrastructure or process of an oil, gas, refinery, petrochemical operation, or any combination thereof, and the source of the feedstock is from an oil, gas, refinery, petrochemical operation, or any combination thereof.

[0169] In some embodiments, distillation may be used to purify the product gas. In one embodiment, gas-to-liquid extraction may be used. In one embodiment, the liquid product isolation may also be concentrated via extraction using an organic phase. In another embodiment, purification may involve other standard techniques selected from ultrafiltration, one or more chromatographic techniques, or any combination thereof.

[0170] The methods of the present disclosure may further include separating a gas fermentation product from the fermentation broth. The gas fermentation product can be separated or purified from the fermentation broth using any method or combination of methods known in the art, including, for example, distillation, simulated moving bed processing, membrane processing, pervaporation, gas stripping, phase separation, ion exchange, or extractive fermentation, including, for example, liquid-liquid extraction. Ethylene can be separated according to a method or combination of methods known in the art, as described in U.S. Pat. No. 2,769,321, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the produced ethylene is harvested from the bioreactor culture vessel.

[0171] In one embodiment, the gas fermentation product may be concentrated from the fermentation broth using reverse osmosis and / or pervaporation (U.S. Pat. No. 5,552,023). Water may be removed by distillation, and the bottoms (containing a high proportion of the gas fermentation product) may then be recovered using distillation or vacuum distillation to produce a high-purity gas stream. Alternatively, the gas fermentation product may be further purified by reactive distillation with aldehydes (Atul, Chem Eng Sci, 59:2881-2890, 2004) or azeotropic distillation with hydrocarbons (U.S. Pat. No. 2,218,234), with or without concentration by reverse osmosis and / or pervaporation. In another approach, the gas fermentation product may be captured from the aqueous solution onto activated carbon or a polymeric absorber (with or without reverse osmosis and / or pervaporation) and recovered using a low-boiling organic solvent (Chinn, Recovery of Glycols, Sugars, and Related Multiple -OH Compounds from Dilute-Aqueous Solution by Regenerable Adsorption onto Activated Carbons, University of California, Berkeley, 1999). The gas fermentation product can then be recovered from the organic solvent by distillation. In certain embodiments, the gas fermentation product is recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating the microbial cells from the broth (conveniently by filtration), and recovering the gas fermentation product from the broth. Co-products, such as alcohols or acids, may also be separated or purified from the broth. Alcohols may be recovered, for example, by distillation. Acids may be recovered, for example, by adsorption on activated carbon. The separated microbial cells may, in certain embodiments, be returned to the bioreactor. Additionally, the separated microbial cells may, in some embodiments, be recycled back to the bioreactor. The cell-free permeate remaining after the target product has been removed is also preferably returned, in whole or in part, to the bioreactor.Additional nutrients (such as vitamin B) can be added to the cell-free permeate to replenish the medium before returning it to the bioreactor.

[0172] Many methods have been demonstrated for the recovery of diols from aqueous media. Simulated moving bed (SMB) technology has been used to recover 2,3-butenediol from aqueous mixtures of ethanol and related oxygenates (U.S. Pat. No. 8,658,845). Reactive separation has also demonstrated effective diol recovery. In some embodiments, ethylene glycol is recovered by reacting a diol-containing stream with an aldehyde, followed by fractionation and regeneration of the diol, final fractionation, and recovery of a concentrated diol stream. See, for example, U.S. Pat. No. 7,951,980.

[0173] In one embodiment, the method includes recovering the ethylene produced as disclosed above. In one embodiment, the method further includes, after recovering the ethylene, converting or using the ethylene in the production of one or more chemicals.

[0174] Ethylene is a high-value gaseous compound widely used in industry. In one embodiment, ethylene can be used as an anesthetic or as a fruit ripening agent, as well as in the production of several other chemicals. In some embodiments, ethylene may be used to produce polyethylene and other polymers such as styrene, polystyrene, ethylene oxide, ethylene dichloride, ethylene dibromide, ethyl chloride, and ethylbenzene. Ethylene oxide is, for example, a key raw material in the production of surfactants and detergents and ethylene glycol, which is used in the automotive industry as an anti-freeze product. In one embodiment, ethylene dichloride, ethylene dibromide, and ethyl chloride may be targeted to produce products such as polyvinyl chloride, trichloroethylene, perchloroethylene, methyl chloroform, polyvinylidene chloride and copolymers, and ethyl bromide. In one embodiment, ethylbenzene is a precursor to styrene, which is used to produce polystyrene (used as an insulation product) and styrene butadiene (a rubber suitable for use in tires and footwear). In another embodiment, the product is ethylene propylene diene monomer (EPDM) rubber, ethylene propylene (EPR / EPM) rubber, or any combination thereof.

[0175] Of course, the methods of the present invention may be integrated or coupled to one or more processes for the production of downstream chemicals from ethylene. In some embodiments, the methods of the present invention may directly or indirectly supply ethylene to chemical processes or reactions sufficient for the conversion or production of other useful chemicals.

[0176] In some embodiments, ethylene is converted to hydrocarbon liquid fuels. In one embodiment, ethylene is oligomerized over a catalyst to selectively produce target products selected from gasoline, condensates, aromatics, heavy oil diluents, distillates, or any combination thereof. In other embodiments, the distillates are selected from diesel, jet fuel, sustainable aviation fuel (SAF), or any combination thereof.

[0177] In one embodiment, ethylene oligomerization is utilized toward a desired product. In one embodiment, the ethylene oligomerization is catalyzed by a homogeneous catalyst, a heterogeneous catalyst, or any combination thereof, and may have a transition metal as an active site. In some embodiments, ethylene is further converted to long-chain hydrocarbons by oligomerization. In other embodiments, linear olefins are the primary product from ethylene oligomerization. In some embodiments, alpha olefins are the primary product from ethylene oligomerization. In one embodiment, olefins undergo an upgrading process. In some embodiments, the olefin upgrading process is hydrogenation. In one embodiment, olefins are subjected to olefin conversion techniques. In some embodiments, ethylene is incorporated into or converted into sustainable aviation fuel (SAF). In one embodiment, ethylene is interconverted to propylene, 2-butene, or any combination thereof. In one embodiment, propylene is converted to polypropylene.

[0178] As a raw material, ethylene can be used to make polymers such as polyethylene (PE), polyethylene terephthalate (PET), and polyvinyl chloride (PVC), as well as fibers and other organic chemicals. These products are used in a wide range of industrial and consumer markets, including packaging, transportation, electrical / electronics, textiles, and construction industries, as well as consumer chemicals, coatings, and adhesives.

[0179] Ethylene can be chlorinated to ethylene dichloride (EDC), which can then be cracked to make vinyl chloride monomer (VCM). Nearly all VCM is used to make polyvinyl chloride, which has its primary use in the construction industry.

[0180] Other ethylene derivatives include alpha olefins used in linear low-density polyethylene (LLDPE) production, surfactant alcohols, and plasticizer alcohols; vinyl acetate monomer (VAM) used in adhesives, paints, paper coatings, and barrier resins; and industrial ethanol used as a solvent or in the production of chemical intermediates such as ethyl acetate and ethyl acrylate. Ethylene may be converted into ethylene vinyl acetate (EVA) or poly(ethylene vinyl acetate) (PEVA). EVA may be converted into thermoplastics. EVA may be incorporated into or used to make hot melt adhesives, hot glue sticks, sock cleats, plastic wrap, craftsman foam sheets, and foam stickers. EVA may be incorporated into or used to make drug delivery devices. In some embodiments, EVA can be used to make foams. In one embodiment, EVA foam is used as padding for sports equipment such as ski boots, bicycle saddles, hockey pads, boxing and mixed martial arts gloves and helmets, wakeboard boots, water ski boots, fishing rods, and fishing reel handles. In some embodiments, EVA foam is used as a shock absorber in sports shoes. EVA may also be used as an EVA-based compression-molded foam. EVA may be incorporated into or used to create floats for commercial fishing gear and floating eyewear. EVA may also be incorporated into or used to create encapsulation materials for crystalline silicon solar cells. In some embodiments, EVA may be incorporated into or used to create coating formulations for anti-skid surfaces, gravel, fishing rods, cork replacements, packaging, textiles, bookbinding, adhesive plastic films, metal surfaces, coated paper, redispersible powders for gypsum and cement renderings, and interior water-based paints. EVA may undergo hydrolysis to provide ethylene vinyl alcohol (EVOH) copolymers.EVA can be used in orthotics, surfboard and skimboard traction pads, car mats, artificial flowers, cold flow improvers for diesel fuel, separators in HEPA filters, thermoplastic mouthguards, conditioning and waterproofing, nicotine transdermal patches, and plastic model kit parts.

[0181] Ethylene can further be used as a monomer base for the production of various polyethylene oligomers by coordination polymerization using metal chloride or metal oxide catalysts. The most common catalyst consists of titanium(III) chloride, the so-called Ziegler-Natta catalyst. Another common catalyst is the Phillips catalyst, which is prepared by depositing chromium(VI) oxide on silica.

[0182] The polyethylene oligomers so produced can be classified according to their density and branching. Furthermore, the mechanical properties are highly dependent on variables such as the degree and type of branching, crystalline structure, and molecular weight. There are several types of polyethylene that can be produced from ethylene, including, but not limited to: Ultra-high molecular weight polyethylene (UHMWPE), Ultra-low molecular weight polyethylene (ULMWPE or PE-WAX), High molecular weight polyethylene (HMWPE), High density polyethylene (HDPE), High density cross-linked polyethylene (HDXLPE), Cross-linked polyethylene (PEX or XLPE), Medium density polyethylene (MDPE), Linear low-density polyethylene (LLDPE), Low-density polyethylene (LDPE), Very low density polyethylene (VLDPE), and Chlorinated polyethylene (CPE).

[0183] Low-density polyethylene (LDPE) and linear low-density polyethylene (LLDPE) are primarily used in film applications such as food and non-food packaging, shrink and stretch films, and non-packaging applications. High-density polyethylene (HDPE) is primarily used in blow-molded and injection-molded applications such as containers, drums, housewares, caps, and pallets. HDPE can also be extruded into film for water, gas, and irrigation pipes, as well as reject bags, carrier bags, and industrial linings.

[0184] According to one embodiment, the ethylene formed from the above disclosure can be converted to ethylene oxide via direct oxidation according to the following equation: C2H4+O2→C2H4O The ethylene oxide thereby produced is a key chemical intermediate in several commercially important processes, including the production of monoethylene glycol. Other EO derivatives include ethoxylates (for use in shampoos, restaurant cleaners, etc.), glycol ethers (solvents, fuels, etc.), and ethanolamines (surfactants, personal care products, etc.).

[0185] According to one embodiment of the present disclosure, ethylene oxide produced as described above can be used to produce commercial quantities of monoethylene glycol according to the following equation: (CH2CH2)O+H2O→HOCH2CH2OH According to another embodiment, the claimed microorganism can be modified to directly produce monoethylene glycol. As disclosed in WO 2019 / 126400, the entirety of which is incorporated herein by reference, the microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. The microorganism can be modified to produce monoethylene glycol directly. the enzyme capable of converting 3-phospho-D-glycerate to 3-phosphonooxypyruvate, an enzyme capable of converting 3-phosphonooxypyruvate to 3-phospho-L-serine, an enzyme capable of converting 3-phospho-L-serine to serine, an enzyme capable of converting serine to glycine, an enzyme capable of converting 5,10-methylenetetrahydrofolate to glycine, an enzyme capable of converting serine to hydroxypyruvate, an enzyme capable of converting D-glycerate to hydroxypyruvate, an enzyme capable of converting malate to glyoxylate, an enzyme capable of converting glyoxylate to glycolate, an enzyme capable of converting hydroxypyruvate to glycolaldehyde, and / or an enzyme capable of converting glycolaldehyde to ethylene glycol.

[0186] In one embodiment, the microorganism comprises one or more of a heterologous enzyme capable of converting oxaloacetate to citrate, a heterologous enzyme capable of converting glycine to glyoxylate, a heterologous enzyme capable of converting isocitrate to glyoxylate, a heterologous enzyme capable of converting glycolate to glycolaldehyde, or any combination thereof. In some embodiments, the heterologous enzyme capable of converting oxaloacetate to citrate is citrate [Si]-synthase [2.3.3.1], ATP-citrate synthase [2.3.3.8], or citrate (Re)-synthase [2.3.3.3], and the heterologous enzyme capable of converting glycine to glyoxylate is alanine-glyoxylate transaminase [2.6.1.44], serine-glyoxylate transaminase [2.6.1.45], serine-pyruvate transaminase [2.6.1.51], glycine-oxaloacetate transaminase [2.6.1.35], glycine transaminase [2.6.1.4], glycine dehydrogenase [1.4.1.10], alanine dehydrogenase [1.4.1.1], or glycine In some embodiments, the heterologous enzyme capable of converting isocitrate to glyoxylate is isocitrate lyase [4.1.3.1], and the heterologous enzyme capable of converting isocitrate to glyoxylate is isocitrate lyase [4.1.3.1]. In some embodiments, the heterologous enzyme capable of converting glycolate to glycolaldehyde is glycolaldehyde dehydrogenase [1.2.1.21], lactaldehyde dehydrogenase [1.2.1.22], succinate-semialdehyde dehydrogenase [1.2.1.24], 2,5-dioxovalerate dehydrogenase [1.2.1.26], aldehyde dehydrogenase [1.2.1.3 / 4 / 5], betaine aldehyde dehydrogenase [1.2.1.8], or aldehyde ferredoxin oxidoreductase [1.2.7.5], or a combination thereof.

[0187] Monoethylene glycol produced according to any of the described processes can be used as a feedstock for producing polyester fibers for textile applications including nonwovens, diaper coverstock, building materials, construction materials, road construction fabrics, filters, fiberfill, felt, transportation upholstery, paper reinforcement, tape reinforcement, tents, rope, string, sails, fishing nets, seat belts, laundry bags, synthetic artery substitutes, carpets, rugs, apparel, sheets and pillowcases, towels, curtains, drapery fabric, bedding, blankets, and a variety of other products.

[0188] MEG can be used alone as a liquid coolant, antifreeze, preservative, dehydrant, drilling fluid, or any combination thereof. The MEG produced can also be used to produce secondary products such as insulation materials, polyester films, de-icing fluids, heat transfer fluids, automotive antifreeze and other liquid coolants, preservatives, dehydrants, drilling fluids, water-based adhesives, latex paint and asphalt emulsions, electrolytic capacitors, paper, and polyester resins for use in synthetic skin care products.

[0189] Importantly, the monoethylene glycol produced can be converted to the polyester resin polyethylene terephthalate (PET) according to one of two main processes. The first process involves esterifying the monoethylene glycol with dimethyl terephthalate according to the following two-step process: First step C6H4(CO2CH3)2+2 HOCH2CH2OH→C6H4(CO2CH2CH2OH)2+2 CH3OH Second step n C6H4(CO2CH2CH2OH)2→ [(CO)C6H4(CO2CH2CH2O)] n +n HOCH2CH2OH

[0190] Alternatively, monoethylene glycol can be subjected to an esterification reaction utilizing terephthalic acid according to the following reaction: n C6H4(CO2H)2+n HOCH2CH2OH→[(CO)C6H4(CO2CH2CH2O)] n +2n H2O

[0191] Polyethylene terephthalate, prepared either by esterification or deesterification of monoethylene glycol, has considerable applicability in the manufacture of bottles and other packaging applications, particularly plastic bottles. It can also be used as high-strength fibers such as Dacron, as part of durable press blends with other fibers such as rayon, wool, and cotton, as fiber fillers used in insulating clothing, furniture, and pillows, as artificial silk, and as carpet fibers. It can also be used to manufacture yarns for automobile tires, conveyor and drive belts, fire and garden hose reinforcement, seat belts, nonwovens for stabilizing drains, culverts, and railroad rights-of-way, and nonwovens used in diaper topsheets and disposable medical garments.

[0192] At its high molecular weight, PET can be made into a high-strength plastic that can be formed by all the common methods used with other thermoplastics. Magnetic recording tape and photographic film are made by extrusion of PET film. Molten PET can be blown into strong, rigid, transparent containers that are virtually impermeable to gases and liquids. In this form, PET is widely used in bottles, especially plastic bottles and jars.

[0193] The present disclosure provides compositions comprising ethylene glycol produced by a microorganism according to the methods described herein. For example, the composition comprising ethylene glycol may be an antifreeze, a preservative, a dehydrating agent, or a drilling fluid.

[0194] The present disclosure also provides polymers containing ethylene glycol produced by microorganisms according to the methods described herein. Such polymers may be homopolymers, such as polyethylene glycol, or copolymers, such as polyethylene terephthalate. Methods for synthesizing these polymers are well known in the art. See, for example, Herzberger et al., Chem Rev., 116(4): 2170-2243 (2016) and Xiao et al., Ind Eng Chem Res. 54(22): 5862-5869 (2015).

[0195] The present disclosure further provides polyethylene glycol conjugates. In some embodiments, the polyethylene glycol (PEG) conjugate comprises PEG conjugated to a biopharmaceutical, a protein, an antibody, an anticancer drug, or any combination thereof. In other embodiments, the PEG conjugate is diethyl terephthalate (DET). In some embodiments, the PEG conjugate is dimethoxyethane.

[0196] The present disclosure further provides a composition comprising a polymer comprising ethylene glycol produced by a microorganism according to the methods described herein. For example, the composition may be a fiber, resin, film, or plastic.

[0197] In one embodiment, ethanol or ethyl alcohol produced according to the methods of the present disclosure is used in antiseptic hand lotions (WO 2014 / 100851), therapeutic treatments for methylene glycol and methanol poisoning (WO 2006 / 088491), pain relievers (WO 2011 / 034887) and oral hygiene products (U.S. Pat. No. 6,811,769), as well as antibacterial preservatives (U.S. Pat. App. No. 2013 / 0230609), engine fuels (U.S. Pat. No. 1,128,549), rocket fuels (U.S. Pat. No. 3,020,708), plastics, fuel cells (U.S. Pat. No. 2,020,708), and other applications. ,405,986), household fire fuel (U.S. Pat. No. 4,692,168), industrial chemical precursor (U.S. Pat. No. 3,102,875), cannabis solvent (WO 2015 / 073854), as a winterization extraction solvent (WO 2017 / 161387), paint masking product (WO 1992 / 008555), paint or tincture (U.S. Pat. No. 1,408,091), DNA and RNA purification and extraction (WO 1997 / 010331), and cooling bath for various chemical reactions (U.S. Pat. No. 2,099,090). In addition to the foregoing, ethanol produced by the disclosed methods can be used in any other application where ethanol may otherwise be applicable.

[0198] In additional embodiments, the isopropanol or isopropyl alcohol (IPA) produced according to the present methods may be used in a number of product applications, including either isolated or as a feedstock for the manufacture of more complex products. Isopropanol may also be used in products such as cosmetics and personal care products, deicers, paints and resins, foods, inks, adhesives, and pharmaceutical tablets, as well as solvents for pharmaceuticals, including disinfectants, sterilants, and skin creams.

[0199] The resulting IPA can be used in the extraction and purification of natural products such as vegetable and animal oils and fats. Other applications include its use as a cleaning and drying agent in the manufacture of electronic components and metals, and as an aerosol solvent in medical and veterinary products. It can also be used as a coolant, coupling agent, polymerization modifier, deicer, and antiseptic in pile manufacturing.

[0200] Alternatively, IPA produced according to the methods of the present disclosure may be used to produce additional useful compounds, including plastics, derivative ketones such as methyl isobutyl ketone (MIBK), isopropylamine, and isopropyl esters. Still further, IPA can be converted to propylene according to the following equation: CH3CH2CH2OH→CH3-CH=CH2

[0201] The propylene produced can be used as a monomer base for the production of various polypropylene oligomers by chain growth polymerization via either gas-phase or bulk reactor systems. The most common catalysts consist of titanium(III) chloride, the so-called Diegler-Natta catalyst, and metallocene catalysts.

[0202] The polypropylene oligomers so produced may be classified according to tactic and may be formed into a number of products, either by extrusion or molding of polypropylene pellets, including expanded polypropylene, which may be used in plumbing products, heat-resistant items such as kettles and food containers, disposable bottles (including plastic bottles), transparent bags, flooring materials such as rugs and mats, ropes, adhesive stickers, and building materials. Polypropylene may also be used in hydrophilic clothing and medical dressings.

[0203] In some embodiments, ethylene is used to make polyethylene: a common plastic used in a variety of consumer products such as plastic bags, plastic film, geomembranes, and containers including bottles.

[0204] In one embodiment, ethylene is used to make ethylene glycol, a raw material in the production of polyester fibers for clothing, upholstery, shirts, and blankets. In another embodiment, ethylene is used in the production of freeze protection in refrigeration and heating systems.

[0205] In another embodiment, ethylene is used in ethylene oxide, which is used to make other chemicals used in the manufacture of products such as detergents, thickeners, solvents, plastics, and various organic chemicals. In some embodiments, ethylene oxide is used as a sterilizing agent and fumigation agent for medical devices.

[0206] In some embodiments, ethylene is used in vinyl acetate, which is used to make paints, adhesives, paper coatings, and other chemicals used in textiles.

[0207] In another embodiment, ethylene is used in ethylene dichloride to produce vinyl chloride, which is used to make polyvinyl chloride (PVC), which is used to make a variety of plastic and vinyl products, including pipes, wire and cable coatings, and packaging materials.

[0208] In some embodiments, ethylene is used with aluminum alkyls as a catalyst to increase the efficiency of making ethylene and other chemicals.

[0209] In some embodiments, ethylene is used in ethylene propylene rubber (EPR), which is used in electrical insulation, roof membranes, radiator hoses in vehicles, and waterproofing.

[0210] In another embodiment, ethylene is used in agriculture as a plant hormone and is used in agriculture to force fruit ripening.

[0211] In some embodiments, ethylene is used to make styrene, which is then used to make polystyrene, which is used in a variety of consumer products such as disposable tableware, CD and DVD cases, and insulating materials.

[0212] In embodiments, ethylene is used to produce alpha olefins, which are used as comonomers in the production of polyethylene, and in the production of surfactants and lubricants.

[0213] In one embodiment, ethylene is used to produce butadiene, hi some embodiments, butadiene is used in rubber tires.

[0214] In one embodiment, a method for the continuous production of ethylene, the process comprises passing a gaseous substrate through a bioreactor containing a culture of a recombinant C1-fixing microorganism capable of producing ethylene in a culture medium, whereby the microorganism converts the gaseous substrate to ethylene, and recovering the ethylene from the bioreactor.

[0215] In other embodiments, the ethylene is converted into components used in the manufacture of tires. In one embodiment, the ethylene is converted into components used in tire screws.

[0216] 10. The method of claim 9, wherein the tire is near the end of its life.

[0217] 10. The method of embodiment 1, wherein the gaseous substrate is derived from a process involving tires.

[0218] 10. The method of embodiment 1, wherein the gaseous substrate is derived from a product recycling process or a sustainable chemical process.

[0219] 10. The method of claim 1, further comprising converting the ethylene into components used to manufacture new tires.

[0220]

[0023] In an embodiment, the method according to the present invention comprises a resin component selected from ethylene and other olefins combined with a synthetic component selected from butadiene and isoprene to form a hybrid polymer for use in manufacturing tires.

[0221] One embodiment is directed to a method for producing a polymer from a gaseous substrate, comprising: a first gas fermentation process producing at least one first product selected from butadiene, isoprene, conjugated dienes, or any combination thereof; a second gas fermentation process producing at least one second product selected from ethylene and olefins, or any combination thereof; and wherein the at least one first product and the at least one second product copolymerize to form the polymer.

[0222]

[0013] 3. The method of embodiment 1, wherein the first gas fermentation process and the second gas fermentation process are carried out in parallel.

[0223]

[0013] 2. The method of claim 1, wherein the first gas fermentation process and the second gas fermentation process are both carried out continuously.

[0224]

[0013] 3. The method of claim 1, wherein a first gas fermentation process produces a rubber component, a second gas fermentation process produces a resin component, and the rubber component and the resin component are copolymerized to form a polymer.

[0225]

[0013] In an embodiment, the method according to the present invention, wherein the rubber component and the resin component are copolymerized by a suitable polymerization catalyst.

[0226] 10. The method of claim 9, wherein the rubber component is selected from butadiene, isoprene, conjugated dienes, or any combination thereof.

[0227] 10. The method of embodiment 1, wherein the resin component is selected from ethylene, olefin, or any combination thereof.

[0228]

[0013] The method of embodiment 1, wherein the suitable polymerization catalyst further comprises another component contained in a typical polymerization catalyst composition containing a metallocene complex.

[0229] 10. The method of embodiment 1, wherein the metallocene complex is a complex compound having one or more cyclopentadienyl groups or derivative cyclopentadienyl groups bonded to the metal center.

[0230] 10. The method of embodiment 1, wherein the central metal is selected from a lanthanide element, scandium, yttrium, or any combination thereof.

[0231] 10. The method of claim 9, wherein the central metal is selected from samarium (Sm), neodymium (Nd), praseodymium (Pr), gadolinium (Gd), cerium (Ce), holmium (Ho), scandium (Sc), and yttrium (Y).

[0232] 10. The method of claim 1, further comprising converting the polymer into a tire.

[0233] One embodiment for the circular production of tires from a gaseous substrate is directed to a first gas fermentation process to produce at least one first product selected from butadiene, isoprene, conjugated dienes, or any combination thereof, and a second gas fermentation process to produce at least one second product selected from ethylene and olefins, or any combination thereof, wherein the at least one first product and the at least one second product are copolymerized to form a polymer, and the substrate is derived from the process comprising tires.

[0234] 10. The method of embodiment 1, wherein the substrate is derived from a process involving end-of-life tires.

[0235] One embodiment is directed to a method for the circular production of tires, the method comprising: 1) passing a gaseous substrate through a first bioreactor containing a culture of a recombinant C1-fixed microorganism capable of producing at least one first product selected from butadiene, isoprene, conjugated dienes, or any combination thereof in a culture medium, whereby the microorganism converts the gaseous substrate to the at least one first product, and recovering the at least one first product from the bioreactor; 2) passing the gaseous substrate through a second bioreactor containing a culture of a recombinant C1-fixed microorganism in a culture medium, whereby the microorganism converts the gaseous substrate to at least one second product selected from ethylene and olefins, or any combination thereof, whereby the microorganism converts the gaseous substrate to at least one second product, and recovering the at least one second product from the bioreactor; 3) polymerizing the at least one first product with at least one second product in the presence of a suitable polymerization catalyst to form a hybrid polymer; and 4) converting the hybrid polymer into a tire.

[0236]

[0013] The method of embodiment 1, wherein the suitable polymerization catalyst further comprises another component contained in a typical polymerization catalyst composition containing a metallocene complex.

[0237] 10. The method of embodiment 1, wherein the metallocene complex is a complex compound having one or more cyclopentadienyl groups or derivative cyclopentadienyl groups bonded to the metal center.

[0238] 10. The method of embodiment 1, wherein the central metal is selected from a lanthanide element, scandium, yttrium, or any combination thereof.

[0239] 10. The method of claim 9, wherein the central metal is selected from samarium (Sm), neodymium (Nd), praseodymium (Pr), gadolinium (Gd), cerium (Ce), holmium (Ho), scandium (Sc), and yttrium (Y).

[0240] 10. The method of claim 1, wherein the first bioreactor and the second bioreactor are run in parallel.

[0241] 10. The method of claim 1, wherein both the first bioreactor and the second bioreactor are operated continuously.

[0242] 10. The method of embodiment 1, wherein the substrate is derived from a process involving end-of-life tires.

[0243]

[0023] 3. The method of claim 1, further comprising converting the isoprenoid into a product selected from synthetic rubber, styrene-containing block polymers, thermoplastic rubber, pressure-sensitive or thermoset adhesives, butyl rubber, citric acid, linalool, ionone, myrcene, L-menthol, N,N-diethylnerylamine, geraniol, nerolidol, flavors, fragrances, fuel additives, plastics, and polyisoprene.

[0244] 10. The method of claim 1, further comprising converting the butadiene to a product selected from styrene butadiene rubber, synthetic rubber, tires, tire components, thermoplastic rubber, shoes, shoe soles, adhesives, sealants, asphalt, polymer-modified components, nylon, ABS resin, chloroprene / neoprene rubber, nitrile rubber, plastics, acrylic resins, acrylonitrile-butadiene-styrene resins, and synthetic elastomers.

[0245] One embodiment is directed to a method for chemical recycling, the method including providing to a pyrolysis, gasification, and / or partial oxidation process, a gas fermentation process, a pharmaceutical manufacturing process to produce products including butadiene, isoprenoids, ethylene, polyethylene terephthalate (PET), or any combination thereof, a synthetic rubber manufacturing process, a tire manufacturing process, a tire-using process, a process for collecting and shredding used tires, and returning to the pyrolysis, gasification, and / or partial oxidation process.

[0246] One embodiment is directed to a method for chemical recycling, the method comprising: 1) subjecting waste to a pyrolysis, gasification, and / or partial oxidation process; 2) subjecting waste to a gas fermentation process; 3) subjecting waste to a pharmaceutical manufacturing process to produce products including butadiene, isoprenoids, ethylene, polyethylene terephthalate (PET), or any combination thereof; 4) subjecting waste to a synthetic rubber manufacturing process; 5) subjecting waste to a tire manufacturing process; 6) subjecting waste to a tire-using process; 7) subjecting waste to a process for collection and shredding of used tires; and 8) subjecting waste to a pyrolysis, gasification, and / or partial oxidation process.

[0247] One embodiment is directed to a method for chemical recycling, the method comprising: 1) providing to a pyrolysis, gasification, and / or partial oxidation process; 2) providing to a gas fermentation process; 3) providing to a pharmaceutical manufacturing process to produce a product; 4) providing to a synthetic rubber manufacturing process; 5) providing to a tire manufacturing process; 6) providing to a tire use process; 7) providing to a process for collecting and shredding used tires; and 8) returning to the pyrolysis, gasification, and / or partial oxidation process.

[0248] Another embodiment is directed to a method for chemical recycling, the method including: 1) pyrolysis, gasification, and / or a partial oxidation process to produce an effluent; 2) passing the effluent through a gas fermentation process to produce a product; 3) passing the gas fermentation product through a chemical manufacturing process to produce a product; 4) passing the product through a synthetic rubber manufacturing process to produce synthetic rubber; 5) passing the synthetic rubber product through a tire manufacturing process to produce tires; 6) providing the tires to a tire-using process; 7) passing the used tires through a process for used tire collection and shredding; and 8) returning the used tires to the pyrolysis, gasification, and / or partial oxidation process.

[0249] One embodiment is directed to providing a method and genetically engineered microorganism capable of producing ethylene from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding an ethylene-forming enzyme (EFE).

[0250] In some embodiments of the methods disclosed herein, the microorganism is a recombinant C1-fixing microorganism capable of producing ethylene from a gaseous substrate comprising a nucleic acid encoding a group of exogenous enzymes including ethylene-forming enzymes (EFEs).

[0251] In some embodiments of the microorganisms disclosed herein, the microorganisms are recombinant C1-fixing microorganisms capable of switching cellular load in the production of ethylene, wherein the microorganisms comprise nucleic acids encoding a group of exogenous enzymes including an ethylene-forming enzyme (EFE) and one or more inducible promoters.

[0252] The microorganism of an embodiment, further comprising a nucleic acid encoding a group of exogenous enzymes including alpha-ketoglutarate permease (AKGP), wherein the nucleic acid is operably linked to a promoter.

[0253] A microorganism of an embodiment, wherein the microorganism is selected from the group consisting of Cupriavidus necator and Ralstonia eutropha.

[0254] A microorganism of an embodiment, wherein the microorganism is Cupriavidus necator.

[0255] The microorganism of an embodiment, further comprising a nucleic acid encoding an alpha-ketoglutarate permease, wherein the nucleic acid is codon optimized for expression in the microorganism.

[0256] The microorganism of an embodiment, wherein the one or more inducible promoters are selected from an H2-inducible promoter, a phosphate-limited inducible promoter, a nitrogen-limited inducible promoter, or any combination thereof.

[0257] The microorganism of an embodiment, wherein the inducible promoter is a phosphate-limited inducible promoter.

[0258] The microorganism of an embodiment, wherein the phosphate concentration is about 0 to 0.5 mM.

[0259] The microorganism of an embodiment, wherein the phosphate concentration is about 0.52 mM.

[0260] 2. The microorganism of embodiment 1, wherein the EFE is codon-optimized for expression in the microorganism.

[0261] 2. The microorganism of embodiment 1, further comprising a disruptive mutation in one or more genes.

[0262]

[0023] The microorganism of an embodiment, wherein ethylene is converted into a derivative material selected from polyethylene (PE), polyethylene terephthalate (PET), polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), sustainable aviation fuel (SAF), or any combination thereof.

[0263] The microorganism of an embodiment, wherein the gaseous substrate comprises CO2 and an energy source.

[0264] The microorganism of an embodiment, wherein the gaseous substrate comprises CO2, and H2, O2, or both.

[0265] One embodiment is directed to a method for the continuous production of ethylene, the process comprising passing a gaseous substrate through a bioreactor containing a culture of the recombinant C1-fixing microorganism of claim 1 in a culture medium such that the microorganism converts the gaseous substrate into ethylene, and recovering the ethylene from the bioreactor.

[0266] In one embodiment, the present invention is directed to a method of culturing a microorganism according to an embodiment, comprising growing the microorganism in a medium comprising a gaseous substrate, wherein the gaseous substrate comprises CO2.

[0267] The method of an embodiment, wherein the gaseous substrate comprises industrial waste or off-gas.

[0268] The method of an embodiment, further comprising an energy source.

[0269] The method of an embodiment, wherein the energy source is provided intermittently.

[0270] The method of the embodiment, wherein the energy source is H2.

[0271] One embodiment is directed to a method comprising growing a microorganism in a medium comprising a gaseous substrate, the gaseous substrate comprising CO2 and an energy source.

[0272] The method of the embodiment further comprises co-producing ethylene and microbial biomass.

[0273] The method of the embodiment, wherein switching the cellular load comprises limiting intracellular oxygen concentration.

[0274] The method of the embodiment, wherein switching the cell load comprises limiting the dissolved oxygen concentration.

[0275] The method of an embodiment, wherein the dissolved oxygen concentration is at least about 0.5% saturation (% saturation) to 1.0% saturation, and up to about 50% saturation to 60% saturation.

[0276] The method of the embodiment, wherein the dissolved oxygen concentration is at least about 0.5% saturation to 1.0% saturation and up to about 60% saturation to 70% saturation.

[0277] The method of the embodiment, wherein the dissolved oxygen concentration is at least about 0.5% saturation to 1.0% saturation and up to about 70% saturation to 80% saturation.

[0278] The method of an embodiment, wherein the dissolved oxygen concentration is at least about 0.5% saturation to 1.0% saturation, and up to about 80% saturation to 90% saturation.

[0279] The method of an embodiment, wherein the dissolved oxygen concentration is at least about 0.5% saturation to 1.0% saturation, and up to about 90% saturation to 100% saturation.

[0280] The method of an embodiment, wherein the dissolved oxygen concentration is at least about 0.01% saturation to 1.0% saturation and up to about 50% saturation to 60% saturation.

[0281] The method of the embodiment, wherein the dissolved oxygen concentration is at least about 0.01% saturation to 1.0% saturation and up to about 60% saturation to 70% saturation.

[0282] The method of the embodiment, wherein the dissolved oxygen concentration is at least about 0.01% saturation to 1.0% saturation and up to about 70% saturation to 80% saturation.

[0283] The method of an embodiment, wherein the dissolved oxygen concentration is at least about 0.01% saturation to 1.0% saturation and up to about 80% saturation to 90% saturation.

[0284] The method of the embodiment, wherein the dissolved oxygen concentration is at least about 0.01% saturation to 1.0% saturation and up to about 90% saturation to 100% saturation.

[0285] The method of the embodiment, wherein O2 is provided to the inlet at about 4% to about 30% by volume.

[0286] The method of the embodiment, wherein O2 is provided to the inlet at about 1% to about 50% by volume.

[0287] The method of the embodiment, wherein switching the cell load comprises limiting the steady-state phosphate concentration.

[0288] The method of embodiments, wherein switching the cell load comprises limiting the steady-state phosphate concentration from about 0 mM to about 0.50 mM.

[0289] The method of embodiments, wherein switching the cell load comprises limiting the steady-state phosphate concentration to about 0.05 mM to about 0.50 mM.

[0290] The method of embodiments, wherein switching the cell load comprises limiting the steady-state phosphate concentration to between about 0.01 mM and about 0.60 mM.

[0291] The method of embodiments, wherein switching the cell load comprises limiting the steady-state phosphate concentration to between about 0.01 mM and about 0.70 mM.

[0292] The method of embodiments, wherein switching the cell load comprises limiting the steady-state phosphate concentration to between about 0.01 mM and about 0.80 mM.

[0293] The method of embodiments, wherein switching the cell load comprises limiting the steady-state phosphate concentration to between about 0.01 mM and about 0.90 mM.

[0294] The method of embodiments, wherein switching the cell load comprises limiting the steady-state phosphate concentration to about 0.01 mM to about 1.0 mM.

[0295] The method of the embodiment, wherein switching the cell load comprises limiting the steady-state phosphate concentration to about 0.52 mM.

[0296] The method of the embodiment, wherein the microbial biomass is suitable as animal feed.

[0297]

[0013] 3. The method of embodiment 1, wherein the gaseous substrate further comprises H2, O2, or both.

[0298] In some embodiments of the microorganisms disclosed herein, the microorganisms produce commodity chemicals, microbial biomass, single cell proteins (SCPs), one or more intermediates, or any combination thereof.

[0299] In some aspects, the microbial biomass has a unit value. In one embodiment, the microbial biomass has a market value.

[0300] In some aspects of the microorganisms disclosed herein, the microorganism is derived from a parent bacterium selected from the group consisting of Cupriavidus necator.

[0301] In some embodiments of the microorganisms disclosed herein, the product is selected from the group consisting of 1-butanol, butyrate, butene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, terpenes, isoprene, fatty acids, fatty alcohols, 2-butanol, 1,2-propanediol 1-propanol, 1-hexanol, 1-octanol, chorismic acid derived products, 3-hydroxybutyric acid, 1,3-butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, isoamyl alcohol, or monoethylene glycol.

[0302] The present disclosure further provides a genetically engineered C1-fixing microorganism comprising a microbial biomass and at least one excipient.

[0303] The present disclosure further provides genetically engineered C1-fixing microorganisms, wherein the animal feed may be suitable for feeding one or more of beef cattle, dairy cattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, banteng, gayal, yaks, chickens, turkeys, ducks, geese, quail, guinea pigs, squabs / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents.

[0304] The present disclosure further provides a genetically engineered C1-fixing microorganism, wherein the microorganism is suitable as a single cell protein (SCP).

[0305] The present disclosure further provides genetically engineered C1-fixing microorganisms, which are suitable as cell-free protein synthesis (CFPS) platforms.

[0306] The present disclosure further provides a genetically engineered C1-fixing microorganism, wherein the product is native to the microorganism.

[0307] In some embodiments of the methods disclosed herein, the substrate comprises one or more of CO, CO2, and H2.

[0308] According to another embodiment, the claimed microorganisms can be modified to directly produce commodity chemicals such as those described in U.S. Patent Application Publication No. 2023 / 0092645A1, the disclosure of which is incorporated herein by reference. In one embodiment, the commodity chemical is selected from ethanol, isopropanol, monoethylene glycol, sulfuric acid, propylene, sodium hydroxide, sodium carbonate, ammonia, benzene, acetic acid, ethylene oxide, formaldehyde, methanol, or any combination thereof. In one embodiment, the commodity chemical is aluminum sulfate, ammonia, ammonium nitrate, ammonium sulfate, carbon black, chlorine, diammonium phosphate, monoammonium phosphate, hydrochloric acid, hydrogen fluoride, hydrogen peroxide, nitric acid, oxygen, phosphoric acid, sodium silicate, titanium dioxide, or any combination thereof. In another embodiment, the commodity chemical is acetic acid, acetone, acrylic acid, acrylonitrile, adipic acid, benzene, butadiene, butanol, caprolactam, cumene, cyclohexane, dioctyl phthalate, ethylene glycol, methanol, octanol, phenol, phthalic anhydride, polypropylene, polystyrene, polyvinyl chloride, polypropylene glycol, propylene oxide, styrene, terephthalic acid, toluene, toluene diisocyanate, urea, vinyl chloride, xylene, or any combination thereof. The method according to an embodiment, wherein the commodity chemical is utilized in a sector selected from plastics, synthetic fibers, synthetic rubber, dyes, pigments, paints, coatings, fertilizers, agrochemicals, pesticides, cosmetics, soaps, cleaning agents, detergents, pharmaceuticals, mining, or any combination thereof.

[0309] In another embodiment, the method comprises converting commodity chemicals into ethanol, acetate, 1-butanol, butyrate, 2,3-butanediol, lactate, butene, butadiene, methyl ethyl ketone (2-butanone), ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, terpenes, isoprene, fatty acids, 2-butanol, 1,2-propanediol, 1-propanol, 1-hexanol, 1-octanol, chorismic acid derived products, 3-hydroxybutyrate, 1,3-butanediol, 2-hydroxyisobutyrate, 2-hydroxyisobutyric acid, isopropyl alcohol, propyl ketone ... Isobutylene, adipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, isoamyl alcohol, monoethylene glycol, disinfectant hand lotion, methylene glycol poisoning treatment, methanol poisoning treatment, pharmaceutical solvent for painkillers, oral hygiene products, antibacterial preservatives, engine fuel, rocket fuel, plastics, fuel cells, home fireplace fuel, precursors to industrial chemicals, cannabis solvent, extraction solvent for winterization, paint masking agent, paints, tinctures, purification and extraction of DNA and RNA, cooling baths for various chemical reactions, raw materials Ethylene in ethanol, anesthetics, ethylene and nitrogen in fruit ripening, fertilizers, safety glass, oxyfuel in metal cutting, welding, high velocity thermal spraying, refrigerants, raw materials for polyethylene, raw materials for polyethylene, raw materials for PET, raw materials for PVC, textiles, packaging, paints, adhesives, ethylene dichloride (EDC), vinyl chloride monomer (VCM), alpha olefins, linear alpha olefins, detergent alcohol, plasticizer alcohol, vinyl acetate monomer (VAM), barrier resins, industrial ethanol, ethyl acetate, ethyl acrylate, polyethylene oligomers, ultra-high molecular weight polyethylene (ULHMPE) UHMWPE), ultra-low molecular weight polyethylene (ULMWPE or PE-WAX), high molecular weight polyethylene (HMWPE), high density polyethylene (HDPE), high density cross-linked polyethylene (HDXLPE), cross-linked polyethylene (PEX or XLPE), medium density polyethylene (MDPE), low density polyethylene (LDPE), very low density polyethylene (VLDPE), chlorinated polyethylene (CPE), film, food packaging, non-food packaging, shrink film, stretch film, containers, drums, household goods, caps, pallets, pipes, garbage bags, carry bags,Industrial linings, ethylene oxide, ethoxylates, shampoos, kitchen detergents, glycol ethers, ethanolamines, surfactants, personal care products, polyester fibers, woven fabrics, nonwoven fabrics, diaper cover stock, road construction fabrics, filters, fiberfill, felt, transportation upholstery, paper reinforcement, tape reinforcement, tents, ropes, string, sails, fishing nets, seat belts, laundry bags, synthetic artery substitutes, carpets, rugs, apparel, sheets, pillowcases, towels, curtains, curtain fabrics, bedsticks, blankets, liquid coolants, antifreeze, preservatives, dehydrating agents, drilling oil, polyester resins, insulation materials, polyester film, deicing fluids, heat transfer fluids, automotive antifreeze, water-based adhesives, latex paints, asphalt emulsions, electrolytic capacitors, synthetic leather, polyester resin PET, bottles, PET bottles, high-strength fibers, Dacron, durable press blends , insulated clothing, furniture stuffing, pillow stuffing, artificial silk yarn, carpet fiber, automobile tire thread, conveyor belts, drive belts, fire and garden hose reinforcement, nonwoven fabrics for drainage ditches, culverts and railway stabilization, nonwoven fabrics for disposable diaper top sheets, disposable medical clothing, high-strength plastics, magnetic recording tape, photographic film, raw materials, cosmetic solvents, inks, pharmaceutical tablets, disinfectants, bactericides, skin creams, refined vegetable oils and fats, refined animal oils and fats, cleaning agents, drying agents, aerosol solvents, derivative ketones, isopropylamine, isopropyl esters, propylene, polypropylene oligomers, polymerization modifiers, coupling agents, heat-resistant products, kettles, food containers, disposable bottles, transparent bags, flooring materials, mats, adhesive seals, expanded polypropylene, building materials, hydrophilic clothing, medical dressings, or combinations thereof.

[0310] In another embodiment, the method comprises converting a commodity chemical into a product selected from a humectant, a filter, a fire sprinkler system, a fuel for heating food, a heat transfer fluid, a non-reactive ingredient in a formulation, a deodorizer or air purifier, a softener, an arts and crafts adhesive or paste, a toy, a children's product, a freezer gel pack, a wood decay or fungus treatment, a tissue or organ preservation agent, an alkyd resin, a resin ester, an enamel, a lacquer, a latex paint, an asphalt emulsion, a thermoplastic resin, a hydration inhibitor, a water vapor scavenger, a shoe polish, a vaccine, a screen cleaning fluid, a water-based hydraulic fluid, a liquid cooling agent. Heat transfer agents for computers, personal lubricants, lubricants, toothpaste, antifoaming agents for the food industry, fire retardant hydraulic fluids, additives for electropolishing belts, industrial solvents, garbage bags, shower curtains, cups, tableware, medical equipment, durable consumer goods, non-durable consumer goods, plastic bags, plastic lids, industrial twine, construction materials, felt, oven trays, frozen food trays, microwave trays, artificial blood vessel scaffolding, artificial blood vessels, textile appliances, polyester artificial blood vessels, vehicle liner materials, soaps, cosmetics, laundry detergent jugs, laundry detergent, soap microplastics, microbeads, Cosmetic microbeads, detergent pods, disinfectants with scrubs, toothpaste with microbeads, facial cleansers, conditioners, body washes, hand cleaners, exfoliating products, bath products, shower gels, powdered laundry detergents, lotions, deodorants, toilet cleaners, sunscreen, shopping bags, mouthwash bottles, peanut butter containers, salad dressing and vegetable oil containers, polar fleece fiber, tote bags, paneling, milk jugs, juice bottles, bleach bottles, lubricating oil bottles, cereal box liners, recycling containers , floor tiles, drain pipes, benches, picnic tables, fences, wire jackets, sliding windows, decks, mudguards, road gutters, speed barriers, squeeze bottles, bread, dry cleaning bags, trash can liners, trash cans, compost bins, shipping envelopes, wood, syrup bottles, ketchup bottles, straws, medical bottles, battery cables, battery cases, disposable plates and cups, egg cartons, take-out containers, compact disc cases, notice boards and signs, synthetic fibers, yarn, stable phase change materials, thermal energy storage materials, nylon 6,6, including incorporating or converting into articles selected from nylon, tires, rubber, adiponitrile, shoes, footwear, or combinations thereof. [Example]

[0311] The following examples further illustrate the present disclosure but, of course, should not be construed as in any way limiting its scope.

[0312] Example 1: Ethylene production from formate as the sole carbon and energy source. A gene encoding an ethylene-forming enzyme was codon-adapted and synthesized for expression in Cupriavidus necator. The adapted gene, along with the constitutive promoter P10, was cloned into the broad-host-range expression vector pBBR1MCS2. The resulting product was used to transform E. coli, and positive clones identified by PCR were confirmed by DNA sequencing. The sequence-confirmed plasmid was then transformed into Cupriavidus necator PHB-4 via electroporation and selected on tryptic soy broth (TSB) agar plates containing 50 mg / L chloramphenicol. Transformants containing the pBBR1-Efe plasmid were confirmed via sequencing and single colonies were then grown in TSB at 30°C overnight and used to generate glycerol stocks for storage at -80°C. Strain recovery was performed by streaking onto TSB plates containing 50 mg / L chloramphenicol with a 72-hour incubation at 30°C.

[0313] A single colony from a freshly streaked TSB plate was used to inoculate 3 mL of TSB containing 50 mg / L chloramphenicol in a 14 mL Falcon round-bottom polystyrene test tube with a snap cap. After overnight incubation at 30°C and 200 rpm in a Thermo MAXQ shaker, 0.25 mL of the culture was used to inoculate 25 mL of formate medium in a 125 mL Erlenmeyer flask.

[0314] The culture was incubated at 30°C and 200 rpm for 48 hours, with 65 mM formic acid added at various times to control the pH between 6.5 and 7.5. After 48 hours, 20 mL of culture was transferred to a 160 mL serum bottle, 65 mM formic acid was added, and the bottle was sealed with an airtight septum. After an additional 16 hours of incubation, a 60 mL headspace volume was removed with an airtight syringe and analyzed for ethylene production via GC. Samples were analyzed for various hydrocarbons and oxyacid salts on a custom Wasson system. Ethylene was separated on a 50 m x 0.53 um Wasson PN 2378 column and analyzed via GC FID.

[0315] As shown in Figure 2 , the Cupriavidus necator strain carrying the Efe expression plasmid (pBBR1-Efe) produced more than 100 ppm ethylene, whereas no ethylene was detected in the control strain containing the empty pBBR1 plasmid.

[0316] As shown in Figure 4, the reaction abbreviations are as follows: ACALD, acetaldehyde dehydrogenase (acetylation); ACONT1, aconitase (citrate hydrolase); ACONT2, aconitase (isocitrate hydrolase); AKGDH, 2-oxogluphosphate dehydrogenase; ALCD2x, alcohol dehydrogenase (ethanol); ASPTA, aspartate transaminase; ATPS4m, ATP synthase (four protons for one ATP); CITt, citrate transporter; CS, citrate synthase; CYTCOBO3, cytochrome oxidase bo3 (ubiquinol-8: 4 protons); EFE, ethylene-forming reaction; ENO, enolase; FBA, fructose bisphosphate aldolase; FBP, fructose bisphosphatase; FDH, formate dehydrogenase; FUM, fumarase; GAPD, glyceraldehyde-3-phosphate dehydrogenase; GLUDC, glutamate decarboxylase; GLUS, glutamate synthase; H2td, hydrogen transport; HYDS, hydrogenase (NADH); ICDHx, isocitrate dehydrogenase (NAD); ICITt, isocitrate transport; LACDH, L-lactate dehydrogenase; MDH, malate dehydrogenase; ME1, malic enzyme (NAD); NADH16, NADH dehydrogenase (ubiquinone-8 and 3 protons); O2t, O2 transport (diffusion); PDH1, pyruvate dehydrogenase E1 component; PDH2, pyruvate dehydrogenase E2 component (dihydrolipoamide acetyltransferase); PDH3, dihydrolipoamide dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PPC, phosphoenolpyruvate carboxylase; PRUK, phosphoribulokinase; RBPC, ribulose bisphosphate carboxylase; RPE, ribulose 5-phosphate 3-epimerase; SUCDi, succinate dehydrogenase (irreversible); SUCOAS, succinate synthase (ADP forming); TKT2, transketolase.

[0317] The genes listed are as follows: ACALD:H16_A1806 or H16_B0596 or H16_A2747 or H16_B0551 ACONT1:H16_A2638 or H16_B0568 or H16_A1907 ACONT2:H16_A2638 or H16_B0568 or H16_A1907 AKGDH: (H16_A2325 and H16_A2324 and H16_B1098) or (H16_A3724 and H16_A2325 and H16_A2324) or (H16_A2325 and H16_A2324 and H16_A1377) or (H16_A2323 and H16_A2325 and H16_A2324) ALCD2x: H16_B2470 or H16_B0517 or H16_A3330 or H16_B1433 or H16_A0757 or H16_B1699 or H16_B1834 or H16_B1745 ASPTA:H16_A2857 ATPS4m: H16_A3643 and H16_A3642 and H16_A3639 and H16_A3636 and H16_A3637 and H16_A3638 and H16_A3640 and H16_A3641 CS: (H16_A2627 and H16_B0357 and H16_B2211) or (H16_A2627 and H16_B0357 and H16_A1229) or (H16_A2627 and H16_B0357 and H16_B0414) CYTCOBO3: H16_A3396 and H16_A3397 and H16_A3398 and H16_A2319 and H16_A2318 and H16_A2316 and H16_B2062 and H16_B2059 and H16_A0342 and H16_A0343 and H16_A0347 and H16_A0345 ENO:H16_A1188 FBA:H16_B0278 or H16_B1384 or H16_A0568 or PHG416 FBP: H16_B1390 or H16_A0999 or PHG422 FDH: (H16_B1700 and H16_B1701) or (H16_A0640 and H16_A0642 and H16_A0641 and H16_A0644) or H16_A3292 or (H16_A2934 and H16_A2937 and H16_A2936 and H16_B1471) or (H16_B1454 and H16_B1452 and H16_B1453) or H16_B1383 FUM:H16_B0103 or H16_A2528 GAPD: H16_B1386 or H16_A3146 or PHG418 GLUDC:H16_A2930 GLUS:H16_B2194 or H16_A3430 or H16_B2192 or H16_A3431 or H16_B2193 HYDS: PHG088 and PHG089 and PHG090 and PHG091 ICDHx:H16_B1016 LACDH:H16_A0666 MDH:H16_B0334 or H16_A2634 ME1:H16_A3153 NADH16: H16_A1051 and H16_A1052 and H16_A1050 and H16_A1055 and H16_A1056 and H16_A1053 and H16_A1054 and H16_A1061 and H16_A1060 and H16_A1063 and H16_A1062 and H16_A1059 and H16_A1058 and H16_A1057 and H16_A0251 PDH1: H16_A1374 or H16_B1300 or H16_B0145 or H16_B2234 or H16_B2233 or H16_A1753 PDH2:H16_A1375 or H16_B0146 PDH3: H16_A3724 or H16_A2323 or H16_A1377 or H16_B1098 PGK:H16_A0566 or H16_B1385 or PHG417 PGM:H16_A0332 or H16_A0493 PPC:H16_A2921 PRUK:H16_B1389 or PHG421 RBPC: (PHG426 and PHG427) or (H16_B1394 and H16_B1395) RPE: (H16_B1391 and H16_A3317) or (PHG423 and H16_A3317) SUCDi: H16_B0204 and H16_A2632 and H16_A2631 and H16_A2630 and H16_A2629 SUCOAS: H16_A0548 and H16_A0547 TKT2: (H16_B1388 and H16_A3147) or (PHG420 and H16_A3147).

[0318] Example 2: Continuous ethylene production from CO with H as the energy source. A gene encoding an ethylene-forming enzyme was codon-adapted and synthesized for expression in Cupriavidus necator. The adapted gene, along with the constitutive promoter P10, was cloned into the broad-host-range expression vector pBBR1MCS2. The resulting product was used to transform E. coli, and positive clones identified by PCR were confirmed by DNA sequencing. The sequence-confirmed plasmid was then transformed into Cupriavidus necator PHB-4 via electroporation and selected on tryptic soy broth (TSB) agar plates containing 50 mg / L chloramphenicol. Transformants containing the pBBR1-Efe plasmid were confirmed via sequencing and single colonies were then grown in TSB at 30°C overnight and used to generate glycerol stocks for storage at -80°C. Strain recovery was performed by streaking onto TSB plates containing 50 mg / L chloramphenicol with a 72-hour incubation at 30°C.

[0319] A single colony from a freshly streaked TSB plate was used to inoculate 3 mL of TSB containing 50 mg / L chloramphenicol in a 14 mL Falcon round-bottom polystyrene test tube with a snap cap. After overnight incubation at 30°C and 200 rpm in a Thermo MAXQ shaker, 1 mL of the culture was used to inoculate 100 mL of LB in a 200 mL Schott bottle. Cells were grown at 30°C and 200 rpm until an optical density of approximately 0.3–0.4 was reached.

[0320] 100 mL of the above culture was used to inoculate a 1.4 L Infors HT Multifors 2 CSTR containing 600 mL of 2x start-up medium. ℃ The cultures were incubated at 0°C for 1 hour and started with 250 rpm agitation and 150 nccm gas flow (3.14% O, 41% H, 3% CO, 52.86% N). Agitation and gas flow were increased to a maximum of 1450 rpm and 750 nccm as the cultures grew. OD 600 When the ΔΨ exceeded 0.5, the culture was diluted with 4x HCl containing 7 μL / hr of Pluronic 31R1 antifoam. The medium was continuously rotated. The feed oxygen rate was gradually increased to promote biomass production, the balance was removed from the nitrogen rate, and the outlet oxygen rate was constrained to remain below 4.5% as a safety measure.

[0321] Gas samples from the reactor were piped through 305 stainless steel to a flow selection valve controlled by a micro GC (manufacturer: Qmicro). The samples were then analyzed on a Rt-U SIP XP PLOT column under isothermal conditions (70 °C) via a thermal conductivity detector (TCD).

[0322] After the cultures were well established, the gas fraction was adjusted from O-limited to H-limited conditions and non-zero dissolved oxygen (DO) concentrations were observed. Ethylene production fluctuated as the system settled to a steady state and gas fractions were adjusted, but production was maintained over an 11-day period (Figure 3). During this period, the H fraction ranged from 11-18%, the O fraction from 5.5-6.6%, CO was held at 3%, and N was maintained as the balance. Upon returning to O-limited conditions, ethylene production ceased, demonstrating the importance of oxygen availability for ethylene production.

[0323] Example 3: Genome-scale modeling of gene deletion strategies to eliminate unwanted by-products during ethylene production from CO and H in Cupriavidus necator. A genome-scale metabolic model of Cupriavidus necator, similar to that described by Park et al., BMC Systems Biology, 5:101, 2011, was used to predict gene deletions to eliminate unwanted by-products during ethylene production from CO and H. Heterologous ethylene-producing reactions were added to the wild-type Cupriavidus necator model structure to represent the incorporation of pathways for the production of unnatural compounds. We used the constraint-based computational modeling techniques Flux Balance Analysis (FBA) and Linear Minimization of Metabolic Regulation (LMOMA) (Maia, Proceedings of the Genetic and Evolutionary Computation Conference Companion on - GECCO '17, New York, NY, ACM Press, pp. 1661-1668, 2017) with cobrapy version 0.8.2 (Ebrahim., COBRApy: CONstraints-Based Reconstruction and Analysis for Python, BMC Syst Biol, 7: 74, 2013) along with optlang version 1.2.3 (Jensen, Optlang: An Algebraic Modeling Language for Mathematical Optimization, The Journal of Open Source Software, 2, doi:10.21105 / joss.00139, 2017) as the solver interface, Gurobi. Optimizer version 7.0.2 was used as the optimization solver to simulate ethylene production.

[0324] [Table 1-1] [Table 1-2]

[0325] Example 4: Generating a scarless deletion at the glutamic acid decarboxylase (GLUDC) H16_A2930 locus. Amplification and assembly of homology arms. To create a scarless deletion of the glutamic acid decarboxylase (GLUDC) open reading frame of Cupriavidus necator H16, 500-bp homology arms were PCR-amplified from C. necator H16 gDNA using Kappa 2x Master Mix and Gibson-assembled into the suicide plasmid pK18mobsacB using Thermo Fisher's GeneArt Seamless Cloning and Assembly Enzyme Mix. Briefly, forward and reverse primers were designed with Geneious Primer to contain 5' and 3' Gibson tails compatible with pK18mobsacB digested with the restriction enzyme BamHI. Primer and homology arm sequences are provided in Table ### below. The correct homology arm amplicon size was verified on a 1% agarose gel and column purified using the Zymo DNA Clean and Concentrator Kit. The purified homology arms were combined at 100 ng each with 100 ng of BamHI-digested pK18mobsacB in a 20 μL reaction using GeneArt's Frontist Cloning Enzyme Mix and incubated at room temperature for 30 minutes. Next, 3 μL of the reaction mixture was used to transform chemically competent DH10B Escherichia coli and plated onto LB agar plates containing 50 ng / μL kanamycin antibiotic. The plates were incubated at 37°C for 24 hours, and colonies were screened by PCR to confirm the presence of each homology arm insert. Positive colonies were grown overnight in 5 mL of LB broth supplemented with 50 ng / μL kanamycin prepared using the Qiagen MINI Prep Kit, and the plasmids were sequence-verified using the Illumina MiSeq System.

[0326] Transformation by Conjugation. The pK18mobsacB plasmid containing the left and right 500-bp homology arms was electroporated into S17-1 E. coli cells and plated on LB supplemented with 50 ng / μL kanamycin. A single colony was picked and inoculated into 5 mL of LB broth supplemented with 50 ng / μL kanamycin to generate the conjugation donor strain. To generate the conjugation recipient strain, a single colony of C. necator H16 grown on LB agar supplemented with 300 ng / μL gentamicin was picked and used to inoculate 5 mL of LB broth supplemented with 300 ng / μL gentamicin. The donor E. coli culture was grown overnight at 37°C with shaking at 250 rpm, while the recipient C. necator culture was grown overnight at 30°C with shaking at 200 rpm. The next morning, cells were harvested by centrifugation at 6000 rpm for 3 minutes at 25°C, and the pellet was resuspended in 50 μL of LB medium. 50 μL of donor and recipient cells were mixed and spotted onto a sterile hydrophilic filter on antibiotic-free LB agar.

[0327] Selection and counter-selection. Plates were incubated overnight at 30°C, and the following morning, cells were removed from the filters by folding them in half using sterile forceps and transferring them to a tube containing 1 mL of LB. Cells were released from the filters by vortexing, and serial dilutions were prepared (10 0 ~10 -3 ), and plated in 100 μL volumes onto LB agar containing 300 ng / μL kanamycin. Plates were incubated at 30°C for 5 days. Colonies were then replicate-patched onto LB agar plates containing 300 ng / μL kanamycin and 20% sucrose + 300 ng / μL kanamycin. Colonies that grew on kanamycin but not on sucrose + kanamycin (primary integrants) were streak-purified onto LB agar plates containing 300 ng / μL kanamycin and then cultured overnight at 30°C in LB broth without antibiotics. The next morning, serial dilutions (10 in 100 μL volumes) were prepared. 0~10 -3 The culture was plated overnight at 30°C onto agar containing 20% sucrose (no antibiotics) to select for secondary recombinants. Sucrose-resistant colonies were patched onto LB agar containing 20% sucrose (with or without 300 ng / μL kanamycin) to select for recombinants that were sucrose-resistant and antibiotic-sensitive. Finally, 12 SucR, KanS colonies were prepared for PCR and sequencing to verify the deletion of the GLUDC H16_A2930 locus.

[0328] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0329] [Table 3-1] [Table 3-2] [Table 3-3]

[0330] After sequence confirmation of the GLUDC H16_A2930 locus deletion, the EEF expression construct described in Example 1 was transformed into this strain via electroporation and selected on tryptic soy broth (TSB) agar plates containing 50 mg / L chloramphenicol. Transformants containing the pBBR1-Efe plasmid were confirmed via sequencing and single colonies were then grown in TSB overnight at 30°C and used to generate glycerol stocks for storage at -80°C. Strain recovery was performed by streaking onto TSB plates containing 50 mg / L chloramphenicol with a 72-hour incubation at 30°C. Fermentation for ethylene production on formate or CO2 / H2 was performed as described in the previous examples.

[0331] Additional gene deletions and chromosomal integrations beneficial for reducing ethylene production and / or by-product formation were performed as described above.

[0332] Example 5: System for generating bubbles in a container FIG. 5 illustrates an example of a system 100 for generating gas bubbles in a vessel. The system 100 comprises a cylindrical reactor 102. Liquid enters an inlet or top section 101 of the reactor 102. The liquid may enter the top section 101 via an external pump in fluid communication with the system 100. According to certain embodiments, the liquid entering the top section 101 is recirculated by an external pump in fluid communication with the system 100. The liquid enters the top section of a perforated plate 104, where the liquid is accelerated by passing through orifices in the plate 104. According to certain embodiments, plate 104 may be configured to accelerate, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99%, or any value up to about 100% of the liquid in reactor 102. Sparger 106 injects gas bubbles from gas source 108 into the liquid. Sparger 106 is positioned within reactor 102 to form a first region where the injected bubbles rise within reactor 102 and encounter accelerated liquid 112 exiting the bottom of plate 104. The accelerated liquid 112 from the plate 104 breaks up the rising bubbles into microbubbles, thereby increasing the surface area required for the desired chemical or biological reaction. The microbubbles may have diameters ranging from about 0.1 mm to about 5 mm, or from about 0.5 mm to about 2 mm. In some embodiments, the microbubbles may have diameters ranging from about 0.2 mm to 1.5 mm.According to another embodiment, the diameter of the microbubbles may be, for example, about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.30, 2.31, 2.32, 2.33, 2.34, 2.35, 2.36, 2.37, 2.38, 2.39, 2.40, 2.41, 2.42, 2.43, 2.44, 2.45, 2.46, 2.47, The thickness of the sparger 106 can be any number between 5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 mm, or greater than, greater than, less than, equal to, or about 5.0 mm. A sparger 106 is further positioned within the reactor 102 to form a second region through which a fluid stream of liquid and microbubbles can flow downward.

[0333] The microbubbles may have a reduced upward velocity compared to the injected bubbles. Due to the accelerated overall flow of the liquid, the fluid 116 containing the liquid and the microbubbles may have a net downward flow. The downward velocity of the fluid 116 is greater than the overall upward velocity of the microbubbles. The fluid 116 may exit the reactor 102 at an outlet 111. The plate 104 may have a thickness (and orifice depth) of about 1 mm to 25 mm. According to another embodiment, the thickness of the plate can be, for example, greater than, greater than, less than, equal to, or any number between about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 mm.

[0334] As shown in FIG. 5, the dimensions of the components of system 100 can vary depending on the required application or process. According to certain embodiments, the diameter of reactor 102 can be, for example, greater than, greater than, less than, equal to, or any number between about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5 meters. According to other embodiments, the length of the reactor 102 may be any of a variety of lengths, such as, for example, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.5, 21.5, 22.0, 22.5, 23.0, It can be any number between 23.5, 24.0, 24.5, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0 meters, greater than, greater than, less than, equal to, or about 50.0 meters.

[0335] The velocity of the accelerated liquid or portion of the liquid from the plate 104 can be determined by the following formula: QL=N×(π / 4)×d2×vj where QL is the volumetric flow rate of the liquid (m3 / s), vj is the jet velocity, N is the total number of orifices on the plate, d is the diameter of the orifice, and π is the mathematical symbol for pi. According to one embodiment, the velocity of the accelerated liquid from the plate 104 can be, for example, greater than, greater than, less than, equal to, or any number between about 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500 mm / s, or about 20000 mm / s. As shown in FIG. 5 , the velocity of the accelerated liquid 112 is important to ensure that the gas bubbles injected into the liquid by the sparger 106 break down into appropriately sized microbubbles and that the liquid and microbubble fluids have sufficient velocity to generate a net downward fluid flow. The liquid superficial velocity VL in the main reactor may be calculated by the following formula: VL = QL / AC, where QL is the volumetric flow rate of the liquid in the reactor (m / s) and AC is the cross-sectional area of the reactor. Therefore, the liquid superficial velocity represents the velocity of the liquid phase when occupying the entire cross-sectional area of the reactor. According to embodiments, the liquid superficial velocity may also include stagnant liquid and microbubble regions and voids, and / or net downward fluid flow. For the same liquid flow rate, the gas flow rate can be varied depending on the actual application. The gas phase superficial velocity VG can be calculated by the following formula: V = QG / AC, where QG is the volumetric flow rate (m3 / s) of gas injected into the liquid from the sparger, and AC is the cross-sectional area of the reaction vessel. According to another embodiment, the superficial velocity of the gas phase in the vessel can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 mm / s, or any value between about 100 mm / s or greater. According to yet another embodiment, the superficial velocity of the gas phase in the vessel can be, for example, about 50-60 mm / s.

[0336] Locating a sparger or spargers 106 within and above the reactor 102 has the added benefit of reducing hydrostatic pressure at the top of the reactor 102, facilitating enhanced gas-to-liquid mass transfer rates while reducing energy requirements. Additionally, the required reactor components are minimized, further reducing reactor size, thereby maximizing gas-to-liquid mass transfer rates in a smaller reactor footprint. In some embodiments, for example, the systems and methods disclosed herein are capable of maximizing gas-to-liquid mass transfer rates in a smaller reactor footprint. 3 In other embodiments, the gas-to-liquid mass transfer rate is greater than, less than, equal to, or up to about 200 m / min, e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or greater. 3 / min. Furthermore, the sparger configuration, the achieved gas and liquid phase superficial velocities, and the increased gas-to-liquid mass transfer rates disclosed herein overcome known obstacles associated with the use of gas and liquid phase systems in previous and conventional reactors, particularly in bioreactors with gaseous substrates and aqueous cultures.

[0337] Example 6: Ethylene production using various genetic sources of ethylene-forming enzymes Genes encoding ethylene-forming enzymes from various organisms (accession numbers: Microcoleus asticus (NQE34890), Myxococcus stipitatus (WP_015351455.1), Nostoc sp. ATCC 43529 (RCJ18531), Ralstonia solanacearum (WP_014618742.1), Scytonema sp. NIES-4073 (WP_096562523.1) were cloned, codon-adapted, and synthesized for expression in Cupriavidus necator. The adapted genes were expressed under the control of a rhamnose-inducible promoter (P rhaBADThe pBBR1-Efe plasmid, along with the pBBR1-Efe and bicistronic RBS elements, was cloned into the broad-host-range expression vector pBBR1MCS2. The resulting product was used to transform E. coli, and positive clones identified by PCR were confirmed by DNA sequencing. The sequence-confirmed plasmid was then transformed into Cupriavidus necator PHBPHB-4 via electroporation and selected on tryptic soy broth (TSB) agar plates containing 50 mg / L chloramphenicol. Transformants containing the pBBR1-Efe plasmid were confirmed via sequencing and single colonies were then grown in TSB overnight at 30°C and used to make glycerol stocks for storage at -80°C. Strain recovery was performed by streaking onto TSB plates containing 50 mg / L chloramphenicol with 72-hour incubation at 30°C.

[0338] A single colony from a freshly streaked TSB plate was used to inoculate 1 mL of J minimal medium containing 10 g / L fructose, 10 g / L tryptone, and 5 g / L yeast extract in a deep-well 96-well plate and grown for 24 hours at 1000 rpm and 30°C. This preculture was used to inoculate 20 mL of J minimal medium containing 10 g / L fructose (1%) in a 160 mL serum bottle. The culture was then grown for 6 hours at 30°C and 200 rpm, at which point 0.5 mM rhamnose was added. After an additional 18 hours of growth, the bottle was sealed with an airtight septum. After an additional 24 hours of incubation, a 60 mL headspace volume was removed with an airtight syringe and analyzed for ethylene production via GC. Samples were analyzed for various hydrocarbons and oxyacid salts on a custom Wasson system. Ethylene was separated on a 50 m x 0.53 um Wasson PN 2378 column and analyzed via GC FID.

[0339] As shown in Figure 7, Cupriavidus necator strains harboring each of the Efe mutants produced detectable ethylene, whereas no ethylene was detected using the control strain containing the empty pBBR1 plasmid. Because these EEF variants range from 63-71% AA similarity to the standard enzyme from Pseudomonas syringae, this demonstrates the ability of diverse EEF enzymes to enable ethylene production.

[0340] Example 7. Use of a condition-dependent promoter for EEF expression during continuous ethylene production from CO with H as the energy source. A gene encoding an ethylene-forming enzyme was codon-adapted and synthesized for expression in Cupriavidus necator. The adapted gene was cloned into the broad-host-range expression vector pBBR1MCS2 along with a phosphate-limited inducible promoter (Ppst-pho) and a bicistronic RBS element. The resulting product was used to transform E. coli, and positive clones identified by PCR were confirmed by DNA sequencing. The sequence-confirmed plasmid was then transformed into Cupriavidus necator PHB-4 via electroporation and selected on tryptic soy broth (TSB) agar plates containing 50 mg / L chloramphenicol. Transformants containing the pBBR1-Efe plasmid were confirmed via sequencing and single colonies were then grown in TSB overnight at 30°C and used to generate glycerol stocks for storage at -80°C. Recovery of the strain was performed by streaking onto TSB plates containing 50 mg / L chloramphenicol with incubation at 30°C for 72 h.

[0341] A single colony from a freshly streaked TSB plate was used to inoculate 3 mL of TSB containing 50 mg / L chloramphenicol in a 14 mL Falcon round-bottom polystyrene test tube with a snap cap. After overnight incubation at 30°C and 200 rpm in a Thermo MAXQ shaker, 1 mL of the culture was used to inoculate 100 mL of LB in a 200 mL Schott bottle. Cells were grown at 30°C and 200 rpm until an optical density of approximately 0.3–0.4 was reached.

[0342] 100 mL of the above culture was used to inoculate a 1.4 L Infors HT Multifors 2 CSTR under conditions similar to those described in Example 2.

[0343] After the cultures were fully established, the medium composition was adjusted to achieve phosphate-limited conditions and further induce ethylene-forming enzyme expression (Figure 8). This shift in medium composition resulted in a roughly five-fold increase in ethylene concentration and production (Figure 8), demonstrating the use of a condition-dependent promoter for ethylene production.

[0344] Similarly, the use of promoters responsive to a gaseous carbon substrate, CO2, and an energy source, H2, can also be used to express ethylene-forming enzymes to enable ethylene production in C. necator. Here, an adapted ethylene-forming enzyme gene and a bicistronic RBS element are inserted into the megaplasmid CbbL promoter (P) that responds to CO2. CbbL,p ) or H2-responsive soluble hydrogenase promoter (P SHThe plasmids were cloned into the broad-host-range expression vector pBBR1MCS2, either in a 5'- or 1'-terminal nucleotide sequence. The resulting products were used to transform E. coli, and positive clones identified by PCR were confirmed by DNA sequencing. The sequence-confirmed plasmids were then transformed into Cupriavidus necator PHB-4 via electroporation and selected on tryptic soy broth (TSB) agar plates containing 50 mg / L chloramphenicol. Transformants containing the pBBR1-Efe plasmid were confirmed via sequencing and single colonies were then grown in TSB overnight at 30°C and used to generate glycerol stocks for storage at -80°C. Strain recovery was performed by streaking onto TSB plates containing 50 mg / L chloramphenicol with a 72-hour incubation at 30°C.

[0345] Following a procedure similar to that of the previous example (Example 2), these strains were run in a 1.4 L Infors HT Multifors 2 CSTR under conditions similar to those described in Example 2. Under these conditions, P CbbL,p Expression of the ethylene-forming enzyme using the promoter resulted in continuous production of ethylene from CO and H for over two weeks (Figure 9). Furthermore, the use of a soluble hydrogenase promoter (P) to drive ethylene-forming enzyme expression was demonstrated. SH The use of ethylene also allows ethylene production to reach concentrations of over 100 ppm in the outlet gas stream (Figure 10).

[0346] All references, including publications, patent applications, and patents, cited herein are incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The reference herein to any prior art is not, and should not be construed as, an acknowledgment that that prior art forms part of the common general knowledge in any country in the field of endeavor.

[0347] In the context of describing this disclosure (particularly in the context of the claims that follow), the use of the terms "a," "an," and "the" and similar referents shall be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" shall be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise noted. The term "consisting essentially of" limits the scope of a composition, process, or method to certain materials or steps, or to those that do not materially affect the basic and novel characteristics of the composition, process, or method. The use of alternatives (e.g., "or") should be understood to mean either one of the alternatives, both, or any combination thereof. As used herein, the term "about" means ±20% of the stated range, value, or structure, unless otherwise indicated.

[0348] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually recited herein. For example, any concentration range, percentage range, ratio range, integer range, size range, or thickness range should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as integer tenths and hundredths), unless otherwise indicated.

[0349] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better elucidate the invention and does not limit the scope of the invention unless otherwise asserted. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0350] Preferred embodiments of the present disclosure are described herein. Variations of those preferred embodiments may become apparent to those skilled in the art upon reading the above description. The inventors anticipate that those skilled in the art will adopt such variations as necessary, and the inventors intend for the present disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0351] Embodiment

[0352] Embodiment 1: A recombinant C1-fixing microorganism capable of producing ethylene from a gaseous substrate comprising nucleic acids encoding a group of exogenous enzymes including ethylene-forming enzymes (EFEs).

[0353] Embodiment 2: A recombinant C1-fixing microorganism capable of switching cell load in the production of ethylene, comprising a nucleic acid encoding a group of exogenous enzymes including an ethylene-forming enzyme (EFE) and one or more inducible promoters.

[0354] Embodiment 3: The microorganism of embodiment 1, wherein the microorganism is selected from the group consisting of Cupriavidus necator and Ralstonia eutropha.

[0355] Embodiment 4: The microorganism of embodiment 4, wherein the microorganism is Cupriavidus necator.

[0356] Embodiment 5: The microorganism of embodiment 2, further comprising a nucleic acid encoding an alpha-ketoglutarate permease, wherein the nucleic acid is codon optimized for expression in the microorganism.

[0357] Embodiment 6: The microorganism of embodiment 2, wherein the one or more inducible promoters are selected from an H2-inducible promoter, a phosphate-limited inducible promoter, a nitrogen-limited inducible promoter, a CO2-inducible promoter, or any combination thereof.

[0358] Embodiment 7: The microorganism of embodiment 2, wherein the EFE is codon-optimized for expression in the microorganism.

[0359] Embodiment 8: The microorganism of embodiment 1, further comprising a disruptive mutation in one or more genes.

[0360] Embodiment 9: The microorganism of embodiment 1, wherein ethylene is converted into a derivative material selected from polyethylene (PE), polyethylene terephthalate (PET), polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), sustainable aviation fuel (SAF), or any combination thereof.

[0361] Embodiment 10: The microorganism of embodiment 1, wherein the gaseous substrate comprises CO2 and an energy source.

[0362] Embodiment 11: The microorganism of embodiment 1, wherein the gaseous substrate comprises CO2 and H2, O2, or both.

[0363] Embodiment 12: A method for the continuous production of ethylene, the process comprising passing a gaseous substrate through a bioreactor containing a culture of the recombinant C1-fixing microorganism of embodiment 1 in a culture medium such that the microorganism converts the gaseous substrate into ethylene, and recovering ethylene from the bioreactor.

[0364] Embodiment 13: The method of embodiment 12, wherein the gaseous substrate comprises industrial waste or off-gas.

[0365] Embodiment 14: The method of embodiment 12, further comprising an energy source.

[0366] Embodiment 15: The method of embodiment 12, wherein the energy source is provided intermittently.

[0367] Embodiment 16: The method of embodiment 12, wherein the gaseous substrate comprises CO2 and an energy source.

[0368] Embodiment 17: The method of embodiment 16, wherein the energy source is H2.

[0369] Embodiment 18: The method of embodiment 16, wherein the gaseous substrate further comprises H2, O2, or both.

[0370] Embodiment 19: The method of embodiment 12, further comprising limiting the dissolved oxygen concentration, thereby switching the cell load.

[0371] Embodiment 20: The method of embodiment 12, further comprising controlling the iron concentration to comprise at least 50 mg / L.

[0372] Embodiment 21: The method of embodiment 12, further comprising converting the ethylene into components used in manufacturing tires.

[0373] Embodiment 22: The method of embodiment 21, wherein the tire is an end-of-life tire.

[0374] Embodiment 23: The method of embodiment 12, wherein the gaseous substrate is derived from a process involving tires.

[0375] Embodiment 24: The method of embodiment 12, wherein the gaseous substrate is derived from a product recycling process or a sustainable chemical process.

[0376] Embodiment 25: The method of embodiment 23, further comprising converting the ethylene into components used in the manufacture of new tires.

Claims

1. A recombinant C1-fixing microorganism capable of producing ethylene from a gaseous substrate comprising a nucleic acid encoding a group of exogenous enzymes including ethylene-forming enzymes (EFEs).

2. A recombinant C1-fixing microorganism capable of switching cell load in the production of ethylene, comprising a nucleic acid encoding a group of exogenous enzymes including an ethylene-forming enzyme (EFE) and one or more inducible promoters.

3. 2. The microorganism of claim 1, wherein the microorganism is selected from the group consisting of Cupriavidus necator and Ralstonia eutropha.

4. The microorganism of claim 3 , wherein the microorganism is Cupriavidus necator.

5. 3. The microorganism of claim 2, further comprising a nucleic acid encoding an alpha-ketoglutarate permease, said nucleic acid being codon optimized for expression in said microorganism.

6. The one or more inducible promoters are ２ Inducible promoter, phosphate-limited inducible promoter, nitrogen-limited inducible promoter, CO ２ 3. The microorganism of claim 2, wherein the promoter is selected from the group consisting of a promoter, an inducible ... and any combination thereof.

7. The microorganism of claim 2 , wherein the EFE is a codon optimized for expression in the microorganism.

8. The microorganism of claim 1, further comprising a disruptive mutation in one or more genes.

9. 10. The microorganism of claim 1, wherein ethylene is converted into a derivative material selected from polyethylene (PE), polyethylene terephthalate (PET), polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), sustainable aviation fuel (SAF), or any combination thereof.

10. The gaseous substrate is CO ２ and an energy source.

11. The gaseous substrate is CO ２ , and H2, O ２ , or both.

12. 1. A method for the continuous production of ethylene, the process comprising passing a gaseous substrate through a bioreactor containing a culture of the recombinant C1-fixed microorganism of claim 1 in a culture medium such that the microorganism converts the gaseous substrate into ethylene, and recovering the ethylene from the bioreactor.

13. 13. The method of claim 12, wherein the gaseous substrate comprises industrial waste or off-gas.

14. The method of claim 12 further comprising an energy source.

15. The method of claim 12 , wherein the energy source is provided intermittently.

16. The gaseous substrate is CO ２ and an energy source.

17. The energy source is H ２ 17. The method of claim 16, wherein:

18. The gaseous substrate is H ２ , O ２ 17. The method of claim 16, further comprising:

19. 13. The method of claim 12, further comprising limiting the dissolved oxygen concentration, thereby switching the cell load.

20. 13. The method of claim 12, further comprising controlling the iron concentration to comprise at least 50 mg / L.

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