Microorganisms and methods for continuous co-production of tandem repeat proteins and chemical products from c1 substrates

Abstract

The microorganisms are genetically engineered to continuously co-produce amino acids, proteins, microbial biomass, chemicals, or any combination thereof by microbial fermentation, in particular by microbial fermentation of gaseous substrates. The microorganism is of a C1 fixed type. The yield of ethylene, microbial biomass and heterologous tandem repeat protein can be improved. This can be achieved by an improved promoter or nutrition limiting means.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 366,756, filed on June 21, 2022, 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 31, 2023 is named "LT228WO1-Sequences.xml" and is 5,763 bytes in size. Technical Field

[0005] The present disclosure relates to genetically engineered microorganisms and methods for the continuous co-production of amino acids, proteins, microbial biomass, chemicals, or any combination thereof, 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 (such as industrial waste gases or synthesis gas) containing carbon dioxide (CO2), carbon monoxide (CO) and / or hydrogen (H2) into various fuels and chemicals. However, recently, gas fermentation has become an alternative platform for the biological fixation of such gases. Specifically, it has been demonstrated that C1 fixed microorganisms convert gases containing CO2, CO and / or H2 into products such as ethanol and 2,3-butanediol. However, the effective co-production of such chemical products and heterologous proteins may be limited by slow microbial growth, limited gas absorption, sensitivity to toxins, or conversion of carbon substrates into unwanted by-products. In addition, there is a growing interest in creating sustainable materials that exhibit multifunctional properties from synthetic and biological processes. Therefore, it is a continuous and unmet need to develop new protein-based materials that can be easily produced from renewable resources and will provide a wide range of useful applications. There is also a need for genetically engineered microorganisms with improved properties for continuous co-production of chemicals, proteins, biomass, or any combination thereof by microbial fermentation of gaseous substrates. 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 the disclosure disclosed herein is not limited to a particular advantage or function, the present disclosure provides a genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one protein having a tandem repeat sequence and a heterologous nucleic acid encoding at least one secreted chemical product.

[0009] In some aspects of the microorganisms disclosed herein, the microorganism produces a commodity chemical product, a tandem repeat protein product, a microbial biomass, a single cell protein (SCP), one or more intermediates, or any combination thereof.

[0010] In some aspects of the microorganisms disclosed herein, the microorganism produces a heterologous protein product. In one embodiment, the heterologous protein product comprises a heterologous nucleic acid encoding at least one protein having a tandem repeat sequence.

[0011] In some aspects of the microorganisms disclosed herein, the microorganism comprises a genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one protein having a tandem repeat sequence and a heterologous nucleic acid encoding at least one secreted chemical product, wherein the microorganism is a C1 fixed bacterium.

[0012] In some aspects of the microorganisms disclosed herein, the microorganism comprises a genetically engineered C1 immobile microorganism capable of co-producing a heterologous protein and a chemical product from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeat sequences and a heterologous nucleic acid encoding at least one chemical product, wherein the microorganism is capable of accumulating the at least one heterologous protein in the cell and secreting the at least one chemical product from the cell.

[0013] In some aspects of the microorganisms disclosed herein, the microorganism comprises one or more heterologous enzymes derived from a genus selected from the group consisting of Bacillus, Clostridium, Cupriavidus, Escherichia, Gluconobacter, Hyphomicrobium, Lysinibacillus, Paenibacillus, Pseudomonas, Sedimenticola, Sporosarcina, Streptomyces, Thermithiobacillus, Thermotoga, Dosidicus, and Zea.

[0014] In some aspects of the microorganisms disclosed herein, the microorganism comprises a genetically engineered C1-fixing microorganism capable of co-producing at least one heterologous functional protein and at least one chemical product having at least two or more carbons from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one protein having a tandem repeat sequence and a heterologous nucleic acid encoding at least one secreted chemical product.

[0015] In some aspects of the microorganisms disclosed herein, the microorganism comprises a genetically engineered C1 fixed microorganism capable of co-producing at least one heterologous functional protein and at least one chemical product having at least two or more carbons from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding a set of genes comprising at least one protein having a tandem repeat sequence and at least one secreted chemical product.

[0016] In some aspects of the microorganisms disclosed herein, the microorganism comprises a genetically engineered C1 immobilized microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprising:

[0017] a) a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeat sequences; and

[0018] b) a heterologous nucleic acid encoding at least one chemical having two or more carbons, wherein the microorganism is capable of accumulating the at least one heterologous protein in a cell and secreting the at least one chemical product from the cell.

[0019] One aspect comprises a method of co-producing at least one heterologous protein and at least one chemical product by culturing the genetically engineered C1 immobile microorganism in the presence of a gaseous substrate comprising one or more of CO, CO2, and H2, wherein the culturing is a continuous fermentation process.

[0020] One aspect comprises a method wherein the gaseous substrate comprises a C1 carbon source comprising one or more of CO, CO2, and H2.

[0021] One aspect comprises a method wherein the gaseous substrate comprises synthesis gas or industrial waste gas.

[0022] One aspect comprises a method for co-producing at least one heterologous protein and at least one chemical product by culturing the genetically engineered C1 immobilized form, wherein the product is one or more of acetone and isopropanol.

[0023] In some aspects of the microorganisms disclosed herein, the microorganism comprises a genetically engineered C1 fixed microorganism, wherein the at least one heterologous protein having one or more tandem repeat sequences is selected from collagen, silk, elastin, keratin, arthropod elastic protein, titin, squid ring tooth (SRT) protein or any combination thereof.

[0024] In some aspects of the microorganisms disclosed herein, the microorganism is a member of a genus selected from the group consisting of Acetobacterium, Alkalibaculum, Blautia, Butyribacterium, Clostridium, Cupriavidus, Eubacterium, Moorella, Oxobacter, Ralstonia, Sporomusa, and Thermoanaerobacter.

[0025] In some aspects of the microorganisms disclosed herein, the microorganism is derived from a parent microorganism selected from the group consisting of Acetobacterium woodi, Alkalibaculumbacchii, Blautia producta, Butyribacteriummethylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium magnum, Clostridium ragsdalei, Clostridium scatologenes, Cupriavidus necator, Eubacterium limosum, Moorella thermautotrophica, Mooreella thermoacetica, Oxobacter pfennigii, Ralstonia eutropha, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, and Thermoanaerobacter kiuvi.

[0026] In some aspects of the microorganisms disclosed herein, the microorganism is derived from a parent bacterium selected from the group consisting of Clostridium ethanologenum, Clostridium lndallii, or Clostridium rassii.

[0027] In some aspects of the microorganisms disclosed herein, the microorganism is derived from a parent bacterium selected from the group consisting of Cupriavidus necrotus.

[0028] A genetically engineered C1 stationary microorganism, wherein the at least one heterologous protein having one or more tandem repeat sequences is selected from silk or SRT proteins.

[0029] In some aspects of the microorganisms disclosed herein, the gaseous fermentation product is selected from alcohols, acids, diacids, olefins, terpenes, isoprene, and alkynes, or any combination thereof.

[0030] In some aspects of the microorganisms disclosed herein, at least one of the secreted chemical products is selected from the group consisting of 1-butanol, butyrate, butene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, terpenes, isoprene, fatty acids, 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.

[0031] In some aspects of the microorganisms disclosed herein, the microorganism further comprises a disruptive mutation in one or more genes.

[0032] The present disclosure further provides a genetically engineered C1 immobilized microorganism further comprising a microbial biomass and at least one excipient.

[0033] The present disclosure further provides a genetically engineered C1 immobilized microorganism, wherein the microbial biomass is suitable as animal feed.

[0034] 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.

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

[0036] The present disclosure further provides a genetically engineered C1 immobilized microorganism, wherein the microorganism is suitable as a cell-free protein synthesis (CFPS) platform.

[0037] The present disclosure further provides a genetically engineered C1 immobilized microorganism, wherein at least one secreted chemical product is native to the microorganism.

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

[0039] In some aspects of the methods disclosed herein, at least a portion of the substrate is industrial waste gas, industrial tail gas, or synthesis gas.

[0040] 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.

[0041] In some embodiments, the present disclosure provides a method for storing energy in the form of a biopolymer, the method comprising intermittently processing at least a portion of electrical energy generated by a renewable and / or non-renewable energy source in an electrolysis process to produce at least H2, O2, or CO; intermittently delivering at least one of the H2, O2, or CO from the electrolysis process to a bioreactor containing a culture, the culture comprising a liquid nutrient medium and a microorganism capable of producing a biopolymer; and fermenting the culture.

[0042] 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.

[0043] 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: delivering 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 delivering 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.

[0044] 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.

[0045] 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.

[0046] In one embodiment, the autotrophic microorganisms intermittently consume part or all of the energy provided by the available energy.

[0047] 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

[0048] 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:

[0049] Figure 1 The expression of tandem repeat proteins in ethanologenic Clostridium is shown by Western blotting (Table 2). The production of tandem repeat proteins was assessed by Western blotting analysis using anti-Strep tag antibodies. The culture was dissolved and clarified; the protein content of the clarified lysate and insoluble precipitate (resuspended in 5M urea) was analyzed separately. The samples were run on Tris-glycine SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-Strep tag antibodies conjugated to alkaline phosphatase for observation. Proteins of expected size were observed in the insoluble precipitates of SRT008, SRT011, SRT012 and SS015. In addition, SS015 was observed in the clarified lysate.

[0050] Figures 2A-2C The performance of strSRT012 in a continuous CSTR fermentation with cell recycle (CR) using a syngas blend (55% CO, 5% H2, 30% CO2 and 10% N2) is shown. Figure 2A : The ethanol yield per biomass and the SRT012 protein content relative to the 5th day (reactor was switched to continuous) were analyzed by HPLC and comparative Western blot, respectively; Figure 2B : Biomass and metabolite profile analysis by HPLC; Figure 2C : Gas generation spectrum analyzed by GC-TCD (negative = uptake).

[0051] Figures 3A-3CThe performance of SRT008 ​​in a batch CSTR fermentation using a syngas blend (55% CO, 5% H2, 30% CO2, and 10% N2) is shown. Figure 3A : Ethanol yield per biomass and SRT008 ​​protein content relative to day 0 were analyzed by HPLC and comparative Western blot, respectively; Figure 3B : Biomass and metabolite profile analysis by HPLC; Figure 3C : Gas generation spectrum analyzed by GC-TCD (negative = uptake).

[0052] Figures 4A-4C The performance of SRT012 in a batch CSTR fermentation using a syngas blend (55% CO, 5% H2, 30% CO2, and 10% N2) is shown. Figure 4A : Ethanol and SRT012 protein content per biomass were analyzed by HPLC and comparative Western blot, respectively, relative to day 0. The last data point for protein content per biomass was obtained after the gas was turned off; Figure 4B : Biomass and metabolite profile analysis by HPLC; Figure 4C : Gas generation spectrum analyzed by GC-TCD (negative = uptake).

[0053] Figures 5A-5C The performance of SRT008 ​​in a batch CSTR fermentation using a high hydrogen synthesis gas blend (10% CO, 50% H2, 30% CO2, and 10% N2) is shown. Figure 5A : Ethanol and SRT008 ​​protein content per biomass relative to day 0 were analyzed by HPLC and comparative Western blot, respectively; Figure 5B : Biomass and metabolite profile analysis by HPLC; Figure 5C : Gas generation spectrum analyzed by GC-TCD (negative = uptake).

[0054] Figures 6A-6C The performance of SRT012 in a batch CSTR fermentation using a high hydrogen synthesis gas blend (10% CO, 50% H2, 30% CO2, and 10% N2) is shown. Fig. 6A : Ethanol and SRT012 protein content per biomass relative to day 0 were analyzed by HPLC and comparative Western blot, respectively; Figure 6B : Biomass and metabolite profile analysis by HPLC; Figure 6C : Gas generation spectrum analyzed by GC-TCD (negative = uptake).

[0055] Figure 7Continuous 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. DETAILED DESCRIPTION

[0056] 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.

[0057] The inventors have surprisingly been able to engineer C1 -fixing microorganisms to co-produce proteins, chemicals or precursors of chemicals, and microbial biomass by fermenting substrates comprising CO and / or CO2.

[0058] Unless otherwise defined, the following terms as used throughout this specification are defined as follows:

[0059] 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.

[0060] 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.

[0061] 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.

[0062] "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.

[0063] "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.

[0064] "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.

[0065] "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.

[0066] "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.

[0067] 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.

[0068] 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.

[0069] 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."

[0070] 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.

[0071] The term "copolymer" is a composition comprising two or more monomer species connected by the same polymer chain of the present disclosure.

[0072] "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.

[0073] "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.

[0074] "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.

[0075] "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.

[0076] Microorganism of the present disclosure can be derived from substantially any parent microorganism. In one embodiment, microorganism of the present disclosure can be derived from a parent microorganism selected from the group consisting of: Clostridium acetobutylicum, Clostridium beijerinckii, Escherichia coli and Saccharomyces cerevisiae. In other embodiments, the microorganism is derived from a parent microorganism, and the parent microorganism is selected from the group consisting of: Acetobacter acetobutylicum, Alkaliphilic Bacteria, Blautia, Methyl-eating Butyric Acid Bacteria, Clostridium acetate, Ethanol-producing Clostridium, Eating Carbon Monoxide Clostridium, Clostridium kluyveri, Clostridium de Shi, Clostridium formicacetic acid, Clostridium yongdaer, Clostridium, Clostridium rabbis, Clostridium feces, Eubacterium mucosum, Thermoautotrophic Moore's Bacteria, Thermoacetate Moore's Bacteria, Prevotella Acetobacter, Ovate Mouse Sporoderm, Forest Soil Acetate Mouse Sporoderm, Spherical Mouse Sporoderm and Kaiwu Thermoanaerobic Bacteria. In one embodiment, the parent microorganism is ethanologenic Clostridium, Ljungdahl Clostridium or Rasslera Clostridium. In another embodiment, the parent microorganism is ethanologenic Clostridium LZ1561, which was deposited on June 7, 2010 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) at D-38124 Inhoffenstraβe 7B, Brunswick, Germany, under the terms of the Budapest Treaty, and has accession number DSM 23693. This strain is described in International Patent Application No. PCT / NZ2011 / 000144, published as WO 2012 / 015317.

[0077] 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 generally include insertion, deletion, mutation or replacement of a nucleic acid or gene. Typically, the microorganism of the present disclosure is derived from a parent microorganism. In one embodiment, the microorganism of the present disclosure is derived from ethanologenic Clostridium, Yondahl Clostridium or Rassler Clostridium. In a preferred embodiment, the microorganism of the present disclosure is derived from ethanologenic Clostridium LZ1561, which is deposited under DSMZ accession number DSM23693.

[0078] 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, 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.

[0079] Table 1 provides a representative list of microorganisms and identifies their functional properties.

[0080]

[0081]

[0082] 1 Acetobacter woodii can produce ethanol from fructose, but not from gas.

[0083] 2 Whether C. major can grow on CO has not been studied.

[0084] 3 Moorella thermoacetica, a strain of Moorella, HUC22-1, has been reported to produce ethanol from gas.

[0085] 4 Whether R. ovale can grow on CO has not been studied.

[0086] 5 Whether forest soil B. aceticola can grow on CO has not been studied.

[0087] 6 Whether R. sphaeroides can grow on CO has not been studied.

[0088] "Wood-Jungdahl" refers to the Wood-Jungdahl pathway of carbon fixation, as described, for example, by Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008. "Wood-Jungdahl microorganism" predictably refers to a microorganism containing the Wood-Jungdahl pathway. Typically, the microorganisms of the present disclosure contain a natural Wood-Jungdahl pathway. In this article, the Wood-Jungdahl pathway can be a natural, unmodified Wood-Jungdahl pathway, or the Wood-Jungdahl pathway can be a Wood-Jungdahl pathway with a certain degree of genetic modification (e.g., overexpression, heterologous expression, knockout, etc.), as long as the Wood-Jungdahl pathway still functions to convert CO, CO2 and / or H2 into acetyl-CoA.

[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 identified in Table 1.

[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 the anaerobic bacteria identified in Table 1.

[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 a preferred embodiment, the microorganisms of the present disclosure are derived from the acetogens identified in Table 1.

[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 identified in Table 1.

[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 the autotrophs identified in Table 1.

[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 the carboxydotrophic bacteria identified in Table 1.

[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] In one embodiment, the microorganisms of the present disclosure are derived from a cluster of Clostridium, comprising ethanologenic Clostridium species, Yondahl Clostridium species, and Rassler Clostridium species. These species were originally reported and characterized by Abrini, Arch Microbiol, 161:345-351, 1994 (ethanologenic Clostridium); Tanner, Int J System Bacteriol, 43:232-236, 1993 (Yondahl Clostridium); and Huhnke, WO 2008 / 028055 (Rassler Clostridium).

[0099] There are many similarities between these three species. Specifically, these species are C1 fixed, anaerobic, acetogenic, ethanologenic and carbon monoxide trophic members of the genus Clostridium. These species have similar genotypes and phenotypes and similar energy conservation and fermentation metabolism patterns. In addition, these species are clustered in the Clostridium rRNA homology group I with more than 99% identical 16S rRNA DNA, have a DNA G+C content of about 22-30 mol%, are Gram-positive, have similar morphology and size (logarithmic growth phase cells are between 0.5-0.7×3-5 μm), are mesophilic (grow best at 30-37°C), have a similar pH range of about 4-7.5 (optimal pH is about 5.5-6), lack cytochrome, and conserve energy through the Rnf complex. In addition, carboxylic acid reduction to its corresponding alcohol has been demonstrated in these species (Perez, "Biotechnol Bioeng", 110:1066-1077, 2012). Importantly, these species also all show robust autotrophic growth on CO-containing gases, producing ethanol and acetate (or acetic acid) as the major fermentation products and, under certain conditions, small amounts of 2,3-butanediol and lactic acid.

[0100] However, there are also many differences between the three species. The species were isolated from different sources: ethanologenic Clostridium from the intestine of rabbits, Clostridium Ljungdahl from chicken waste, and Clostridium ragmerii from freshwater sediments. The species differ in the utilization of various sugars (e.g., rhamnose, arabinose), acids (e.g., gluconate, citrate), amino acids (e.g., arginine, histidine), and other substrates (e.g., betaine, butanol). In addition, the species differ in auxotrophy of certain vitamins (e.g., thiamine, biotin). The species differ in the nucleic acid and amino acid sequences of the Wood-Ljungdahl pathway genes and proteins, although the general structure and numbers of these genes and proteins have been found to be the same in all species ( Curr Opin Biotechnol, 22:320-325, 2011).

[0101] Thus, in general, many of the properties of C. ethanologenicus, C. jundal or C. ragsbyii are not specific to the species, but are general properties of this cluster of C1-fixed, anaerobic, acetogenic, ethanologenic and carboxydotrophic members of the genus Clostridium. However, because these species are actually different, a genetic modification or manipulation of one of these species may not have the same effect in another of these species. For example, differences in growth, performance or product production may be observed.

[0102] Microorganisms disclosed herein can also be derived from isolates or mutants of ethanologenic Clostridium, Yondahl Clostridium or Rassner Clostridium. Isolates and mutants of ethanologenic Clostridium include JA1-1 (DSM10061) (Abrini, "Microbiology Literature Collection", 161: 345-351, 1994), LBS1560 (DSM19630) (WO 2009 / 064200) and LZ1561 (DSM23693) (WO2012 / 015317). Isolates and mutants of Clostridium ljungdahlii include ATCC 49587 (Tanner, Int J Syst Bacteriol, 43:232-236, 1993), PETCT (DSM 13528, ATCC 55383), ERI-2 (ATCC 55380) (US 5,593,886), C-01 (ATCC 55988) (US 6,368,819), O-52 (ATCC 55989) (US 6,368,819) and OTA-1 (Tirado-Acevedo, Production of bioethanol from synthesis gas using Clostridium ljungdahlii, PhD thesis, North Carolina State University, 2010). Isolates and mutants of Clostridium raginsii include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO 2008 / 028055).

[0103] However, as described above, the microorganisms of the present disclosure may also be derived from essentially any parent microorganism, such as a parent microorganism selected from the group consisting of Clostridium acetobutylicum, Clostridium baylyllinii, Escherichia coli, and Saccharomyces cerevisiae.

[0104] 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.

[0105] 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.

[0106] In some embodiments, the microorganism is Cupriavidus necrotus DSM248 or DSM541.

[0107] 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.

[0108] 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.

[0109] 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. The microorganism of the embodiment further comprises a nucleic acid encoding a set of exogenous enzymes, wherein the exogenous enzymes include at least one α-ketoglutarate permease (AKGP).

[0110] The microorganism of the embodiment, wherein the nucleic acid encoding a set of exogenous enzymes comprises at least one EFE, at least one AKGP or any combination thereof. The microorganism of the embodiment, wherein the nucleic acid encoding a set of exogenous enzymes comprises at least one EFE and at least one AKGP. The microorganism of the embodiment, wherein the nucleotides encoding a set 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. The microorganism of the embodiment, wherein one or more inducible promoters are selected from H2 inducible promoters, phosphate-limited type inducible promoters, nitrogen-limited type inducible promoters or any combination thereof. 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 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] One or more of the enzymes of the present disclosure may be overexpressed. "Overexpression" refers to increased expression of a nucleic acid or protein in a microorganism of the present disclosure compared to a wild-type or parent microorganism from which the microorganism of the present disclosure was derived. Overexpression may be achieved by any means known in the art, including modifying gene copy number, gene transcription rate, gene translation rate, or enzyme degradation rate. As described above, the catalytic Figure 1 One or more of the enzymes of reactions 2, 5, 6, 8, 9, 10, 19, 20, 24 or 25 may be overexpressed.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] "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.

[0123] "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.

[0124] 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.

[0125] In addition, nucleic acid 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. Ideally, the promoter is a Wood-Yongdahl 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.

[0126] 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.

[0127] These homologous genes include homologous genes in species such as Clostridium jundal, Chloroflexus aurantiacus, Metallosphaera or Sulfolobus, detailed information of which is publicly available on websites such as Genbank or NCBI. The phrase "functionally equivalent variants" should also include nucleic acids whose sequences vary due to codon optimization of a particular organism. The "functionally equivalent variants" of the nucleic acids 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] The methylation construct / vector comprises a nucleic acid sequence encoding a methyltransferase.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] In a specific embodiment, both constructs / vectors are isolated in parallel.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] In a specific embodiment, the expression construct / vector and / or the methylation construct / vector is a plasmid.

[0143] 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.

[0144] Any number of constructs / vectors suitable for allowing expression of a methyltransferase gene may be used to generate a methylation construct / vector.

[0145] 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.

[0146] "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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] In certain embodiments, the fermentation is performed in the absence of a carbohydrate substrate such as sugars, starch, lignin, cellulose, or hemicellulose.

[0163] In addition to the tandem repeat proteins and chemical products, the microorganisms of the disclosure can be cultured to produce one or more co-products. 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), 2017 / 066498), chorismate-derived products (WO 2016 / 191625), 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), 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 to another product, product, 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.

[0164] 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.

[0165] "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%.

[0166] 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.

[0167] 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.

[0168] "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.

[0169] "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.

[0170] " 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.

[0171] "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.

[0172] "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.

[0173] 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).

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] Ethylene is a high-value gaseous compound that is widely used in industry.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] In one embodiment, ethylene oligomerization is used for desired product. 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 hydrocarbon by oligomerization. In other embodiments, straight chain olefin is the main product of ethylene oligomerization. In certain embodiments, alpha olefin is the main product of ethylene oligomerization. In one embodiment, olefin is subjected to upgrading process. In certain embodiments, the upgrading process of olefin is hydrogenation. In one embodiment, olefin is subjected to olefin conversion technology. In one embodiment, ethylene is mutually converted into propylene, 2-butene or any combination thereof. In one embodiment, propylene is converted into polypropylene.

[0191] As a raw material, ethylene can be used to make polymers such as polyethylene (PE), polyethylene terephthalate (PET) and polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), 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.

[0192] 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.

[0193] Other ethylene derivatives include alpha olefins, detergent alcohols and plasticizer alcohols used in linear low-density polyethylene (LLDPE) production; vinyl acetate monomer (VAM) used in adhesives, paints, paper coatings and barrier resins; and industrial ethanol used as a solvent or in the manufacture of chemical intermediates such as ethyl acetate and ethyl acrylate.

[0194] 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.

[0195] 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:

[0196] Ultra-high molecular weight polyethylene (UHMWPE);

[0197] Ultra-low molecular weight polyethylene (ULMWPE or PE-WAX);

[0198] High molecular weight polyethylene (HMWPE);

[0199] High-density polyethylene (HDPE);

[0200] High-density cross-linked polyethylene (HDXLPE);

[0201] Cross-linked polyethylene (PEX or XLPE);

[0202] Medium density polyethylene (MDPE);

[0203] Linear low-density polyethylene (LLDPE);

[0204] Low-density polyethylene (LDPE);

[0205] Very low density polyethylene (VLDPE); and

[0206] Chlorinated polyethylene (CPE).

[0207] 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.

[0208] 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:

[0209] C2H4+O2→C2H4O

[0210] 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.).

[0211] 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:

[0212] (CH2CH2)O+H2O→HOCH2CH2OH

[0213] 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. 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.

[0214] 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.

[0215] 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.

[0216] 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:

[0217] Step 1

[0218] C6H4(CO2CH3)2+2HOCH2CH2OH→C6H4(CO2CH2CH2OH)2+2CH3OH

[0219] Step 2

[0220] n C6H4(CO2CH2CH2OH)2→[(CO)C6H4(CO2CH2CH2O)] n +n HOCH2CH2OH

[0221] Alternatively, monoethylene glycol can be the subject of an esterification reaction with terephthalic acid according to the following reaction:

[0222] n C6H4(CO2H)2+n HOCH2CH2OH→[(CO)C6H4(CO2CH2CH2O)] n +2n H2O

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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).

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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:

[0233] CH3CH2CH2OH→CH3-CH=CH2

[0234] 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.

[0235] 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.

[0236] In one embodiment, the tandem repeat protein is a squid ring tooth (SRT) protein. In one embodiment, the tandem repeat protein is an insect silk protein. In some embodiments, the tandem repeat protein is used to manufacture personal care products, textiles, plastics, biomedical products, or any combination thereof. In another embodiment, the tandem repeat protein comprises at least one polypeptide of the present disclosure, silk fiber and / or copolymer of the present disclosure, one or more acceptable carriers, or any combination thereof. In one embodiment, the product further comprises a drug. In another embodiment, the product is used as a drug for medical devices, cosmetics, or any combination thereof. In one embodiment, the tandem repeat protein comprises a silk fiber, a copolymer, a drug for the manufacture of a drug for the treatment or prevention of a disease. In some embodiments, the tandem repeat protein, fiber, copolymer, or any combination thereof can be used for a wide variety of medical, military, industrial, and commercial applications. In one embodiment, the tandem repeat protein can be used to manufacture a medical device, comprising sutures, skin grafts, cell growth matrices, replacement ligaments, surgical meshes, or any combination thereof. In other embodiments, the tandem repeat protein can be used in industrial and commercial products, including cables, ropes, nets, fishing lines, clothing fabrics, bulletproof vest linings, container fabrics, backpacks, shoulder bags, bags or wallet straps, adhesive bonding materials, non-adhesive bonding materials, strapping materials, tent fabrics, tarpaulins, pool covers, vehicle covers, fence materials, sealants, building materials, weatherproof materials, flexible partition materials, sports equipment or any combination thereof. In one embodiment, the tandem repeat protein can be used in any fiber or fabric that requires high tensile strength and elasticity. In one embodiment, the tandem repeat protein can be used in native form, modified form, derived form or any combination thereof. In certain embodiments, the tandem repeat protein can be spun and / or bundled or woven together with other fiber types. The present disclosure contemplates that the generation of such combinations of the present disclosure can be easily implemented to enhance any desired characteristics, including but not limited to appearance, softness, weight, durability, waterproof performance, improved manufacturing costs, which can usually be sought in the manufacture and production of fibers for medical, industrial or commercial applications. In some embodiments, the tandem repeat protein is a cosmetic and skin care composition, which comprises an anhydrous composition having an effective amount of tandem repeat protein in a cosmetically acceptable medium. In one embodiment, the composition includes but is not limited to skin care, cleansing, makeup, anti-wrinkle products or any combination thereof. In another embodiment, the composition comprises beauty soap, facial cleanser, shampoo, rinse, hair dye, hair cosmetics, ordinary cream, lotion, shaving cream, conditioner, cologne, shaving lotion, cosmetic oil, facial mask, foundation, eyebrow pencil, eye cream, eye shadow, mascara, perfume, tanning and sunscreen cosmetics, sunscreen lotion, nail cosmetics, eyeliner cosmetics, lip cosmetics, oral care products, toothpaste or any combination thereof. In another embodiment, the tandem repeat protein is used to promote wound healing in a bandage, bandage material, porous cloth or a coating on any combination thereof.In one implementation, the tandem repeat protein can be used in a film comprising a wound dressing material, an amorphous film, or any combination thereof. In one embodiment, the tandem repeat protein is used for a stent, a stent graft, or any combination thereof. In one embodiment, the tandem repeat protein can be used in the form of a line, a braid, a sheet, a powder, or any combination thereof. In one embodiment, the stent graft can contain a coating on some or all of the tandem repeat proteins, wherein the coating degrades after the stent graft is inserted into the host, and the coating delays the contact between the tandem repeat protein and the host. Suitable coatings include, but are not limited to, gelatin, degradable polyesters (e.g., PLGA, PLA, MePEG-PLGA, PLGA-PEG-PLGA, and copolymers and mixtures thereof), cellulose and cellulose derivatives (e.g., hydroxypropyl cellulose), polysaccharides (e.g., hyaluronic acid, dextran, dextran sulfate, chitosan), lipids, fatty acids, sugar esters, nucleic acid esters, polyanhydrides, polyorthoesters, and polyvinyl alcohol (PVA). In one embodiment, the stent graft containing tandem repeat proteins can contain a bioactive agent (drug), wherein the agent is released from the stent graft and then induces an enhanced cellular response (e.g., cell or extracellular matrix deposition) and / or fibrotic response in a host into which the stent graft has been inserted. In some embodiments, the tandem repeat proteins can also be used in a matrix for producing ligaments and tendons in vitro. In one embodiment, the tandem repeat proteins are used in hydrogels. In one embodiment, the tandem repeat proteins of the present disclosure can be applied to fiber surfaces for use in textiles. In one embodiment, the fiber material includes, but is not limited to, cotton textile fibers, such as rayon and Lycra. TMPolyester, nylon, wool and other natural fibers including natural silk. In some embodiments, the composition suitable for applying silk protein to the fiber may include a cosolvent, such as ethanol, isopropanol, hexafluoroalcohol, isothiocyanate and other polar solvents that can be mixed with water to form a solution or microemulsion. The solution containing the tandem repeat protein can be sprayed on the fiber, or the fiber can be immersed in the solution. In some embodiments, the rapid drying of the coated material is utilized. In another embodiment, the tandem repeat protein composition is applied to the woven fiber. In one embodiment, the tandem repeat protein is used to coat a stretchable fabric, which includes stretchable clothing, stockings or any combination thereof. In one embodiment, the tandem repeat protein can be added to polyurethane, other resins or thermoplastic fillers to prepare panels and other building materials, or as molded furniture and workbenches that replace wood and particle boards. In one embodiment, composite materials can also be used in building and automotive construction, especially roofs and door panels. In other embodiments, the tandem repeat protein fiber reinforced resin makes the material stronger, including a lightweight structure, and its strength is equal to or better than other particle boards and composite materials. In some embodiments, tandem repeat protein fibers are isolated and added to synthetic composite forming resins for use in combination with plant-derived proteins, starches, and oils to produce bio-based composites. In one embodiment, the tandem repeat protein is a paper additive. In another embodiment, the tandem repeat protein is used for technical and smart textiles. In some embodiments, the technical and smart textiles do not change properties when wet and maintain strength and ductility. In one embodiment, the tandem repeat protein is used for functional clothing for sports and leisure clothing, work clothes, protective clothing, or any combination thereof. In some embodiments, the tandem repeat protein is used in clothing, equipment, materials to withstand long-term exposure, heavy wear, personal protection from external environmental influences, resistance to ballistic projectiles, fire resistance and chemical resistance, or any combination thereof.

[0237] In one embodiment, ethylene is used to produce butadiene. In some embodiments, butadiene is used in rubber tires.

[0238] In one embodiment, a method for continuously producing ethylene comprises: delivering a gaseous substrate to 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.

[0239] In other embodiments, ethylene is converted into components used to make tires. In one embodiment, ethylene is converted into components used in tire treads.

[0240] The method according to an embodiment, wherein the tire is a scrap tire.

[0241] The method according to an embodiment, wherein the gaseous substrate originates from a process involving tires.

[0242] The method according to an embodiment, wherein the gaseous substrate originates from a product recycling process or a sustainable chemical process.

[0243] According to an embodiment, the method further comprises converting the ethylene into a component for manufacturing new tires.

[0244] 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.

[0245] 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.

[0246] The method according to an embodiment, wherein the first gas fermentation process and the second gas fermentation process are run in parallel.

[0247] The method according to an embodiment, wherein the first gas fermentation process and the second gas fermentation process are both operated continuously.

[0248] 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.

[0249] The method according to an embodiment, wherein the rubber component and the resin component are copolymerized by a suitable polymerization catalyst.

[0250] The method according to an embodiment, wherein the rubber component is selected from butadiene, isoprene, conjugated diene or any combination thereof.

[0251] The method according to an embodiment, wherein the resin component is selected from ethylene, olefins or any combination thereof.

[0252] 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.

[0253] 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.

[0254] The method according to an embodiment, wherein the central metal is selected from lanthanides, scandium, yttrium or any combination thereof.

[0255] 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).

[0256] The method according to an embodiment further comprises converting the polymer into a tire.

[0257] 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.

[0258] The method according to an embodiment, wherein the substrate is derived from a process involving scrap tires.

[0259] One embodiment relates to a method for cyclically producing a tire, the method comprising: 1) transferring a gaseous substrate to a first bioreactor containing a culture of a recombinant C1 immobile microorganism capable of producing at least one first product selected from butadiene, isoprene, a conjugated diene, or any combination thereof in a culture medium, such 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) transferring the gaseous substrate to 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, such 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.

[0260] 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.

[0261] 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.

[0262] The method according to an embodiment, wherein the central metal is selected from lanthanides, scandium, yttrium or any combination thereof.

[0263] 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).

[0264] The method according to an embodiment, wherein the first bioreactor and the second bioreactor are operated in parallel.

[0265] The method according to an embodiment, wherein both the first bioreactor and the second bioreactor are operated continuously.

[0266] The method according to an embodiment, wherein the substrate is derived from a process involving scrap tires.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] One embodiment relates to a genetically engineered microorganism capable of producing a commodity chemical product, a tandem repeat protein product, microbial biomass, a single cell protein (SCP), one or more intermediates, or any combination thereof.

[0274] The microorganism according to the embodiments, wherein the microorganism produces a heterologous protein product, wherein the microorganism comprises a heterologous nucleic acid encoding at least one protein having a tandem repeat sequence.

[0275] The microorganism according to the embodiments, wherein the microorganism comprises a genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one protein having a tandem repeat sequence and a heterologous nucleic acid encoding at least one secreted chemical product, wherein the microorganism is a C1 fixed bacterium.

[0276] The microorganism according to an embodiment, wherein the microorganism comprises a genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one protein having one or more tandem repeat sequences and a heterologous nucleic acid encoding at least one secreted chemical product, wherein the microorganism is a C1 fixed bacterium.

[0277] A microorganism according to an embodiment, wherein the microorganism comprises a genetically engineered C1 immobilized microorganism capable of co-producing a heterologous protein and a chemical product from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeat sequences and a heterologous nucleic acid encoding at least one chemical product.

[0278] The microorganism according to the embodiment, wherein the microorganism comprises a genetically engineered C1 immobilized microorganism capable of co-producing a heterologous protein and a chemical product from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeat sequences and a heterologous nucleic acid encoding at least one chemical product, wherein the microorganism is capable of accumulating the at least one heterologous protein in the cell and secreting the at least one chemical product from the cell.

[0279] The microorganism according to the embodiments, wherein the microorganism comprises one or more heterologous enzymes, wherein the one or more heterologous enzymes are derived from a genus selected from the group consisting of: Bacillus, Clostridium, Cupriavidus, Escherichia, Gluconobacter, Mycetomicrobium, Lysinibacillus, Paenibacillus, Pseudomonas, Sedimentobacillus, Sporosarcina, Streptomyces, Thermothiobacillus, Thermotoga, and Zea mays.

[0280] A microorganism according to an embodiment, wherein the microorganism comprises a genetically engineered C1 fixed microorganism capable of co-producing at least one heterologous functional protein and at least one chemical product having at least two or more carbons from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one protein having a tandem repeat sequence and a heterologous nucleic acid encoding at least one secreted chemical product.

[0281] A microorganism according to an embodiment, wherein the microorganism comprises a genetically engineered C1 fixed microorganism capable of co-producing at least one heterologous functional protein and at least one chemical product having at least two or more carbons from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding a set of genes comprising at least one protein having a tandem repeat sequence and at least one secreted chemical product.

[0282] According to the microorganism described in the embodiment, a genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprises a heterologous nucleic acid encoding at least one protein having one or more tandem repeat sequences and a heterologous nucleic acid encoding the at least one chemical product, wherein the microorganism is a C1 fixed bacterium.

[0283] The microorganism according to the embodiment, wherein the microorganism comprises a genetically engineered C1 immobilized microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprising:

[0284] a) a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeat sequences; and

[0285] b) a heterologous nucleic acid encoding at least one chemical having two or more carbons, wherein the microorganism is capable of accumulating the at least one heterologous protein in a cell and secreting the at least one chemical product from the cell.

[0286] The method according to an embodiment, wherein the method co-produces at least one heterologous protein and at least one chemical product by culturing the genetically engineered C1 immobile microorganism in the presence of a gaseous substrate comprising one or more of CO, CO2 and H2, wherein the culturing is a continuous fermentation process.

[0287] According to the method described in the embodiment, at least one heterologous protein having one or more tandem repeat sequences and at least one chemical product are co-produced by the following method: culturing the genetically engineered microorganism according to claim 1 in the presence of a gaseous substrate comprising one or more of CO, CO2 and H2, wherein the culturing is a continuous fermentation process.

[0288] The method according to the embodiment, wherein the gaseous substrate comprises a C1 carbon source, and the C1 carbon source comprises one or more of CO, CO2 and H2.

[0289] The method according to an embodiment, wherein the gaseous substrate comprises synthesis gas or industrial waste gas.

[0290] The method according to the embodiment, wherein the method co-produces at least one heterologous protein having one or more tandem repeat sequences and at least one chemical product by culturing a genetically engineered C1 stationary form, wherein the chemical product is one or more of ethylene, ethanol, acetone, isopropanol, or any combination thereof.

[0291] The microorganism according to the embodiments, wherein the microorganism comprises a genetically engineered C1 fixed microorganism, wherein the at least one heterologous protein having one or more tandem repeat sequences is selected from collagen, silk, elastin, keratin, arthropod elastic protein, titin, squid ring tooth (SRT) protein, sucker protein or any combination thereof.

[0292] The microorganism according to the embodiment, wherein the microorganism is a member of a genus selected from the group consisting of Acetobacter, Alkaliphilus, Blautia, Butyribacter, Clostridium, Cuprobacter, Eubacterium, Moorella, Acetobacter, Ralstonia, Murine Spore and Thermoanaerobacter.

[0293] In some aspects of the microorganisms disclosed herein, the microorganism is derived from a parent microorganism selected from the group consisting of: Acetobacter wurderi, Alkaliphilus pasteurianus, Blautia producus, Butyrobacter methylotrophicus, Clostridium aceticum, Clostridium ethanologenicum, Clostridium carboxyvorans, Clostridium kluyveri, Clostridium delbrueckii, Clostridium formicaceticum, Clostridium yendarius, Clostridium magna, Clostridium rabbis, Clostridium faecalis, Cupribotium nematophilum, Eubacterium mucosum, Moorella thermoautotrophicum, Moorella thermoacetica, Acetobacter prausnitzii, Ralstonia eutropha, Ovomorpha, Acetobacter sphaeroides, Mouse spores, and Thermoanaerobic Bacteria kevlarii.

[0294] The microorganism according to the embodiment, wherein the microorganism is derived from a parent bacterium selected from the group consisting of Clostridium ethanologenum, Clostridium ljungdahlii, or Clostridium ragsbyii.

[0295] The microorganism according to the embodiment, wherein the microorganism is derived from a parent bacterium selected from the group consisting of: Cupriavidus necrotus.

[0296] The microorganism according to the embodiments, wherein the at least one heterologous protein having one or more tandem repeat sequences is selected from silk or SRT protein.

[0297] According to the microorganism described in the embodiment, the gaseous fermentation product is selected from alcohols, acids, diacids, olefins, terpenes, isoprene and alkynes or any combination thereof.

[0298] According to the microorganism described in the embodiment, at least one secreted chemical product is selected from the following group: 1-butanol, butyrate, butene, butadiene, methyl ethyl ketone, 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 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.

[0299] According to the microorganism of the embodiment, at least one secreted chemical product is selected from 1-butanol, butyrate, butene, butadiene, methyl ethyl ketone, 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 or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-butene-1-ol, isovalerate, isopentanol, monoethylene glycol, or any combination thereof.

[0300] The microorganism according to the embodiments, wherein the microorganism further comprises a disruptive mutation in one or more genes.

[0301] The microorganism according to the embodiments, wherein the genetically engineered C1 immobilized microorganism further comprises microbial biomass and at least one excipient.

[0302] The microorganism according to the embodiment, wherein the genetically engineered C1 immobilized microorganism, wherein the microbial biomass is suitable as animal feed.

[0303] According to the microorganisms described in the embodiments, the C1 fixed-type microorganisms are genetically engineered, and 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, white-tailed gaur, big-headed cattle, yaks, chickens, turkeys, ducks, geese, quails, guinea fowl, chicks / pigeons, fish, shrimps, crustaceans, cats, dogs and rodents.

[0304] The microorganism according to the embodiment, wherein the genetically engineered C1 immobilized microorganism, wherein the microorganism is suitable as a single cell protein (SCP).

[0305] In some aspects, the microbial biomass has a unit value. In one embodiment, the microbial biomass has a market value.

[0306] According to the microorganism described in the embodiment, the genetically engineered C1 immobilized microorganism, wherein the microorganism is suitable as a cell-free protein synthesis (CFPS) platform.

[0307] According to the microorganism of the embodiment, wherein the genetically engineered C1 immobilized microorganism, at least one secreted chemical product is native to the microorganism.

[0308] The microorganism according to an embodiment, the genetically engineered microorganism according to claim 1, wherein the at least one heterologous protein is a sepia ring tooth protein and the at least one chemical product is ethylene.

[0309] The microorganism according to the embodiments, wherein the at least one heterologous protein is silk protein and the at least one chemical product is ethylene.

[0310] The microorganism according to the embodiments, wherein the at least one chemical product is ethylene.

[0311] The method according to the embodiment, wherein the substrate comprises one or more of CO, CO2 and H2.

[0312] According to the microorganism of the embodiment, at least a portion of the substrate is industrial waste gas, industrial tail gas or synthesis gas.

[0313] According to an embodiment, a method is described wherein both anaerobic and aerobic gases may 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.

[0314] Examples

[0315] The following examples further illustrate the present disclosure, but should of course not be construed as limiting its scope in any way.

[0316] Example 1: Production of tandem repeat proteins in Clostridium autoethanologenum.

[0317] Genes encoding tandem repeat proteins (Table 2) were synthesized and assembled into the Clostridium-E. coli shuttle vector pMTL8225 (Heap, J Microbiol Methods 78:79-85, 2009). The gene contains DNA encoding an N-terminal double strep tag as a procedure for protein detection by protein blotting and / or affinity purification (Schmidt, Protein Expr Purif 92:54-61, 2013). These vectors have pre-cloned Clostridium promoters and terminators. The promoter sequence is described in Karim et al. Synthetic Biology 2020; 5(1):ysaa019. The resulting plasmid carries the ermB antibiotic selectable marker. After transformation into Clostridium, the sequence-verified strains were grown autotrophically in 6-well plates.

[0318] Protein expression experiments were started in 6-well plates with 3 mL of minimal medium containing yeast extract? and 200 kPa of synthetic gas mixture (55% CO, 5% H2, 30% CO2 and 10% N2) and cultured at 37°C until the strain reached a biomass concentration of 0.20-0.43 gDCW / L. The strain was then subcultured to 0.006-0.03 gDCW / L in a 1 L Schott bottle containing 200 mL of minimal medium in the presence of a 150 kPa synthetic gas mixture (55% CO, 5% H2, 30% CO2 and 10% N2) at 37°C. The biomass concentration was monitored until it reached 0.13-0.32 gDCW / L, and then the biomass was harvested for protein detection.

[0319] The production of tandem repeat proteins was assessed by Western blot analysis using anti-Strep tag antibodies. The culture was dissolved and clarified; the protein content of the clarified lysate and insoluble precipitate (resuspended in 5M urea) was analyzed separately. The samples were run on Tris-glycine SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-Strep tag antibodies conjugated to alkaline phosphatase for observation. Proteins of expected size were observed in the insoluble precipitates of SRT008, SRT011, SRT012 and SS015. In addition, SS015 was observed in the clarified lysate.

[0320] Table 2: Tandemly repeated proteins expressed in ethanologenic Clostridium.

[0321]

[0322]

[0323]

[0324] Example 2: Production of SRT008 ​​and SRT012 from syngas fermentation in a batch CSTR.

[0325] The strains SRT008 ​​and SRT012 (Table 2) containing tandem repeat proteins were characterized in batch mode in a CSTR to characterize protein production and chemical production. An actively growing (early exponential) culture from a Schott bottle was used as an inoculum for a 2L CSTR at atmospheric pressure with a syngas blend (55% CO, 5% H2, 30% CO2, and 10% N2). A gas interruption occurred during the run, which resulted in a disruption of the culture and ended SRT008 ​​earlier than expected.

[0326] SRT008 ​​reached a peak biomass concentration of 2.5 gDCW / L ( Figure 3B ), and reached a peak CO uptake of 1331 mmol / L / day ( Figure 3C ). In addition to the peak ethanol concentration of 26.88 g / L ( Figure 3A , 3B ), this strain reached a peak acetic acid titer of 4.14 mg / L and a peak butanediol titer of 4.73 mg / L ( Figure 3B ). SRT008 ​​production was observed by Western blotting at days 0, 2.78, and 5.81, with the highest relative protein content at day 0.

[0327] SRT012 reached a peak biomass concentration of 1.31 gDCW / L before experiencing mechanical problems on day 2.73 ( Figure 4B ), and reached a peak CO uptake of 1537 mmol / L / day. In addition to the peak ethanol concentration of 9.57 g / L ( Figure 4A , 4B ), this strain reached a peak acetic acid titer of 8.27 mg / L and a peak butanediol titer of 0.57 mg / L ( Figure 4B ). SRT012 production was observed by Western blotting at days 0, 1.78, and 5.02, with the highest relative protein content at day 1.78 ( Figure 4C ). The last data point for protein content per biomass was obtained after the gas was turned off.

[0328] Example 3: Production of SRT012 from syngas fermentation in a continuous CSTR.

[0329] Under continuous CSTR conditions with strain SRT012, using a syngas mixture (55% CO, 5% H2, 30% CO2, and 10% N2), a 3-L reactor, and a cell recirculating membrane (CRM), a dilution (D) rate of 1.2 vessels / day (v / d) was started on day 5, then increased to 1.5 v / d of D on day 12.9, and finally reached 2.5 v / d of D between day 12.9 and day 60. The biomass concentration accumulated to a peak concentration of 20.85 gDCW / L ( Figure 2B ), and CO gas uptake reached a peak of 9300 mmol / L / day ( Figure 2C ). The maximum ethanol concentration of this strain was 34.82 g / L, and the maximum acetic acid concentration was 11.79 g / L ( Figure 2B ).

[0330] SRT008 ​​protein was observed in all samples analyzed ( Figure 2A ). The production of SRT008 ​​was evaluated by Western blot analysis using an anti-Strep tag antibody. Samples were taken from the CSTR and frozen for later analysis. After the samples were thawed, the cells were lysed and the insoluble precipitate was resuspended in 5M urea. The samples were diluted with Laemmli sample buffer and run on a tris-glycine SDS-PAGE protein gel. The samples were transferred to a nitrocellulose membrane, stained with Ponceau S to show total protein, and then probed with an anti-Strep tag antibody conjugated to horseradish peroxidase to show specific proteins. The specific protein content was measured and normalized to the total protein content using densitometry analysis. The specific protein content normalized to the total protein content is reported relative to day 5 (reactor continuous operation); the highest relative protein content was on day 0, and another peak occurred between day 45 and day 55, about the same time that ethanol production decreased and the cell recycling membrane was replaced.

[0331] Example 4: Production of SRT008 ​​and SRT012 from high hydrogen syngas fermentation in a batch CSTR.

[0332] The strains SRT008 ​​and SRT012 (Table 2) containing tandem repeat proteins were characterized in batch mode in a CSTR to characterize protein production and chemical production. An actively growing (early exponential) culture from a Schott bottle was used as an inoculum for a 2L CSTR at atmospheric pressure with a syngas blend (55% CO, 5% H2, 30% CO2, and 10% N2). The culture was grown in a reactor using a high hydrogen gas blend (10% CO, 50% H2, 30% CO2, and 10% N2).

[0333] SRT008 ​​achieved a peak biomass concentration of 1.32 gDCW / L ( Figure 5B ), and reached a peak CO uptake of 590 mmol / L / day and a peak H2 uptake of 2060 mmol / L / day ( Figure 5C ). In addition to the peak ethanol concentration of 45.06 g / L ( Figure 5B ), this strain reached a peak acetic acid titer of 5.47 g / L ( Figure 5B ). As shown in all figures, just before day 4, the agitator was turned off for approximately 45 minutes and malfunctioned; the cultures were affected but recovered quickly. SRT008 ​​production was observed by Western blotting at days 0, 1.7, 2.7, 3.7, and 6.8, with peak protein content on day 0 ( Figure 5A ).

[0334] SRT012 achieved a peak biomass concentration of 1.75 gDCW / L ( Figure 6B ), and reached a peak CO uptake of 535 mmol / L / day and a maximum H2 uptake of 2074 mmol / L / day ( Figure 6C ). In addition to the peak ethanol concentration of 52.46 g / L ( Figure 6B ), this strain reached a peak acetic acid titer of 5.47 g / L ( Figure 6B SRT012 production was observed by Western blot at days 0, 1.7, 3.7, 5.8, and 10.7, with peak protein content measured at day 10.7 ( Fig. 6A ).

[0335] Example 5: Continuous production of ethylene from CO2 using H2 as energy source.

[0336] 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.

[0337] 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.

[0338] 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.

[0339] 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).

[0340] 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 7 ). 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.

[0341] 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.

[0342] 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.

[0343] 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).

[0344] 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.

[0345] 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.

[0346] Embodiments of the present disclosure

[0347] Embodiment 1. A genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one protein having one or more tandem repeat sequences and a heterologous nucleic acid encoding the at least one chemical product, wherein the microorganism is a C1 fixed bacterium.

[0348] Embodiment 2. A genetically engineered microorganism according to embodiment 1, wherein the at least one heterologous protein having one or more tandem repeat sequences is selected from collagen, silk, elastin, keratin, arthropod elastic protein, titin, squid ring tooth (SRT) protein, sucker protein or any combination thereof.

[0349] Embodiment 3. The genetically engineered microorganism of embodiment 2, wherein the at least one heterologous protein is an SRT protein and the at least one chemical product is ethylene.

[0350] Embodiment 4. The genetically engineered microorganism of embodiment 2, wherein the at least one heterologous protein is silk protein and the at least one chemical product is ethylene.

[0351] Embodiment 5. The genetically engineered microorganism of embodiment 1, wherein the at least one chemical product is ethylene.

[0352] Embodiment 6. The genetically engineered microorganism according to embodiment 2, wherein the at least one heterologous protein having one or more tandem repeat sequences is selected from silk or SRT protein.

[0353] Embodiment 7. The genetically engineered microorganism of embodiment 1, wherein at least one secreted chemical product is selected from 1-butanol, butyrate, butene, butadiene, methyl ethyl ketone, 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 or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-butene-1-ol, isovalerate, isopentanol, monoethylene glycol, or any combination thereof.

[0354] Embodiment 8. The genetically engineered microorganism of embodiment 1, further comprising a disruptive mutation in one or more genes.

[0355] Embodiment 9. The genetically engineered microorganism according to embodiment 1, wherein the parent microorganism is selected from the group consisting of: Acetobacter wurderi, Alkaliphilus pasteurianus, Blautia producis, Butyrobacter methylotrophicus, Clostridium aceticum, Clostridium ethanologenicum, Clostridium carbon monoxide-eating, Clostridium kluyveri, Clostridium delbrueckii, Clostridium formicaceticum, Clostridium yendarius, Clostridium magna, Clostridium rabbis, Clostridium faecalis, Eubacterium mucosum, Moorella thermoautotrophicum, Moorella thermoacetica, Acetobacter prausnitzii, Murine spores of ovale, Murine spores of forest soil, Murine spores of spherical shape, Cupricobacter nebulosum and Thermoanaerobic Bacteria of Kaiwu.

[0356] Embodiment 10. A method for co-producing at least one heterologous protein having one or more tandem repeat sequences and at least one chemical product by culturing the genetically engineered microorganism according to embodiment 1 in the presence of a gaseous substrate comprising one or more of CO, CO2 and H2, wherein the culturing is a continuous fermentation process.

[0357] Embodiment 11. The method according to embodiment 10, wherein the gaseous substrate comprises a C1 carbon source, and the C1 carbon source comprises one or more of CO, CO2 and H2.

[0358] Embodiment 12. The method of embodiment 10, wherein the gaseous substrate comprises synthesis gas or industrial waste gas.

[0359] Embodiment 13. The method of embodiment 10, wherein the chemical product is one or more of: ethylene, ethanol, acetone, isopropanol, or any combination thereof.

[0360] Embodiment 14. The genetically engineered microorganism of embodiment 1, further comprising microbial biomass and at least one excipient.

[0361] Embodiment 15. The genetically engineered microorganism of embodiment 14, wherein the microbial biomass is suitable as animal feed.

[0362] Embodiment 16. The genetically engineered microorganism of embodiment 15, wherein the animal feed is suitable for feeding one or more of: 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.

[0363] Embodiment 17. The genetically engineered microorganism according to embodiment 1, wherein the microorganism is suitable as a single cell protein (SCP).

[0364] Embodiment 18. The genetically engineered microorganism according to embodiment 1, wherein the microorganism is suitable as a cell-free protein synthesis (CFPS) platform.

[0365] Embodiment 19. The genetically engineered microorganism of embodiment 1, wherein the at least one chemical product is native to the microorganism.

[0366] Embodiment 20. A genetically engineered C1 immobilized microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprising:

[0367] a) a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeat sequences; and

[0368] b) a heterologous nucleic acid encoding at least one chemical having two or more carbons, wherein the microorganism is capable of accumulating the at least one heterologous protein in a cell and secreting the at least one chemical product from the cell.

Claims

1. A genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprising a heterologous nucleic acid encoding at least one protein having one or more tandem repeat sequences and a heterologous nucleic acid encoding the at least one chemical product, wherein the microorganism is a C1 fixed bacterium.

2. The genetically engineered microorganism of claim 1, wherein the at least one heterologous protein having one or more tandem repeat sequences is selected from collagen, silk, elastin, keratin, elastic protein, titin, squid ring tooth (SRT) protein, suckerin, or any combination thereof.

3. The genetically engineered microorganism of claim 2, wherein the at least one heterologous protein is an SRT protein and the at least one chemical product is ethylene.

4. The genetically engineered microorganism of claim 2, wherein the at least one heterologous protein is silk protein and the at least one chemical product is ethylene.

5. The genetically engineered microorganism of claim 1, wherein the at least one chemical product is ethylene.

6. The genetically engineered microorganism of claim 2, wherein the at least one heterologous protein having one or more tandem repeat sequences is selected from silk or SRT proteins.

7. The genetically engineered microorganism of claim 1, wherein at least one secreted chemical product is selected from 1-butanol, butyrate, butene, butadiene, methyl ethyl ketone, 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 or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-butene-1-ol, isovalerate, isopentanol, monoethylene glycol, or any combination thereof.

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

9. The genetically engineered microorganism of claim 1, wherein the parent microorganism is selected from the group consisting of: Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium magnum, Clostridium ragsdalei, Clostridium scatologenes, Eubacterium limosum, Moorella thermautotrophica, Moorellathermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Cupriavidus necator, and Thermoanaerobacter kivui.

10. A method for co-producing at least one heterologous protein having one or more tandem repeat sequences and at least one chemical product by culturing the genetically engineered microorganism according to claim 1 in the presence of a gaseous substrate comprising one or more of CO, CO2 and H2, wherein the culturing is a continuous fermentation process.

11. The method of claim 10, wherein the gaseous substrate comprises a C1 carbon source comprising one or more of CO, CO2 and H2.

12. The method of claim 10, wherein the gaseous substrate comprises synthesis gas or industrial waste gas.

13. The method of claim 10, wherein the chemical product is one or more of: ethylene, ethanol, acetone, isopropanol, or any combination thereof.

14. The genetically engineered microorganism of claim 1, further comprising microbial biomass and at least one excipient.

15. The genetically engineered microorganism of claim 14, wherein the microbial biomass is suitable as animal feed.

16. The genetically engineered microorganism of claim 15, wherein the animal feed is suitable for feeding one or more of: beef cattle, dairy cows, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, Gaur, Gaur, yaks, chickens, turkeys, ducks, geese, quail, guinea fowl, chicks / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents.

17. The genetically engineered microorganism of claim 1, wherein the microorganism is suitable as a single cell protein (SCP).

18. The genetically engineered microorganism of claim 1, wherein the microorganism is suitable as a cell-free protein synthesis (CFPS) platform.

19. The genetically engineered microorganism of claim 1, wherein the at least one chemical product is native to the microorganism.

20. A genetically engineered C1 immobilized microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, the microorganism comprising: a) a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeat sequences; and b) a heterologous nucleic acid encoding at least one chemical having two or more carbons, wherein the microorganism is capable of accumulating the at least one heterologous protein in a cell and secreting the at least one chemical product from the cell.

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