Microorganisms and methods for continuous production of ethylene from C1 substrates

Abstract

The methods and microorganisms are genetically engineered to continuously produce ethylene by microbial fermentation, in particular by microbial fermentation of gaseous substrates. The microorganism is of a C1 fixed type. In addition, the gaseous substrate comprises CO2 and an energy source. The yield of ethylene can be increased by altering the promoter or by means of nutrition restriction.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 366,758 filed on June 21, 2022, U.S. Provisional Patent Application No. 63 / 506,350 filed on June 5, 2023, and U.S. Provisional Patent Application No. 63 / 506,351 filed on June 5, 2023, the entire contents of which are incorporated herein by reference.

[0003] Reference to a sequence listing

[0004] This application contains a sequence listing that has been electronically submitted in the ST.26 sequence listing XML format and is hereby incorporated 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. Technical Field

[0005] 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 Art

[0006] It has long been recognized that catalytic methods (such as the Fischer-Tropsch process) can be used to convert gases containing carbon dioxide (CO2), carbon monoxide (CO) and / or hydrogen (H2), such as industrial waste gases or synthesis gas, into various fuels and chemicals. However, recently, gas fermentation has become an alternative platform for the biological fixation of such gases. In particular, C1-fixing microorganisms have been demonstrated to convert gases containing CO2, CO and / or H2 into products such as ethanol and 2,3-butanediol. Ethylene is the most widely produced organic compound in the world and is used in a wide range of industries including plastics, solvents and textiles. Ethylene is currently produced by steam cracking fossil fuels or dehydrogenating ethane. However, with millions of metric tons of ethylene produced each year, the excess carbon dioxide produced by such processes has greatly increased the global carbon footprint. Therefore, the production of ethylene by renewable methods will help meet the huge needs of the energy and chemical industries, while also helping to protect the environment. However, the efficient production of such chemical products may be limited by slow microbial growth, limited gas uptake, sensitivity to toxins, or conversion of carbon substrates into undesirable byproducts. Therefore, there is a continuing and unmet need to develop a method for the efficient production of ethylene by microbial fermentation of gaseous substrates that can be readily produced from renewable resources and will provide a wide range of useful applications. Summary of the invention

[0007] It is against the above background that the present disclosure provides certain advantages and advances over the prior art.

[0008] Although this disclosure is not limited to a particular advantage or function disclosed herein, the present disclosure provides a method and a genetically engineered microorganism capable of producing ethylene from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding an ethylene forming enzyme (EFE).

[0009] In some aspects of the methods disclosed herein, the microorganism is a recombinant C1 immobile microorganism capable of producing ethylene from a gaseous substrate, the recombinant C1 immobile microorganism comprising a nucleic acid encoding a set of exogenous enzymes comprising an ethylene forming enzyme (EFE).

[0010] In some aspects of the microorganisms disclosed herein, the microorganism relates to a recombinant C1 fixed microorganism capable of switching the cell load in the production of ethylene, the microorganism comprising a nucleic acid encoding a set of exogenous enzymes comprising an ethylene forming enzyme (EFE) and one or more inducible promoters.

[0011] The microorganism according to the embodiment, further comprising a nucleic acid encoding a group of exogenous enzymes comprising α-ketoglutarate permease (AKGP), wherein the nucleic acid is operably linked to a promoter.

[0012] The microorganism according to the embodiment, wherein the microorganism is selected from the group consisting of Cupriavidus necator and Ralstonia eutropha.

[0013] The microorganism according to the embodiment, wherein the microorganism is Cupriavidus necrotus.

[0014] According to the microorganism of the embodiments, it further comprises a nucleic acid encoding α-ketoglutarate, wherein the nucleic acid is codon-optimized for expression in the microorganism.

[0015] The microorganism according to the embodiments, 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.

[0016] The microorganism according to the embodiment, wherein the CO2-inducible promoter is CBB.

[0017] The microorganism according to the embodiments, wherein the EFE is codon-optimized for expression in the microorganism.

[0018] The microorganism according to the embodiment further comprises a disruptive mutation in one or more genes.

[0019] According to the microorganism described in the embodiment, ethylene is converted into a derived material, and the derived material is selected from polyethylene (PE), polyethylene terephthalate (PET), polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), sustainable aviation fuel (SAF) or any combination thereof.

[0020] The microorganism according to the embodiment, wherein the gaseous substrate comprises CO2 and an energy source.

[0021] The microorganism according to the embodiments, wherein the gaseous substrate comprises CO2 and H2, O2 or both.

[0022] One embodiment relates to a method for continuously producing ethylene, the method comprising: passing a gaseous substrate into a bioreactor containing a culture of the recombinant C1 immobile 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.

[0023] One embodiment relates to a method of culturing the microorganism of claim 1, the method comprising culturing the microorganism in a culture medium comprising a gaseous substrate, wherein the gaseous substrate comprises CO2.

[0024] The method according to an embodiment, wherein the gaseous substrate comprises industrial waste products or exhaust gases.

[0025] The method according to an embodiment further comprises an energy source.

[0026] The method according to an embodiment, wherein the energy source is provided intermittently.

[0027] The method according to an embodiment, wherein the energy source is H2.

[0028] One embodiment relates to a method comprising culturing the microorganism in a culture medium comprising a gaseous substrate, wherein the gaseous substrate comprises CO2 and an energy source.

[0029] The method according to an embodiment further comprises co-producing ethylene and the microbial biomass.

[0030] The method according to an embodiment, wherein switching the cell load comprises the step of limiting intracellular oxygen concentration.

[0031] The method according to an embodiment, wherein the microbial biomass is suitable as animal feed.

[0032] The method according to an embodiment, wherein the gaseous substrate further comprises H2, O2, or both.

[0033] In some aspects of the microorganisms disclosed herein, the microorganism produces a commodity chemical product, a microbial biomass, a single cell protein (SCP), one or more intermediates, or any combination thereof.

[0034] In some aspects of the microorganisms disclosed herein, the microorganism is derived from a parent bacterium selected from the group consisting of Cupriavidus necrotus.

[0035] In some aspects of the microorganisms disclosed herein, the product is selected from the group consisting of 1-butanol, butyrate, butylene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, terpenes, isoprene, fatty acids, fatty alcohols, 2-butanol, 1,2-propylene glycol, 1-propanol, 1-hexanol, 1-octanol, chorismate-derived products, 3-hydroxybutyrate, 1,3-butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-butene-1-ol, isovalerate, isopentanol, or monoethylene glycol.

[0036] The present disclosure further provides a genetically engineered C1 immobilized microorganism further comprising a microbial biomass and at least one excipient.

[0037] The present disclosure further provides a genetically engineered C1 stationary microorganism, wherein the animal feed is suitable for feeding one or more of the following: beef cattle, dairy cows, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, gaur, giraffes, yaks, chickens, turkeys, ducks, geese, quail, guinea fowl, chicks / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents.

[0038] The present disclosure further provides a genetically engineered C1 immobilized microorganism, wherein the microorganism is suitable as a single cell protein (SCP).

[0039] The present disclosure further provides a genetically engineered C1 immobilized microorganism, wherein the microorganism is suitable as a cell-free protein synthesis (CFPS) platform.

[0040] The present disclosure further provides a genetically engineered C1 immobilized microorganism, wherein the chemical product is native to the microorganism.

[0041] In some aspects of the methods disclosed herein, the substrate comprises one or more of CO, CO2, and H2.

[0042] In some embodiments, both anaerobic and aerobic gases can be used to feed separate cultures (eg, an anaerobic culture and an aerobic culture) in two or more different bioreactors that are both integrated into the same process stream.

[0043] In some embodiments, the present disclosure provides a method for storing energy in the form of a biopolymer, the method comprising intermittently treating at least a portion of electrical energy generated by a renewable and / or non-renewable energy source in an electrolysis process to produce at least H2, O2, or CO; intermittently passing at least one of H2, O2, or CO from the electrolysis process into a bioreactor containing a culture, the culture comprising a liquid nutrient medium and a microorganism capable of producing a biopolymer; and fermenting the culture.

[0044] In one embodiment, the present disclosure also provides a system for storing energy in the form of biopolymers, 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 H2, O2 or CO; an industrial device for producing at least a C1 feedstock; a bioreactor in intermittent fluid communication with the electrolysis process and / or in continuous fluid communication with the industrial device, comprising a reaction vessel suitable for intermittent growth, fermentation and / or cultivation and containing microorganisms capable of producing biopolymers.

[0045] In some embodiments, the present 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, wherein the method comprises: passing a C1 feedstock comprising one or both of CO and CO2 from an industrial process to the bioreactor, wherein the C1 feedstock has a unit cost, intermittently passing at least one of H2, O2, or CO from the electrolysis process to the bioreactor, wherein the electrolysis process has a unit cost, and fermenting the culture to produce one or more fermentation products, wherein each of the one or more fermentation products has a unit value. In certain instances, multiple electrolysis processes are utilized to provide one or all of CO, CO2, and H2 to the bioreactor.

[0046] In another embodiment, the local grid provides electricity delivered intermittently as electrical energy generated by the power 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.

[0047] In one embodiment, the present disclosure can be operated intermittently by storing energy in the form of biopolymers, where product conversion can be performed intermittently during periods of excess grid power supply, or idled during power shortages or power demands. The present disclosure provides a method that can be fine-tuned to help balance the grid system by storing energy in the form of biopolymers.

[0048] In one embodiment, the autotrophic microorganisms intermittently consume part or all of the energy provided by the available energy.

[0049] In one embodiment, the system disclosed herein relates to the generation of microbubbles, and may include a container for holding a liquid, a plate containing a plurality of holes positioned in the upper portion of the container and configured to accelerate at least a portion of the liquid in the container, and at least one ejector positioned in the container, wherein the surface of the ejector is positioned at about 50 mm to about 300 mm, 500 mm, or 1000 mm from the bottom of the plate. The ejector may be configured to inject bubbles into the liquid. In some embodiments, the ejector may be positioned in the container to generate a first zone for bubbles to rise in the container, and to generate a second zone for the accelerated liquid to break the bubbles into microbubbles and for the fluid to flow through the container. The fluid may include the accelerated portion of the liquid and microbubbles. In still other examples, the superficial velocity of the gas phase in the container may be at least 30 mm / sec. The ejector may be a sintered ejector or an orifice ejector. The thickness of the plate may be about 1 mm to about 25 mm. The velocity of the accelerated liquid may be about 8000 mm / sec to about 17000 mm / sec. In other examples, the speed of the accelerated liquid can be about 12000 mm / sec to about 17000 mm / sec. In some examples, the diameter of the bubbles injected into the liquid from the ejector can be about 2 mm to about 20 mm. In another example, the diameter of the bubbles injected into the liquid from the ejector can be about 5 mm to about 15 mm or about 7 mm to about 13 mm. The diameter of the tiny bubbles can be about 0.1 mm to about 5 mm or about 2 mm to about 1.5 mm. The plurality of holes can also be configured to accelerate at least 90% of the liquid in the container.

[0050] In another embodiment, the method disclosed herein relates to the generation of tiny bubbles, which may include injecting gas into a container containing liquid by at least one injector, the injector is positioned in the container and is configured to inject bubbles into the liquid, and accelerating a portion of the liquid in the container by a perforated plate positioned in the upper portion of the container, wherein the liquid can be accelerated from the plate to break the bubbles into tiny bubbles. In some instances, the superficial velocity of the gas phase in the container can be at least 30 mm / sec. In other instances, the superficial velocity of the gas phase in the container can be about 30 mm / sec to about 80 mm / sec. The injector can be a sintered injector or a perforated plate injector. The liquid can be accelerated from the perforated plate at a speed of about 8000 mm / sec to about 17000 mm / sec. In some instances, the liquid can be accelerated from the perforated plate at a speed of about 12000 mm / sec to about 17000 mm / sec. The diameter of the bubble injected into the liquid from the injector can be about 2 mm to about 20 mm, or greater than 5 mm to about 15 mm or about 7 mm to about 13 mm. The bubbles injected into the liquid from the sprayer are not usually spherical. The bubbles injected can be referred to as coarse bubbles. In contrast, the diameter of the tiny bubbles can be about 0.1mm to about 5mm or about 0.2mm to about 1.5mm. The tiny bubbles are usually spherical. The liquid stream can be introduced at a position close to the plate. The ejector can be positioned vertically or parallel to the plate, and the top surface or side surface of the ejector can be positioned at about 50mm to about 300mm, 500mm or 1000mm from the bottom of the plate.

[0051] In yet another embodiment, the system disclosed herein relates to a bioreactor, which may include a container containing a liquid growth medium, a plate that may include a plurality of holes positioned at an upper portion of the container and configured to accelerate at least a portion of the liquid growth medium in the container, a substrate that may include at least one C1 carbon source, and at least one injector positioned in the container, wherein the surface of the injector may be positioned at about 50 mm to about 300 mm, 500 mm, or 1000 mm from the bottom of the plate, and the injector is configured to inject substrate bubbles into the liquid growth medium. The injector positioned in the container may create a first zone for the substrate bubbles to rise in the container, and a second zone for the accelerated liquid growth medium to break the substrate bubbles into substrate micro-bubbles and for the fluid to flow through the container. The fluid may have an accelerated portion of the liquid growth medium, and may have substrate micro-bubbles and a culture of at least one microorganism in the liquid growth medium. The culture of at least one microorganism may anaerobically ferment the substrate to produce at least one fermentation product.

[0052] In still another embodiment, the method disclosed herein relates to producing substrate microbubbles in a bioreactor, and may include ejecting substrate bubbles of at least one C1 carbon source into a container containing a liquid growth medium by at least one ejector positioned in the container, and accelerating a portion of the liquid growth medium in the container by a perforated plate positioned in the upper portion of the container. The liquid growth medium accelerated from the plate can break the substrate bubbles into substrate microbubbles. The superficial velocity of the gas phase in the container can be at least 30 mm / sec. A culture of at least one microorganism can be included in the liquid growth medium, and the substrate can be anaerobically fermented to produce at least one fermentation product.

[0053] 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. Note that the scope of the claims is defined by the recitation therein rather than by the specific discussion of the features and advantages set forth in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] These and other aspects of the present disclosure, which should 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 in which:

[0055] Figure 1 Schematic diagram showing the pathways of CO2 fixation, central carbon metabolism and TCA cycle in Cupriavidus necrotica heterologously expressing ethylene-forming enzymes for ethylene production.

[0056] Figure 2 Shown is ethylene production by Cupriavidus necator strains with expression of ethylene-forming enzymes (pBBR1-Efe) and a blank vector control (pBBR1) when grown on formate as the sole carbon and energy source.

[0057] Figure 3 Continuous ethylene production over an 11-day period by a Cupriavidus necrotizer strain with expression of ethylene-forming enzymes (pBBR1-Efe) in a CSTR with CO2 as the sole carbon source is shown.

[0058] Figure 4 The gene knockout strategy predicted by flux balance analysis (red arrows) is shown to eliminate unwanted byproducts during ethylene production from CO2 and H2 in C. necator. Gene annotations are provided below each enzyme name (see Table 1 for additional details).

[0059] Figure 5 A system for generating bubbles within a container according to the systems and methods disclosed herein is schematically depicted.

[0060] Figure 6Shown are ethylene production by Cupriavidus necator strains with ethylene-forming enzyme (Efe) expressed by either a constitutive or phosphate-limited inducible promoter and a blank vector control (pBBR1) when grown in phosphate-limited minimal medium.

[0061] Figure 7 Shown is ethylene production by Cupriella necator strains with ethylene forming enzyme variants from various organisms expressed by a chemically inducible promoter (rhamnose). Accession numbers for EFE variants: Pseudomonas syringae (AAD16440.1), Microcoleus asticus (NQE34890), Myxococcus petiolus (WP_015351455.1), Nostoc spp. ATCC 43529 (RCJ18531), Ralstonia solanacearum (WP_014618742.1), Pseudoclase NIES-4073 (WP_096562523.1).

[0062] Figure 8 Continuous ethylene production over a 5.5 day period by a Cupriavidus necator strain with an ethylene-forming enzyme expressed by a phosphate-limited inducible promoter in a CSTR with CO2 as the sole carbon source is shown. Starting from day 14.7, the CSTR was operated under phosphate-limited conditions.

[0063] Fig. 9 Continuous ethylene production over a 14-day period by a Cupriavidus necrotizer strain with ethylene-forming enzymes expressed by a synthetic CbbL promoter in a CSTR with CO2 as the sole carbon source is shown.

[0064] Fig.10 Shown is ethylene production from CO2 as the sole carbon source during CSTR startup by Cupriavidus necrotica with ethylene-forming enzymes expressed from a synthetic soluble hydrogenase promoter.

[0065] Fig.11 It was shown that increasing FeSO4x7H2O concentrations resulted in increased ethylene production in cells grown on fructose under PO4 limiting conditions. DETAILED DESCRIPTION

[0066] The following examples are described in general terms. The present disclosure is further illustrated by the disclosure given below under the heading "Examples" herein, which provides experimental data supporting the present disclosure, specific examples of various aspects of the present disclosure, and ways of carrying out the present disclosure.

[0067] The inventors have surprisingly been able to engineer C1 -fixing microorganisms to continuously produce ethylene.

[0068] Unless otherwise defined, the following terms as used throughout this specification are defined as follows:

[0069] The present disclosure provides microorganisms for bio-co-production of proteins, chemicals and microbial biomass. A "microorganism" is a microscopic organism, specifically a bacterium, archaea, virus or fungus. In one embodiment, the microorganism of the present disclosure is a bacterium.

[0070] The term "non-naturally occurring" when used to refer to a microorganism is intended to mean that the microorganism has at least one genetic modification 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 laboratories or research institutions. The microorganisms of the present disclosure are non-naturally occurring.

[0071] The term "genetic modification", "genetic alteration" or "genetic engineering" refers broadly to the manipulation of the genome or nucleic acid of a microorganism by hand. Similarly, the term "genetically modified", "genetically altered" or "genetically engineered" refers to a microorganism containing such genetic modification, genetic alteration or genetic engineering. These terms can be used to distinguish between laboratory-produced microorganisms and naturally occurring microorganisms. The method of genetic modification includes, 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 methods, gene shuffling and codon optimization. The microorganism of the present disclosure is genetically engineered.

[0072] "Recombinant" indicates that a nucleic acid, protein or microorganism is the product of genetic modification, genetic engineering or genetic recombination. Generally, the term "recombinant" refers to a nucleic acid, protein or microorganism containing or encoded by genetic material derived from multiple sources, such as two or more different microbial strains or species. The microorganisms of the present disclosure are typically recombinant.

[0073] "Wild type" refers to the typical form of an organism, strain, gene, or its characteristics as found in nature, as distinguished from mutant or variant forms.

[0074] "Endogenous" refers to a nucleic acid or protein present or expressed in the wild-type or parent microorganism from which the microorganism of the present disclosure is derived. For example, an endogenous gene is a gene naturally present in the wild-type or parent microorganism from which the microorganism of the present disclosure is derived. In one embodiment, the expression of an endogenous gene can be controlled by an exogenous regulatory element, such as an exogenous promoter.

[0075] "Exogenous" refers to nucleic acids or proteins that originate from outside the microorganism of the present disclosure. For example, an exogenous gene or enzyme can be artificially or recombinantly produced and introduced into the microorganism of the present disclosure or expressed in the microorganism of the present disclosure. An exogenous gene or enzyme can also be isolated from a heterologous microorganism and introduced into the microorganism of the present disclosure or expressed in the microorganism of the present disclosure. Exogenous nucleic acids can be adjusted to be integrated into the genome of the microorganism of the present disclosure or to remain in an extrachromosomal state in the microorganism of the present disclosure, such as in a plasmid.

[0076] "Heterologous" refers to nucleic acids or proteins that do not exist in the wild type or parent microorganism from which the microorganism of the present disclosure is derived. For example, a heterologous gene or enzyme can be derived from different strains or species and introduced into the microorganism of the present disclosure or expressed in the microorganism of the present disclosure. A heterologous gene or enzyme can be introduced into the microorganism of the present disclosure or expressed in the microorganism of the present disclosure in the form in which it exists in different strains or species. Alternatively, a heterologous gene or enzyme can be modified in some way, for example, by codon optimization for expression in the microorganism of the present disclosure or by engineering it to change function, such as to reverse the direction of enzyme activity or to change substrate specificity.

[0077] In particular, the heterologous nucleic acid or protein expressed in the microorganisms described herein can be derived from Bacillus, Clostridium, Cupriavidus, Escherichia, Gluconobacter, Hyphomicrobium, Lysinibacillus, Paenibacillus, Pseudomonas, Sedimenticola, Sporosarcina, Streptomyces. Streptomyces, Thermithiobacillus, Thermotoga, Zea mays, Klebsiella, Mycobacterium, Salmonella, Mycobacteroides, Staphylococcus, Burkholderia, Listeria, Acinetobacter, Shigella, Neisseria ), Bordetella, Streptococcus, Enterobacter, Vibrio, Legionella, Xanthomonas, Serratia, Cronobacter, Cupriavidus, Helicobacter, Yersinia, Cutibacterium, Francisella, Pectobacterium, Vibrio Arcobacter, Lactobacillus, Shewanella, Erwinia, Sulfurospirillum, Peptococcaceae, Thermococcus, Saccharomyces, Pyrococcus, Glycine, Homo, Ralstonia, Brevibacterium, Methylobacterium,Geobacillus, bos, gallus, Anaerococcus, Xenopus, Amblyrhynchus, rattus, mus, sus, Rhodococcus, Rhizobium, Megasphaera, Mesorhizobium, Peptococcus, Agrobacterium, Campylobacter, Acetobacterium, Alkalibaculum, Blautia, Butyric acid bacteria, Eubacterium, Moorella, Oxobacter, Sporomusa, Thermoanaerobacter, Schizosaccharomyces cerevisiae haromyces, Paenibacillus, Fictibacillus, Lysinibacillus, Ornithinibacillus, Halobacillus, Kurthia, Lentibacillus, Anoxybacillus, Solibacillus, Virgibacillus, Alicyclobacillus, Sporosarcina, Salimicrobium, Sporosarcina, Planococcus, Corynebacterium, Thermaerobacter, Sulfobacillus, or Symbiobacterium.

[0078] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. The terms refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) of any length or their analogs. A polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci defined by linkage analysis (locus), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (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 may contain one or more modified nucleotides, such as methylated nucleotides or nucleotide analogs. If present, modifications to the nucleotide structure may be made before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0079] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., transcribed into mRNA or other RNA transcripts) and / or the process by which mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as "gene products."

[0080] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation to a labeling component. As used herein, the term "amino acid" includes natural and / or non-natural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptide mimetics.

[0081] The term "copolymer" is a composition comprising two or more monomer species connected by the same polymer chain of the present disclosure.

[0082] "Enzyme activity" or simply "activity" refers broadly to enzymatic activity, including but not limited to the activity of an enzyme, the amount of an enzyme, or the availability of an enzyme-catalyzed 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-catalyzed reaction. Similarly, "reducing" enzyme activity includes reducing the activity of an enzyme, reducing the amount of an enzyme, or reducing the availability of an enzyme-catalyzed reaction.

[0083] "Mutation" refers to a nucleic acid or protein that has been 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 can be a deletion, insertion or substitution in a gene encoding an enzyme. In another embodiment, the mutation can be a deletion, insertion or substitution of one or more amino acids in an enzyme.

[0084] "Destroyed gene" refers to a gene that is modified in some way to reduce or eliminate gene expression, gene regulatory activity, or encoded protein or enzyme activity. Destruction can partially inactivate, completely inactivate or delete genes or enzymes. Destruction can be a knockout (KO) mutation that completely eliminates the expression or activity of a gene, protein or enzyme. This destruction may also be a knockout that reduces but does not completely eliminate the expression or activity of a gene, protein or enzyme. Destruction can be anything that reduces, prevents or blocks the biosynthesis of a product produced by an enzyme. The destruction may include, for example, mutations in genes encoding proteins or enzymes, mutations in gene regulatory elements that participate in the expression of genes encoding enzymes, introduction of nucleic acids that produce proteins that reduce or inhibit enzyme activity, or introduction of nucleic acids (e.g., antisense nuclear RNA, RNAi, TALEN, siRNA, CRISPR or CRISPRi) or proteins that inhibit protein or enzyme expression. Destruction can be introduced using any method known in the art. In order to achieve the purposes of this disclosure, destruction is laboratory-generated, rather than naturally occurring.

[0085] "Parent microorganism" is a microorganism used to produce the disclosed microorganism. The parent microorganism can be a naturally occurring microorganism (i.e., a wild-type microorganism) or a previously modified microorganism (i.e., a mutant or recombinant microorganism). The disclosed microorganism can be modified to express or overexpress one or more enzymes that are not expressed or overexpressed in the parent microorganism. Similarly, the disclosed microorganism can be modified to contain one or more genes that the parent microorganism does not contain. The disclosed microorganism can also be modified to not express or express one or more enzymes of a lower amount expressed in the parent microorganism.

[0086] The microorganisms of the present disclosure can be derived from essentially any parent microorganism.

[0087] The term "derived from" means that a nucleic acid, protein or microorganism is modified or transformed from a different (e.g., parent or wild-type) nucleic acid, protein or microorganism to produce a new nucleic acid, protein or microorganism. Such modifications or adaptations typically include insertion, deletion, mutation or substitution of a nucleic acid or gene.

[0088] Microorganisms of the present disclosure can be further classified based on functional properties. For example, microorganisms of the present disclosure can be or can be derived from C1 fixed microorganisms, aerobic bacteria / anaerobic bacteria, acetogenic bacteria, ethanologenic bacteria (ethanologen), carbon monoxide nutrient bacteria (carboxydotroph), autotrophic bacteria and / or methanotrophs. Microorganisms of the present disclosure can be selected from chemoautotrophs, hydrogen nutrient type organisms, hydrogen and oxygen mixed gas organisms, methanotrophs or any combination thereof. In certain embodiments, microorganisms can be hydrogen oxidation type, carbon monoxide oxidation type, hydrogen and oxygen mixed gas type or any combination thereof, with the ability of growing and synthesizing biomass on gaseous carbon sources such as synthesis gas and / or CO2, so that the production microorganism synthesizes the target chemical product under gas culture. Microorganisms and methods of the present disclosure can realize the low-cost synthesis of biochemicals, and the biochemicals can compete with amino acids, proteins and other biological nutrients from petrochemical products and higher plant sources in price. In certain embodiments, the price of these amino acids, proteins and other biological nutrients can be much lower than the price of amino acids, proteins and other biological nutrients produced by heterotrophic or microbial phototrophic synthesis. More generally, oxyhydrogen microorganisms, hydrogenotrophs, carboxydotrophs, and chemoautotrophs are able to capture CO2 or CO as their sole carbon source to support biological growth. In some embodiments, this growth includes the biosynthesis of amino acids and proteins. Oxyhydrogen microorganisms and other hydrogenotrophs can use H2 as a source of reduced electrons to achieve respiration and biochemical synthesis. In some embodiments of the present invention, oxyhydrogen microorganisms and / or hydrogenotrophs and / or carboxydotrophs and / or other chemoautotrophs grow on a gas stream, which includes, but is not limited to, one or more of the following: CO2; CO; H2; and inorganic minerals dissolved in an aqueous solution. In some embodiments, oxyhydrogen microorganisms and / or hydrogenotrophs and / or carboxydotrophs and / or other chemoautotrophs and / or methanotrophs convert greenhouse gases into biomolecules including amino acids and proteins.

[0089] "C1" refers to a single carbon molecule, such as CO, CO2, CH4 or CH3OH. "C1 oxide" refers to a single carbon molecule that also contains at least one oxygen atom, such as CO, CO2 or CH3OH. "C1 carbon source" refers to a single carbon molecule used as part or the only carbon source for the microorganism of the present disclosure. For example, the C1 carbon source can contain one or more of the following: CO, CO2, CH4, CH3OH or CH2O2. Preferably, the C1 carbon source contains one or both of CO and CO2. "C1 fixed microorganism" is a microorganism capable of producing one or more products from a C1 carbon source. The microorganism of the present disclosure is typically a C1 fixed bacteria. In a preferred embodiment, the microorganism of the present disclosure is derived from a C1 fixed microorganism.

[0090] "Anaerobic bacteria" are microorganisms that do not require oxygen to grow. If oxygen is present above a certain threshold, the anaerobic bacteria may have adverse reactions or even die. However, some anaerobic bacteria are able to tolerate low levels of oxygen (e.g., 0.000001-5% oxygen), sometimes referred to as "microaerobic conditions." Typically, the microorganisms of the present disclosure are anaerobic bacteria. In a preferred embodiment, the microorganisms of the present disclosure are derived from anaerobic bacteria.

[0091] "Acetogens" are obligate anaerobes 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, Acta Biochimica et Biophysica Sinica, 1784: 1873-1898, 2008). Specifically, acetogens use the Wood-Ljungdahl pathway as a mechanism for (1) synthesis of acetyl-CoA from CO2 reduction, (2) a terminal electron acceptance and energy conservation process, and (3) CO2 fixation (assimilation) in cellular carbon synthesis (Drake, "Acetogenic Prokaryotes", The Prokaryotes, 3rd ed., p. 354, New York, NY, 2006). All naturally occurring acetogens are C1-fixing, anaerobic, autotrophic, and non-methane-oxidizing. Typically, the microorganisms of the present disclosure are acetogens. In preferred embodiments, the microorganisms of the present disclosure are derived acetogens.

[0092] An "ethanologen" is a microorganism that produces or is capable of producing ethanol. Typically, the microorganism of the present disclosure is an ethanologen. In a preferred embodiment, the microorganism of the present disclosure is derived from an ethanologen.

[0093] "Autotrophs" are microorganisms that can grow in the absence of organic carbon. In contrast, autotrophs use inorganic carbon sources, such as CO and / or CO2. Typically, the microorganisms of the present disclosure are autotrophs. In a preferred embodiment, the microorganisms of the present disclosure are derived from autotrophs.

[0094] A "carboxydotrophic bacterium" is a microorganism that is able to utilize CO as the sole source of carbon and energy. Typically, the microorganisms of the present disclosure are carboxydotrophic bacteria. In a preferred embodiment, the microorganisms of the present disclosure are derived from carboxydotrophic bacteria.

[0095] "Methanotrophs" are microorganisms that are able to utilize methane as a 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.

[0096] The term "oxyhydroxide" refers to a mixture of molecular hydrogen and oxygen. "Oxyhydroxide microorganisms" are microorganisms that can use hydrogen as an electron donor and oxygen as an electron acceptor in respiration to produce intracellular energy carriers such as adenosine-5'-triphosphate (ATP).

[0097] The terms "oxyhydrogen" and "oxyhydrogen microorganism" may be used synonymously with "oxyhydrogen" and "oxyhydrogen microorganism", respectively. Oxyhydrogen microorganisms typically use molecular hydrogen via hydrogenases, with some of the electrons being used to reduce NAD. + (and / or other intracellular reducing equivalents), and some electrons come from the H2 used for aerobic respiration. Hydrogen-oxygen mixed gas microorganisms typically autotrophically fix CO2 through pathways including, but not limited to, the Calvin cycle or the reverse citric acid cycle.

[0098] However, as described above, the microorganism of the present disclosure may also be derived from essentially any parent microorganism, such as a parent microorganism selected from the group consisting of: Escherichia coli and Saccharomyces cerevisiae.

[0099] In another embodiment, the microorganism of the present disclosure is an aerobe. In one embodiment, the microorganism of the present disclosure comprises an aerobic hydrogen bacterium. In one embodiment, the aerobe comprises at least one disrupted gene.

[0100] Many aerobic bacteria are known to be able to ferment the disclosed methods and systems. Examples of such bacteria suitable for the present invention include bacteria of the genera Cupriavidus and Ralstonia. In some embodiments, the aerobic bacteria is Cupriavidus hookworm or Ralstonia eutropha. In some embodiments, the aerobic bacteria is Cupriavidus alkaliphilus. In some embodiments, the aerobic bacteria is Cupriavidus basilensis. In some embodiments, the aerobic bacteria is Cupriavidus campinensis. In some embodiments, the aerobic bacteria is Cupriavidus gilardii. In some embodiments, the aerobic bacteria is Cupriavidus laharis. In some embodiments, the aerobic bacteria is Cupriavidus metallidurans. In some embodiments, the aerobic bacteria is Cupriavidus nantongensis. In some embodiments, the aerobic bacteria is Cupriavidus numazuensis. In some embodiments, the aerobic bacteria is Cupriavidus oxalaticus. In some embodiments, the aerobic bacteria is Cupriavidus oxalaticus. In some embodiments, the aerobic bacteria is Cupriavidus pauculus. In some embodiments, the aerobic bacteria is Cupriavidus pinatubonensis. In some embodiments, the aerobic bacteria is Cupriavidus plantarum. In some embodiments, the aerobic bacteria is Cupriavidus respiraculi. In some embodiments, the aerobic bacteria is Cupriavidus taiwanensis. In some embodiments, the aerobic bacteria is Cupriavidus yeoncheonensis.

[0101] In some embodiments, the microorganism is Cupriavidus necrotus DSM248 or DSM541.

[0102] In some embodiments, the aerobic bacteria comprises one or more exogenous nucleic acid molecules encoding naturally-occurring polypeptides, wherein the polypeptide is a ribulose bisphosphate carboxylase, an acetyl-CoA acetyltransferase, a 3-hydroxybutyryl-CoA dehydratase, a butyryl-CoA dehydrogenase, a butanol dehydrogenase, an electron transfer flavoprotein large subunit, a 3-hydroxybutyryl-CoA dehydrogenase, a bifunctional acetaldehyde-CoA / alcohol dehydrogenase, an acetaldehyde dehydrogenase, an aldehyde decarbonylase, an acyl-ACP reductase, an L-1,2-propanediol oxidoreductase, an acyltransferase, a 3-oxoacyl-ACP synthase, a 3-hydroxybutyryl-CoA epimerase / delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase / enoyl-CoA hydratase / 3-hydroxyacyl-CoA dehydrogenase, a short-chain dehydrogenase, a trans-2-enoyl-CoA reductase, or any combination thereof.

[0103] In the microorganism of the present disclosure, carbon flux is strategically turned to the product of concern from non-essential or undesirable product.In certain embodiments, these destroyed genes make carbon flux shift and transfer through target metabolism node from non-essential or undesirable metabolism node, to improve the product output of those target metabolism node downstreams.In one embodiment, the restriction that is selected from nutrients, dissolved oxygen or its any combination transfers carbon flux to the desired product.

[0104] In one embodiment, fermented liquid comprises the feed stream combined with the aerobic microorganism in the bioreactor.In certain embodiments, feed stream, for example carbon source feed stream, stream containing combustible gas and oxygen-containing gas feed stream, react with the microorganism in the bioreactor to form fermented liquid (it also can comprise other products, byproducts and other substratums that are fed to the bioreactor) at least in part. Unreacted oxygen or the oxygen not consumed by microorganism is present in the dispersed gas phase in the fermented liquid in the form of dissolved oxygen and gaseous oxygen. Other soluble gases are also like this. The dispersed gas phase containing unreacted components (for example, oxygen, nitrogen, hydrogen, carbon dioxide and / or water vapor) rises to the head space of bioreactor.

[0105] In some embodiments, oxygen-containing gas, such as air, can be directly fed into the fermentation broth. In one embodiment, the oxygen-containing gas can be an oxygen-enriched source, such as oxygen-enriched air or pure oxygen. In one embodiment, the oxygen-containing gas can contain greater than 6.0 vol.% oxygen, such as greater than 10.0 vol.%, greater than 20.0 vol.%, greater than 40.0 vol.%, greater than 60.0 vol.%, greater than 80.0 vol.% or greater than 90.0 vol.%. In some embodiments, the oxygen-containing gas can be pure oxygen.

[0106] In one embodiment, the microorganism of the present disclosure is capable of producing ethylene. One embodiment relates to a recombinant C1 fixed microorganism capable of producing ethylene from a carbon source, comprising a nucleic acid encoding a set of exogenous enzymes, wherein the exogenous enzymes include at least one ethylene forming enzyme (EFE). In some embodiments, the EFE is derived from Pseudomonas syringae. In one embodiment, the EFE has an EC number 1.13.12.19. According to the microorganism described in the embodiment, it includes at least one EFE with EC number 1.13.12.19. According to the microorganism described in the embodiment, it further includes a nucleic acid encoding a set of exogenous enzymes, wherein the exogenous enzymes include at least one α-ketoglutarate permease (AKGP).

[0107] According to the microorganism described in the embodiment, the nucleic acid encoding a group of exogenous enzymes includes at least one EFE, at least one AKGP or any combination thereof. According to the microorganism described in the embodiment, the nucleic acid encoding a group of exogenous enzymes includes at least one EFE and at least one AKGP. According to the microorganism described in the embodiment, the nucleotides encoding a 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 of a homologous recombination system, a CRISPR Cas system or any combination thereof. In one embodiment, the promoter is an inducible promoter limited by phosphate. In some embodiments, the promoter is a promoter limited by nitrogen. In some embodiments, the promoter is a promoter activated by NtrC-P. In some embodiments, the promoter is an H2 inducible promoter. In one embodiment, the microorganism comprises an intracellular oxygen concentration limit. In another embodiment, the method limits intracellular oxygen concentration. In one embodiment, the method comprises a step of controlling dissolved oxygen. In one embodiment, the method comprises reducing ethylene production as the dissolved oxygen concentration decreases. In some embodiments, the microorganism comprises a molecular switch. In some embodiments, the microorganism comprises the ability to switch cell load under variable conditions.

[0108] In some embodiments, the microorganism is a natural or engineered microorganism capable of converting a gaseous substrate into a carbon source and / or an energy source. In one embodiment, the gaseous substrate comprises CO2 as a carbon source. In some embodiments, the gaseous substrate comprises H2 and / or O2 as an energy source. In one embodiment, the gaseous substrate comprises a mixture of gases, and the gas comprises H2 and / or CO2 and / or CO.

[0109] In some embodiments, the gaseous fermentation products are selected from alcohols, acids, diacids, olefins, terpenes, isoprene and alkynes. In some embodiments, the methods and microorganisms disclosed herein are used to increase ethylene production. In one embodiment, the methods and microorganisms disclosed herein are used to improve the production of gaseous fermentation products.

[0110] In one embodiment, the aerobic bacteria can produce 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-hydroxybutyraldehyde, 1,3-butanediol, 2-hydroxyisobutyryl-CoA, 2-hydroxyisobutyrate, butyryl-CoA, butyrate, butanol, hexanoate, hexanol, octanoate, octanol, 1,3-hexanediol, 2-butene-1-ol, isovaleryl-CoA, isovalerate, isopentanol, methacrolein, methyl methacrylate, or any combination thereof.

[0111] In another embodiment, the bacteria of the present disclosure can produce 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-propylene glycol, 1 propanol, 1 hexanol, 1 octanol, chorismate-derived products, 3 hydroxybutyrate, 1,3 butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipate, 1,3 hexanediol, 3-methyl-2-butanol, 2-butene-1-ol, isovalerate, isopentanol, and monoethylene glycol, or any combination thereof.

[0112] 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.

[0113] Enzyme of the present invention can be codon optimized to express in microorganism of the present invention. "Codon optimization" refers to the mutation of nucleic acid such as gene, which is used to optimize or improve the translation of nucleic acid in a specific strain or species. Codon optimization can produce faster translation rate or higher translation accuracy. In a preferred embodiment, gene of the present invention is codon optimized to express in microorganism of the present invention. Although codon optimization refers to potential genetic sequence, codon optimization usually makes translation improved, thereby improving the expression of enzyme. Therefore, enzyme of the present invention can also be described as codon optimized.

[0114] One or more enzymes in the enzyme of the present disclosure can be overexpressed. "Overexpression" refers to an increase in the expression of nucleic acids or proteins in the 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 modifying gene copy number, gene transcription rate, gene translation rate or enzyme degradation rate.

[0115] Enzymes of the present disclosure may include destructive mutations. "Destructive mutations" refer to mutations that reduce or eliminate (i.e., "destruct") the expression or activity of a gene or enzyme. Destructive mutations may partially inactivate, completely inactivate, or delete a gene or enzyme. Destructive mutations may be knockout (KO) mutations. Destructive mutations may be any mutations that reduce, prevent, or block the biosynthesis of a product produced by an enzyme. The destructive mutations may include, for example, mutations in genes encoding enzymes, mutations in gene regulatory elements involved in the expression of genes encoding enzymes, introduction of nucleic acids that produce proteins that reduce or inhibit enzyme activity, or introduction of nucleic acids that inhibit protein or enzyme expression (e.g., antisense RNA, siRNA, CRISPR). Destructive mutations may be introduced using any method known in the art.

[0116] The introduction of destructive mutations causes the microorganism of the present disclosure to not produce the target product or not produce the target product substantially, or the amount of the target product is reduced compared with the parent microorganism from which the microorganism of the present disclosure is derived. For example, the microorganism of the present disclosure may not produce the target product, or may produce a target product less than the parent microorganism by at least about 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. For example, the microorganism of the present disclosure may produce a target product less than about 0.001, 0.01, 0.10, 0.30, 0.50 or 1.0 g / L.

[0117] Although exemplary sequences and sources of enzymes are provided herein, the disclosure is by no means limited to these sequences and sources, and variants are also encompassed in the disclosure. The term "variant" includes nucleic acids and proteins whose sequences are different from the sequences of reference nucleic acids and proteins such as disclosed in the prior art or exemplified herein. The disclosure can be practiced using variant nucleic acids or proteins that perform substantially the same functions as reference nucleic acids or proteins. For example, variant proteins can perform substantially the same functions as reference proteins or catalyze substantially the same reactions as reference proteins. Variant genes can encode proteins that are identical or substantially identical to reference genes. Variant promoters can have substantially the same ability as reference promoters to promote the expression of one or more genes.

[0118] Such nucleic acids or proteins may be referred to herein as "functional equivalent variants". For example, functional equivalent variants of nucleic acids may include allelic variants, gene fragments, mutant genes, polymorphisms, etc. Homologous genes from other microorganisms are also examples of functional equivalent variants. These homologous genes include homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii or Clostridium jundal, and their detailed information can be publicly available on websites such as Genbank or NCBI. Functional equivalent variants also include nucleic acids whose sequences vary due to codon optimization of specific microorganisms. Functional equivalent variants of nucleic acids will preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or higher nucleic acid sequence identity (homology percentage) with the reference nucleic acid. Functional equivalent variants of proteins preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or higher amino acid identity (homology percentage) with the reference protein. The functional equivalence of variant nucleic acids or proteins can be assessed using any method known in the art.

[0119] "Complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional types. The complementarity percentage represents the percentage of residues in a nucleic acid molecule that are able to 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 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Complete complementarity" means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of 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 to two nucleic acids that hybridize under stringent conditions.

[0120] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between nucleotide residue bases. Hydrogen bonding can occur by Watson Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex can include two chains that form a double-stranded structure, three or more chains that form a multi-chain complex, a single self-hybridizing chain, or any combination of these. The hybridization reaction can constitute a step in a broader process (such as starting PCR, or cutting polynucleotides with an enzyme). A sequence that can hybridize with a given sequence is called the "complementary sequence" of a given sequence.

[0121] Nucleic acid can be delivered to the microorganism of the present disclosure using any method known in the art. For example, nucleic acid can be delivered as naked nucleic acid or can be prepared with one or more reagents such as liposomes. When appropriate, nucleic acid can be DNA, RNA, cDNA or a combination thereof. Restriction inhibitors can be used in certain embodiments. Other carriers can include plasmids, viruses, phages, cosmids and artificial chromosomes. In a preferred embodiment, nucleic acid is delivered to the microorganism of the present disclosure using plasmids. For example, conversion (including transduction or transfection) can be achieved by electroporation, ultrasonic treatment, polyethylene glycol-mediated conversion, chemical or natural competence, protoplast transformation, prophage induction or conjugation. In certain embodiments with an active restriction enzyme system, it may be necessary to methylate the nucleic acid before introducing the nucleic acid into the microorganism.

[0122] In addition, nucleic acids can be designed to include regulatory elements such as promoters to increase or otherwise control the expression of specific nucleic acids. The promoter can be a constitutive promoter or an inducible promoter. The promoter can be a Wood-Yungdahl pathway promoter, a ferredoxin promoter, a pyruvate ferredoxin oxidoreductase promoter, a Rnf composite operon promoter, an ATP synthase operon promoter, or a phosphotransacetylase / acetate kinase operon promoter.

[0123] It should be understood that the disclosure can be implemented using nucleic acids whose sequences are different from the sequences specifically exemplified herein, provided that the nucleic acids perform substantially the same function. For nucleic acid sequences encoding proteins or peptides, this means that the encoded proteins or peptides have substantially the same function. For nucleic acid sequences representing promoter sequences, variant sequences will be able to promote the expression of one or more genes. Such nucleic acids may be referred to as "functional equivalent variants" in this article. For example, functional equivalent variants of nucleic acids include allelic variants, fragments of genes, genes including mutations (deletions, insertions, nucleotide substitutions, etc.) and / or polymorphisms, etc. Homologous genes from other microorganisms may also be considered as examples of functional equivalent variants of sequences specifically exemplified herein.

[0124] The phrase "functionally equivalent variants" should also include nucleic acids whose sequences vary due to codon optimization for a particular organism. 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 identified nucleic acid.

[0125] It should also be understood that the present disclosure can be implemented using polypeptides whose sequences are different 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 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 within their scope fragments of proteins or peptides, wherein the fragments contain truncated forms of polypeptides, wherein the deletions may be 1 to 5, to 10, to 15, to 20, to 25 amino acids, and may extend from residues 1 to 25 at either end of the polypeptide, and wherein the deletions may be any length within the region; or may be in an internal position. Functionally equivalent variants of specific polypeptides herein should also be considered to include polypeptides expressed by homologous genes in other bacterial species, such as those exemplified in the previous paragraph.

[0126] The microorganism of the present disclosure can be prepared by 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, ultrasonic treatment, polyethylene glycol-mediated transformation, chemical or natural competence or conjugation. Suitable transformation is described in, for example, Sambrook J, Fritsch EF, Maniatis T: "Molecular Cloning: A laboratory Manual" Cold Spring Harbor Press (Cold Spring Harbour Laboratory Press, Cold Spring Harbour), 1989.

[0127] In certain embodiments, due to the restriction system active in the microorganism to be transformed, it is necessary to methylate the nucleic acid to be introduced into the microorganism. This can be accomplished using a variety of techniques, including those described below, and further illustrated in the Examples section below.

[0128] For example, in one embodiment, the recombinant microorganism of the present disclosure is produced by a method comprising the following steps: introducing (i) of the expression construct / vector as 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 target microorganism.

[0129] In one embodiment, the methyltransferase gene of step B is constitutively expressed. In another embodiment, the expression of the methyltransferase gene of step B is induced.

[0130] The shuttle microorganism is a microorganism that promotes methylation of the nucleic acid sequence constituting the expression construct / vector, preferably a restriction-negative microorganism. In a specific embodiment, the shuttle microorganism is a restriction-negative Escherichia coli, Bacillus subtilis or Lactococcus lactis.

[0131] The methylation construct / vector comprises a nucleic acid sequence encoding a methyltransferase.

[0132] Once the expression construct / vector and the methylation construct / vector are introduced into the shuttle microorganism, the methyltransferase gene present on the methylation construct / vector will be induced. Induction can be carried out by any suitable promoter system, but in a specific embodiment of the present disclosure, the methylation construct / vector comprises an inducible lac promoter and is induced by adding lactose or its analogs, more preferably isopropyl-β-D-thiogalactoside (IPTG). Other suitable promoters include ara, tet or T7 systems. In other embodiments of the present disclosure, the methylation construct / vector promoter is a constitutive promoter.

[0133] In a particular embodiment, the methylation construct / vector has a replication origin specific to the identity of the shuttle microorganism, such that any gene present on the methylation construct / vector is expressed in the shuttle microorganism. Preferably, the expression construct / vector has a replication origin specific to the identity of the target microorganism, such that any gene present on the expression construct / vector is expressed in the target microorganism.

[0134] The expression of the methyltransferase causes methylation of genes present on the expression construct / vector. The expression construct / vector can then be separated from the shuttle microorganism according to any of a variety of known methods. By way of example only, the methods described in the Examples section described below can be used to separate the expression construct / vector.

[0135] In a specific embodiment, both constructs / vectors are isolated in parallel.

[0136] The expression construct / vector can be introduced into the target microorganism using any of a variety of known methods. However, for example, the method described in the example section below can be used. Because the expression construct / vector is methylated, the nucleotide sequence present on the expression construct / vector can be incorporated into the target microorganism and successfully expressed.

[0137] It is contemplated that the methyltransferase gene can be introduced into the shuttle microorganism and overexpressed. Therefore, in one embodiment, the resulting methyltransferase can be collected using known methods and used in vitro to methylate the expression plasmid. The expression construct / vector can then be introduced into the target microorganism for expression. In another embodiment, the methyltransferase gene is introduced into the genome of the shuttle microorganism, the expression construct / vector is then introduced into the shuttle microorganism, one or more constructs / vectors are separated from the shuttle microorganism, and the expression construct / vector is then introduced into the target microorganism.

[0138] It is contemplated that the expression constructs / vectors and methylation constructs / vectors as defined above can be combined to provide a composition of matter. Such a composition has particular utility in circumventing restrictive barrier mechanisms to produce the recombinant microorganisms of the present disclosure.

[0139] In a specific embodiment, the expression construct / vector and / or the methylation construct / vector is a plasmid.

[0140] Those of ordinary skill in the art will appreciate many suitable methyltransferases for producing the microorganisms of the present disclosure. However, by way of example, the Bacillus subtilis phage ΦT1 methyltransferase and the methyltransferases described in the Examples below may be used. Nucleic acids encoding suitable methyltransferases will be readily understood given the sequence and genetic code of the desired methyltransferase.

[0141] Any number of constructs / vectors suitable for allowing expression of a methyltransferase gene may be used to generate a methylation construct / vector.

[0142] In one embodiment, the substrate comprises CO2 and an energy source. In some embodiments, the substrate comprises CO2 and an energy source. In one embodiment, the substrate comprises CO2, H2, and O2. In some embodiments, the substrate comprises CO2 and any suitable energy source. In one embodiment, the substrate comprises CO. In one embodiment, the substrate comprises CO2 and CO. In another embodiment, the substrate comprises CO2 and H2. In another embodiment, the substrate comprises CO2 and CO and H2.

[0143] "Substrate" refers to the carbon source and / or energy source of the microorganism of the present disclosure. Generally, the substrate is gaseous and comprises a C1 carbon source, such as CO, CO2 and / or CH4. Preferably, the substrate comprises a C1 carbon source of CO or CO+CO2. The substrate may further comprise other non-carbon components, such as H2, N2 or electrons. However, in other embodiments, the substrate may be a carbohydrate (such as 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 certain embodiments, the substrate does not comprise (D)-xylose (Alkim, "Microb Cell Fact", 14: 127, 2015). In certain embodiments, the substrate does not comprise pentose, such as xylose (Pereira, "Metab Eng", 34: 80-87, 2016). In certain embodiments, the substrate may comprise both a gaseous substrate and a carbohydrate substrate (mixed nutrient fermentation). The substrate may further comprise other non-carbon components, such as H2, N2 or electrons.

[0144] In some embodiments, the gaseous substrate generally comprises at least a certain 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 may comprise a range of CO, such as about 20-80, 30-70 or 40-60 mol % CO. Preferably, the gaseous substrate comprises 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 may comprise a relatively low amount of CO, such as about 1-10 or 1-20 mol % CO. The microorganisms of the present disclosure generally convert at least a portion of the CO in the substrate into a gaseous substrate. In some embodiments, the gaseous substrate does not comprise or substantially does not comprise (<1 mol %) CO.

[0145] The gaseous substrate may contain a certain amount of H2. For example, the gaseous substrate may contain about 1, 2, 5, 10, 15, 20, or 30 mol% H2. In some embodiments, the gaseous substrate may contain a relatively high amount of H2, such as about 60, 70, 80, or 90 mol% H2. In other embodiments, the gaseous substrate contains no or substantially no (<1 mol%) H2.

[0146] The gaseous substrate may contain a certain amount of CO2. For example, the gaseous substrate may contain about 1-80 or 1-30 mol% CO2. In some embodiments, the gaseous substrate may contain less than about 20, 15, 10 or 5 mol% CO2. In another embodiment, the gaseous substrate contains no or substantially no (<1 mol%) CO2.

[0147] The gaseous substrate may also be provided in alternative forms. For example, the gaseous substrate may be dissolved in a liquid or adsorbed on a solid support.

[0148] The gaseous substrate and / or C1 carbon source may be a waste gas obtained as a byproduct of an industrial process or from some other source, such as from automobile exhaust or biomass gasification. In certain embodiments, the industrial process is selected from the group consisting of: ferrous metal product manufacturing (such as steel mill manufacturing), non-ferrous metal product manufacturing, 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 collected from the industrial process using any suitable method before it is discharged into the atmosphere.

[0149] The gaseous substrate and / or C1 carbon source may be synthesis gas, such as synthesis gas obtained by gasification of coal or refinery residues, gasification of biomass or lignocellulosic materials, or reforming of natural gas. In another embodiment, the synthesis gas may be obtained by gasification of municipal solid waste or industrial solid waste.

[0150] The term "feedstock" when used in the context of a stream flowing into a gas fermentation bioreactor (i.e., a gas fermentor) or a "gas fermentation feedstock" 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 the gas fermentor or bioreactor, either directly or after processing of the feedstock.

[0151] The terms "waste gas" or "waste stream" may be used to refer to any gas stream that is either directly discharged, combusted without additional value capture, or combusted for energy recovery purposes.

[0152] The term "synthesis gas" or "syngas" refers to a gaseous mixture containing at least one carbon source, such as carbon monoxide (CO), carbon dioxide (CO2), or any combination thereof, and optionally hydrogen (H2), which can be used as a feedstock for the disclosed gas fermentation process and can be produced from a variety of solid and liquid carbonaceous materials.

[0153] The substrate and / or C1 carbon source can be a waste gas obtained as a by-product of an industrial process or a waste gas from another source, such as automobile exhaust, biogas or landfill gas, direct air capture, or waste gas from electrolysis. The substrate and / or C1 carbon source can be a synthesis gas generated by pyrolysis, torrefaction, or gasification. In other words, the peak carbon in the waste material can be recycled by pyrolysis, torrefaction, or gasification to produce a synthesis gas for use as a substrate and / or C1 carbon source. The substrate and / or C1 carbon source can be a gas comprising methane.

[0154] In certain embodiments, the industrial process is selected from the manufacture of ferrous metal products, such as steel manufacturing, non-ferrous metal product manufacturing, petroleum refining, power generation, carbon black production, paper and pulp manufacturing, ammonia production, methanol production, coke manufacturing, petrochemical production, carbohydrate fermentation, cement manufacturing, aerobic digestion, anaerobic digestion, catalytic process, natural gas extraction, cellulose fermentation, oil extraction, geological reservoirs, gas from fossil resources such as natural gas coal and petroleum or any combination thereof. Examples of specific processing steps in industrial processes include catalyst regeneration, fluid catalyst cracking and catalyst regeneration. Air separation and direct air capture are other suitable industrial processes. Specific examples in steel and ferroalloy manufacturing include direct reduction of blast furnace gas, alkaline oxygen furnace gas, coke oven gas, iron furnace top gas and residual gas from iron smelting. In these embodiments, any known method can be used to capture substrate and / or C1 carbon source from industrial processes, and then discharge it into the atmosphere.

[0155] Substrate and / or C1 carbon source can be the synthesis gas referred to as synthesis gas, and it can be obtained from reforming, partial oxidation or gasification method.The example of gasification method comprises the gasification of coal, the gasification of refinery residue, the gasification of petroleum coke, the gasification of biomass, the gasification of lignocellulosic material, the gasification of waste wood, the gasification of black liquor, the gasification of urban solid waste, the gasification of urban liquid waste, the gasification of industrial solid waste, the gasification of industrial liquid waste, the gasification of fuel from garbage source, the gasification of sewage, the gasification of sewer sludge, the gasification of sludge from wastewater treatment, the gasification of biogas.The example of reforming process comprises steam methane reforming, steam naphtha reforming, natural gas reforming, biogas reforming, landfill gas reforming, naphtha reforming and dry methane reforming.The example of partial oxidation process comprises heat and catalytic partial oxidation process, catalytic partial oxidation of natural gas, partial oxidation of hydrocarbon.The example of urban solid waste comprises tires, plastics, fibers such as in shoes, clothing and textiles. Municipal solid waste may be landfill type waste only. Municipal solid waste may be sorted or unsorted. Examples of biomass may include lignocellulosic materials, and may also include microbial biomass. Lignocellulosic materials may include agricultural waste and forest waste.

[0156] The substrate and / or C1 carbon source can be a gas stream containing methane. Such methane-containing gas can 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 can be burned to generate electricity or heat, and the C1 byproduct can be used as a substrate or carbon source.

[0157] The composition of the gaseous substrate may have a significant impact on the efficiency and / or cost of the reaction. For example, the presence of oxygen (O2) can reduce the efficiency of the anaerobic fermentation process. Depending on the composition of the substrate, it may be necessary 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 the desired component.

[0158] Regardless of the source or exact content of the gas used as feedstock, the feedstock can be metered (e.g., for carbon credit calculations or mass balance of sustainable carbon with total product) into the bioreactor to maintain control over the rate and amount of carbon provided to the culture. Similarly, the output of the bioreactor can be metered (e.g., for carbon credit calculations or mass balance of sustainable carbon with total product) or contain valve connections that can control the flow of output and products (e.g., ethylene, ethanol, acetate, 1-butanol, etc.) produced by fermentation. Such valves or metering mechanisms can be used for a variety of purposes, including but not limited to throttling of products through connected pipes and measuring the output from a given bioreactor so that if the product is mixed with other gases or liquids, the resulting mixture can then be mass balanced to determine the percentage of the product produced from the bioreactor.

[0159] In certain embodiments, the fermentation is performed in the absence of a carbohydrate substrate such as sugars, starch, lignin, cellulose, or hemicellulose.

[0160]

[00136] The microorganisms of the present disclosure can be cultured to produce one or more co-products in addition to ethylene. For example, in addition to ethylene, the microorganisms of the present disclosure can also produce or can also be engineered to 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), lactate (WO 2011 / 112103), butene (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 / 036147), 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-propylene glycol (WO 2014 / 036152), 1-propanol (WO 2017 / 066498), 1-hexanol (WO 2017 / 066498), 1-octanol (WO 2017 / 066498), chorismate-derived products (WO 2017 / 066498), 3-hydroxybutyrate (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), 1,3-hexanediol (WO 2017 / 066498), 3-methyl-2-butanol (WO 2017 / 066498), 2-butene-1-ol (WO 2017 / 066498), isovalerate (WO 2017 / 066498), isopentanol (WO 2017 / 066498) and / or monoethylene glycol (WO 2019 / 126400). In certain embodiments, the microbial biomass itself can be considered a product. These products can be further converted to produce at least one component of diesel, jet fuel, sustainable aviation fuel (SAF) and / or gasoline. In certain embodiments, ethylene can be catalytically converted into another product, an article, 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 increase the yield of a product, protein, microbial biomass, or any combination thereof.

[0161] A "natural product" is a product produced by a microorganism that has not been genetically modified. For example, ethanol, acetate, and 2,3-butanediol are natural products of Clostridium ethanologenum, Clostridium ljungdahlii, and Clostridium raginsii. A "non-natural product" is a product produced by a genetically modified microorganism that is not produced by the non-genetically modified microorganism from which the genetically modified microorganism was derived. As is well known, ethylene is not produced by any naturally occurring microorganism and is therefore a non-natural product of all microorganisms.

[0162] "Selectivity" refers to the ratio of the output of the target product to the output of all fermentation products produced by the microorganism. The microorganism of the present disclosure can be engineered to produce products with a specific selectivity or a minimum 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 all fermentation products produced by the microorganism of the present disclosure. In one embodiment, ethylene accounts for at least 10% of the total fermentation products produced by the microorganism of the present disclosure, so that the selectivity of ethylene glycol of the microorganism of the present disclosure is at least 10%. In another embodiment, ethylene accounts for at least 30% of the total fermentation products produced by the microorganism of the present disclosure, so that the selectivity of ethylene of the microorganism of the present disclosure is at least 30%.

[0163] At least one of the one or more fermentation products can be biomass produced by the culture. At least a portion of the microbial biomass can be converted into single cell protein (SCP). At least a portion of the single cell protein can be utilized as a component of animal feed.

[0164] In one embodiment, the present disclosure provides an animal feed comprising 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.

[0165] "Single cell protein" (SCP) refers to a microbial biomass that can be used in protein-rich human and / or animal feeds, typically replacing conventional sources of protein supplements, such as soybean meal or fish meal. In order 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. The method may also include reducing the nucleic acid content of the microbial biomass using any method known in the art, because the intake of a diet high in nucleic acid content may lead to accumulation of nucleic acid degradation products and / or gastrointestinal discomfort. The single cell protein may be suitable for feeding animals such as livestock or pets. Specifically, animal feed can be suitable for feeding one or more beef cattle, dairy cows, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, white-hipped bison, large-fronted cattle, yaks, chickens, turkeys, ducks, geese, quails, guinea fowls, nestlings / pigeons, fish, shrimps, crustaceans, cats, dogs and rodents. The composition of animal feed can be customized according to the nutritional requirements of different animals. In addition, the process can include blending or combining microbial biomass with one or more excipients.

[0166] "Microbial biomass" refers to biological material comprising microbial cells. For example, microbial biomass can comprise or consist of a pure or substantially pure culture of bacteria, archaea, viruses, or fungi. When initially isolated from a fermentation broth, microbial biomass typically contains a significant amount of water. This water can be removed or reduced by drying or processing the microbial biomass.

[0167] " excipient " can refer to any material that can be added to microbial biomass to enhance or change the form, property or nutritional content of animal feed. For example, excipient can include one or more of the following: carbohydrate, fiber, fat, protein, vitamin, mineral, water, flavoring agent, sweetener, antioxidant, enzyme, preservative, probiotic or antibiotic. In certain embodiments, excipient can be hay, straw, silage, cereal, oil or fat or other plant materials. Excipient can be in Chiba, Section 18: "Dietary ingredients and common feed raw materials (Diet Formulation and CommonFeed Ingredients)", Animal Nutrition Handbook (Animal Nutrition Handbook), the 3rd revised edition, 575-633 pages, any feed raw materials identified in 2014.

[0168] "Biopolymer" refers to a natural polymer produced by cells of a living organism. In certain embodiments, the biopolymer is PHA. In certain embodiments, the biopolymer is PHB.

[0169] "Bioplastics" refers to plastic materials produced from renewable biomass sources. Bioplastics can be produced from renewable resources such as vegetable fats and oils, corn starch, straw, wood chips, sawdust or recycled food waste.

[0170] In this context, reference to an acid (eg, acetic acid or 2-hydroxyisobutyric acid) is to be understood as also including the corresponding salts (eg, acetate or 2-hydroxyisobutyrate).

[0171] Typically, culture is carried out in a bioreactor. The term "bioreactor" includes a culture / fermentation device composed of one or more containers, towers or piping arrangements, such as a continuous stirred tank reactor (CSTR), an immobilized cell reactor (ICR), a trickle bed reactor (TBR), a bubble column, an airlift fermenter, a static mixer or other containers or other devices suitable for gas-liquid contact. In certain embodiments, the bioreactor may 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 "culture" and "fermentation" are used interchangeably. These terms cover both the growth phase and the product biosynthesis phase of the culture / fermentation process.

[0172] Cultivation is usually maintained in an aqueous medium containing nutrients, vitamins and / or minerals sufficient to allow microbial growth. Preferably, the aqueous medium is an anaerobic microbial growth medium, such as a minimal anaerobic microbial growth medium. Suitable culture media are well known in the art.

[0173] Cultivation / fermentation should ideally be carried out under appropriate conditions to produce ethylene glycol. Typically, cultivation / fermentation is carried out under anaerobic conditions. The reaction conditions to be considered 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 a limiting factor, and maximum product concentration to avoid product inhibition. Specifically, the introduction rate of the substrate can be controlled to ensure that the concentration of the gas in the liquid phase does not become a limiting factor, because culturing under gas limiting conditions consumes product.

[0174] Operating a bioreactor under high pressure allows to increase the mass transfer rate of gas from gas phase to liquid phase. Therefore, it is generally preferred to perform cultivation / fermentation under a pressure higher than atmospheric pressure. Similarly, since a given gas conversion rate is partly a function of the substrate retention time and the retention time indicates the required volume of the bioreactor, the volume of the required bioreactor can be greatly reduced using a pressurized system, and therefore the capital cost of the cultivation / fermentation equipment is reduced. This then means that when the bioreactor is maintained at an elevated pressure rather than atmospheric pressure, the retention time can be reduced, and the retention time is defined as the liquid volume in the bioreactor divided by the input gas flow rate. Optimal reaction conditions will depend in part on the specific microorganism used. However, in general, it is preferred to operate fermentation under a pressure higher than atmospheric pressure. In addition, since a given gas conversion rate changes with the substrate retention time to a certain extent, and obtains the required retention time and indicates the required volume of the bioreactor, the volume of the required bioreactor can be greatly reduced using a pressurized system, and therefore the capital cost of the fermentation equipment is reduced.

[0175] "Ejectors" may include devices for introducing gas into a liquid (injected in the form of bubbles) to agitate the liquid or dissolve the gas in the liquid. Examples of injectors may include orifice plate injectors, sintered injectors, and drilled tube injectors. In some configurations, drilled tube injectors may be mounted horizontally. In other examples, the injector may be mounted vertically or horizontally. In some examples, the injector may be a perforated plate or ring, sintered glass, sintered steel, porous rubber tube, porous metal tube, porous ceramic or stainless steel, drilled tube, stainless steel drilled tube, polymer drilled tube, etc. Sprayers may be of different grades (porosity) or may include holes of a specific size to produce bubbles of a specific size or a range of bubble sizes.

[0176] "Container", "reaction vessel" or "tower" can be a vessel or container in which one or more gas and liquid streams or flows can be introduced to generate bubbles and / or generate microbubbles, and used for subsequent gas-liquid contact, gas absorption, biological or chemical reaction or adsorption of surfactant materials. In the reaction vessel, the gas phase and the liquid phase can flow in the vertical direction. In the reaction vessel, larger bubbles from the ejector may rise upward, and their buoyancy is greater than the resistance applied by the liquid. Smaller microbubbles with buoyancy less than or equal to the resistance applied by the liquid can flow downward with the liquid, as described in the systems and methods disclosed herein. The tower or reaction vessel may not be limited to any specific aspect ratio (ratio of height to diameter). The tower or reaction vessel may also not be limited to any specific material, and may be composed of any material suitable for the process (such as stainless steel, PVC, carbon steel or polymer material). The tower or reaction vessel may contain internal components, such as one or more static mixers commonly used in biological and chemical engineering processes. The reaction vessel may also be composed of external or internal heating or cooling elements (such as water jackets, heat exchangers or cooling coils). The reaction vessel may also be in fluid contact with one or more pumps to circulate liquid, bubbles, microbubbles, and / or one or more fluids of the system.

[0177] A "perforated plate" or "plate" may include a plate or similar device designed to facilitate the introduction of a liquid or another liquid into a container, which may be in the form of multiple liquid jets (i.e., accelerated liquid flow). The perforated plate may have a plurality of orifices or holes evenly or unevenly distributed on the plate, allowing the liquid to flow from the top of the plate to the bottom of the plate. In some examples, the hole may be spherical, rectangular, hexagonal, conical, pentagonal, cylindrical, truncated conical or circular. In other examples, the plate may include one or more nozzles suitable for generating a liquid jet, which flows into the tower. The plate may also contain any distribution or arrangement of channels, wherein such channels are suitable for receiving liquid and facilitating the liquid to flow into the reaction vessel. The plate may be made of stainless steel with a predetermined number of laser-burned, machined or drilled orifices or holes. The specific hole size may depend on the desired micro-bubble size and the desired liquid, micro-bubble and / or fluid velocity. A specific hole shape may be required to achieve the appropriate liquid acceleration and velocity from the plate to break or shear the ejector bubbles into the desired micro-bubble size and produce sufficient overall fluid downflow to carry the micro-bubbles and liquid down into the reaction vessel. The shape of the hole may also affect the ease of manufacturing and the associated costs. According to one embodiment, a straight hole may be optimal due to ease of manufacturing.

[0178] As disclosed herein, the system and method use multiple liquid jets or the part of the accelerated liquid flow produced by the perforated plate in the container to accelerate the liquid and break the bubble into smaller micro-bubbles with a larger apparent surface area than the original bubble. The original bubble is produced by injecting gas with an ejector that is completely located in the reaction vessel. In one example, the diameter of the original bubble injected into the liquid from the ejector can be about 2mm to about 20mm. In another example, the diameter of the original bubble injected into the liquid from the ejector can be about 5mm to about 15mm. In other examples, the diameter of the original bubble injected into the liquid from the ejector can be about 7mm to about 13mm. When injected, the original bubble migrates upwards through the liquid subsequently, and encounters multiple liquid jets or the part of the accelerated liquid flow, which breaks the original bubble into micro-bubbles. The resulting micro-bubbles and liquid flow downward along the reactor vessel in the downward fluid flow. The micro-bubbles of the substrate provide a carbon source and an optional energy source for the microorganism, and then the microorganism produces one or more desired products. The ejector is positioned in the container to create a first zone for primary bubbles to rise in the container and a second zone for accelerated liquid to break the primary bubbles into tiny bubbles and for fluid to flow through the container, wherein the fluid contains the accelerated portion of the liquid and the tiny bubbles.

[0179] Due to the nature of multiphase systems, a method for maximizing product production is to increase gas-liquid mass transfer. The more gas substrates transferred to the reaction liquid, the more desired products are produced. The smaller microbubbles disclosed herein provide an increased apparent surface area, resulting in an increase in the gas-liquid mass transfer rate, overcoming known solubility problems. In addition, the downflow reactor system disclosed herein effectively increases the residence time of the microbubbles. The increase in the time that the microbubbles are retained in the reaction liquid will generally provide an increased amount of the reaction products produced, as well as an increased surface area in contact with the microorganisms. Therefore, the system and method disclosed herein improves the previous system by generating microbubbles, and the microbubbles maximize the gas-liquid apparent surface area, thereby generating a high gas-liquid mass transfer rate. Further, the system and method disclosed herein provide apparent gas and liquid velocities that the previous system and method cannot achieve, so that microbubbles with a high gas phase residence time are generated, thereby effectively generating chemical and biological reaction products.

[0180] In certain embodiments, fermentation is performed in the absence of light or in the presence of an insufficient amount of light to meet the energy requirements of the photosynthetic microorganism. In certain embodiments, the microorganisms of the present disclosure are non-photosynthetic microorganisms.

[0181] 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, fractionation, 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 fermentation broth from the bioreactor, separating microbial cells from the fermentation broth (preferably by filtration), and recovering one or more target products from the fermentation broth. Alcohol and / or acetone can be recovered, for example, by distillation. Acids can be recovered, for example, by adsorption on activated carbon. The separated microbial cells are preferably returned to the bioreactor. The free permeate remaining after the removal of the target product is also preferably returned to the bioreactor. Additional nutrients (such as vitamin B) can be added to the free permeate before it is returned to the bioreactor to supplement the culture medium. Purification techniques can include affinity tag purification (e.g., His, Twin-Strep, and FLAG), bead-based systems, tip-based methods, 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) are also disclosed.

[0182] In some embodiments, the chemical products produced can be separated and enriched (including purification) using any suitable separation and / or purification technology known in the art. In one embodiment, the chemical products produced are gaseous. In one embodiment, the chemical products are liquid. In one embodiment, the gaseous chemical products can pass through a filter, a gas separation membrane, a gas purifier, or any combination thereof. In one embodiment, the chemical products are separated by an absorption column. In another embodiment, the chemical products are stored in one or more cylinders after separation. In one embodiment, the chemical products are integrated into the infrastructure or process of oil, natural gas, refineries, petrochemical operations, or any combination thereof. The infrastructure or process may be existing or new. In one embodiment, the gas fermentation product is integrated into oil and gas production, transportation and refining and / or chemical complexes. In another embodiment, the source of the raw material comes from oil, natural gas, refineries, petrochemical operations, or any combination thereof. In one embodiment, the gas fermentation product is integrated into the infrastructure or process of oil, natural gas, refineries, petrochemical operations, or any combination thereof, and the raw material comes from oil, natural gas, refineries, petrochemical operations, or any combination thereof.

[0183] In some embodiments, distillation can be used to purify product gases. In one embodiment, gas-liquid extraction can be used. In one embodiment, liquid product isolates can also be enriched by extraction using an organic phase. In another embodiment, purification can involve other standard techniques selected from ultrafiltration, one or more chromatographic techniques, or any combination thereof.

[0184] The method of the present disclosure may further comprise separating the gaseous fermentation product from the fermentation broth. Any method or combination of methods known in the art may be used to separate or purify the gaseous fermentation product from the fermentation broth, including, for example, distillation, simulated moving bed process, membrane treatment, evaporation, pervaporation, gas stripping, phase separation, ion exchange or extractive fermentation (including, for example, liquid-liquid extraction). As described in U.S. Patent No. 2,769,321 (the disclosure of which is incorporated herein by reference in its entirety), ethylene may be separated according to methods known in the art or combinations of methods. In one embodiment, the ethylene produced is collected from a bioreactor culture vessel.

[0185] In one embodiment, the gaseous fermentation product can be concentrated from the fermentation broth using reverse osmosis and / or pervaporation (US 5,552,023). Water can be removed by distillation, and then the bottoms (containing a high proportion of gaseous fermentation products) can be recovered using distillation or vacuum distillation to produce a high-purity stream. Alternatively, with or without reverse osmosis and / or pervaporation, the gaseous fermentation product can be further purified by reactive distillation with aldehydes (Atul, "Chemical Engineering (ChemEng Sci)", 59:2881-2890, 2004) or using azeotropic distillation of hydrocarbons (US2,218,234). In another approach, the gaseous fermentation products can be captured from aqueous solutions on activated carbon or polymer absorbents (with or without reverse osmosis and / or pervaporation) and recovered using low boiling point organic solvents (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 gaseous fermentation products can then be recovered from the organic solvent by distillation. In certain embodiments, the gaseous fermentation products are recovered from the fermentation broth by continuously removing a portion of the fermentation broth from the bioreactor, separating the microbial cells from the fermentation broth (preferably by filtration), and recovering the gaseous fermentation products from the fermentation broth. Byproducts (such as alcohols or acids) can also be separated or purified from the fermentation broth. Alcohol can be recovered, for example, by distillation. Acids can be recovered, for example, by adsorption to activated carbon. In certain embodiments, the separated microbial cells can be returned to the bioreactor. Further, in some embodiments, the separated microbial cells can be recycled to the bioreactor. The cell-free permeate remaining after removing the target product is also preferably returned to the bioreactor in whole or in part. Additional nutrients (such as vitamin B) can be added to the free permeate before it is returned to the bioreactor to supplement the culture medium.

[0186] A variety of methods have been demonstrated to recover glycols from aqueous media. Simulated moving bed (SMB) technology has been used to recover 2,3-butanediol from aqueous mixtures of ethanol and related oxygenates (U.S. Pat. No. 8,658,845). Reactive separation has also been shown to be effective in recovering glycols. In some embodiments, ethylene glycol recovery is performed by reaction of a glycol-containing stream with an aldehyde, fractionation and regeneration of the glycol, and final fractionation to recover a concentrated glycol stream. See, for example, U.S. Pat. No. 7,951,980.

[0187] In one embodiment, the method comprises recovering the ethylene produced as described above. In one embodiment, the method further comprises, after recovering the ethylene, converting or using the ethylene in the production of one or more chemical products.

[0188] Ethylene is a high-value gaseous compound that is widely used in industry.

[0189] In one embodiment, ethylene can be used as an anesthetic or fruit ripening agent, and for producing many other chemical products. In certain embodiments, ethylene can be used to produce polyethylene and other polymers, such as styrene, polystyrene, ethylene oxide, ethylene dichloride, ethylene dibromide, ethyl chloride and ethylbenzene. For example, ethylene oxide is a key raw material for producing surfactants and detergents and producing ethylene glycol, which is used as an antifreeze product in the automotive industry. In an embodiment involving ethylene dichloride, ethylene dibromide and ethyl chloride, they can be used 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 of styrene, for producing polystyrene (as an insulating product) and styrene-butadiene (it is a rubber suitable for tires and footwear). In another embodiment, the product is ethylene propylene diene monomer (EPDM) rubber, ethylene propylene (EPR / EPM) rubber or any combination thereof.

[0190] It should be understood that the methods of the present invention can be integrated or linked with one or more methods for producing downstream chemical products from ethylene. In some embodiments, the methods of the present invention can feed ethylene directly or indirectly to a chemical process or reaction sufficient to convert or produce other useful chemical products.

[0191] In some embodiments, ethylene is converted to a hydrocarbon liquid fuel. In one embodiment, ethylene is oligomerized over a catalyst to selectively produce a target product selected from gasoline, condensate, aromatics, heavy oil diluent, distillate, or any combination thereof. In other embodiments, the distillate is selected from diesel, jet fuel, sustainable aviation fuel (SAF), or any combination thereof.

[0192] In one embodiment, ethylene oligomerization is used for desired products. In one embodiment, the oligomerization of ethylene can be catalyzed by homogeneous catalyst, heterogeneous catalyst or any combination thereof, and has transition metal as active site. In certain embodiments, ethylene is further converted into long chain hydrocarbons by oligomerization. In other embodiments, straight chain olefins are the main products of ethylene oligomerization. In certain embodiments, alpha olefins are the main products of ethylene oligomerization. In one embodiment, olefins are subjected to upgrading process. In certain embodiments, the upgrading process of olefins is hydrogenation. In one embodiment, olefins are subjected to olefin conversion technology. In certain embodiments, ethylene is incorporated into or converted into sustainable aviation fuel (SAF). In one embodiment, ethylene is mutually converted into propylene, 2-butene or any combination thereof. In one embodiment, propylene is converted into polypropylene.

[0193] 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 widely used in industrial and consumer markets such as packaging, transportation, electrical / electronics, textiles and construction industries as well as consumer chemicals, coatings and adhesives.

[0194] Ethylene can be chlorinated to ethylene dichloride (EDC), which can then be cracked to vinyl chloride monomer (VCM). Almost all VCM is used to make polyvinyl chloride, which is mainly used in the construction industry.

[0195] Other ethylene derivatives include alpha olefins, detergent alcohols and plasticizer alcohols for the production of linear low-density polyethylene (LLDPE); vinyl acetate monomer (VAM) for adhesives, paints, paper coatings and barrier resins; and industrial ethanol used as a solvent or for the manufacture of chemical intermediates such as ethyl acetate and ethyl acrylate. Ethylene can be converted into ethylene-vinyl acetate (EVA) or poly (ethylene-vinyl acetate) (PEVA). EVA can be converted into thermoplastic materials. EVA can be incorporated into or used to make hot melt adhesives, hot melt glue sticks, football shoes, plastic packaging, craft foam boards and foam stickers. EVA can be incorporated into or used to make drug delivery devices. In some embodiments, EVA can be used to make foam. In one embodiment, EVA foam is used as a pad in sports equipment, including 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 can be used as a compression molded foam based on EVA. EVA can be incorporated into or used to make the float of commercial fishing gear and floating goggles. EVA can be incorporated into or used to make the encapsulation material of crystalline silicon solar cells. In certain embodiments, EVA can be incorporated into or used to make the coating formulation in slippers, sandals, fishing rods, cork substitutes, packaging, textiles, book binding, adhesive plastic film, metal surfaces, coated paper, redispersible powders in gypsum and cement plaster, and indoor water-based paint. EVA can undergo hydrolysis to provide ethylene-vinyl alcohol (EVOH) copolymer. EVA can be used for orthotics, surfboards and surfboard traction pads, car mats, artificial flowers, diesel low temperature fluidity improvers, HEPA filter isolation agents, thermoplastic protective mouthpieces, leather conditioning and waterproofing, nicotine transdermal patches and plastic model kits.

[0196] 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, prepared by depositing chromium (VI) oxide on silica.

[0197] The polyethylene oligomers thus produced can be classified according to their density and branching. Further, mechanical properties depend significantly on variables such as the degree and type of branching, crystal structure and molecular weight. There are several types of polyethylene that can be produced from ethylene, including but not limited to:

[0198] Ultra-high molecular weight polyethylene (UHMWPE);

[0199] Ultra-low molecular weight polyethylene (ULMWPE or PE-WAX);

[0200] High molecular weight polyethylene (HMWPE);

[0201] High-density polyethylene (HDPE);

[0202] High-density cross-linked polyethylene (HDXLPE);

[0203] Cross-linked polyethylene (PEX or XLPE);

[0204] Medium density polyethylene (MDPE);

[0205] Linear low-density polyethylene (LLDPE);

[0206] Low-density polyethylene (LDPE);

[0207] Very low density polyethylene (VLDPE); and

[0208] Chlorinated polyethylene (CPE).

[0209] Low-density polyethylene (LDPE) and linear low-density polyethylene (LLDPE) are mainly used in film applications such as food and non-food packaging, shrink and stretch films, and non-packaging uses. High-density polyethylene (HDPE) is mainly used in blow molding and injection molding applications such as containers, drums, household goods, lids and trays. HDPE can also be extruded into water pipes, gas pipes and irrigation pipes as well as films for garbage bags, shopping bags and industrial liners.

[0210] According to one embodiment, the ethylene formed by the disclosure described above can be converted to ethylene oxide by direct oxidation according to the following formula:

[0211] C2H4+O2→C2H4O

[0212] The resulting ethylene oxide is a key chemical intermediate in many important commercial processes, including the manufacture of monoethylene glycol. Other EO derivatives include ethoxylates (used in shampoos, kitchen cleaners, etc.), glycol ethers (solvents, fuels, etc.) and ethanolamines (surfactants, personal care products, etc.).

[0213] According to one embodiment of the present disclosure, the ethylene oxide produced as described above can be used to produce commercial quantities of monoethylene glycol via the following formula:

[0214] (CH2CH2)O+H2O→HOCH2CH2OH

[0215] According to another embodiment, the claimed microorganism can be modified to directly produce monoethylene glycol. As described in WO2019 / 126400 (the disclosure of which is incorporated herein by reference in its entirety), the microorganism further comprises one or more of the following: an enzyme capable of converting acetyl-CoA into pyruvate; an enzyme capable of converting pyruvate into oxaloacetate; an enzyme capable of converting pyruvate into malate; an enzyme capable of converting pyruvate into phosphoenolpyruvate; an enzyme capable of converting oxaloacetate into citric acid-CoA; an enzyme capable of converting citric acid-CoA into citrate; an enzyme capable of converting citrate into aconitate and converting aconitate into isocitrate; an enzyme capable of converting phosphoenolpyruvate into oxaloacetate; an enzyme capable of converting phosphoenolpyruvate into 2-phospho-D-glycerate; an enzyme capable of converting 2- an enzyme capable of converting phospho-D-glycerate to 3-phospho-D-glycerate; an enzyme capable of converting 3-phospho-D-glycerate to 3-phospho-pyruvate; an enzyme capable of converting 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.

[0216] In one embodiment, the microorganism comprises one or more of the following: a heterologous enzyme capable of converting oxaloacetate into citrate; a heterologous enzyme capable of converting glycine into glyoxylate; a heterologous enzyme capable of converting isocitrate into glyoxylate; a heterologous enzyme capable of converting glycolate into 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]; the heterologous enzyme capable of converting glycine to glyoxylate is alanine-glyoxylate aminotransferase [2.6.1.44], serine-glyoxylate aminotransferase [2.6.1.45], serine-pyruvate aminotransferase [2.6.1.51], glycine-oxaloacetate aminotransferase [2.6.1.35], glycine aminotransferase [2.6.1.4], glycine dehydrogenase [1.4.1.10], alanine acid dehydrogenase [1.4.1.1]; or glycine dehydrogenase [1.4.2.1]; the heterologous enzyme capable of converting isocitrate to glyoxylate is isocitrate lyase [4.1.3.1]; the heterologous enzyme capable of converting glycolate to glycolaldehyde is glycolaldehyde dehydrogenase [1.2.1.21], lactate aldehyde 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 any combination thereof.

[0217] The monoethylene glycol produced according to any of the described methods can be used as a component of a variety of products, including as a raw material for making polyester fibers for textile applications including nonwovens, diaper covers, building materials, construction materials, road construction fabrics, filters, fiberfill, felt, traffic upholstery, paper and tape reinforcements, tents, ropes and cords, sails, fishing nets, safety belts, laundry bags, synthetic artery replacements, carpets, floor mats, clothing, sheets and pillowcases, towels, curtains, draperies, bedding, and blankets.

[0218] MEG can be used alone as a liquid coolant, antifreeze, preservative, dehydrating agent, drilling fluid or any combination thereof. The produced MEG can also be used to produce secondary products such as polyester resins for insulation materials, polyester films, deicing fluids, heat transfer fluids, automotive antifreeze and other liquid coolants, preservatives, dehydrating agents, drilling fluids, water-based adhesives, latex paints and asphalt emulsions, electrolytic capacitors, paper and synthetic leather.

[0219] Importantly, the monoethylene glycol produced can be converted into the polyester resin polyethylene terephthalate ("PET") according to one of two main processes. The first process involves transesterification of monoethylene glycol with dimethyl terephthalate according to the following two-step process:

[0220] Step 1

[0221] C6H4(CO2CH3)2+2HOCH2CH2OH→C6H4(CO2CH2CH2OH)2+2CH3OH

[0222] Step 2

[0223] n C6H4(CO2CH2CH2OH)2→[(CO)C6H4(CO2CH2CH2O)] n +n HOCH2CH2OH

[0224] Alternatively, monoethylene glycol can be the subject of an esterification reaction with terephthalic acid according to the following reaction:

[0225] n C6H4(CO2H)2+n HOCH2CH2OH→[(CO)C6H4(CO2CH2CH2O)] n +2n H2O

[0226] Polyethylene terephthalate produced from the transesterification or esterification of monoethylene glycol has significant suitability for many packaging applications such as jars, and particularly for producing bottles, including plastic bottles. It can also be used to produce high-strength textile fibers such as polyester, as part of durable press blends with other fibers such as rayon, wool and cotton, for fiberfill in insulating clothing, furniture and pillows, for rayon, as carpet fiber, automotive tire yarn, conveyor belts and drive belts, for fire and garden hoses, reinforcement materials for seat belts, for nonwoven fabrics used to stabilize drains, culverts and railroad bed, and for use as diaper topsheets and disposable medical garments.

[0227] At higher molecular weights, PET can be made into high-strength plastics that can be molded using all the usual methods for other thermoplastics. Magnetic recording tapes and photographic films are produced by extrusion of PET films. Molten PET can be blown into high-strength and rigid transparent containers that are also virtually impermeable to air and liquids. In this form, PET has been widely used in bottles (especially plastic bottles) and jars.

[0228] The present disclosure provides compositions comprising ethylene glycol produced by a microorganism and according to the methods described herein. For example, the composition comprising ethylene glycol can be an antifreeze, an antiseptic, a dehydrating agent, or a drilling fluid.

[0229] The present disclosure also provides polymers comprising ethylene glycol produced by microorganisms and according to the methods described herein. Such polymers can be, for example, homopolymers, such as polyethylene glycol or copolymers (such as polyethylene terephthalate). The synthetic methods of these polymers are well known in the art. See, for example, Herzberger et al., Chemical Review (ChemRev.), 116 (4): 2170-2243 (2016) and Xiao et al., Industrial and Engineering Chemistry Research (Ind Eng ChemRes.) 54 (22): 5862-5869 (2015).

[0230] The present disclosure further provides polyethylene glycol conjugates. In some embodiments, polyethylene glycol (PEG) conjugates include 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.

[0231] The present disclosure further provides a composition comprising a polymer comprising ethylene glycol produced by a microorganism and according to the methods described herein. For example, the composition can be a fiber, a resin, a film, or a plastic.

[0232] In one embodiment, ethanol or ethyl alcohol produced according to the methods of the present disclosure can be used in a variety of product applications, including disinfectant hand wipes (WO 2014 / 100851), therapeutic treatments for methylene glycol and methanol poisoning (WO 2006 / 088491), as a pharmaceutical solvent for applications such as pain medication (WO 2011 / 034887) and oral hygiene products (U.S. Patent No. 6,811,769), as an antimicrobial preservative (U.S. Patent Application No. 2013 / 0230609), motor fuel (U.S. Patent No. 1,128,549), rocket fuel (U.S. Patent No. 3,020,708), plastics, fuel cells (U.S. Patent No. 2,405,986), household fireplace fuel (U.S. Patent No. 4,692,168), as an industrial chemical precursor (U.S. Patent No. 3,102,875), cannabis solvent (WO 2015 / 073854), as an antifreeze extraction solvent (WO 2017 / 161387), as a paint masking product (WO 1992 / 008555), as a paint or tincture (U.S. Pat. No. 1,408,091), purification and extraction of DNA and RNA (WO 1997 / 010331), and as a cooling bath for various chemical reactions (U.S. Pat. No. 2,099,090). In addition to the foregoing, the ethanol produced by the disclosed method can be used in any other application for which ethanol might otherwise be suitable.

[0233] In other embodiments, isopropyl alcohol or isopropyl alcohol (IPA) produced according to the methods can be used in many product applications, including use alone or as a raw material to produce more complex products. Isopropyl alcohol can also be used in solvents for cosmetics and personal care products, de-icers, paints and resins, foods, inks, adhesives and pharmaceuticals, including products such as tablets and disinfectants, sterilants and skin creams.

[0234] The IPA produced can be used to extract and purify natural products such as plant and animal fats and oils. Other applications include use as a cleaning agent and drying agent in electronic components and metal manufacturing, and as an aerosol solvent in medical and veterinary products. It can also be used as a coolant in beer manufacturing, a coupling agent, a polymerization modifier, a deicer, and a preservative.

[0235] Alternatively, the IPA produced according to the methods of the present disclosure can be used to make other useful compounds, including plastics, derivative ketones such as methyl isobutyl ketone (MIBK), isopropylamine and isopropyl esters. Further, IPA can be converted to propylene according to the following formula:

[0236] CH3CH2CH2OH→CH3-CH=CH2

[0237] The propylene produced can be used as the monomer base for producing various polypropylene oligomers by chain growth polymerization in gas phase or bulk reactor systems. The most common catalysts consist of titanium(III) chloride, so-called Ziegler-Natta catalysts and metallocene catalysts.

[0238] The polypropylene oligomer thus produced can be classified according to stereoregularity, and can be formed into a variety of products by extrusion or molding of polypropylene pellets, including pipe products, heat-resistant products such as kettles and food containers, disposable bottles (including plastic bottles), transparent bags, flooring such as mats and cushions, ropes, self-adhesive labels, and foamed polypropylene that can be used for building materials. Polypropylene can also be used for hydrophilic clothing and medical dressings.

[0239] In some embodiments, ethylene is used to produce polyethylene: a common plastic used in a variety of consumer products, such as plastic bags, plastic films, geomembranes, containers including bottles, etc.

[0240] In one embodiment, ethylene is used to make ethylene glycol: a raw material for making polyester fibers used in clothing, upholstery, carpets, and pillows. In another embodiment, ethylene is used to produce antifreeze in cooling and heating systems.

[0241] In another embodiment, ethylene is used in Ethylene Oxide: used to make other chemicals used to make products such as detergents, thickeners, solvents, plastics, and various organic chemicals. In some embodiments, ethylene oxide is used as a disinfectant and fumigant for medical equipment.

[0242] In some embodiments, ethylene is used in vinyl acetate: used to make other chemicals for paints, adhesives, paper coatings, and textiles.

[0243] In other embodiments, ethylene is used in ethylene dichloride: used to produce vinyl chloride, which is used to make polyvinyl chloride (PVC). PVC is used to make a variety of plastic and vinyl products, including pipes, wire and cable coatings, and packaging materials.

[0244] In some embodiments, ethylene is used in aluminum alkyls: used as catalysts to increase the efficiency of making ethylene and other chemicals.

[0245] In some embodiments, ethylene is used in ethylene propylene rubber (EPR): used in electrical insulation, roofing membranes, radiator hoses in vehicles, and flashing.

[0246] In other embodiments, ethylene is used in agriculture: as a plant hormone, and in agriculture to ripen fruits.

[0247] In some embodiments, ethylene is used to make styrene, which is then used to make polystyrene. Polystyrene is used in a variety of consumer products, such as disposable cutlery, CD and DVD cases, and insulation.

[0248] In an embodiment, ethylene is used to produce alpha olefins: as a comonomer in the production of polyethylene, and as a comonomer in the production of detergents and lubricants.

[0249] In one embodiment, ethylene is used to produce butadiene. In some embodiments, butadiene is used in rubber tires.

[0250] In one embodiment, a method for continuously producing ethylene comprises: passing a gaseous substrate into a bioreactor containing a culture of a recombinant C1 immobilized microorganism capable of producing ethylene in a culture medium, such that the microorganism converts the gaseous substrate into ethylene; and recovering the ethylene from the bioreactor.

[0251] In other embodiments, ethylene is converted into components used to make tires. In one embodiment, ethylene is converted into components used in tire treads.

[0252] The method according to an embodiment, wherein the tire is a scrap tire.

[0253] The method according to an embodiment, wherein the gaseous substrate originates from a process involving tires.

[0254] The method according to an embodiment, wherein the gaseous substrate originates from a product recycling process or a sustainable chemical process.

[0255] According to an embodiment, the method further comprises converting the ethylene into a component for manufacturing new tires.

[0256] According to the method described in the embodiment, a resin component comprising ethylene and other olefins is combined with a synthetic component selected from butadiene and isoprene to form a mixed polymer for manufacturing a tire.

[0257] One embodiment relates to a method for producing a polymer from a gaseous substrate, the method comprising: a first gas fermentation process produces at least one first product selected from butadiene, isoprene, a conjugated diene, or any combination thereof, and a second gas fermentation process produces 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 are copolymerized to form a polymer.

[0258] The method according to an embodiment, wherein the first gas fermentation process and the second gas fermentation process are run in parallel.

[0259] The method according to an embodiment, wherein the first gas fermentation process and the second gas fermentation process are both operated continuously.

[0260] The method according to an embodiment comprises: a first gas fermentation process produces a rubber component, and a second gas fermentation process produces a resin component, and wherein the rubber component and the resin component are copolymerized to form a polymer.

[0261] The method according to an embodiment, wherein the rubber component and the resin component are copolymerized by a suitable polymerization catalyst.

[0262] The method according to an embodiment, wherein the rubber component is selected from butadiene, isoprene, conjugated diene or any combination thereof.

[0263] The method according to an embodiment, wherein the resin component is selected from ethylene, olefins or any combination thereof.

[0264] The method according to the embodiment, wherein the suitable polymerization catalyst further comprises another component contained in a common polymerization catalyst composition containing a metallocene complex.

[0265] The method according to an embodiment, wherein the metallocene complex is a complex having one or more cyclopentadienyl groups or derived cyclopentadienyl groups bonded to a central metal.

[0266] The method according to an embodiment, wherein the central metal is selected from lanthanides, scandium, yttrium or any combination thereof.

[0267] According to the method of an embodiment, the central metal is selected from samarium (Sm), neodymium (Nd), praseodymium (Pr), gadolinium (Gd), cerium (Ce), holmium (Ho), scandium (Sc) and yttrium (Y).

[0268] The method according to an embodiment further comprises converting the polymer into a tire.

[0269] One embodiment for producing tires from a gaseous substrate cycle involves a first gas fermentation process for producing at least one first product selected from butadiene, isoprene, conjugated dienes, or any combination thereof, and a second gas fermentation process for producing 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 wherein the substrate is derived from a process comprising a tire.

[0270] The method according to an embodiment, wherein the substrate is derived from a process involving scrap tires.

[0271] One embodiment relates to a method for cyclically producing a tire, the method comprising: 1) passing a gaseous substrate into a first bioreactor containing a culture of a recombinant C1 immobile microorganism capable of producing at least one first product selected from butadiene, isoprene, conjugated dienes or any combination thereof in a culture medium, so that the microorganism converts the gaseous substrate into the at least one first product; and recovering the at least one first product from the bioreactor; 2) passing a gaseous substrate into a second bioreactor containing a culture of a recombinant C1 immobile microorganism capable of producing at least one second product selected from ethylene and olefins or any combination thereof in a culture medium, so that the microorganism converts the gaseous substrate into the 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 the at least one second product in the presence of a suitable polymerization catalyst to form a mixed polymer; and 4) converting the mixed polymer into a tire.

[0272] The method according to the embodiment, wherein the suitable polymerization catalyst further comprises another component contained in a common polymerization catalyst composition containing a metallocene complex.

[0273] The method according to an embodiment, wherein the metallocene complex is a complex having one or more cyclopentadienyl groups or derived cyclopentadienyl groups bonded to a central metal.

[0274] The method according to an embodiment, wherein the central metal is selected from lanthanides, scandium, yttrium or any combination thereof.

[0275] According to the method of an embodiment, the central metal is selected from samarium (Sm), neodymium (Nd), praseodymium (Pr), gadolinium (Gd), cerium (Ce), holmium (Ho), scandium (Sc) and yttrium (Y).

[0276] The method according to an embodiment, wherein the first bioreactor and the second bioreactor are operated in parallel.

[0277] The method according to an embodiment, wherein both the first bioreactor and the second bioreactor are operated continuously.

[0278] The method according to an embodiment, wherein the substrate is derived from a process involving scrap tires.

[0279] The method according to the embodiment further comprises converting the isoprenoid into a product selected from the group consisting of synthetic rubber, block polymers containing styrene, thermoplastic rubber, pressure-sensitive or thermosetting adhesives, butyl rubber, terpenes selected from the group consisting of citral, linalool, ionone, myrcene, L-menthol, N,N-diethylaniline, geraniol, nerolidol, flavors, fragrances, fuel additives, plastics, polyisoprene.

[0280] The method according to an embodiment further comprises converting the butadiene into a product selected from the group consisting of styrene-butadiene rubber, synthetic rubber, tires, tire components, thermoplastic rubber, footwear, shoe soles, adhesives, sealants, asphalt, polymer modification components, nylon, ABS resin, chloroprene / chloroprene rubber, nitrile rubber, plastics, acrylics, acrylonitrile-butadiene-styrene resins, and synthetic elastomers.

[0281] One embodiment relates to a method for chemical recycling, the method comprising: a pyrolysis, gasification and / or partial oxidation process; provided to a gas fermentation process; provided to a chemical product manufacturing process to produce a product comprising butadiene, isoprenoids, ethylene, polyethylene terephthalate (PET), or any combination thereof; provided to a synthetic rubber production process; provided to a tire manufacturing process; provided to a process that uses tires; a process for collecting and shredding used tires is provided; and provided back to the pyrolysis, gasification and / or partial oxidation process.

[0282] One embodiment relates to a method for chemical recycling, the method comprising: 1) a pyrolysis, gasification and / or partial oxidation process; 2) provided to a gas fermentation process; 3) provided to a chemical product manufacturing process to produce a product comprising butadiene, isoprenoids, ethylene, polyethylene terephthalate (PET), or any combination thereof; 4) provided to a synthetic rubber production process; 5) provided to a tire manufacturing process; 6) provided to a process that uses tires; 7) provided to a process for collecting and shredding used tires; and 8) provided back to the pyrolysis, gasification and / or partial oxidation process.

[0283] One embodiment relates to a method for chemical recycling, the method comprising: 1) a pyrolysis, gasification and / or partial oxidation process; 2) provided to a gas fermentation process; 3) provided to a chemical product manufacturing process to produce a commodity product; 4) provided to a synthetic rubber production process; 5) provided to a tire manufacturing process; 6) provided to a process that uses tires; 7) provided to a process for collecting and shredding used tires; and 8) provided back to the pyrolysis, gasification and / or partial oxidation process.

[0284] Another embodiment relates to a method for chemical recycling, the method comprising: 1) a pyrolysis, gasification and / or partial oxidation process that produces an effluent stream; 2) passing the effluent stream to a gas fermentation process to produce products; 3) passing the gas fermentation products to a chemical product manufacturing process to produce commodity products; 4) passing the commodity products to a synthetic rubber production process to produce synthetic rubber; 5) passing the synthetic rubber products to a tire manufacturing process to produce tires; 6) providing the tires to a process that uses the tires; 7) passing the used tires to a process for collecting and shredding the used tires; and 8) recycling the used tires back to the pyrolysis, gasification and / or partial oxidation process.

[0285] One embodiment provides a method and a genetically engineered microorganism capable of producing ethylene from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding an ethylene-forming enzyme (EFE).

[0286] In some aspects of the methods disclosed herein, the microorganism is a recombinant C1 immobile microorganism capable of producing ethylene from a gaseous substrate, the recombinant C1 immobile microorganism comprising a nucleic acid encoding a set of exogenous enzymes comprising an ethylene forming enzyme (EFE).

[0287] In some aspects of the microorganisms disclosed herein, the microorganism relates to a recombinant C1 fixed microorganism capable of switching the cell load in the production of ethylene, the microorganism comprising a nucleic acid encoding a set of exogenous enzymes comprising an ethylene forming enzyme (EFE) and one or more inducible promoters.

[0288] The microorganism according to the embodiment, further comprising a nucleic acid encoding a group of exogenous enzymes comprising α-ketoglutarate permease (AKGP), wherein the nucleic acid is operably linked to a promoter.

[0289] The microorganism according to the embodiment, wherein the microorganism is selected from the group consisting of: Cupriavidus necrotus and Ralstonia eutropha.

[0290] The microorganism according to the embodiment, wherein the microorganism is Cupriavidus necrotus.

[0291] According to the microorganism of the embodiments, it further comprises a nucleic acid encoding an alpha-ketoglutarate permease, wherein the nucleic acid is codon-optimized for expression in the microorganism.

[0292] The microorganism according to the embodiments, 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.

[0293] The microorganism according to the embodiments, wherein the inducible promoter is a phosphate-limited inducible promoter.

[0294] The microorganism according to the embodiments, wherein the phosphate concentration is about 0-0.5 mM.

[0295] The microorganism according to an embodiment, wherein the phosphate concentration is about 0.52 mM.

[0296] The microorganism according to the embodiments, wherein the EFE is codon-optimized for expression in the microorganism.

[0297] The microorganism according to the embodiment further comprises a disruptive mutation in one or more genes.

[0298] According to the microorganism described in the embodiment, ethylene is converted into a derived material, and the derived material is selected from polyethylene (PE), polyethylene terephthalate (PET), polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), sustainable aviation fuel (SAF) or any combination thereof.

[0299] The microorganism according to the embodiment, wherein the gaseous substrate comprises CO2 and an energy source.

[0300] The microorganism according to the embodiments, wherein the gaseous substrate comprises CO2 and H2, O2 or both.

[0301] One embodiment relates to a method for continuously producing ethylene, the method comprising: passing a gaseous substrate into a bioreactor containing a culture of the recombinant C1 immobile 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.

[0302] One embodiment relates to a method of culturing a microorganism according to an embodiment, the method comprising culturing the microorganism in a culture medium comprising a gaseous substrate, wherein the gaseous substrate comprises CO2.

[0303] The method according to an embodiment, wherein the gaseous substrate comprises industrial waste products or exhaust gases.

[0304] The method according to an embodiment further comprises an energy source.

[0305] The method according to an embodiment, wherein the energy source is provided intermittently.

[0306] The method according to an embodiment, wherein the energy source is H2.

[0307] One embodiment relates to a method comprising culturing the microorganism in a culture medium comprising a gaseous substrate, wherein the gaseous substrate comprises CO2 and an energy source.

[0308] The method according to an embodiment further comprises co-producing ethylene and the microbial biomass.

[0309] The method according to an embodiment, wherein switching the cell load comprises the step of limiting intracellular oxygen concentration.

[0310] The method according to an embodiment, wherein switching the cell load comprises the step of limiting dissolved oxygen concentration.

[0311] According to the method of an embodiment, the dissolved oxygen concentration is at least about 0.5% saturation (% sat.) to 1.0% sat., and at most about 50% sat. to 60% sat.

[0312] According to the method of an embodiment, the dissolved oxygen concentration is at least about 0.5% sat. to 1.0% sat., and at most about 60% sat. to 70% sat.

[0313] According to the method of an embodiment, the dissolved oxygen concentration is at least about 0.5% sat. to 1.0% sat., and at most about 70% sat. to 80% sat.

[0314] According to the method of an embodiment, the dissolved oxygen concentration is at least about 0.5% sat. to 1.0% sat., and at most about 80% sat. to 90% sat.

[0315] According to the method of an embodiment, the dissolved oxygen concentration is at least about 0.5% sat. to 1.0% sat., and at most about 90% sat. to 100% sat.

[0316] According to the method of an embodiment, the dissolved oxygen concentration is at least about 0.01% sat. to 1.0% sat., and at most about 50% sat. to 60% sat.

[0317] According to the method of an embodiment, the dissolved oxygen concentration is at least about 0.01% sat. to 1.0% sat., and at most about 60% sat. to 70% sat.

[0318] According to the method of an embodiment, the dissolved oxygen concentration is at least about 0.01% sat. to 1.0% sat., and at most about 70% sat. to 80% sat.

[0319] According to the method of an embodiment, the dissolved oxygen concentration is at least about 0.01% sat. to 1.0% sat., and at most about 80% sat. to 90% sat.

[0320] According to the method of an embodiment, the dissolved oxygen concentration is at least about 0.01% sat. to 1.0% sat., and at most about 90% sat. to 100% sat.

[0321] The method according to an embodiment, wherein about 4 vol. % to about 30 vol. % O2 is fed into the inlet.

[0322] The method according to an embodiment, wherein about 1 vol. % to about 50 vol. % O2 is fed into the inlet.

[0323] The method according to an embodiment, wherein switching the cell load comprises a step of limiting steady-state phosphate concentration.

[0324] According to the method of an embodiment, switching the cell load comprises the step of limiting the steady-state phosphate concentration to about 0 mM to about 0.50 mM.

[0325] According to the method of an embodiment, switching the cell load comprises the step of limiting the steady state phosphate concentration to about 0.05 mM to about 0.50 mM.

[0326] According to the method of an embodiment, switching the cell load comprises the step of limiting the steady-state phosphate concentration to about 0.01 mM to about 0.60 mM.

[0327] According to the method of an embodiment, switching the cell load comprises the step of limiting the steady-state phosphate concentration to about 0.01 mM to about 0.70 mM.

[0328] According to the method of an embodiment, switching the cell load comprises the step of limiting the steady-state phosphate concentration to about 0.01 mM to about 0.80 mM.

[0329] According to the method of an embodiment, switching the cell load comprises the step of limiting the steady-state phosphate concentration to about 0.01 mM to about 0.90 mM.

[0330] According to the method of an embodiment, switching the cell load comprises the step of limiting the steady-state phosphate concentration to about 0.01 mM to about 1.0 mM.

[0331] According to the method of an embodiment, wherein switching the cell load comprises the step of limiting the steady-state phosphate concentration to about 0.52 mM.

[0332] The method according to an embodiment, wherein the microbial biomass is suitable as animal feed.

[0333] The method according to an embodiment, wherein the gaseous substrate further comprises H2, O2, or both.

[0334] In some aspects of the microorganisms disclosed herein, the microorganism produces a commodity chemical product, a microbial biomass, a single cell protein (SCP), one or more intermediates, or any combination thereof.

[0335] In some aspects, the microbial biomass has a unit value. In one embodiment, the microbial biomass has a market value.

[0336] In some aspects of the microorganisms disclosed herein, the microorganism is derived from a parent bacterium selected from the group consisting of Cupriavidus necrotus.

[0337] In some aspects of the microorganisms disclosed herein, the product is selected from the group consisting of 1-butanol, butyrate, butylene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, terpenes, isoprene, fatty acids, fatty alcohols, 2-butanol, 1,2-propylene glycol, 1-propanol, 1-hexanol, 1-octanol, chorismate-derived products, 3-hydroxybutyrate, 1,3-butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-butene-1-ol, isovalerate, isopentanol, or monoethylene glycol.

[0338] The present disclosure further provides a genetically engineered C1 immobilized microorganism further comprising a microbial biomass and at least one excipient.

[0339] The present disclosure further provides a genetically engineered C1 stationary microorganism, wherein the animal feed is suitable for feeding one or more of the following: beef cattle, dairy cows, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, gaur, giraffes, yaks, chickens, turkeys, ducks, geese, quail, guinea fowl, chicks / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents.

[0340] The present disclosure further provides a genetically engineered C1 immobilized microorganism, wherein the microorganism is suitable as a single cell protein (SCP).

[0341] The present disclosure further provides a genetically engineered C1 immobilized microorganism, wherein the microorganism is suitable as a cell-free protein synthesis (CFPS) platform.

[0342] The present disclosure further provides a genetically engineered C1 immobilized microorganism, wherein the chemical product is native to the microorganism.

[0343] In some aspects of the methods disclosed herein, the substrate comprises one or more of CO, CO2, and H2.

[0344] According to another embodiment, as described in U.S. Patent Application Publication No. 2023 / 0092645A1 (the disclosure of which is incorporated herein by reference), the claimed microorganisms can be modified to directly produce commodity chemicals. In one embodiment, the commodity chemicals are 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 chemicals are 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. According to an embodiment of the method, the commodity chemical is used in an industry selected from plastics, synthetic fibers, synthetic rubbers, dyes, pigments, paints, coatings, fertilizers, agricultural chemicals, pesticides, cosmetics, soaps, cleaners, detergents, pharmaceuticals, mining, or any combination thereof.

[0345] In another embodiment, the method comprises incorporating the commodity chemical into one or more articles of manufacture or converting the commodity chemical into a product selected from the group consisting of ethanol, acetate, 1-butanol, butyrate, 2,3-butanediol, lactate, butylene, butadiene, methyl ethyl ketone (2-butanone), ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, terpenes, isoprene, fatty acids, 2-butanol, 1,2-propylene glycol, 1 propanol, 1 hexanol, 1 octanol, chorismate-derived products, 3-hydroxybutyrate, 1,3-butanediol, 2-hydroxyisobutyrate, 2-hydroxyisobutyric acid, isobutylene, adipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-butene-1-ol, isovalerate, isopentanol, monoethylene glycol, preservatives hand sanitizers, therapeutic treatment for methylene glycol poisoning, therapeutic treatment for methanol poisoning, pharmaceutical solvents for pain relievers, oral hygiene products, antimicrobial preservatives, engine fuels, rocket fuels, plastics, fuel cells, fuel for home fireplaces, industrial chemical precursors, cannabis solvents, winterizing extraction solvents, paint masking products, paints, tinctures, purification and extraction of DNA and RNA, cooling baths for various chemical reactions, raw ethylene, anesthetics, ethylene and nitrogen in fruit ripening, fertilizers, safety glass, metal cutting, welding, oxygen fuels in high-velocity thermal spraying, refrigerants, polyethylene raw materials, PET raw materials, PVC raw materials, fibers, packaging, coatings, adhesives, ethylene dichloride (EDC), vinyl chloride monomer (VCM), alpha olefins, linear alpha olefins, detergent alcohols, plasticizer alcohols, vinyl acetate monomer (VAM), barrier resins, industrial ethanol, ethyl acetate, ethyl acrylate, polyethylene oligomers, 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), low density polyethylene (LDPE), very low density polyethylene (VLDPE), chlorinated polyethylene (CPE), films, food packaging, non-food packaging, shrink film, stretch film, containers, barrels, household goods, lids, pallets, pipes, Garbage bags, shopping bags, industrial linings, ethylene oxide, ethoxylates, shampoo, kitchen cleaners, glycol ethers, ethanolamines, surfactants, personal care products, polyester fibers, textiles, nonwovens, diaper covers, road construction fabrics, filters, fiber fillers, felt, transportation decorations, paper reinforcements, tape reinforcements, tents, ropes, fiber ropes, sails, fishing nets, seat belts, laundry bags, synthetic artery substitutes, carpets, blankets, clothing, sheets, pillowcases, towels, curtains, draperies, bedding, blankets, coolants, antifreeze, preservatives, dehydrating agents, drilling fluids, polyester resins, insulation materials, polyester films, deicing fluids, heat transfer fluids, automotive antifreeze, water-based adhesives, latex paints, asphalt emulsions, electrolytic capacitors, synthetic leather,Polyester resin PET, jars, bottles, plastic bottles, high-strength fibers, polyester, durable press compounds, insulating clothing, furniture fillings, pillow fillings, rayon, carpet fibers, automotive tire yarns, conveyor belts, drive belts, reinforcements for fire and garden hoses, non-woven fabrics for stabilizing drains, culverts, railway roadbeds, non-woven fabrics for diaper surfaces, disposable medical clothing, high-strength plastics, magnetic recording tapes, photographic films, as raw materials, cosmetic solvents, inks, tablets, disinfectants, sterilizers, skin creams, purification of vegetable oils and fats, purification of animal oils and fats, detergents, desiccants, aerosol solvents, derivative ketones, isopropylamine, isopropyl esters, propylene, polypropylene oligomers, polymerization modifiers, coupling agents, heat-resistant products, kettles, food containers, disposable bottles, transparent bags, floors, mats, self-adhesive labels, foamed polypropylene, building materials, hydrophilic clothing, medical dressings, or any combination thereof.

[0346] In another embodiment, the method comprises incorporating a commodity chemical into an article or converting a commodity chemical into a product selected from the group consisting of: wetting agents, filters, fire sprinkler systems, fuel for heating food, heat transfer fluids, unreacted ingredients in formulations, deodorizing or air purifying, softeners, arts / craft glues / pastes, toys, children's products, frozen gel packaging, treating wood rot and fungi, preserving biological tissues and organs, alkyd resins, resin esters, enamels, varnishes, latex paints, asphalt emulsions, thermoplastic resins, hydrate inhibitors, removing water vapor aerosol reagents, shoe polish, vaccines, screen cleaning solutions, water-based hydraulic fluids, heat transfer for liquid-cooled computers, personal lubricants, lubricants, toothpaste, defoamers in food industry applications, flame retardant hydraulic fluids, additives for electropolishing tapes, industrial solvents, garbage bags, shower curtains, cups, utensils, medical devices, durable goods, non-durable goods, plastic bags, plastic covers, industrial strapping, building materials, felt, heatable trays, frozen food trays, microwaveable trays, artificial blood vessel stents, artificial blood vessels, braided devices, polyester-based prostheses, automotive Car lining materials, soap, cosmetics, laundry detergent cans, laundry detergent, soap microplastics, microbeads, cosmetic microbeads, detergent boxes, disinfectants containing detergent, toothpaste containing microbeads, facial cleansers, conditioners, shower gels, hand soaps, exfoliating products, bath products, shower gels, powdered laundry detergents, lotions, deodorants, toilet cleaners, sunscreens, shopping bags, mouthwash bottles, peanut butter containers, salad dressing and vegetable oil containers, fleece fibers, tote bags, paneling, milk jugs, juice bottles, bleach bottles, motor oil bottles, cereal box liners, recycling containers, floor tiles, drainpipes, benches, picnic tables, fences, wire sheathing, sliding windows, decks, fenders, road gutters, speed bumps, squeezable bottles, bread, dry cleaning bags, trash can liners, garbage cans, compost bins, shipping envelopes, lumber, syrup bottles, ketchup bottles, straws, medicine bottles, battery cables, battery cases, disposable plates and cups, egg cartons, takeout containers, compact disc cases, signs and displays, synthetic fibers, yarns, stabilized phase change materials, thermal energy storage materials, nylon 6,6, nylon, tires, rubber, adiponitrile, shoes, footwear, or any combination thereof.

[0347] Examples

[0348] The following examples further illustrate the present disclosure, but should of course not be construed as limiting its scope in any way.

[0349] Example 1: Production of ethylene using formate as the sole carbon and energy source.

[0350] The gene encoding ethylene-forming enzyme is codon-adapted and synthesized for expression in Copper Bacteria. The adapted gene is cloned into the wide host range expression vector pBBR1MCS2 together with the constitutive promoter P10. The resulting product is used to transform Escherichia coli, and the positive clones identified by PCR are confirmed by DNA sequencing. The plasmid confirmed by the sequence is then transformed into Copper Bacteria PHB-4 by electroporation, and selected on a tryptic soy fermentation broth (TSB) agar plate containing 50mg / L chloramphenicol. The transformant containing the pBBR1-Efe plasmid is confirmed by sequencing, and a single colony is grown overnight at 30°C in TSB, and used to prepare a glycerol stock solution to store at -80°C. The strain is revived by streaking on a TSB plate containing 50mg / L chloramphenicol and incubating at 30°C for 72 hours.

[0351] 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 tube with a snap-on cap. After incubation overnight 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.

[0352] This culture was incubated at 30°C and 200rpm for 48 hours, adding 65mM formic acid to control pH between 6.5-7.5 at different times. After 48 hours, 20mL of the culture was transferred to a 160mL serum bottle, 65mM formic acid was added, and the bottle was sealed with an airtight septum. After an additional 16 hours of incubation, 60mL of the headspace volume was removed with an airtight syringe, and ethylene production was analyzed by GC. Various hydrocarbons and oxygenates in the sample were analyzed on a customized Wasson system. Ethylene was separated on a 50m x 0.53um Wasson PN 2378 post, and analyzed by GC FID.

[0353] like Figure 2 As shown, the C. necrotica strain with the Efe expression plasmid (pBBR1-Efe) produced more than 100 ppm of ethylene, while the control strain containing the empty pBBR1 plasmid had no detectable ethylene.

[0354] like Figure 4The reactions are shown in the figure, and the abbreviations are as follows: ACALD, acetaldehyde dehydrogenase (acetylation); ACONT1, aconitase (citrate hydrolase); ACONT2, aconitase (isocitrate hydrolase); AKGDH, 2-oxoglutamate dehydrogenase; ALCD2x, alcohol dehydrogenase (ethanol); ASPTA, aspartate aminotransferase; ATPS4m, ATP synthase (four protons for one ATP); CITt, citrate transporter; CS, citrate 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 transporter; HYDS, hydrogenase ( NADH); ICDHx, isocitrate dehydrogenase (NAD); ICITt, isocitrate transporter; LACDH, L-lactate dehydrogenase; MDH, malate dehydrogenase; ME1, malic enzyme (NAD); NADH16, NADH dehydrogenase (ubiquinone-8 and 3 protons); O2t, O2 transporter (diffusion); PDH1, pyruvate dehydrogenase E1 component; PDH2, pyruvate dehydrogenase E2 component (dihydrolipoamide acetyltransferase) enzyme); PDH3, dihydrolipoamide dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PPC, phosphoenolpyruvate carboxylase; PRUK, phosphoribosylkinase; RBPC, ribulose bisphosphate carboxylase; RPE, ribulose 5-phosphate 3-epimerase; SUCDi, succinate dehydrogenase (irreversible); SUCOAS, succinyl-CoA synthetase (ADP forming); TKT2, transketolase.

[0355] The genes listed are as follows:

[0356] ACALD: H16_A1806 or H16_B0596 or H16_A2747 or H16_B0551

[0357] ACONT1: H16_A2638 or H16_B0568 or H16_A1907

[0358] ACONT2: H16_A2638 or H16_B0568 or H16_A1907

[0359] 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)

[0360] ALCD2x: H16_B2470 or H16_B0517 or H16_A3330 or H16_B1433 or H16_A0757 or H16_B1699 or H16_B1834 or H16_B1745

[0361] ASPTA:H16_A2857

[0362] ATPS4m: H16_A3643 and H16_A3642 and H16_A3639 and H16_A3636 and H16_A3637 and H16_A3638 and H16_A3640 and H16_A3641

[0363] 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)

[0364] 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

[0365] ENO:H16_A1188

[0366] FBA: H16_B0278 or H16_B1384 or H16_A0568 or PHG416

[0367] FBP: H16_B1390 or H16_A0999 or PHG422

[0368] 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

[0369] FUM: H16_B0103 or H16_A2528

[0370] GAPD: H16_B1386 or H16_A3146 or PHG418

[0371] GLUDC:H16_A2930

[0372] GLUS: H16_B2194 or H16_A3430 or H16_B2192 or H16_A3431 or H16_B2193

[0373] HYDS: PHG088 and PHG089 and PHG090 and PHG091

[0374] ICDHx:H16_B1016

[0375] LACDH:H16_A0666

[0376] MDH: H16_B0334 or H16_A2634

[0377] ME1:H16_A3153

[0378] 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

[0379] PDH1: H16_A1374 or H16_B1300 or H16_B0145 or H16_B2234 or H16_B2233 or H16_A1753

[0380] PDH2: H16_A1375 or H16_B0146

[0381] PDH3: H16_A3724 or H16_A2323 or H16_A1377 or H16_B1098

[0382] PGK: H16_A0566 or H16_B1385 or PHG417

[0383] PGM: H16_A0332 or H16_A0493

[0384] PPC: H16_A2921

[0385] PRUK: H16_B1389 or PHG421

[0386] RBPC: (PHG426 and PHG427) or (H16_B1394 and H16_B1395)

[0387] RPE: (H16_B1391 and H16_A3317) or (PHG423 and H16_A3317)

[0388] SUCDi: H16_B0204 and H16_A2632 and H16_A2631 and H16_A2630 and H16_A2629

[0389] SUCOAS: H16_A0548 and H16_A0547

[0390] TKT2: (H16_B1388 and H16_A3147) or (PHG420 and H16_A3147).

[0391] Example 2: Continuous production of ethylene from CO2 using H2 as energy source.

[0392] The gene encoding ethylene-forming enzyme is codon-adapted and synthesized for expression in Copper Bacteria. The adapted gene is cloned into the wide host range expression vector pBBR1MCS2 together with the constitutive promoter P10. The resulting product is used to transform Escherichia coli, and the positive clones identified by PCR are confirmed by DNA sequencing. The plasmid confirmed by the sequence is then transformed into Copper Bacteria PHB-4 by electroporation, and selected on a tryptic soy fermentation broth (TSB) agar plate containing 50mg / L chloramphenicol. The transformant containing the pBBR1-Efe plasmid is confirmed by sequencing, and a single colony is grown overnight at 30°C in TSB, and used to prepare a glycerol stock solution to store at -80°C. The strain is revived by streaking on a TSB plate containing 50mg / L chloramphenicol and incubating at 30°C for 72 hours.

[0393] Single colonies from freshly streaked TSB plates were used to inoculate 3 mL of TSB containing 50 mg / L chloramphenicol in 14 mL Falcon round-bottom polystyrene tubes with snap-on caps. After overnight incubation at 30° C. and 200 rpm in a Thermo MAXQ shaker, 1 mL of 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 about 0.3-0.4 was reached.

[0394] 100 mL of the above culture was used to inoculate a 1.4-LInfors HTMultifors 2CSTR containing 600 mL of 2x start-up medium. The reactor was incubated at 30°C and started with 250 rpm agitation and 150 nccm gas flow (3.14% O2, 41% H2, 3% CO2, 52.86% N2). As the culture grew, agitation and gas flow were gradually increased to 1450 rpm and 750 nccm. When OD 600 Above 0.5, the culture was switched to continuous using 4x medium and 7 μl / hr Pluronic 31R1 antifoam. The feed oxygen percentage was gradually increased to promote biomass production, with the balance being nitrogen removal percentage, limited by keeping the outlet oxygen percentage below 4.5% as a safety measure.

[0395] The gas sample from the reactor was connected to a stream selection valve controlled by a micro GC (manufacturer: Qmicro) through 305 stainless steel. The sample was then analyzed on a Rt-U BOND XP PLOT column under isothermal conditions (70°C) by a thermal conductivity detector (TCD).

[0396] Once the culture was well established, the gas fraction was adjusted from O2-limited to H2-limited conditions such that non-zero dissolved oxygen (DO) concentrations were observed. Ethylene production varied as the system entered steady state and the gas fraction was adjusted, but production was maintained for more than 11 days ( Figure 3 ). During this period, the H2 fraction was between 11-18%, and the O2 fraction was between 5.5-6.6%, with CO2 maintained at 3% and the rest being N2. After switching back to O2-limited conditions, ethylene production ceased, indicating the importance of oxygen availability for ethylene production.

[0397] Example 3: Genome-scale modeling of a gene deletion strategy to eliminate unwanted byproducts during ethylene production from CO2 and H2 in Cupriavidus necrotus.

[0398] A genome-scale metabolic model of C. necator such as that described by Park et al., BMC Systems Biology, 5:101, 2011 was used to predict gene deletions to eliminate unwanted byproducts during ethylene production from CO2 and H2. Heterologous ethylene formation reactions were added to the wild-type C. necator model structure to represent the integration of non-natural compound production pathways. Ethylene production was simulated as follows: Constraint-based computational modeling techniques flux balance analysis (FBA) and linear minimization of metabolic regulation (LMOMA) (Maia, GECCO Proceedings of the Genetic and Evolutionary Computation Conference Companionon-GECCO ), ACM Press, New York, NY, pp. 1661-1668, 2017) using cobrapy version 0.8.2 (Ebrahim, COBRApy: Constraints-Based Reconstruction and Analysis for Python, BMC Systems Biology, 7:74, 2013), 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 and Gurobi Optimizer version 7.0.2 as the optimization solver.

[0399] Table 1: Gene deletions predicted to remove unwanted byproducts while still allowing biomass formation and ethylene production:

[0400]

[0401] Example 4: Generation of a scarless deletion at the glutamate decarboxylase (GLUDC) H16_A2930 locus.

[0402] Homology arm amplification and assembly. To generate a scarless deletion of the glutamate decarboxylase (GLUDC) open reading frame in C. 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 GeneArt Seamless Cloning and Assembly Enzyme Mix from Thermo Fisher Scientific. In brief, forward and reverse primers were designed in Geneious Prime to include 5' and 3' Gibson tails compatible with pK18mobsacB digested with the restriction enzyme BamHI. The 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. In a 20uL reaction, a mass of 100ng of purified homology arms was combined with 100ng of BamHI-digested pK18mobsacB using a GeneArt seamless cloning enzyme mixture and incubated at room temperature for 30 minutes. Next, 3uL of the reaction mixture was used to transform chemically competent DH10B Escherichia coli and plated on LB agar medium containing 50ng / uL kanamycin antibiotics. The plate was incubated at 37°C for 24 hours, and colonies were screened by PCR to verify the presence of each homology arm insert. Positive colonies were grown overnight in 5mL LB fermentation broth supplemented with 50ng / uL kanamycin, prepared using a Qiagen MINI preparative kit, and sequenced for plasmids using an Illumina MiSeq system.

[0403] Transformation was performed by conjugation. The sequence-verified pK18mobsacB plasmid containing left and right 500bp homology arms was electroporated into S17-1 E. coli cells and plated on LB supplemented with 50ng / uL kanamycin. Single colonies were picked and used to inoculate 5mL LB fermentation broth supplemented with 50ng / uL kanamycin to produce conjugated donor strains. In order to produce conjugated recipient strains, single colonies of H. arguta H16 grown on LB agar supplemented with 300ng / uL gentamicin were picked and used to inoculate 5mL LB fermentation broth supplemented with 300ng / uL gentamicin. The donor E. coli culture was grown overnight at 37°C with shaking at 250rpm, while the recipient H. arguta culture was grown overnight at 30°C with shaking at 200rpm. The next morning, cells were collected by centrifugation at 6000 rpm for 3 minutes at 25°C and the pellet was resuspended in 50uL LB medium. 50uL of donor and recipient cells were mixed and spotted on a sterile hydrophilic filter on LB agar without antibiotics.

[0404] Selection and counter-selection. The plates were incubated overnight at 30°C and the cells were removed from the filters the next morning using sterile forceps to fold the filters in half and transfer to a test tube containing 1 mL of LB. The cells were removed from the filters by vortexing and a series of dilutions (10 0 Up to 10 -3 ) and plated on LB agar containing 300ng / uL kanamycin in 100uL volumes. The plates were incubated at 30°C for 5 days. Next, the colonies were replicated on LB agar plates containing 300ng / uL kanamycin and LB agar plates containing 20% ​​sucrose plus 300ng / uL kanamycin. Colonies that grew on kanamycin instead of sucrose plus kanamycin (primary integrants) were streaked out on LB agar plates containing 300ng / uL kanamycin and then cultured overnight at 30°C in LB fermentation broth without antibiotics. The next morning, serial dilutions (10 0 Up to 10 -3 ) The culture was plated on agar containing 20% ​​sucrose (without antibiotics) at 30°C overnight to select secondary recombinants. Sucrose-resistant colonies were affixed on LB agar containing 20% ​​sucrose (with and without 300 ng / uL kanamycin) to select recombinants with sucrose resistance and antibiotic sensitivity. Finally, 12 SucR, KanS colonies were prepared for PCR and sequencing to verify the deletion of the GLUDC H16_A2930 locus.

[0405] Table 2: Primers and homology arm sequences for the GLUDC H16_A2930 locus:

[0406]

[0407]

[0408]

[0409] Table 3: Efe sequences:

[0410]

[0411]

[0412] After sequence confirmation GLUDC H16_A2930 locus deletion, the construct expressing EFE described in example 1 was transformed into this bacterial strain by electroporation, and selected on the tryptic soy fermentation broth (TSB) agar plate containing 50mg / L chloramphenicol.Confirm the transformant containing pBBR1-Efe plasmid by sequencing, and single colony is grown overnight at 30 ℃ in TSB, and is used to prepare glycerol stock solution to store at-80 ℃.By streaking on the TSB plate containing 50mg / L chloramphenicol and incubating at 30 ℃ for 72 hours, strain resurrection was carried out.Based on formate or CO2 / H2, the fermentation of producing ethylene is carried out as described in the above-mentioned example.

[0413] Additional gene deletions and chromosomal integrations that benefit ethylene production and / or reduce byproduct formation were performed as described above.

[0414] Example 5: A system for generating bubbles in a container

[0415] Example of a system for generating bubbles in a container 100 ( Figure 5). System 100 includes a cylindrical reactor 102. Liquid enters the inlet or top portion 101 of reactor 102. Liquid can enter top portion 101 by an external pump in fluid communication with system 100. According to certain embodiments, liquid entering top portion 101 is recirculated by an external pump in fluid communication with system 100. Liquid enters the top of perforated plate 104, and the liquid is accelerated by passing through the holes in plate 104. According to certain embodiments, plate 104 can be configured to accelerate, for example, at least, greater than, less than, equal to 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 to about 100% of the liquid in reactor 102, or any amount therebetween. Ejector 106 injects bubbles into the liquid from gas source 108. The ejector 106 is positioned within the reactor 102 so as to create a first zone in which the injected bubbles rise within the reactor 102 and encounter the accelerated liquid 112 leaving the bottom of the plate 104. The accelerated liquid 112 from the plate 104 breaks the rising bubbles into tiny bubbles, thereby increasing the apparent surface area required for the desired chemical or biological reaction. The diameter of the tiny bubbles can be in the range of about 0.1 mm to about 5 mm or about 0.5 mm to about 2 mm. In some examples, the diameter of the tiny bubbles can include about .2 mm to 1.5 mm. According to another embodiment, the diameter of the microbubbles may be, for example, at least, greater than, less than, or equal to 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, 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 to about 5.0 mm or any number therebetween. The ejector 106 is further positioned within the reactor 102 so as to create a second zone in which the fluid stream of liquid and microbubbles can flow downward.

[0416] Compared with the injected bubbles, the rising speed of the micro-bubbles may be reduced. Due to the overall flow of the accelerated liquid, the fluid 116 containing the liquid and the micro-bubbles can have a net downward flow. The downward speed of the fluid 116 is greater than the total rising speed of the micro-bubbles. The fluid 116 can leave the reactor 102 at the outlet 111. The thickness of the plate 104 (and the depth of the hole) can be about 1mm to 25mm. According to another embodiment, the thickness of the plate can be, for example, at least, greater than, less than, equal to, 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 to about 50 mm or any amount therebetween.

[0417] like( Figure 5 ), the dimensions of the components of system 100 may vary depending on the desired application or process. According to certain embodiments, the diameter of reactor 102 may be, for example, at least, greater than, less than, equal to, 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 to about 20.0 meters or any number therebetween. According to other embodiments, the length of the reactor 102 can be, for example, at least, greater than, less than, or equal to about 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, 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 to about 50.0 meters or any amount therebetween.

[0418] The velocity of the liquid or portion of the liquid accelerated from the plate 104 can be determined by the following equation:

[0419] QL=N x(π / 4)x d2 x vj

[0420] Where QL is the liquid volume flow rate (m3 / s), vj is the jet velocity, N is the total number of holes in the plate, d is the diameter of the hole, 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, at least, greater than, less than, equal to, 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 to about 20000 mm / s or any amount therebetween. Figure 5 As depicted, the velocity of the accelerated liquid 112 is crucial for breaking the bubbles injected into the liquid by the ejector 106 into micro-bubbles of appropriate size, and ensuring that the fluid of the liquid and micro-bubbles has sufficient velocity to produce a net downward fluid flow. The apparent liquid velocity VL in the main reaction vessel can be calculated by the following equation: VL=QL / AC, where QL is the volume flow rate (cubic meters / second) of the liquid in the reaction vessel, and AC is the cross-sectional area of ​​the reaction vessel. Therefore, the apparent liquid velocity represents the velocity of the liquid phase if it occupies the entire cross-sectional area of ​​the reaction vessel. According to an embodiment, the apparent liquid velocity may also include a zone or void of stagnant liquid and micro-bubbles, and / or a net downward fluid flow. For the same liquid flow rate, the gas flow rate may vary according to the actual application. The apparent velocity VG of the gas phase can be determined by the following equation: VG=QG / AC, where QG is the volume flow rate (cubic meters / second) of the gas injected into the liquid from the ejector, and AC is the cross-sectional area of ​​the reaction vessel. According to another embodiment, the superficial velocity of the gas phase in the container can be, for example, at least, greater than, less than, equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 to about 100 mm / sec or any amount therebetween. According to still another embodiment, the superficial velocity of the gas phase in the container can be, for example, about 50-60 mm / sec.

[0421] Positioning one or more ejectors 106 within the reactor 102 and on the upper portion of the reactor 102 has the additional advantage of reducing the hydrostatic pressure at the top of the reactor 102, thereby contributing to the additional advantage of increasing the gas-liquid mass transfer rate while reducing energy requirements. Further, the required reactor components are minimized, yet the gas-liquid mass transfer rate is maximized in a smaller reactor footprint due to the reduced reactor size. For example, in some embodiments, the systems and methods disclosed herein achieve a gas-liquid mass transfer rate of at least 125 cubic meters per minute. In other examples, the gas-liquid mass transfer rate can be, for example, at least, greater than, less than, equal to about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 to about 200 cubic meters per minute or any number therebetween. Additionally, the sparger configurations, resulting superficial velocities of the gas and liquid phases, and increased gas-liquid mass transfer rates disclosed herein overcome known obstacles associated with gas and liquid phase systems using previous and conventional reactors, particularly in bioreactors with gas substrates and aqueous cultures.

[0422] Example 6: Ethylene production using different genetic sources of ethylene-forming enzymes

[0423] Genes encoding ethylene-forming enzymes from various organisms (accession numbers: Microcoleus metropolitanus (NQE34890), Myxococcus petiolus (WP_015351455.1), Nostoc ATCC 43529 (RCJ18531), Ralstonia solanacearum (WP_014618742.1), Pseudoclase NIES-4073 (WP_096562523.1)) were cloned, codon-adapted and synthesized for expression in C. necator. The adapted genes were coupled to a rhamnose-inducible promoter (P rhaBAD ) and bicistronic RBS elements were cloned into the wide host range expression vector pBBR1MCS2. The resulting product was used to transform Escherichia coli, and the positive clones identified by PCR were confirmed by DNA sequencing. The plasmid confirmed by the sequence was then transformed into the hookworm greedy copper bacteria PHB-4 by electroporation, and selected on the tryptic soy fermentation broth (TSB) agar plate containing 50mg / L chloramphenicol. The transformant containing the pBBR1-Efe plasmid was confirmed by sequencing, and the single colony was grown overnight at 30°C in TSB, and used to prepare a glycerol stock solution to store at -80°C. The strain was revived by streaking on a TSB plate containing 50mg / L chloramphenicol and incubated at 30°C for 72 hours.

[0424] Use single colony from freshly streaked TSB plate to inoculate 1mL J minimal medium containing 10g / L fructose, 10g / L tryptone and 5g / L yeast extract in deep well 96-well plate, and grow 24 hours at 1000rpm and 30°C.Use this pre-culture to inoculate (1%) 20mL J minimal medium containing 10g / L fructose in 160mL serum bottle.Then culture is grown 6 hours at 30°C and 200rpm, now adding 0.5mM rhamnose.After another 18 hours of growth, the bottle is sealed with an airtight septum.After another 24 hours of incubation, remove the headspace volume of 60mL with an airtight syringe, and analyze ethylene production by GC.Various hydrocarbons and oxides in the sample are analyzed on a customized Wasson system.Ethylene is separated on a 50m x 0.53um Wasson PN 2378 post, and analyzed by GC FID.

[0425] like Figure 7 As shown, the C. necator strains with each Efe variant produced detectable ethylene, whereas no ethylene was detected using the control strain containing the empty pBBR1 plasmid. Since these EFE variants had AA similarities ranging from 63-71% with the typical enzyme from Pseudomonas syringae, this demonstrated the ability of the different EFE enzymes to produce ethylene.

[0426] Example 7: Use of a conditionally dependent promoter for EFE expression during continuous ethylene production from CO2 using H2 as an energy source.

[0427] The gene encoding ethylene-forming enzyme is codon-adapted and synthesized for expression in hookworm greedy copper bacteria. The adapted gene is cloned into a wide host range expression vector pBBR1MCS2 together with an inducible promoter (Ppst-pho) and a bicistronic RBS element limited by phosphate. The resulting product is used to transform Escherichia coli, and the positive clones identified by PCR are confirmed by DNA sequencing. The plasmid confirmed by the sequence is then transformed into hookworm greedy copper bacteria PHB-4 by electroporation, and selected on a tryptic soy fermentation broth (TSB) agar plate containing 50mg / L chloramphenicol. The transformant containing the pBBR1-Efe plasmid is confirmed by sequencing, and a single colony is grown overnight at 30°C in TSB, and used to prepare a glycerol stock solution to store at -80°C. The strain is revived by streaking on a TSB plate containing 50mg / L chloramphenicol and incubating at 30°C for 72 hours.

[0428] Single colonies from freshly streaked TSB plates were used to inoculate 3 mL of TSB containing 50 mg / L chloramphenicol in 14 mL Falcon round-bottom polystyrene tubes with snap-on caps. After overnight incubation at 30° C. and 200 rpm in a Thermo MAXQ shaker, 1 mL of 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 about 0.3-0.4 was reached.

[0429] Under conditions similar to those described in Example 2, 100 mL of the above culture was used to inoculate a 1.4-LInfors HTMultifors 2CSTR.

[0430] Once the culture was well established, the medium composition was adjusted to achieve phosphate-limiting conditions and further induce the expression of ethylene-forming enzymes ( Figure 8 This change in medium composition resulted in an approximately 5-fold increase in ethylene concentration and production ( Figure 8 ), demonstrating the application of conditional-dependent promoters in ethylene production.

[0431] Similarly, the use of promoters that respond to the gaseous carbon substrate CO2 and the energy source H2 can also be used to express ethylene-forming enzymes to enable ethylene production in C. necator. Here, the adapted ethylene-forming enzyme gene and the bicistronic RBS element were cloned into the broad host range expression vector pBBR1MCS2, which has a large plasmid CbbL promoter (P CbbL,p ) or a soluble hydrogenase promoter (P SH ). The resulting product is used to transform Escherichia coli, and the positive clones identified by PCR are confirmed by DNA sequencing. The plasmid confirmed by the sequence is then transformed into the hookworm greedy copper bacteria PHB-4 by electroporation, and selected on a tryptic soy fermentation broth (TSB) agar plate containing 50mg / L chloramphenicol. The transformant containing the pBBR1-Efe plasmid is confirmed by sequencing, and a single colony is grown overnight at 30°C in TSB, and used to prepare a glycerol stock solution to store at -80°C. The strain is revived by streaking on a TSB plate containing 50mg / L chloramphenicol and incubating at 30°C for 72 hours.

[0432] These strains were run in a 1.4-LInfors HT Multifors 2CSTR following a procedure similar to that described in the previous example (Example 2). Under these conditions, P CbbL,p The promoter expressed ethylene-forming enzymes, resulting in continuous production of ethylene from CO2 and H2 for more than 2 weeks ( Fig. 9In addition, a soluble hydrogenase promoter (P SH ) to drive the expression of ethylene-forming enzymes was also able to produce ethylene, reaching concentrations exceeding 100 ppm in the outlet gas stream ( Fig.10 ).

[0433] All references (including publications, patent applications and patents) cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The reference to any prior art in this specification is not, and should not be taken as, an admission that the prior art forms part of the common general knowledge in the field of endeavor in any country.

[0434] Unless otherwise indicated herein or clearly contradicted by context, the terms "a / an" and "the" and similar referents used in the context of describing the present disclosure (particularly in the context of the following claims) should be interpreted as covering both the singular and the plural. Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including but not limited to"), unless otherwise indicated. The term "consisting essentially of..." limits the scope of the composition, process or method to specific materials or steps, or to those materials or steps that have no substantial effect on the basic and novel characteristics of the composition, process or method. The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination thereof in the alternatives. As used herein, the term "about" refers to ±20% of the specified range, value, or structure, unless otherwise indicated.

[0435] Unless otherwise indicated herein, the recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of individually indicating each individual value falling within the range and each individual value is incorporated into the specification as if the value were individually recited herein. For example, unless otherwise indicated, any concentration range, percentage range, ratio range, integer range, size range, or thickness range should be understood to include the value of any integer within the recited range and, where appropriate, include fractions thereof (such as tenths and hundredths of integers).

[0436] Unless otherwise indicated herein or clearly contradicted by context, all methods described herein may be performed in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present disclosure, and does not limit the scope of the present disclosure. Any language in this specification should not be interpreted as indicating any unclaimed element as necessary for practicing the present disclosure.

[0437] Preferred embodiments of the present disclosure are described herein. After reading the foregoing description, variations of those preferred embodiments may become apparent to those of ordinary skill in the art. The inventors anticipate that the skilled person may adopt such variations when appropriate, and the inventors intend to practice the present disclosure in other ways different from the ways specifically described herein. Therefore, where applicable law permits, the present disclosure includes all modifications and equivalents of the subject matter recited in the appended claims. In addition, the present disclosure encompasses any combination of the above-mentioned elements in all possible variations thereof, unless otherwise indicated herein or otherwise clearly contradictory to the context.

[0438] Embodiments of the present disclosure

[0439] Example 1. A recombinant C1 immobile microorganism capable of producing ethylene from a gaseous substrate, the recombinant C1 immobile microorganism comprising a nucleic acid encoding a set of exogenous enzymes comprising an ethylene forming enzyme (EFE).

[0440] Example 2. A recombinant C1 fixed microorganism capable of switching the cell load in the production of ethylene, the microorganism comprising a nucleic acid encoding a set of exogenous enzymes comprising an ethylene forming enzyme (EFE) and one or more inducible promoters.

[0441] Embodiment 3. The microorganism according to embodiment 1, wherein the microorganism is selected from the group consisting of: Cupriavidus necrotus and Ralstonia eutropha.

[0442] Embodiment 4. The microorganism according to embodiment 4, wherein the microorganism is Cupriavidus necrotus.

[0443] Embodiment 5. The microorganism according to embodiment 2, further comprising a nucleic acid encoding an α-ketoglutarate permease, wherein the nucleic acid is codon-optimized for expression in the microorganism.

[0444] Embodiment 6. A microorganism according to 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.

[0445] Embodiment 7. The microorganism according to embodiment 2, wherein the EFE is codon optimized for expression in the microorganism.

[0446] Embodiment 8. The microorganism of embodiment 1, further comprising a disruptive mutation in one or more genes.

[0447] Embodiment 9. A microorganism according to 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.

[0448] Embodiment 10. A microorganism according to embodiment 1, wherein the gaseous substrate comprises CO2 and an energy source.

[0449] Embodiment 11. A microorganism according to embodiment 1, wherein the gaseous substrate comprises CO2 and H2, O2, or both.

[0450] Embodiment 12. A method for continuously producing ethylene, the method comprising: passing a gaseous substrate into a bioreactor containing a culture of a recombinant C1 immobile microorganism according to embodiment 1 in a culture medium, such that the microorganism converts the gaseous substrate into ethylene; and recovering the ethylene from the bioreactor.

[0451] Embodiment 13. The method of embodiment 12, wherein the gaseous substrate comprises industrial waste products or exhaust gases.

[0452] Embodiment 14. The method according to embodiment 12, further comprising an energy source.

[0453] Embodiment 15. The method of embodiment 12, wherein the energy source is provided intermittently.

[0454] Embodiment 16. The method of embodiment 12, wherein the gaseous substrate comprises CO2 and an energy source.

[0455] Embodiment 17. The method of embodiment 16, wherein the energy source is H2.

[0456] Embodiment 18. The method of embodiment 16, wherein the gaseous substrate further comprises H2, O2, or both.

[0457] Example 19. The method according to Example 12, further comprising the step of limiting dissolved oxygen concentration, thereby switching the cell load.

[0458] Embodiment 20. The method of embodiment 12, further comprising controlling the iron concentration to comprise at least 50 mg / L.

[0459] Embodiment 21. The method of embodiment 12, further comprising converting the ethylene into components for manufacturing tires.

[0460] Embodiment 22. The method of embodiment 21, wherein the tire is a scrap tire.

[0461] Embodiment 23. The method of embodiment 12, wherein the gaseous substrate is derived from a process involving tires.

[0462] Embodiment 24. The method of embodiment 12, wherein the gaseous substrate is derived from a product recycling process or a sustainable chemical process.

[0463] Embodiment 25. The method of embodiment 23, further comprising converting the ethylene into a component for making new tires.

Claims

1. A recombinant C1 immobile microorganism capable of producing ethylene from a gaseous substrate, the recombinant C1 immobile microorganism comprising a nucleic acid encoding a set of exogenous enzymes comprising an ethylene forming enzyme (EFE).

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

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

4. The microorganism according to claim 3, wherein the microorganism is Cupriavidus necrotus.

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

6. The microorganism of claim 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.

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

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

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

10. The microorganism of claim 1, wherein the gaseous substrate comprises CO2 and an energy source.

11. The microorganism of claim 1, wherein the gaseous substrate comprises CO2 and H2, O2, or both.

12. A method for continuously producing ethylene, the method comprising: passing a gaseous substrate into a bioreactor containing a culture of the recombinant C1 immobile 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. The method of claim 12, wherein the gaseous substrate comprises an industrial waste product or exhaust 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 method of claim 12, wherein the gaseous substrate comprises CO2 and an energy source.

17. The method of claim 16, wherein the energy source is H2.

18. The method of claim 16, wherein the gaseous substrate further comprises H2, O2, or both.

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

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

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