Microorganisms and methods for continuous co-production of tandem repeat proteins and chemical products from C1-substrates

Abstract

Microorganisms are genetically engineered to continuously co-produce amino acids, proteins, microbial biomass, chemicals, or any combination thereof through microbial fermentation, particularly microbial fermentation of gaseous substrates. The microorganisms are C1-fixing. Improved production of ethylene, microbial biomass, and heterologous tandem repeat proteins can be achieved. This can be achieved by promoter modification or nutrient limitation.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 366,756, filed June 21, 2022, which is incorporated by reference herein in its entirety. INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING This application contains an ST.26 Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The ST.26 Sequence Listing XML, created on May 31, 2023, is named LT228WO1-Sequences.BMS and is 5,763 bytes in size.

[0002] 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 microbial fermentation of gaseous substrates. [Background technology]

[0003] It has long been recognized that catalytic processes such as the Fischer-Tropsch process can be used to convert gases containing carbon dioxide (CO), carbon monoxide (CO), and / or hydrogen (H), such as industrial waste gases or syngas, into a variety of fuels and chemicals. However, gas fermentation has recently emerged as an alternative platform for the biological fixation of such gases. Specifically, C1-fixing microorganisms have been shown to convert gases containing CO, CO, and / or H into products such as ethanol and 2,3-butanediol. However, efficient coproduction of such chemical products with heterologous proteins can be limited by factors such as slow microbial growth, limited gas uptake, susceptibility to toxins, and the diversion of carbon substrates into undesirable byproducts. Furthermore, there is growing interest in creating sustainable materials from synthetic and biological processes that exhibit multifunctional properties. Therefore, there is an ongoing and unmet need to develop novel protein-based materials that can be easily produced from renewable resources and would offer a wide range of useful applications. There also remains a need for genetically engineered microorganisms with improved characteristics for the continuous co-production of chemicals, proteins, biomass, or any combination thereof, by microbial fermentation of gaseous substrates. Summary of the Invention

[0004] It is against this background that the present disclosure offers certain advantages and advancements over the prior art.

[0005] Although the disclosure disclosed herein is not limited to a particular benefit or functionality, the disclosure provides a genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one secreted chemical product from a gaseous substrate, wherein the microorganism comprises a heterologous nucleic acid encoding at least one protein having tandem repeats, and a heterologous nucleic acid encoding at least one secreted chemical product.

[0006] In some embodiments of the microorganisms disclosed herein, the microorganisms produce a commodity chemical, a tandem repeat protein product, a microbial biomass, a single cell protein (SCP), one or more intermediates, or any combination thereof.

[0007] 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 tandem repeats.

[0008] In some embodiments of the microorganisms disclosed herein, the microorganisms include genetically engineered microorganisms capable of co-producing at least one heterologous protein and at least one secreted chemical product from a gaseous substrate, the microorganisms include a heterologous nucleic acid encoding at least one protein having tandem repeats and a heterologous nucleic acid encoding at least one secreted chemical product, and the microorganisms are C1-fixing bacteria.

[0009] In some embodiments of the microorganisms disclosed herein, the microorganisms include genetically engineered C1-fixing microorganisms capable of co-producing a heterologous protein and a chemical product from a gaseous substrate, wherein the microorganisms comprise a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeats and a heterologous nucleic acid encoding at least one chemical product, and the microorganisms are capable of accumulating the at least one heterologous protein intracellularly and secreting the at least one chemical product from the cell.

[0010] In some embodiments of the microorganism, the microorganism comprises one or more heterologous enzymes from a genus selected from the group consisting of Bacillus, Clostridium, Cupriavidus, Escherichia, Gluconobacter, Hyphomicrobium, Lysinibacillus, Paenibacillus, Pseudomonas, Sedimenticola, Sporosarcina, Strreptomyces, Thermithiobacillus, Thermotoga, Dosidicus, and Zea.

[0011] In some embodiments of the microorganisms disclosed herein, the microorganisms include genetically engineered C1-fixing microorganisms capable of co-producing at least one heterologous functional protein and at least one chemical product having two or more carbons from a gaseous substrate, wherein the microorganisms include a heterologous nucleic acid encoding at least one protein having tandem repeats and a heterologous nucleic acid encoding at least one secreted chemical product.

[0012] In some embodiments of the microorganisms disclosed herein, the microorganisms include genetically engineered C1-fixing microorganisms capable of co-producing at least one heterologous functional protein and at least one chemical product having two or more carbons from a gaseous substrate, wherein the microorganisms comprise heterologous nucleic acids encoding a group of genes comprising at least one protein having tandem repeats and at least one secreted chemical product.

[0013] In some embodiments of the microorganisms disclosed herein, the microorganisms comprise genetically engineered C1-fixing microorganisms capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, wherein the microorganisms comprise: a) a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeats; b) a heterologous nucleic acid encoding at least one chemical product having two or more carbon atoms, which heterologous nucleic acid enables the microorganism to accumulate the at least one heterologous protein within the cell and secrete the at least one chemical product from the cell.

[0014] One embodiment includes a method for co-producing at least one heterologous protein and at least one chemical product by culturing a genetically engineered C1-fixing microorganism in the presence of a gaseous substrate comprising one or more of CO, CO, and H, wherein the culturing is a continuous fermentation process.

[0015] One embodiment includes a method, wherein the gaseous substrate includes a C1 carbon source comprising one or more of CO, CO2, and H2.

[0016] One embodiment includes a method, wherein the gaseous substrate comprises synthesis gas or industrial waste gas.

[0017] One embodiment includes a method for co-producing at least one heterologous protein and at least one chemical product by genetically engineered C1 immobilized culture, wherein the product is one or more of acetone and isopropanol.

[0018] In some embodiments of the microorganisms disclosed herein, the microorganism comprises a genetically engineered C1-fixing microorganism, and the at least one heterologous protein having one or more tandem repeats is selected from collagen, silk, elastin, keratin, resilin, titin, squid ring tooth (SRT) protein, or any combination thereof.

[0019] 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.

[0020] In some embodiments of the microorganisms disclosed herein, the microorganism is 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, Cupriavidus necator, Eubacterium limosum, Moorella thermautotrophica, Moorella thermoacetica, Oxobacter pfennigii, Ralstonia eutropha, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, and Thermoanaerobacter kiuvi.

[0021] In some aspects of the microorganisms disclosed herein, the microorganisms are derived from a parent bacterium selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei.

[0022] In some aspects of the microorganisms disclosed herein, the microorganism is derived from a parent bacterium selected from the group consisting of Cupriavidus necator.

[0023] A genetically engineered C1-fixing microorganism, wherein the at least one heterologous protein having one or more tandem repeats is selected from a silk or SRT protein.

[0024] In some aspects of the microorganisms disclosed herein, the gas fermentation products are selected from alcohols, acids, diacids, alkenes, terpenes, isoprene, and alkynes, or any combination thereof.

[0025] In some embodiments of the microorganisms disclosed herein, the at least one chemical product is 1-butanol, butyrate, butene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, terpenes, isoprene, fatty acids, 2-butanol, 1,2-propanediol, 1-propanol, 1-hexanol, 1-octanol, chorismic acid-derived products, 3-hydroxybutyric acid, 1,3-butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, isoamyl alcohol, or monoethylene glycol.

[0026] In some embodiments of the microorganisms disclosed herein, the microorganism further comprises a disruptive mutation in one or more genes.

[0027] The present disclosure further provides a genetically engineered C1-fixing microorganism comprising a microbial biomass and at least one excipient.

[0028] The present disclosure further provides genetically engineered C1-fixing microorganisms, wherein the microbial biomass is suitable as animal feed.

[0029] The present disclosure further provides genetically engineered C1-fixing microorganisms, wherein the animal feed may be suitable for feeding one or more of beef cattle, dairy cattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, banteng, gayal, yaks, chickens, turkeys, ducks, geese, quails, guinea fowl, pigeons / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents.

[0030] The present disclosure further provides a genetically engineered C1-fixing microorganism, wherein the microorganism is suitable as a single cell protein (SCP).

[0031] The present disclosure further provides genetically engineered C1-fixing microorganisms, which are suitable as cell-free protein synthesis (CFPS) platforms.

[0032] The present disclosure further provides a genetically engineered C1-fixing microorganism, wherein at least one secreted chemical product is native to the microorganism.

[0033] In some embodiments of the methods disclosed herein, the substrate comprises one or more of CO, CO2, and H2.

[0034] In some aspects of the methods disclosed herein, at least a portion of the substrate is industrial waste gas, industrial off-gas, or syngas.

[0035] In some embodiments, both anaerobic and aerobic gases can be used to feed separate cultures (e.g., an anaerobic culture and an aerobic culture) in two or more different bioreactors that are both incorporated into the same process stream.

[0036] In some embodiments, the present disclosure provides a method for storing energy in the form of a biopolymer, the method comprising: intermittently processing at least a portion of electrical energy generated from a renewable and / or non-renewable energy source in an electrolysis process to produce at least H, O, or CO; intermittently passing at least one of the H, O, or CO from the electrolysis process to a bioreactor containing a culture comprising a liquid nutrient medium and a microorganism capable of producing the biopolymer; and fermenting the culture.

[0037] In embodiments, 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 H, O, or CO; an industrial plant for producing at least a C feedstock; and a bioreactor in intermittent fluid communication with the electrolysis process and / or in continuous fluid communication with the industrial plant, the bioreactor comprising a reaction vessel suitable for intermittently growing, fermenting, and / or culturing and housing a microorganism capable of producing the biopolymers.

[0038] In some embodiments, the disclosure provides a method for improving the performance and / or economics of a fermentation process, the fermentation process defining a bioreactor containing a bacterial culture in a liquid nutrient medium, the method including: passing a C1 feedstock comprising one or both of CO and CO2 from an industrial process to the bioreactor (the C1 feedstock having a per-unit cost); intermittently passing at least one of H2, O2, or CO2 from an electrolysis process to the bioreactor (the electrolysis process having a per-unit cost); and fermenting the culture to produce one or more fermentation products (each of the one or more fermentation products having a per-unit value). In certain instances, multiple electrolysis processes are utilized to provide one or all of CO2, CO2, and H2 to the bioreactor.

[0039] In another embodiment, the local grid supplies electricity delivered intermittently as electric energy generated by the grid based on the availability of electricity or the availability of electricity below a threshold price, where the price of electricity decreases as demand decreases or is set by the local grid.

[0040] In embodiments, the present disclosure can be operated intermittently by storing energy in the form of biopolymers, and product conversion can be intermittent during periods when the power grid is oversupplied with electricity, or can be on standby when there is a power shortage or demand. By storing energy in the form of biopolymers, the present disclosure provides a process that can be fine-tuned to assist in balancing the power grid system.

[0041] In one embodiment, the autotrophic microorganisms intermittently consume energy provided, in part or in whole, by the availability of electrical power.

[0042] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description taken in conjunction with the appended claims. It should be noted that the scope of the claims is defined by the description therein, and not by the specific discussion of the features and advantages set forth herein.

[0043] These and other aspects of the present disclosure, which is to be considered in all its novel aspects, will become apparent from the following description, given by way of example only, with reference to the accompanying drawings. [Brief explanation of the drawings]

[0044] [Figure 1]Figure 1 shows the expression of tandem repeat proteins in C. autoethanogenum via Western blot (Table 2). Tandem repeat protein production was assessed by Western blot analysis using an anti-Strep tag antibody. Cultures were lysed and clarified, and the clarified lysate and insoluble pellet (resuspended in 5 M urea) were analyzed separately for protein content. Samples were run on Tris-glycine SDS-PAGE, transferred to nitrocellulose membranes, and visualized by probing with an anti-Strep tag antibody conjugated to alkaline phosphatase. Proteins of the expected size were observed in the insoluble pellet for SRT008, SRT011, SRT012, and SS015. Additionally, SS015 was observed in the clarified lysate. [Figure 2A] Figures 2A-2C show the performance of strSRT012012 in continuous CSTR fermentation with cell recycle (CR) using a syngas blend (55% CO, 5% H, 30% CO, and 10% N). Figure 2A: Ethanol production per biomass and SRT012 protein content on day 5 (reactor became continuous) analyzed by HPLC and comparative Western blot, respectively; Figure 2B: Biomass and metabolite profile analyzed by HPLC; Figure 2C: Gas production profile analyzed by GC-TCD (negative = uptake). [Figure 2B] (As mentioned above.) [Figure 2C] (As mentioned above.) [Figure 3A] Figures 3A-3C show the performance of SRT008 in batch CSTR fermentation using a syngas blend (55% CO, 5% H, 30% CO, and 10% N). Figure 3A: Ethanol production per biomass and SRT008 protein content on day 0 analyzed by HPLC and comparative Western blot, respectively; Figure 3B: Biomass and metabolite profile analyzed by HPLC; Figure 3C: Gas production profile analyzed by GC-TCD (negative = uptake). [Figure 3B] (As mentioned above.) [Figure 3C](As mentioned above.) [Figure 4A] Figures 4A-4C show the performance of SRT012 in batch CSTR fermentations using a syngas blend (55% CO, 5% H, 30% CO, 10% N). Figure 4A: Ethanol and SRT012 protein content per biomass on day 0 analyzed by HPLC and comparative Western blot, respectively. The last data point for protein content per biomass was taken after gas shutoff. Figure 4B: Biomass and metabolite profile analyzed by HPLC; Figure 4C: Gas production profile analyzed by GC-TCD (negative = uptake). [Figure 4B] (As mentioned above.) [Figure 4C] (As mentioned above.) [Figure 5A] Figures 5A-5C show the performance of SRT008 in batch CSTR fermentations using a high-hydrogen syngas blend (10% CO, 50% H, 30% CO, and 10% N). Figure 5A: Ethanol per biomass and SRT008 protein content on day 0 analyzed by HPLC and comparative Western blot, respectively; Figure 5B: Biomass and metabolite profile analyzed by HPLC; Figure 5C: Gas production profile analyzed by GC-TCD (negative = uptake). [Figure 5B] (As mentioned above.) [Figure 5C] (As mentioned above.) [Figure 6A] Figures 6A-6C show the performance of SRT012 in batch CSTR fermentations using a high-hydrogen syngas blend (10% CO, 50% H, 30% CO, 10% N). Figure 6A: Ethanol per biomass and SRT012 protein content on day 0 analyzed by HPLC and comparative Western blot, respectively; Figure 6B: Biomass and metabolite profile analyzed by HPLC; Figure 6C: Gas production profile analyzed by GC-TCD (negative = uptake). [Figure 6B] (As mentioned above.) [Figure 6C] (As mentioned above.) [Figure 7] FIG. 7 shows continuous ethylene production from CO2 as the sole carbon source in a CSTR for 11 days by Cupriavidus necator strains carrying ethylene-forming enzyme expression (pBBR1-Efe). DETAILED DESCRIPTION OF THE INVENTION

[0045] The following description of the embodiments is presented in general terms, and the present disclosure will be further elucidated from the disclosure set forth herein below under the heading "Examples," which provides supporting experimental data for the disclosure, specific examples of various aspects of the disclosure, and means for carrying out the disclosure.

[0046] The inventors were surprisingly able to engineer C1-fixing microorganisms to co-produce proteins, chemicals or chemical precursors, and microbial biomass by fermentation of substrates containing CO and / or CO2.

[0047] Unless otherwise specified, the following terms used throughout this specification are defined as follows:

[0048] The present disclosure provides microorganisms for the biological co-production of proteins, chemicals, and microbial biomass. A "microorganism" is a microscopic organism, particularly a bacterium, archaea, virus, or fungus. In one embodiment, the microorganism of the present disclosure is a bacterium.

[0049] The term "non-naturally occurring," when used with respect to a microorganism, is intended to mean that the microorganism has at least one genetic modification that is not found in naturally occurring strains of the referenced species, including wild-type strains of the referenced species. Non-naturally occurring microorganisms are typically developed in a laboratory or research facility. The microorganisms of the present disclosure are non-naturally occurring.

[0050] The terms "genetic modification," "genetic alteration," or "genetic engineering" broadly refer to the manipulation of the genome or nucleic acid of a microorganism by man. Similarly, the terms "genetically modified," "genetically altered," or "genetically engineered" refer to a microorganism that includes such a genetic modification, genetic alteration, or genetic engineering. These terms may be used to distinguish between laboratory-produced and naturally occurring microorganisms. Methods of genetic modification include, for example, heterologous gene expression, gene or promoter insertion or deletion, nucleic acid mutation, altered gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis, gene shuffling, and codon optimization. The microorganisms of the present disclosure are genetically engineered.

[0051] "Recombinant" indicates that a nucleic acid, protein, or microorganism is the product of genetic modification, manipulation, or recombination. Generally, the term "recombinant" refers to a nucleic acid, protein, or microorganism that contains or is encoded by genetic material derived from multiple sources, such as two or more different strains or species of a microorganism. The microorganisms of the present disclosure are generally recombinant.

[0052] "Wild-type" refers to the typical form of an organism, strain, gene, or trait that occurs in nature, as distinguished from mutant or variant forms.

[0053] "Endogenous" refers to a nucleic acid or protein present or expressed in the wild-type or parental microorganism from which the microorganism of the present disclosure is derived. For example, an endogenous gene is a gene that naturally occurs in the wild-type or parental microorganism from which the microorganism of the present disclosure is derived. In one embodiment, expression of an endogenous gene can be controlled by an exogenous regulatory element, such as an exogenous promoter.

[0054] "Exogenous" refers to a nucleic acid or protein that originates outside the microbial organism of the present disclosure. For example, an exogenous gene or enzyme can be artificially or recombinantly created and introduced into or expressed in a microbial organism of the present disclosure. An exogenous gene or enzyme can also be isolated from a heterologous microbial organism and introduced into or expressed in a microbial organism of the present disclosure. An exogenous nucleic acid can be adapted to integrate into the genome of a microbial organism of the present disclosure or to remain extrachromosomally in a microbial organism of the present disclosure, for example, a plasmid.

[0055] "Heterologous" refers to a nucleic acid or protein that is not present in the wild-type or parental microorganism from which the microorganism of the present disclosure is derived. For example, a heterologous gene or enzyme can be from a different strain or species and introduced into or expressed in a microorganism of the present disclosure. A heterologous gene or enzyme can be introduced into or expressed in a microorganism of the present disclosure in a form that occurs in the different strain or species. Alternatively, a heterologous gene or enzyme can be modified in several ways, for example, by codon-optimizing it for expression in a microorganism of the present disclosure, or by engineering it to change its function, for example, to reverse the direction of enzymatic activity or change its substrate specificity.

[0056] In order to provide a slightly less expensive range of products Bacillus Clostridium Cupriavi dus、Escherichia、Gluconobacter、Hyphomicrobium、Lysinibacillus、Pa enibacillus, Pseudomonas, Sedimenticola, Sporosarcina, Streptomyces, Thermithiobacillus, Thermotoga, Zea, Klebsiella, Mycobacterium, Sa lmonella、Mycobacteroides、Staphylococcus、Burkholderia、Listeria、Acinetobacter、Shigella、Neisseria、Bordetella、Streptococcus、Enter obacter, Vibrio, Legionella, Xanthomonas, Serratia, Cronobacter, Cupriavidus, Helicobacter, Yersinia, Cutibacterium, Francisella, Pectob acterium、Arcobacter、Lactobacillus、Shewanella、Erwinia、Sulfurospirillum、Peptococcaceae、Thermococcus、Saccharomyces、Pyrococcus、Gl ycine, Homo, Ralstonia, Brevibacterium, Methylobacterium, Geobacillus, bos, gallus, Anaerococcus, Xenopus, Amblyrhynchus, rattus, mouse, sus 、Rhodococcus、Rhizobium、Megasphaera、Mesorhizobium、Peptococcus、Agrobacterium、Campylobacter、Acetobacterium、Alkalibaculum、Blautia Butyribacterium Eubacterium Moorella Oxobacter Sporomusa Thermoanaerobacter Schizosaccharomyces Paenibacillus FictibacillusIt may be derived from Lysinibacillus, Ornithinibacillus, Halobacillus, Kurthia, Lentibacillus, Anoxybacillus, Solibacillus, Virgibacillus, Alicyclobacillus, Sporosarcina, Salimicrobium, Sporosarcina, Planococcus, Corynebacterium, Thermaerobacter, Sulfobacillus, or Symbiobacterium. ,

[0057] The terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can contain one or more modified nucleotides, such as methylated nucleotides or nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0058] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product."

[0059] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may be linear or branched, may comprise modified amino acids, or may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine, both D- and L-optical isomers, and amino acid analogs and peptidomimetics.

[0060] The term "copolymer" refers to a composition of the present disclosure that includes two or more types of monomers linked in the same polymer chain.

[0061] "Enzyme activity" or simply "activity" broadly refers to enzymatic activity, including, but not limited to, the activity of an enzyme, the amount of an enzyme, or the availability of an enzyme to catalyze a reaction. Thus, "increasing" enzyme activity includes increasing the activity of an enzyme, increasing the amount of an enzyme, or increasing the availability of an enzyme to catalyze a reaction. Similarly, "reducing" enzyme activity includes decreasing the activity of an enzyme, decreasing the amount of an enzyme, or decreasing the availability of an enzyme to catalyze a reaction.

[0062] "Mutated" refers to a nucleic acid or protein that is modified in a microorganism of the present disclosure compared to the wild-type or parent microorganism from which the microorganism of the present disclosure is derived. In one embodiment, the mutation may be a deletion, insertion, or substitution in a gene encoding an enzyme. In another embodiment, the mutation may be a deletion, insertion, or substitution of one or more amino acids in an enzyme.

[0063] A "disrupted gene" refers to a gene that has been modified in some way to reduce or eliminate gene expression, regulatory activity of the gene, or activity of the encoded protein or enzyme. A disruption can partially inactivate, completely inactivate, or delete a gene or enzyme. A disruption can be a knockout (KO) mutation that completely eliminates the expression or activity of a gene, protein, or enzyme. A disruption can also be a knockdown that reduces but does not completely eliminate the expression or activity of a gene, protein, or enzyme. A disruption can reduce, prevent, or block the biosynthesis of a product produced by the enzyme. A disruption can include, for example, a mutation in a gene encoding a protein or enzyme, a mutation in a genetic regulatory element involved in the expression of a gene encoding an enzyme, the introduction of a protein-producing nucleic acid that reduces or inhibits the activity of the enzyme, or the introduction of a nucleic acid (e.g., antisense RNA, RNAi, TALEN, siRNA, CRISPR, CRISPRi) or protein that inhibits the expression of a protein or enzyme. The disruption can be introduced using any method known in the art. For purposes of this disclosure, a disruption is created in a laboratory and not naturally occurring.

[0064] A "parental microorganism" is a microorganism used to generate a microorganism of the present disclosure. A parental microorganism can be a naturally occurring microorganism (i.e., a wild-type microorganism) or a microorganism that has been previously modified (i.e., a mutant or recombinant microorganism). A microorganism of the present disclosure can be modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parental microorganism. Similarly, a microorganism of the present disclosure can be modified to include one or more genes that were not included by the parental microorganism. A microorganism of the present disclosure can also be modified to not express or to express a lower amount of one or more enzymes than were expressed in the parental microorganism.

[0065] The microorganisms of the present disclosure can be derived from essentially any parental microorganism, hi one embodiment, the microorganisms of the present disclosure can be derived from a parental 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 selected from the group consisting of Acetobacterium woodii, Alkalibaculum bacchii, Blautia product, 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, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, and Thermoanaerobacter kivui. In one embodiment, the parental microorganism is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei.In another embodiment, the parental microorganism is Clostridium autoethanogenum LZ1561, which was deposited under the terms of the Budapest Treaty on June 7, 2010, with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstrabe 7B, D-38124 Braunschweig, Germany, and has been assigned accession number DSM 23693. This strain is described in International Patent Application No. PCT / NZ2011 / 000144, published as WO2012 / 015317.

[0066] The term "derived from" indicates that a nucleic acid, protein, or microorganism is modified or adapted from a different (e.g., parent or wild-type) nucleic acid, protein, or microorganism to produce a new nucleic acid, protein, or microorganism. Such modifications or adaptations typically include insertions, deletions, mutations, or substitutions of nucleic acids or genes. Generally, the microorganisms of the present disclosure are derived from a parent microorganism. In one embodiment, the microorganisms of the present disclosure are derived from Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the microorganisms of the present disclosure are derived from Clostridium autoethanogenum LZ1561, deposited under DSMZ accession number DSM23693.

[0067] The microorganisms of the present disclosure can be further classified based on their organoleptic properties. For example, the microorganisms of the present disclosure can be or be derived from C1-fixing microorganisms, anaerobes, acetogens, ethanologens, carboxydotrophs, autotrophs, and / or methanotrophs. The microorganisms of the present disclosure can be selected from chemoautotrophs, hydrogenotrophs, hydrogenotrophs, methanotrophs, or any combination thereof. In some embodiments, the microorganisms can be hydrogen-oxidizing, carbon monoxide-oxidizing, hydrogenotrophs, or any combination thereof, capable of growing and synthesizing biomass on a gaseous carbon source, such as syngas and / or CO2, such that the producing microorganisms synthesize target chemical products under gaseous cultivation. The microorganisms and methods of the present disclosure can enable low-cost synthesis of biochemical agents, which can be price-competitive with petrochemical agents and higher-quality plant-derived amino acids, proteins, and other biological nutrients. In certain embodiments, these amino acids, proteins, and other biological nutrients may have a substantially lower price than amino acids, proteins, and other biological nutrients produced through heterotrophic or microbial phototrophic synthesis. Hydrogenobacterial microorganisms, hydrogenotrophs, carboxydotrophs, and chemoautotrophs can capture CO or CO as their sole carbon source to support biological growth. In some embodiments, this growth includes amino acid and protein biosynthesis. Hydrogenobacterial microorganisms and other hydrogenotrophs can use H as a source of reduced electrons for respiration and biochemical synthesis. In some embodiments of the present invention, hydrogenobacterial microorganisms and / or hydrogenotrophs and / or carboxydotrophs and / or other chemoautotrophic microorganisms grow on a gas stream including, but not limited to, one or more of the following: CO, CO, H, and inorganic minerals dissolved in an aqueous solution. In some embodiments, hydrogenobacterial microorganisms and / or hydrogenotrophs and / or carboxydotrophs and / or other chemoautotrophs and / or methanotrophs convert greenhouse gases into biomolecules, including amino acids and proteins.

[0068] Table 1 provides a representative list of microorganisms and identifies their functional properties.

[0069] [Table 1-1] [Table 1-2]

[0070] "Wood-Ljungdahl" refers to, for example, Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008. "Wood-Ljungdahl microorganism" refers to a microorganism that, as expected, contains the Wood-Ljungdahl pathway. Often, the microorganisms of the present disclosure contain a native Wood-Ljungdahl pathway. As used herein, the Wood-Ljungdahl pathway can be a native, unmodified Wood-Ljungdahl pathway or a Wood-Ljungdahl pathway with some degree of genetic modification (e.g., overexpression, heterologous expression, knockout, etc.), so long as it still functions to convert CO, CO, and / or H to acetyl-CoA.

[0071] "C1" refers to a one-carbon molecule, e.g., CO, CO2, CH4, or CH3OH. "C1 oxygenate" refers to a one-carbon molecule that also contains at least one oxygen atom, e.g., CO, CO2, or CH3OH. "C1 carbon source" refers to a single carbon molecule that serves as a partial or sole carbon source for the microorganisms of the present disclosure. For example, a C1 carbon source can include one or more of CO, CO2, CH4, CH3OH, or CH2O2. Preferably, a C1 carbon source includes one or both of CO and CO2. "C1-fixing microorganism" is a microorganism that has the ability to produce one or more products from a C1 carbon source. Often, the microorganisms of the present disclosure are C1-fixing bacteria. In a preferred embodiment, the microorganisms of the present disclosure are derived from the C1-fixing microorganisms identified in Table 1.

[0072] An "anaerobe" is a microorganism that does not require oxygen for growth. Anaerobes may react negatively or die if oxygen is present above a certain threshold. However, some anaerobes can tolerate low levels of oxygen (e.g., 0.000001-5% oxygen), sometimes referred to as "micro-oxic conditions." Often, the microorganisms of the present disclosure are anaerobes. In a preferred embodiment, the microorganisms of the present disclosure are derived from the anaerobes identified in Table 1.

[0073] "Acetogens" are obligate anaerobic bacteria that use the Wood-Ljungdahl pathway as their primary mechanism for energy conservation and synthesis of acetyl-CoA and acetyl-CoA-derived products, such as acetate (Ragsdale, Biochim Biophys Acta, 1784:1873-1898, 2008). Specifically, acetogens use the Wood-Ljungdahl pathway as (1) a mechanism for the reductive synthesis of acetyl-CoA from CO2, (2) a terminal electron acceptor, an energy-saving process, and (3) a mechanism for the fixation (assimilation) of CO2 in the synthesis of cellular carbon (Drake, Acetogenic Prokaryotes, In: The Prokaryotes, 3 rd (Eds., 1999, p. 354, New York, NY, 2006). All naturally occurring acetogens are C1-fixing, anaerobic, autotrophic, and non-methanotrophic. Often, the microorganisms of the present disclosure are acetogens. In preferred embodiments, the microorganisms of the present disclosure are derived from the acetogens identified in Table 1.

[0074] An "ethanologen" is a microorganism that produces or is capable of producing ethanol. Often, the microorganisms of the present disclosure are ethanologens. In preferred embodiments, the microorganisms of the present disclosure are derived from the ethanologens identified in Table 1.

[0075] An "autotroph" is a microorganism that is capable of growth in the absence of organic carbon. Instead, autotrophs use inorganic carbon sources, such as CO and / or CO. Often, the microorganisms of the present disclosure are autotrophs. In preferred embodiments, the microorganisms of the present disclosure are derived from the autotrophs identified in Table 1.

[0076] A "carboxydotroph" is a microorganism capable of utilizing CO as a sole source of carbon and energy. Often, the microorganisms of the present disclosure are carboxydotrophs. In preferred embodiments, the microorganisms of the present disclosure are derived from the carboxydotrophs identified in Table 1.

[0077] A "methanotroph" is a microorganism that is capable of utilizing methane as its sole source of carbon and energy. In certain embodiments, the microorganisms of the present disclosure are methanotrophs or are derived from methanotrophs. In other embodiments, the microorganisms of the present disclosure are not methanotrophs or are not derived from methanotrophs.

[0078] The term "hydrogen bacteria" refers to a mixture of molecular hydrogen and oxygen gas. A "hydrogen bacteria microorganism" is a microorganism that can use hydrogen as an electron donor and oxygen as an electron acceptor in respiration for the production of intracellular energy carriers such as adenosine-5'-triphosphate (ATP).

[0079] The terms "acid hydrogen" and "acid hydrogen microorganism" can be used synonymously with "hydrogen bacteria" and "hydrogen bacterial microorganism," respectively. Hydrogen bacterial microorganisms generally use molecular hydrogen through hydrogenases, and some of the electrons donated by H2 are converted to NAD + (and / or other intracellular reducing equivalents), and some of the electrons from H are used for aerobic respiration. Hydrogen-producing microorganisms generally fix CO autonomously through pathways including, but not limited to, the Calvin cycle or the reverse citric acid cycle.

[0080] In one embodiment, the microorganism of the present disclosure is from the Clostridia cluster, which includes the species Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, which were first reported and characterized by Abrini, Arch Microbiol, 161: 345-351, 1994 (Clostridium autoethanogenum), Tanner, Int J System Bacteriol, 43: 232-236, 1993 (Clostridium ljungdahlii), and Huhnke, WO2008 / 028055 (Clostridium ragsdalei).

[0081] These three species share many similarities. In particular, they are all C1-fixing, anaerobic, acetogenic, ethanologenic, and carboxydotrophic members of the genus Clostridium. They share similar genotypes and phenotypes, as well as energy-conserving and fermentative modes of metabolism. Furthermore, they are clustered within the Clostridial rRNA homology group I, with 16S rRNA DNA sequences that are greater than 99% identical, a DNA G+C content of approximately 22-30 mol%, Gram-positive, have similar morphology and size (logarithmically growing cells of 0.5-0.7 × 3-5 μm), are mesophilic (optimal growth at 30-37°C), have a similar pH range of approximately 4-7.5 (optimal pH of approximately 5.5-6), lack cytochromes, and conserve energy via the Rnf complex. Reduction of carboxylic acids to their corresponding alcohols has also been demonstrated in these species (Perez, Biotechnol Bioeng, 110:1066-1077, 2012). Importantly, these species also all exhibit robust autotrophic growth on CO2-containing gas, producing ethanol and acetate (or acetic acid) as the primary fermentation products, with smaller amounts of 2,3-butanediol and lactic acid under certain conditions.

[0082] However, these three species also share some differences. These species were isolated from different sources: Clostridium autoethanogenum from rabbit intestines, Clostridium ljungdahlii from poultry waste, and Clostridium ragsdalei from freshwater sediments. These species differ in their 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). Furthermore, these species differ in their nutritional requirements for certain vitamins (e.g., thiamine, biotin). Although these species have differences in the nucleic acid and amino acid sequences of Wood-Ljungdahl pathway genes and proteins, the general organization and number of these genes and proteins have been found to be the same in all species (Kopke, Curr Opin Biotechnol, 22: 320-325, 2011).

[0083] Thus, in summary, many of the characteristics of Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei a are not specific to that species, but rather are general characteristics of this cluster of C1-fixing, anaerobic, acetogenic, ethanologenic, and carboxydotrophic members of the genus Clostridium. However, because these species are, in fact, quite different, 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.

[0084] The microorganisms of the present disclosure may also be derived from isolates or mutants of Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. Isolates and mutants of Clostridium autoethanogenum include JA1-1 (DSM10061) (Abrini, Arch Microbiol, 161: 345-351, 1994), LBS1560 (DSM19630) (WO2009 / 064200), and LZ1561 (DSM23693) (WO 2012 / 015317). Isolates and variants of Clostridium ljungdahlii include ATCC 49587 (Tanner, Int J Syst Bacteriol, 43: 232-236, 1993), PETCT (DSM13528, ATCC 55383), ERI-2 (ATCC 55380) (US Pat. No. 5,593,886), C-01 (ATCC 55988) (US Pat. No. 6,368,819), O-52 (ATCC 55989) (US Pat. No. 6,368,819), and OTA-1 (Tirado-Acevedo, Production of bioethanol from synthetic gas using Clostridium ljungdahlii, PhD thesis, North Carolina State University, 2010). Isolates and mutants of Clostridium ragsdalei include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO2008 / 028055).

[0085] However, as noted above, the microorganisms of the present disclosure can also be derived from essentially any microorganism, such as a parent microorganism selected from the group consisting of Clostridium acetobutylicum, Clostridium beijerinckii, Escherichia coli, and Saccharomyces cerevisiae.

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

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

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

[0089] In some embodiments, the aerobic bacterium comprises one or more exogenous nucleic acid molecules encoding naturally occurring polypeptides, wherein the polypeptides are selected from the group consisting of ribolose bisphosphate carboxylase, acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, butanol dehydrogenase, electron transfer flavoprotein large subunit, 3-hydroxybutyryl-CoA dehydrogenase, bifunctional acetaldehyde-CoA / alcohol dehydrogenase, acetaldehyde ... The enzymes are selected from the group consisting of 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxybutyryl-CoA epimerase / delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase / enoyl-CoA hydratase / 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxybutyr ...

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

[0091] In one embodiment, the microorganism of the present disclosure is capable of producing ethylene. One embodiment is directed to a recombinant C1-fixing microorganism capable of producing ethylene from a carbon source comprising a nucleic acid encoding a group of exogenous enzymes including at least one ethylene-forming enzyme (EFE). In some embodiments, the EEF is derived from Pseudomonas syringae. The microorganism of the embodiment further comprises a nucleic acid encoding a group of exogenous enzymes including at least one alpha-ketoglutarate permease (AKGP).

[0092] The microorganism of an embodiment, wherein the nucleic acid encoding the group of exogenous enzymes comprises at least one EFE, at least one AKGP, or any combination thereof. The microorganism of an embodiment, wherein the nucleic acid encoding the group of exogenous enzymes comprises at least one EFE and at least one AKGP. The microorganism of an embodiment, wherein the nucleotides encoding the group of exogenous enzymes are inserted into a bacterial vector plasmid, a high copy number bacterial vector plasmid, a bacterial vector plasmid with an inducible promoter, a nucleotide guide for a homologous recombination system, a CRISPR-Cas system, or any combination thereof. In one embodiment, the promoter is a phosphate-limited inducible promoter. The microorganism of an embodiment, wherein the one or more inducible promoters are selected from an H2-inducible promoter, a phosphate-limited inducible promoter, a nitrogen-limited inducible promoter, or any combination thereof. In some embodiments, the promoter is an NtrC-P-activated promoter. In some embodiments, the promoter is an H2-inducible promoter. In one embodiment, the microorganism comprises intracellular oxygen concentration limitation. In another embodiment, the method limits intracellular oxygen concentration. In one embodiment, the method comprises controlling dissolved oxygen. In one embodiment, the method comprises reducing ethylene production with a decrease in dissolved oxygen concentration. In some embodiments, the microorganism comprises a molecular switch. In some embodiments, the microorganism comprises the ability to switch cellular load under variable conditions.

[0093] In some embodiments, the microorganism is a natural or engineered microorganism capable of converting a gaseous substrate as a carbon and / or energy source. In one embodiment, the gaseous substrate comprises CO as a carbon source. In some embodiments, the gaseous substrate comprises H and / or O as an energy source. In one embodiment, the gaseous substrate comprises a mixture of gases including H and / or CO and / or CO.

[0094] In some embodiments, the gas fermentation product is selected from an alcohol, an acid, a diacid, an alkene, a terpene, an isoprene, and an alkyne. In some embodiments, the methods and microorganisms disclosed herein are for the improved production of ethylene. In one embodiment, the methods and microorganisms disclosed herein are for the improved production of gas fermentation products.

[0095] In one embodiment, the aerobic bacteria can produce products such as acetone, isopropanol, 3-hydroxyisovaleryl-CoA, 3-hydroxyisovalerate, isobutylene, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, isoprene, farnesene, 3-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxybutyric acid, 3-hydroxybutyrylaldehyde, 1,3-butanediol, 2-hydroxyisobutyryl-CoA, 2-hydroxyisobutyrate, butyryl-CoA, butyrate, butanol, caproate, hexanol, octanoate, octanol, 1,3-hexanediol, 2-buten-1-ol, isovaleryl-CoA, isovalerate, isoamyl alcohol, or any combination thereof.

[0096] In another embodiment, the bacteria of the present disclosure 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-propanediol, 1 propanol, 1 hexanol, 1 octanol, chorismic acid-derived products, 3 hydroxybutyrate, 1,3 butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, 1,3 hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, isoamyl alcohol, and monoethylene glycol, and any combination thereof.

[0097] The present disclosure provides microorganisms that produce ethylene by culturing the disclosed microorganisms in the presence of a substrate.

[0098] The enzymes of the present disclosure may be codon optimized for expression in the microorganisms of the present disclosure. "Codon optimization" refers to the mutation of a nucleic acid, such as a gene, for optimizing or improving translation of the nucleic acid in a particular strain or species. Codon optimization can result in a faster translation rate or higher translation accuracy. In a preferred embodiment, the genes of the present disclosure are codon optimized for expression in the microorganisms of the present disclosure. Although codon optimization refers to the underlying gene sequence, codon optimization often results in improved translation and therefore improved enzyme expression. Therefore, the enzymes of the present disclosure may also be described as being codon optimized.

[0099] One or more of the enzymes of the present disclosure may be overexpressed. "Overexpressed" refers to increased expression of a nucleic acid or protein in a microorganism of the present disclosure compared to the wild-type or parent microorganism from which the microorganism of the present disclosure is derived. Overexpression can be achieved by any means known in the art, including altering gene copy number, gene transcription rate, gene translation rate, or enzyme degradation rate. As described above, one or more of the enzymes catalyzing reactions 2, 5, 6, 8, 9, 10, 19, 20, 24, or 25 in Figure 1 may be overexpressed.

[0100] Enzymes of the present disclosure may include disruptive mutations. A "disruptive mutation" is a mutation that reduces or eliminates (i.e., "disrupts") the expression or activity of a gene or enzyme. A disruptive mutation may partially inactivate, completely inactivate, or delete a gene or enzyme. A disruptive mutation may be a knockout (KO) mutation. A disruptive mutation may be any mutation that reduces, prevents, or inhibits the biosynthesis of a product produced by the enzyme. A disruptive mutation may include, for example, a mutation in a gene encoding the enzyme, a mutation in a genetic regulatory element involved in the expression of a gene encoding the enzyme, the introduction of a nucleic acid that produces a protein that reduces or inhibits the activity of the enzyme, or the introduction of a nucleic acid (e.g., antisense RNA, siRNA, CRISPR) or protein that inhibits the expression of the enzyme. A disruptive mutation may be introduced using any method known in the art.

[0101] Introduction of a disruptive mutation results in a microorganism of the present disclosure that does not produce the target product, or produces substantially no product, or a reduced amount of the target product, compared to the parent microorganism from which the microorganism of the present disclosure is derived. For example, a microorganism of the present disclosure may not produce the target product, or may produce at least about 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% less target product than the parent microorganism. For example, a microorganism of the present disclosure may produce less than about 0.001, 0.01, 0.10, 0.30, 0.50, or 1.0 g / L of product.

[0102] Although exemplary sequences and sources of enzymes are provided herein, the present disclosure is in no way limited to these sequences and sources and encompasses variants. The term "variant" includes nucleic acids and proteins whose sequences vary from those of reference nucleic acids and proteins, such as those disclosed in the prior art or exemplified herein. The present disclosure can be practiced using variant nucleic acids or proteins that perform substantially the same function as the reference nucleic acid or protein. For example, the variant protein may perform substantially the same function or catalyze substantially the same reaction as the reference protein. A variant gene may encode the same or substantially the same protein as the reference gene. A variant promoter may have substantially the same ability to promote expression of one or more genes as the reference promoter.

[0103] Such nucleic acids or proteins may be referred to herein as "functionally equivalent variants." By way of example, functionally equivalent variants of nucleic acids may include allelic variants, gene fragments, mutated genes, polymorphisms, and the like. Homologous genes from other microorganisms are also examples of functionally equivalent variants. These include homologous genes from species such as Clostridium acetobutylicum, Clostridium beijerinckii, or Clostridium ljungdahlii, details of which are publicly available on websites such as Genbank or NCBI. Functionally equivalent variants also include nucleic acids whose sequences have been altered as a result of codon optimization for a particular microorganism. Functionally equivalent variants of nucleic acids preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98%, or more nucleic acid sequence identity (percent homology) with the reference nucleic acid. Functionally equivalent variants of a protein preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98%, or more amino acid identity (percent homology) with the reference protein. Functional equivalence of a variant nucleic acid or protein can be assessed using any method known in the art.

[0104] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by conventional Watson-Crick or other non-conventional methods. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10, which are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0105] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR or the cleavage of a polynucleotide by an enzyme. A sequence that can hybridize to a given sequence is referred to as the "complement" of the given sequence.

[0106] Nucleic acids can be delivered to the microorganisms of the present disclosure using any method known in the art. For example, nucleic acids can be delivered as naked nucleic acids or formulated with one or more agents, such as liposomes. Nucleic acids can be DNA, RNA, cDNA, or combinations thereof, as needed. In certain embodiments, restriction inhibitors can be used. Additional vectors can include plasmids, viruses, bacteriophages, cosmids, and artificial chromosomes. In preferred embodiments, nucleic acids are delivered to the microorganisms of the present disclosure using plasmids. For example, transformation (including transduction or transfection) can be achieved by electroporation, sonication, polyethylene glycol-mediated transformation, chemical or natural competence, protoplast transformation, prophage induction, or conjugation. In certain embodiments with active restriction enzyme systems, it may be necessary to methylate the nucleic acid before introducing it into the microorganism.

[0107] Additionally, nucleic acids may be designed to include regulatory elements, such as promoters, to increase or otherwise control expression of a particular nucleic acid. The promoter may be a constitutive promoter or an inducible promoter. Ideally, the promoter is a Wood-Ljungdahl pathway promoter, a ferredoxin promoter, a pyruvate ferredoxin oxidoreductase promoter, an Rnf complex operon promoter, an ATP synthase operon promoter, or a phosphotransacetylase / acetate kinase operon promoter.

[0108] It should be understood that the present disclosure can be practiced using nucleic acids whose sequences vary from those specifically exemplified herein, provided that the sequences perform substantially the same function. For nucleic acid sequences encoding proteins or peptides, this means that the encoded protein or peptide has substantially the same function. For nucleic acid sequences representing promoter sequences, the variant sequence has the ability to promote expression of one or more genes. Such nucleic acids may be referred to herein as "functionally equivalent variants." By way of example, functionally equivalent variants of nucleic acids include allelic variants, fragments of genes, genes containing mutations (deletions, insertions, nucleotide substitutions, etc.), and / or polymorphisms. Homologous genes from other microorganisms may also be considered examples of functionally equivalent variants of the sequences specifically exemplified herein.

[0109] These include homologous genes in species such as Clostridium ljungdahlii, Chloroflexus aurantiacus, Metallosphaera, or Sulfolobus spp, details of which are publicly available on websites such as Genbank or NCBI. The phrase "functionally equivalent variants" should also be taken to include nucleic acids whose sequences vary as a result of codon optimization of a particular microorganism. "Functionally equivalent variants" of 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 specified nucleic acid.

[0110] It should also be understood that the present disclosure can be practiced using polypeptides whose sequences vary from the amino acid sequences specifically exemplified herein. These variants may be referred to herein as "functionally equivalent variants." Functionally equivalent variants of proteins or peptides include those proteins or peptides that share at least 40%, preferably 50%, preferably 60%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95%, or more amino acid identity with the identified protein or peptide and have substantially the same function as the peptide or protein of interest. Such variants include fragments of proteins or peptides within that range, where fragments include truncated forms of the polypeptide, and deletions may be 1 to 5, 10, 15, 20, or 25 amino acids, or may extend from residues 1 to 25 at either end of the polypeptide, or may be of any length within the region or at an internal location. Functionally equivalent variants of particular polypeptides herein should also be taken to include polypeptides expressed by homologous genes in other species of bacteria, for example, as exemplified in the previous paragraph.

[0111] The microorganisms of the present disclosure can be prepared from a parental microorganism and one or more exogenous nucleic acids using any number of techniques known in the art for producing recombinant microorganisms. For example, transformation (including transduction or transfection) can be achieved by electroporation, sonication, polyethylene glycol-mediated transformation, chemical or natural competence, or conjugation. Suitable transformation techniques are described, for example, in Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, 1989.

[0112] In certain embodiments, the nucleic acid introduced into the microorganism must be methylated by a restriction system that is active in the microorganism to be transformed. This can be done using a variety of techniques, including those described below and further exemplified in the Examples section later herein.

[0113] By way of example, in one embodiment, a recombinant microorganism of the present disclosure is produced by a method comprising the following steps: introducing (i) an expression construct / vector described herein and (ii) a methylation construct / vector comprising a methyltransferase gene into a shuttle microorganism; expressing the methyltransferase gene; separating one or more constructs / vectors from the shuttle microorganism; and introducing one or more constructs / vectors into a destination microorganism.

[0114] In one embodiment, the methyltransferase gene of step B is constitutively expressed. In another embodiment, expression of the methyltransferase gene of step B is inducible.

[0115] The shuttle microorganism is a microorganism, preferably a restriction-negative microorganism, that promotes methylation of the nucleic acid sequences that make up the expression construct / vector. In particular embodiments, the shuttle microorganism is a restriction-negative E. coli, Bacillus subtilis, or Lactococcus lactis.

[0116] The methylation construct / vector comprises a nucleic acid sequence encoding a methyltransferase.

[0117] Upon introduction of the expression construct / vector and the methylation construct / vector into the shuttle microorganism, the methyltransferase gene present on the methylation construct / vector is induced. Induction may be by any suitable promoter system, but in one particular embodiment of the present disclosure, the methylation construct / vector contains an inducible lac promoter and is induced by the addition of lactose or an analog thereof, more preferably isopropyl-β-D-thiogalactoside (IPTG). Other suitable promoters include the ara, tet, or T7 systems. In a further embodiment of the present disclosure, the methylation construct / vector promoter is a constitutive promoter.

[0118] In certain embodiments, the methylation construct / vector has an origin of replication specific to the identity of the shuttle microorganism, such that any genes present on the methylation construct / vector are expressed in the shuttle microorganism. Preferably, the expression construct / vector has an origin of replication specific to the identity of the destination microorganism, such that any genes present on the expression construct / vector are expressed in the destination microorganism.

[0119] Expression of the methyltransferase enzyme results in methylation of the genes present on the expression construct / vector. The expression construct / vector may then be isolated from the shuttle microorganism according to any one of several known methods. By way of example only, the expression construct / vector may be isolated using the methodology described in the Examples section below.

[0120] In one particular embodiment, both constructs / vectors are isolated simultaneously.

[0121] The expression construct / vector may be introduced into the destination microorganism using any number of known methods. However, by way of example, the methodology described in the Examples section below may be used. Because the expression construct / vector is methylated, the nucleic acid sequences present on the expression construct / vector can be incorporated into and successfully expressed in the destination microorganism.

[0122] It is envisioned that the methyltransferase gene can be introduced into the shuttle microorganism and overexpressed. Thus, in one embodiment, the resulting methyltransferase enzyme can be harvested using known methods and used in vitro to methylate an expression plasmid. The expression construct / vector can then be introduced into a destination microorganism for expression. In another embodiment, the methyltransferase gene is introduced into the genome of the shuttle microorganism, followed by introducing the expression construct / vector into the shuttle microorganism, isolating one or more constructs / vectors from the shuttle microorganism, and then introducing the expression construct / vector into the destination microorganism.

[0123] It is envisioned that the expression constructs / vectors and methylation constructs / vectors defined above may be combined to provide compositions that have particular utility in circumventing restriction barrier mechanisms for generating recombinant microorganisms of the present disclosure.

[0124] In one particular embodiment, the expression construct / vector and / or the methylation construct / vector is a plasmid.

[0125] Those skilled in the art will recognize the use of several suitable methyltransferases in generating the microorganisms of the present disclosure. However, by way of example, the Bacillus subtilis phage ΦT1 methyltransferase and methyltransferases described in the Examples below may be used. Nucleic acids encoding suitable methyltransferases will be readily recognized given the sequence and genetic code of the desired methyltransferase.

[0126] Any number of constructs / vectors adapted to allow expression of a methyltransferase gene may be used to generate the methylation construct / vector.

[0127] In one embodiment, the substrate comprises CO. In one embodiment, the substrate comprises CO and CO. In another embodiment, the substrate comprises CO and H. In another embodiment, the substrate comprises CO, CO, and H.

[0128] "Substrate" refers to a carbon and / or energy source for the microorganisms of the present disclosure. Often, the substrate is gaseous and includes a C1 carbon source, such as CO, CO, and / or CH. Preferably, the substrate includes a C1 carbon source of CO or CO + CO. The substrate may further include other non-carbon components, such as H, N, or electrons. However, in other embodiments, the substrate may be a carbohydrate, such as a sugar, starch, fiber, lignin, cellulose, or hemicellulose, or a combination thereof. For example, the carbohydrate may be fructose, galactose, glucose, lactose, maltose, sucrose, xylose, or some combination thereof. In some embodiments, the substrate does not include (D)-xylose (Alkim, Microb Cell Fact, 14:127, 2015). In some embodiments, the substrate does not include a pentose, such as xylose (Pereira, Metab Eng, 34: 80-87, 2016). In some embodiments, the substrate may include both gaseous and carbohydrate substrates. (Mixotrophic fermentation) The substrate may further include other non-carbon components such as H, N, or electrons.

[0129] Gaseous substrates generally contain at least some amount of CO, such as about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mol% CO. Gaseous substrates can contain a range of CO, such as about 20-80, 30-70, or 40-60 mol% CO. Preferably, the gaseous substrate contains about 40-70 mol% CO (e.g., steel mill or blast furnace gas), about 20-30 mol% CO (e.g., basic oxygen furnace gas), or about 15-45 mol% CO (e.g., synthesis gas). In some embodiments, the gaseous substrate can contain relatively low amounts of CO, such as about 1-10 or 1-20 mol% CO. Microorganisms of the present disclosure typically convert at least a portion of the CO in the gaseous substrate to products. In some embodiments, the gaseous substrate is free or substantially free (<1 mol%) of CO.

[0130] The gaseous substrate may contain some amount of H. For example, the gaseous substrate may contain about 1, 2, 5, 10, 15, 20, or 30 mol% H. In some embodiments, the gaseous substrate may contain relatively large amounts of H, such as about 60, 70, 80, or 90 mol% H. In further embodiments, the gaseous substrate is free or substantially free (<1 mol%) of H.

[0131] The gaseous substrate may contain some amount of CO. For example, the gaseous substrate may contain about 1-80 or 1-30 mol% CO. In some embodiments, the gaseous substrate may contain less than about 20, 15, 10, or 5 mol% CO. In other embodiments, the gaseous substrate is free or substantially free (<1 mol%) of CO.

[0132] The gaseous substrate may also be provided in alternative forms, for example, the gaseous substrate may be dissolved in a liquid or adsorbed onto a solid support.

[0133] The gaseous substrate and / or C1 carbon source may be obtained as a by-product of an industrial process, such as from automobile exhaust or biomass gasification, or may be a waste gas or off-gas from some other source. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal product production, such as steel mill production, non-ferrous metal product production, petroleum refining, coal gasification, power generation, carbon black production, ammonia production, methanol production, and coke production. In these embodiments, the gaseous substrate and / or C1 carbon source may be captured from the industrial process before it is released into the atmosphere using any convenient method.

[0134] The gaseous substrate and / or C1 carbon source may be a synthesis gas, such as synthesis gas obtained by gasification of coal or refinery residues, gasification of biomass or lignocellulosic material, or reforming of natural gas. In another embodiment, the synthesis gas may be obtained from the gasification of municipal or industrial solid waste.

[0135] The term "feedstock," when used in the context of a stream flowing to a gas fermentation bioreactor (i.e., gas fermentor) or "gas fermentation feedstock," should be understood to encompass any material (solid, liquid, or gas) or stream that can provide a substrate and / or C1 carbon source to the gas fermentor or bioreactor directly or after processing the feedstock.

[0136] The terms "waste gas" or "waste gas stream" may be used to refer to any gas stream that is either directly radiated, flared without additional value capture, or combusted for the purposes of energy recovery.

[0137] The term synthesis gas or syngas refers to a gaseous mixture containing at least one carbon source, such as carbon monoxide (CO), carbon dioxide (CO), or any combination thereof, and optionally hydrogen (H), which can be used as a feedstock for the disclosed gas fermentation processes and can be produced from a wide range of carbonaceous materials, both solid and liquid.

[0138] The substrate and / or C1 carbon source may be waste gas obtained as a by-product of an industrial process, or waste gas from another source, such as from automobile exhaust, biogas, landfill gas, direct air capture, or waste gas from electrolysis. The substrate and / or C1 carbon source may be syngas produced by pyrolysis, torrefaction, or gasification. In other words, carbon in waste may be recycled by pyrolysis, torrefaction, or gasification to produce syngas for use as the substrate and / or C1 carbon source. The substrate and / or C1 carbon source may be a gas containing methane.

[0139] In certain embodiments, the industrial process is selected from ferrous metals manufacturing, such as steel production, non-ferrous manufacturing, petroleum refining, power production, carbon black production, paper and pulp manufacturing, ammonia production, methanol production, coke production, petrochemical production, carbohydrate fermentation, cement manufacturing, aerobic digestion, anaerobic digestion, catalytic processes, natural gas extraction, cellulose fermentation, oil extraction, geological reservoirs, natural gas, gas from fossil sources such as coal and petroleum, or any combination thereof. Examples of specific processing steps within industrial processes include catalyst regeneration, fluid catalytic cracking, and catalyst regeneration. Air separation and direct air recovery are other suitable industrial processes. Specific examples in steel and ferroalloy production include blast furnace gas, basic oxygen furnace gas, coke oven gas, direct reduction of iron furnace top gas, and residual gas from iron smelting. In these embodiments, the substrate and / or C1 carbon source may be captured from the industrial process before it is released into the atmosphere using any known method.

[0140] The substrate and / or C1 carbon source may be synthesis gas, also known as syngas, which may be obtained from a reforming, partial oxidation, or gasification process. Examples of gasification processes include coal gasification, refinery residue gasification, petroleum coke gasification, biomass gasification, lignocellulosic material gasification, waste wood gasification, black liquor gasification, municipal solid waste gasification, municipal liquid waste gasification, industrial solid waste gasification, industrial liquid waste gasification, waste fuel gasification, sewage gasification, sewage sludge gasification, wastewater treatment sludge gasification, and biogas gasification. Examples of reforming processes include steam methane reforming, steam naphtha reforming, natural gas reforming, biogas reforming, landfill gas reforming, naphtha reforming, and dry methane reforming. Examples of partial oxidation processes include thermal and catalytic partial oxidation processes, catalytic partial oxidation of natural gas, and partial oxidation of hydrocarbons. Examples of municipal solid waste include tires, plastics, shoes, apparel, and textile fibers. Municipal solid waste may simply be landfill-type waste. Municipal solid waste may be sorted or unsorted. Examples of biomass may include lignocellulosic material and may include microbial biomass. Lignocellulosic material may include agricultural waste and forestry waste.

[0141] The substrate and / or C1 carbon source may be a gas stream containing methane. Such methane-containing gas may be obtained from fossil methane emissions, such as during fracking, wastewater treatment, livestock, agriculture, and municipal solid waste landfills. It is also contemplated that methane may be combusted to generate electricity or heat, and the C1 by-product may be used as a substrate or carbon source.

[0142] The composition of the gaseous substrate can significantly affect the efficiency and / or cost of the reaction. For example, the presence of oxygen (O2) can reduce the efficiency of an anaerobic fermentation process. Depending on the composition of the substrate, it may be desirable to treat, scrub, or filter the substrate to remove any undesirable impurities, such as toxins, undesirable components, or dust particles, and / or to increase the concentration of desired components.

[0143] Regardless of the source or exact content of gas used as a feedstock, the feedstock may be metered into the bioreactor to maintain control of the driven rate and amount of carbon provided to the culture (e.g., for carbon credit calculations or mass balancing of sustainable carbon and overall products). Similarly, the output of the bioreactor (e.g., for carbon credit calculations or mass balancing of sustainable carbon and overall products) may be metered or equipped with valve connections that can control the flow of the output and products (e.g., ethylene, ethanol, acetate, 1-butanol, etc.) produced via fermentation. Such valves or metering mechanisms may be useful for a variety of purposes, including, but not limited to, measuring the amount of output from a given bioreactor, as well as slugs of product through connected pipelines, so that if the product is mixed with other gases or liquids, the resulting mixture can later be mass balanced to determine the percentage of product produced from the bioreactor.

[0144] In certain embodiments, the fermentation is carried out in the absence of a carbohydrate substrate, such as sugar, starch, fiber, lignin, cellulose, or hemicellulose.

[0145] In addition to the tandem repeat proteins and chemical products, the microorganisms of the present disclosure may be cultured to produce one or more co-products. For example, the microorganisms of the present disclosure may produce ethanol (WO 2007 / 117157), acetate (WO 2007 / 117157), 1-butanol (WO 2008 / 115080, WO 2012 / 053905, and WO 2017 / 066498), butyrate (WO 2008 / 115080), 2,3-butanediol (WO 2009 / 151342 and WO 2016 / 094334), 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-propanediol (WO 2014 / 036152), 1-propanol (WO 2017 / 066498), 1-hexanol (WO 2017 / 066498), 1-octan ... Ctanol (WO 2017 / 066498), chorismic acid-derived products (WO 2016 / 191625), 3-hydroxybutyric acid (WO 2017 / 066498), 1,3-butanediol (WO 2017 / 066498), 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid (WO 2017 / 066498), isobutylene (WO 2017 / 066498), adipic acid (WO 2017 / 066498), 1,The microbial biomass may be capable of, or may be engineered to, produce 3-hexanediol (WO 2017 / 066498), 3-methyl-2-butanol (WO 2017 / 066498), 2-buten-1-ol (WO 2017 / 066498), isovalerate (WO 2017 / 066498), isoamyl alcohol (WO 2017 / 066498), and / or monoethylene glycol (WO 2019 / 126400). In certain embodiments, the microbial biomass itself may be considered a product. These products may be further converted to produce at least one component of diesel, jet fuel, sustainable aviation fuel (SAF), and / or gasoline. In certain embodiments, ethylene may be catalytically converted to another product, commodity, or any combination thereof. Additionally, the microbial biomass can be further processed to produce single-cell protein (SCP) by any method or combination of methods known in the art. In addition to one or more target chemical products, the microorganisms of the present disclosure can also produce ethanol, acetate, and / or 2,3-butanediol. In another embodiment, the microorganisms and methods of the present disclosure improve the production of products, proteins, microbial biomass, or any combination thereof.

[0146] A "natural product" is a product produced by a non-genetically modified microorganism. For example, ethanol, acetate, and 2,3-butanediol are natural products of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei. A "non-natural product" is a product produced by a genetically engineered microorganism but not by the non-genetically engineered microorganism from which the genetically engineered microorganism is derived. Ethylene is not known to be produced by any naturally occurring microorganism, and is therefore a non-natural product of all microorganisms.

[0147] "Selectivity" refers to the ratio of target product production to total fermentation products produced by a microorganism. The microorganisms of the present disclosure can be engineered to produce products with a particular selectivity or with minimal selectivity. In one embodiment, the target product, such as ethylene glycol, accounts for at least about 5%, 10%, 15%, 20%, 30%, 50%, or 75% of the total fermentation products produced by the microorganisms of the present disclosure. In one embodiment, ethylene accounts for at least 10% of the total fermentation products produced by the microorganisms of the present disclosure, such that the microorganisms of the present disclosure have a selectivity for at least 10% ethylene glycol. In another embodiment, ethylene accounts for at least 30% of the total fermentation products produced by the microorganisms of the present disclosure, such that the microorganisms of the present disclosure have a selectivity for at least 30% ethylene.

[0148] At least one of the one or more fermentation products may be biomass produced by the culture. At least a portion of the microbial biomass may be converted to single cell protein (SCP). At least a portion of the single cell protein may be utilized as an ingredient in animal feed.

[0149] In one embodiment, the present disclosure provides an animal feed comprising a microbial biomass and at least one excipient, wherein the microbial biomass comprises microorganisms grown on a gaseous substrate comprising one or more of CO, CO2, and H2.

[0150] "Single-cell protein" (SCP) refers to microbial biomass that can be used in protein-rich human and / or animal feeds, often replacing traditional protein supplement sources such as soybean or fish meal. To produce single-cell protein or other products, the process may include additional separation, processing, or treatment steps. For example, the method may include sterilizing the microbial biomass, centrifuging the microbial biomass, and / or drying the microbial biomass. In certain embodiments, the microbial biomass is dried using spray drying or paddle drying. Because ingestion of a diet high in nucleic acid content can lead to the accumulation of nucleic acid degradation products and / or gastrointestinal disorders, the method may also include reducing the nucleic acid content of the microbial biomass using any method known in the art. The single-cell protein may be suitable for feeding to animals, such as livestock or pets. Specifically, the animal feed may be suitable for feeding one or more of beef cattle, dairy cattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, banteng, gayal, yaks, chickens, turkeys, ducks, geese, quails, guinea fowl, pigeons / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents. The composition of the animal feed may be tailored to meet the nutritional requirements of different animals. Additionally, the process may include mixing or combining the microbial biomass with one or more excipients.

[0151] "Microbial biomass" refers to biological material containing microbial cells. For example, microbial biomass can comprise or consist of pure or substantially pure cultures of bacteria, archaea, viruses, or fungi. When initially separated from a fermentation broth, microbial biomass generally contains large amounts of water. This water can be removed or reduced by drying or processing the microbial biomass.

[0152] "Excipient" may refer to any substance that can be added to microbial biomass to enhance or modify the form, characteristics, or nutritional content of animal feed. For example, an excipient may include one or more of carbohydrates, fiber, fat, protein, vitamins, minerals, water, flavorings, sweeteners, antioxidants, enzymes, preservatives, probiotics, or antibiotics. In some embodiments, an excipient may be hay, straw, silage, grain, oil or fat, or other plant material. An excipient may be any feed ingredient identified in Chiba, Section 18: Diet Formulation and Common Feed Ingredients, Animal Nutrition Handbook, 3rd revision, pages 575-633, 2014.

[0153] "Biopolymer" refers to a natural polymer produced by cells of living organisms. In certain embodiments, the biopolymer is a PHA. In certain embodiments, the biopolymer is a PHB.

[0154] "Bioplastic" refers to plastic materials produced from renewable biomass sources. Bioplastics can be produced from renewable sources such as vegetable fats and oils, corn starch, straw, wood chips, sawdust, or recycled food waste.

[0155] Any reference herein to an acid (eg, acetic acid or 2-hydroxyisobutyric acid) should be interpreted as also including the corresponding salt (eg, acetate or 2-hydroxyisobutyrate).

[0156] Typically, culturing is carried out in a bioreactor. The term "bioreactor" includes a culturing / fermentation device consisting of one or more vessels, columns, or piping arrangements, such as a continuous stirred tank reactor (CSTR), immobilized cell reactor (ICR), trickle bed reactor (TBR), bubble column, gas lift fermenter, static mixer, or other vessel or device suitable for gas-liquid contact. In some embodiments, a bioreactor can include a first growth reactor and a second culture / fermentation reactor. Substrate may be provided to one or both of these reactors. As used herein, the terms "culturing" and "fermenting" are used interchangeably. These terms encompass both the growth phase and the product biosynthesis phase of the culturing / fermentation process.

[0157] Cultures are generally maintained in an aqueous medium containing sufficient nutrients, vitamins, and / or minerals to allow microbial growth. Preferably, the aqueous medium is an anaerobic microbial medium, such as a minimal anaerobic microbial growth medium. Suitable media are known in the art.

[0158] The culture / fermentation should desirably be carried out under conditions suitable for the production of ethylene glycol. If necessary, the culture / fermentation is carried out under anaerobic conditions. Reaction conditions to consider include pressure (or partial pressure), temperature, gas flow rate, liquid flow rate, medium pH, medium redox potential, agitation rate (if a continuous stirred tank reactor is used), inoculum level, maximum gas substrate concentration to ensure that the gas in the liquid phase does not become limiting, and maximum product concentration to avoid product inhibition. Specifically, the rate of substrate introduction may be controlled to ensure that the gas concentration in the liquid phase does not become limiting.

[0159] Operating a bioreactor at elevated pressure allows for an increased rate of gas mass transfer from the gas phase to the liquid phase. Therefore, it is generally preferable to conduct the culture / fermentation at a pressure higher than atmospheric pressure. Furthermore, because a given gas conversion rate is in part a function of substrate retention time, and retention time dictates the required volume of the bioreactor, the use of a pressurized system can significantly reduce the required bioreactor volume and, consequently, the capital costs of the culture / fermentation equipment. This means that the retention time, defined as the liquid volume in the bioreactor divided by the input gas flow rate, can be reduced when the bioreactor is maintained at a pressure higher than atmospheric pressure. Optimal reaction conditions depend in part on the specific microorganism used. However, it is generally preferable to conduct the fermentation at a pressure higher than atmospheric pressure. Furthermore, because a given gas conversion rate is in part a function of substrate retention time, and achieving a desired retention time further dictates the required volume of the bioreactor, the use of a pressurized system can significantly reduce the required bioreactor volume and, consequently, the capital costs of the fermentation equipment.

[0160] In certain embodiments, fermentation is carried out in the absence of light or in the presence of an amount of light insufficient to meet the energy requirements of the photosynthetic microorganisms, hi certain embodiments, the microorganisms of the present disclosure are non-photosynthetic microorganisms.

[0161] The target product can be separated or purified from the fermentation broth using any method or combination of methods known in the art, including, for example, fractional distillation, evaporation, pervaporation, gas stripping, phase separation, and extractive fermentation, including, for example, liquid-liquid extraction. In certain embodiments, the target product is recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating the microbial cells from the broth (conveniently by filtration), and recovering one or more target products from the broth. Alcohol and / or acetone may be recovered, for example, by distillation. Acids may be recovered, for example, by adsorption on activated carbon. The separated microbial cells are preferably returned to the bioreactor. The cell-free permeate remaining after the target product is removed is also preferably returned to the bioreactor. Additional nutrients (such as vitamin B) may be added to the cell-free permeate to replenish the medium before being returned to the bioreactor. Purification techniques can include affinity tag purification (e.g., His, Twin-Strep, and FLAG), bead-based systems, chip-based approaches, and FPLC systems for larger-scale automated purification. Purification methods that do not rely on affinity tags (e.g., salting out, ion exchange, and size exclusion) have also been described.

[0162] In some embodiments, the produced chemical products may be isolated and concentrated, including purified, using any suitable separation and / or purification technique known in the art. In one embodiment, the produced chemical products are gaseous. In one embodiment, the chemical products are liquid. In one embodiment, the gaseous chemical products may be passed through a filter, a gas separation membrane, a gas purifier, or any combination thereof. In one embodiment, the chemical products are separated using 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 an infrastructure or process of an oil, gas, refining, petrochemical operation, or any combination thereof. The infrastructure or process may be existing or new. In one embodiment, the gaseous fermentation product is integrated into an oil and gas production, transportation and refining, and / or chemical complex. In another embodiment, the source of the feedstock is from an oil, gas, refining, petrochemical operation, or any combination thereof. In one embodiment, the gas fermentation product is integrated into the infrastructure or process of an oil, gas, refining, petrochemical operation, or any combination thereof, and the source of the feedstock is from an oil, gas, refining, petrochemical operation, or any combination thereof.

[0163] In some embodiments, distillation may be used to purify the product gas. In one embodiment, gas-to-liquid extraction may be used. In one embodiment, the liquid product isolate may also be concentrated via extraction using an organic phase. In another embodiment, purification may involve other standard techniques selected from ultrafiltration, one or more chromatographic techniques, or any combination thereof.

[0164] The methods of the present disclosure may further include separating the gas fermentation product from the fermentation broth. The gas fermentation product can be separated or purified from the fermentation broth using any method or combination of methods known in the art, including, for example, distillation, simulated moving bed processing, membrane processing, pervaporation, gas stripping, phase separation, ion exchange, or extractive fermentation, including, for example, liquid-liquid extraction. Ethylene can be separated according to a method or combination of methods known in the art, as described in U.S. Pat. No. 2,769,321, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the produced ethylene is harvested from the bioreactor culture vessel.

[0165] In one embodiment, the gas fermentation product may be concentrated from the fermentation broth using reverse osmosis and / or pervaporation (U.S. Pat. No. 5,552,023). Water may be removed by distillation, and the bottoms (containing a high proportion of the gas fermentation product) may then be recovered using distillation or vacuum distillation to produce a high-purity gas stream. Alternatively, the gas fermentation product may be further purified by reactive distillation with aldehydes (Atul, Chem Eng Sci, 59:2881-2890, 2004) or azeotropic distillation with hydrocarbons (U.S. Pat. No. 2,218,234), with or without concentration by reverse osmosis and / or pervaporation. In another approach, gas fermentation products may be captured from aqueous solutions onto activated carbon or polymeric absorbers (with or without reverse osmosis and / or pervaporation) and recovered using low-boiling 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 gas fermentation products can then be recovered from the organic solvent by distillation. In certain embodiments, the gas fermentation products are recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating the microbial cells from the broth (conveniently by filtration), and recovering one or more gas fermentation products from the broth. Co-products, such as alcohols or acids, may also be separated or purified from the broth. Alcohols may be recovered, for example, by distillation. Acids may be recovered, for example, by adsorption on activated carbon. The separated microbial cells may, in certain embodiments, be returned to the bioreactor. Additionally, the separated microbial cells may, in some embodiments, be recycled to the bioreactor. The cell-free permeate remaining after the target product has been removed is also preferably returned, in whole or in part, to the bioreactor.Additional nutrients (such as vitamin B) can be added to the cell-free permeate to replenish the medium before returning it to the bioreactor.

[0166] Many methods have been demonstrated for the recovery of diols from aqueous media. Simulated moving bed (SMB) technology has been used to recover 2,3-butenediol from aqueous mixtures of ethanol and related oxygenates (U.S. Pat. No. 8,658,845). Reactive separation has also demonstrated effective diol recovery. In some embodiments, ethylene glycol is recovered by reacting a diol-containing stream with an aldehyde, followed by fractionation and regeneration of the diol, final fractionation, and recovery of a concentrated diol stream. See, for example, U.S. Pat. No. 7,951,980.

[0167] In one embodiment, the method includes recovering the ethylene produced as disclosed above. In one embodiment, the method further includes, after recovering the ethylene, converting or using the ethylene in the production of one or more chemicals.

[0168] Ethylene is a high-value gaseous compound widely used in industry. In one embodiment, ethylene can be used as an anesthetic or as a fruit ripening agent, as well as in the production of several other chemical products. In some embodiments, ethylene may be used to produce polyethylene and other polymers such as styrene, polystyrene, ethylene oxide, ethylene dichloride, ethylene dibromide, ethyl chloride, and ethylbenzene. Ethylene oxide is, for example, a primary raw material in the production of surfactants and detergents and ethylene glycol, which is used in the automotive industry as an anti-freeze product. In one embodiment, ethylene dichloride, ethylene dibromide, and ethyl chloride may be targeted to produce products such as polyvinyl chloride, trichloroethylene, perchloroethylene, methyl chloroform, polyvinylidene chloride and copolymers, and ethyl bromide. In one embodiment, ethylbenzene is a precursor to styrene, which is used to produce polystyrene (used as an insulation product) and styrene butadiene (a rubber suitable for use in tires and footwear). In another embodiment, the product is ethylene propylene diene monomer (EPDM) rubber, ethylene propylene (EPR / EPM) rubber, or any combination thereof.

[0169] Of course, the methods of the present invention may be integrated or coupled to one or more processes for the production of downstream chemical products from ethylene. In some embodiments, the methods of the present invention may directly or indirectly supply ethylene to chemical processes or reactions sufficient for the conversion or production of other useful chemical products.

[0170] In some embodiments, ethylene is converted to hydrocarbon liquid fuels. In one embodiment, ethylene is oligomerized over a catalyst to selectively produce target products selected from gasoline, condensates, aromatics, heavy oil diluents, distillates, or any combination thereof. In other embodiments, the distillates are selected from diesel, jet fuel, sustainable aviation fuel (SAF), or any combination thereof.

[0171] In one embodiment, ethylene oligomerization is utilized to direct the desired product. In one embodiment, the ethylene oligomerization is catalyzed by a homogeneous catalyst, a heterogeneous catalyst, or any combination thereof, and may have a transition metal as an active site. In some embodiments, ethylene is further converted to long-chain hydrocarbons by oligomerization. In other embodiments, linear olefins are the primary product from ethylene oligomerization. In some embodiments, alpha olefins are the primary product from ethylene oligomerization. In one embodiment, olefins undergo an upgrading process. In some embodiments, the olefin upgrading process is hydrogenation. In one embodiment, olefins are subjected to an olefin conversion technology. In one embodiment, ethylene is interconverted to propylene, 2-butene, or any combination thereof. In one embodiment, propylene is converted to polypropylene.

[0172] 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 used in a wide range of industrial and consumer markets, including packaging, transportation, electrical / electronics, textiles, and construction industries, as well as consumer chemicals, coatings, and adhesives.

[0173] Ethylene can be chlorinated to ethylene dichloride (EDC), which can then be cracked to make vinyl chloride monomer (VCM). Nearly all VCM is used to make polyvinyl chloride, which has its primary use in the construction industry.

[0174] Other ethylene derivatives include alpha olefins used in linear low density polyethylene (LLDPE) production, surfactant alcohols and plasticizer alcohols, vinyl acetate monomer (VAM) used in adhesives, paints, paper coatings and barrier resins, and industrial ethanol used as a solvent or in the production of chemical intermediates such as ethyl acetate and ethyl acrylate.

[0175] Ethylene can further be used as a monomer base for the production of various polyethylene oligomers by coordination polymerization using metal chloride or metal oxide catalysts. The most common catalyst consists of titanium(III) chloride, the so-called Ziegler-Natta catalyst. Another common catalyst is the Phillips catalyst, which is prepared by depositing chromium(VI) oxide on silica.

[0176] The polyethylene oligomers so produced can be classified according to their density and branching. Furthermore, the mechanical properties are highly dependent on variables such as the degree and type of branching, crystalline structure, and molecular weight. There are several types of polyethylene that can be produced from ethylene, including, but not limited to: Ultra-high molecular weight polyethylene (UHMWPE), Ultra-low molecular weight polyethylene (ULMWPE or PE-WAX), High molecular weight polyethylene (HMWPE); High density polyethylene (HDPE), High density cross-linked polyethylene (HDXLPE), Cross-linked polyethylene (PEX or XLPE), Medium density polyethylene (MDPE), Linear low-density polyethylene (LLDPE), Low-density polyethylene (LDPE), Ultra-low density polyethylene (VLDPE) and Chlorinated polyethylene (CPE).

[0177] Low-density polyethylene (LDPE) and linear low-density polyethylene (LLDPE) are primarily used in film applications such as food and non-food packaging, shrink and stretch films, and non-packaging applications. High-density polyethylene (HDPE) is primarily used in blow-molded and injection-molded applications such as containers, drums, housewares, caps, and pallets. HDPE can also be extruded into film for water, gas, and irrigation pipes, as well as reject bags, carrier bags, and industrial linings.

[0178] According to one embodiment, the ethylene formed from the above disclosure can be converted to ethylene oxide via direct oxidation according to the following equation: C2H4+O2→C2H4O The ethylene oxide thereby produced is a key chemical intermediate in several commercially important processes, including the production of monoethylene glycol. Other EO derivatives include ethoxylates (for use in shampoos, restaurant cleaners, etc.), glycol ethers (solvents, fuels, etc.), and ethanolamines (surfactants, personal care products, etc.).

[0179] According to one embodiment of the present disclosure, ethylene oxide produced as described above can be used to produce commercial quantities of monoethylene glycol according to the following equation: (CH2CH2)O+H2O→HOCH2CH2OH According to another embodiment, the claimed microorganisms can be modified to directly produce monoethylene glycol. In some aspects of the microorganisms disclosed in WO 2019 / 126400, the entire contents of which are incorporated by reference herein, the microorganisms can be modified to produce monoethylene glycol directly ... The microorganism further comprises one or more of the following enzymes: an enzyme capable of converting 3-phospho-D-glycerate to 3-phosphonooxypyruvate, an enzyme capable of converting 3-phosphonooxypyruvate to 3-phospho-L-serine, an enzyme capable of converting 3-phospho-L-serine to serine, an enzyme capable of converting serine to glycine, an enzyme capable of converting 5,10-methylenetetrahydrofolate to glycine, an enzyme capable of converting serine to hydroxypyruvate, an enzyme capable of converting D-glycerate to hydroxypyruvate, an enzyme capable of converting malate to glyoxylate, an enzyme capable of converting glyoxylate to glycolate, an enzyme capable of converting hydroxypyruvate to glycolaldehyde, and / or an enzyme capable of converting glycolaldehyde to ethylene glycol. In one embodiment, the microorganism comprises one or more heterologous enzymes capable of converting oxaloacetate to citrate, a heterologous enzyme capable of converting glycine to glyoxylate, a heterologous enzyme capable of converting isocitrate to glyoxylate, a heterologous enzyme capable of converting glycolic acid to glycolaldehyde, or any combination thereof.Heterologous enzymes capable of converting oxaloacetate to citrate are citrate [Si]-synthase [2.3.3.1], ATP-citrate synthase [2.3.3.8], or citrate (Re)-synthase [2.3.3.3], and heterologous enzymes capable of converting glycine to glyoxylate are alanine-glyoxylate transaminase [2.6.1.44], serine-glyoxylate transaminase [2.6.1.45], serine-pyruvate transaminase [2.6.1.51], glycine-oxaloacetate transaminase [2.6.1.35], glycine transaminase [2.6.1.4], glycine dehydrogenase [1.4.1.10], alanine dehydrogenase [1.4.1.1], or glycine dehydrogenase [1.4.1.2]. In some embodiments, the heterologous enzyme capable of converting isocitrate to glyoxylate is isocitrate lyase [4.1.3.1], and the heterologous enzyme capable of converting glycolate to glycolaldehyde is glycolaldehyde dehydrogenase [1.2.1.21], lactaldehyde dehydrogenase [1.2.1.22], succinate-semialdehyde dehydrogenase [1.2.1.24], 2,5-dioxovalerate dehydrogenase [1.2.1.26], aldehyde dehydrogenase [1.2.1.3 / 4 / 5], betaine aldehyde dehydrogenase [1.2.1.8], or aldehyde ferredoxin oxidoreductase [1.2.7.5], or a combination thereof.

[0180] Monoethylene glycol produced by either method can be used as a component in a variety of products, such as nonwovens, diaper cover stock, building materials, construction materials, road fabrics, filters, fiberfill, felt, transportation interior materials, paper and tape reinforcement, tents, ropes and lines, sails, fishing nets, seat belts, laundry bags, artificial blood vessels, carpets, rugs, clothing, sheets and pillowcases, towels, draperies, curtains, bed sheets, blankets, and the like.

[0181] MEG can be used alone as a liquid coolant, antifreeze, preservative, dehydrant, drilling fluid, or any combination thereof. The MEG produced can also be used to manufacture secondary products such as insulation materials, polyester films, de-icing fluids, heat transfer fluids, automotive antifreeze and other liquid coolants, preservatives, dehydrant, drilling fluids, water-based adhesives, latex paint and asphalt emulsions, electrolytic capacitors, paper, and polyester resins for use in synthetic skin care products.

[0182] Importantly, the monoethylene glycol produced can be converted to the polyester resin polyethylene terephthalate (PET) according to one of two main processes. The first process involves esterifying the monoethylene glycol with dimethyl terephthalate according to the following two-step process: First step C6H4(CO2CH3)2+2 HOCH2CH2OH→C6H4(CO2CH2CH2OH)2+2 CH3OH Second step n C6H4(CO2CH2CH2OH)2→ [(CO)C6H4(CO2CH2CH2O)] n +n HOCH2CH2OH

[0183] Alternatively, monoethylene glycol can be subjected to an esterification reaction utilizing terephthalic acid according to the following reaction: n C6H4(CO2H)2+n HOCH2CH2OH → [(CO)C6H4(CO2CH2CH2O)] n + 2n H2O

[0184] Polyethylene terephthalate, produced either by esterification or deesterification of monoethylene glycol, has considerable applicability in the manufacture of bottles and other packaging applications, particularly plastic bottles. It is also used in the production of high-strength fibers such as Dacron, and may be used as part of durable blends with other fibers, such as rayon, wool, and cotton, for insulating clothing, furniture, pillow stuffing, artificial silk, carpet fibers, yarn for automobile tires, conveyor and drive belts, fire and garden hose reinforcement, seat belts, nonwovens for drains, culverts, railroad stabilization, diaper topsheets, and disposable medical garments.

[0185] At its high molecular weight, PET can be made into a high-strength plastic that can be formed by all the common methods used with other thermoplastics. Magnetic recording tape and photographic film are made by extrusion of PET film. Molten PET can be blown into strong, rigid, transparent containers that are virtually impermeable to gases and liquids. In this form, PET is widely used in bottles, especially plastic bottles and jars.

[0186] The present disclosure provides compositions comprising ethylene glycol produced by a microorganism according to the methods described herein. For example, the composition comprising ethylene glycol may be an antifreeze, a preservative, a dehydrating agent, or a drilling fluid.

[0187] The present disclosure also provides polymers containing ethylene glycol produced by microorganisms according to the methods described herein. Such polymers may be homopolymers, such as polyethylene glycol, or copolymers, such as polyethylene terephthalate. Methods for synthesizing these polymers are well known in the art. See, for example, Herzberger et al., Chem Rev., 116(4): 2170-2243 (2016) and Xiao et al., Ind Eng Chem Res. 54(22): 5862-5869 (2015).

[0188] The present disclosure further provides polyethylene glycol conjugates. In some embodiments, the polyethylene glycol (PEG) conjugate comprises PEG conjugated to a biopharmaceutical, a protein, an antibody, an anticancer drug, or any combination thereof. In other embodiments, the PEG conjugate is diethyl terephthalate (DET). In some embodiments, the PEG conjugate is dimethoxyethane.

[0189] The present disclosure further provides a composition comprising a polymer comprising ethylene glycol produced by a microorganism according to the methods described herein. For example, the composition may be a fiber, resin, film, or plastic.

[0190] In one embodiment, ethanol or ethyl alcohol produced according to the methods of the present disclosure is used in antiseptic hand lotions (WO 2014 / 100851), therapeutic treatments for methylene glycol and methanol poisoning (WO 2006 / 088491), pain relievers (WO2011 / 034887) and oral hygiene products (U.S. Patent No. 6,811,769), as well as antibacterial preservatives (U.S. Patent Application No. 2013 / 0230609), engine fuels (U.S. Patent No. 1,128,549), rocket fuels (U.S. Patent No. 3,020,708), plastics, fuel cells (U.S. Patent No. 2,405,986), domestic fire fuels (U.S. Patent No. 4,692,168), industrial chemical precursors (U.S. Patent No. 3,102,875), cannabis solvents (WO2015 / 073854), as winterization extraction solvents (WO2017 / 161387), paint masking products (WO 1992 / 008555), paints or tinctures (U.S. Pat. No. 1,408,091), DNA and RNA purification and extraction (WO 1997 / 010331), cooling baths for various chemical reactions (U.S. Pat. No. 2,099,090), and numerous other product applications. In addition to the foregoing, the ethanol produced by the disclosed methods can be used in any other application where ethanol may otherwise be applicable.

[0191] In additional embodiments, the isopropanol or isopropyl alcohol (IPA) produced according to the present methods may be used in a number of product applications, including either isolated or as a feedstock for the manufacture of more complex products. Isopropanol may also be used in products such as cosmetics and personal care products, deicers, paints and resins, foods, inks, adhesives, and pharmaceutical tablets, as well as solvents for pharmaceuticals, including disinfectants, sterilants, and skin creams.

[0192] The resulting IPA can be used in the extraction and purification of natural products such as vegetable and animal oils and fats. Other applications include its use as a cleaning and drying agent in the manufacture of electronic components and metals, and as an aerosol solvent in medical and veterinary products. It can also be used as a coolant, coupling agent, polymerization modifier, deicer, and antiseptic in pile manufacturing.

[0193] Alternatively, IPA produced according to the methods of the present disclosure may be used to produce additional useful compounds, including plastics, derivative ketones such as methyl isobutyl ketone (MIBK), isopropylamine, and isopropyl esters. Still further, IPA can be converted to propylene according to the following equation:

[0194] CH3CH2CH2OH→CH3-CH=CH2

[0195] The propylene produced can be used as a monomer base for the production of various polypropylene oligomers by chain growth polymerization via either gas-phase or bulk reactor systems. The most common catalysts consist of titanium(III) chloride, the so-called Diegler-Natta catalyst, and metallocene catalysts.

[0196] The polypropylene oligomers so produced may be classified according to tactic and may be formed into a number of products, either by extrusion or molding of polypropylene pellets, including expanded polypropylene, which may be used in plumbing products, heat-resistant items such as kettles and food containers, disposable bottles (including plastic bottles), transparent bags, flooring materials such as rugs and mats, ropes, adhesive stickers, and building materials. Polypropylene may also be used in hydrophilic clothing and medical dressings.

[0197] 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 in the manufacture of a personal care product, a textile, a plastic, a biomedical product, or any combination thereof. In another embodiment, the tandem repeat protein comprises at least one polypeptide of the present disclosure, silk fibers and / or copolymers of the present disclosure, one or more acceptable carriers, or any combination thereof. In one embodiment, the product further comprises a pharmaceutical agent. In another embodiment, the product is used as a pharmaceutical, a medical device, a cosmetic, or any combination thereof. In one embodiment, the tandem repeat protein comprises silk fibers, copolymers, drugs used in the manufacture of a pharmaceutical for treating or preventing disease. In some embodiments, the tandem repeat proteins, fibers, copolymers, or any combination thereof may be used in a wide and diverse array of medical, military, industrial, and commercial applications. In one embodiment, the tandem repeat proteins can be used in the manufacture of medical devices, including sutures, skin grafts, cell growth matrices, replacement ligaments, surgical meshes, or any combination thereof. In other embodiments, the tandem repeat proteins can be used in industrial and commercial products, including cables, ropes, netting, fishing lines, clothing fabrics, bulletproof vest linings, container fabrics, backpacks, knapsacks, bag and purse straps, adhesive bonding materials, non-adhesive bonding materials, strapping materials, tent fabrics, tarpaulins, pool covers, vehicle covers, fencing materials, sealants, building materials, weatherproofing materials, flexible partition materials, sporting goods, or combinations thereof. In one embodiment, the tandem repeat proteins can be used in any fiber or textile where high tensile strength and elasticity are desirable. In one embodiment, the tandem repeat proteins can be used in their native, modified, or derivative forms, or any combination thereof. In some embodiments, the tandem repeat proteins can be spun together and / or bundled or braided with other fiber types.The present disclosure contemplates that such combinations of the present disclosure can be readily manufactured to enhance any desired property, including, but not limited to, improved appearance, softness, weight, durability, water repellency, and manufacturing costs, which may be commonly sought in the production and manufacturing of fibers for medical, industrial, or commercial applications. In some embodiments, the tandem repeat protein is a cosmetic or skin care composition comprising an anhydrous composition having an effective amount of the tandem repeat protein in a cosmetically acceptable medium. In one embodiment, the composition includes, but is not limited to, a skin care, skin cleanser, prosthetic, anti-wrinkle product, or any combination thereof. In another embodiment, the composition includes, but is not limited to, a cosmetic soap, a facial cleanser, a shampoo, a rinse, a hair dye, a hair cosmetic, a general cream, an emulsion, a shaving cream, a conditioner, a cologne, a shaving lotion, a cosmetic oil, a face mask, a foundation, an eye brow pencil, an eye cream, an eye shadow, a mascara, a perfume, a tan and sunscreen cosmetic, a sunscreen lotion, a nail cosmetic, an eyeliner, a lip cosmetic, an oral care product, a toothpaste, or any combination thereof. In another embodiment, the tandem repeat protein is used as a coating on a bandage to promote wound healing, a bandage material, a porous fabric, or any combination thereof. In one embodiment, the tandem repeat protein may be used in a film, including a wound dressing material, an amorphous film, or any combination thereof. In one embodiment, the tandem repeat protein is used in a stent, a stent graft, or any combination thereof. In one embodiment, the tandem repeat protein may be used in a thread, a braid, a sheet, a powder, or any combination thereof. In one embodiment, the stent graft may contain a coating on some or all of the tandem repeat protein, which coating degrades upon insertion of the stent graft into a host, thereby delaying 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 tandem repeat protein containing stent graft may contain a bioactive agent (drug), where the drug is released from the stent graft and subsequently induces an enhanced cellular response (e.g., cellular or extracellular matrix deposition) and / or a fibrotic response in the host into which the stent graft is inserted. In some embodiments, tandem repeat proteins may also be used in matrices for producing ligaments and tendons ex vivo. In one embodiment, the tandem repeat protein is used in a hydrogel. In one embodiment, the tandem repeat proteins of the present disclosure can be applied to the surface of fibers for use in textiles. In one embodiment, textile materials include, but are not limited to, cotton textile fibers, polyesters such as rayon and Lycra™, nylon, wool, and other natural fibers, including natural silk. In some embodiments, compositions suitable for applying silk proteins to fibers can include cosolvents such as ethanol, isopropanol, hexafluoranol, isothiocyanoalaninate, and other polar solvents that can be mixed with water to form a solution or microemulsion. The tandem repeat protein-containing solution can be sprayed onto the fibers, or the fibers can be immersed in the solution. In some embodiments, rapid drying of the coated material is utilized. In another embodiment, the tandem repeat protein composition is applied onto woven fibers. In one embodiment, the tandem repeat protein is used to coat stretch weaves, including stretch garments, stockings, or any combination thereof.In one embodiment, tandem repeat proteins can be added to polyurethanes, other resins, or thermoplastic fillers to prepare panelboards and other structural materials, or as molded furniture and benchtops to replace wood and particleboard. In one embodiment, composites can also be used in building and automotive construction, particularly rooftops and door panels. In other embodiments, tandem repeat protein fibers reinforce resins, making them much stronger, including lightweight structures with equal or superior strength to other particleboards and composites. In some embodiments, tandem repeat protein fibers are isolated and added to synthetic composite-forming resins, and used in combination with plant-derived proteins, starches, and oils to create biologically based composites. In one embodiment, tandem repeat proteins are paper additives. In another embodiment, tandem repeat proteins are used in technical and intelligent textiles. In some embodiments, technical and intelligent textiles do not change properties when wet, maintaining their strength and extensibility. In one embodiment, the tandem repeat proteins are used in functional garments for sports and leisure wear, work wear, protective clothing, or any combination thereof, hi some embodiments, the tandem repeat proteins are used in garments, equipment, materials for durability against prolonged exposure, heavy wear, personal protection from the external environment, ballistic projectile resistance, fire and chemical resistance, or any combination thereof.

[0198] In one embodiment, ethylene is used to produce butadiene, hi some embodiments, butadiene is used in rubber tires.

[0199] In one embodiment, a method for the continuous production of ethylene, the process comprises passing a gaseous substrate through a bioreactor containing a culture of a recombinant C1-fixing microorganism capable of producing ethylene in a culture medium, whereby the microorganism converts the gaseous substrate to ethylene, and recovering the ethylene from the bioreactor.

[0200] In other embodiments, the ethylene is converted into components used in the manufacture of tires. In one embodiment, the ethylene is converted into components used in tire screws.

[0201] 10. The method of claim 9, wherein the tire is an end-of-life tire.

[0202] 10. The method of embodiment 1, wherein the gaseous substrate is derived from a process involving tires.

[0203]

[0013] 3. The method of embodiment 1, wherein the gaseous substrate is derived from a product recycling process or a sustainable chemical process.

[0204] 10. The method of claim 1, further comprising converting the ethylene into components used to manufacture new tires.

[0205] 10. The method of claim 1, comprising a resin component selected from ethylene and other olefins coupled to a synthetic component selected from butadiene and isoprene to form a hybrid polymer for use in manufacturing tires.

[0206] One embodiment is directed to a method for producing a polymer from a gaseous substrate, comprising: a first gas fermentation process producing at least one first product selected from butadiene, isoprene, conjugated dienes, or any combination thereof; a second gas fermentation process producing at least one second product selected from ethylene and olefins, or any combination thereof; and wherein the at least one first product and the at least one second product copolymerize to form the polymer.

[0207]

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

[0208]

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

[0209]

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

[0210]

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

[0211] 10. The method of claim 9, wherein the rubber component is selected from butadiene, isoprene, conjugated dienes, or any combination thereof.

[0212] 10. The method of embodiment 1, wherein the resin component is selected from ethylene, olefin, or any combination thereof.

[0213]

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

[0214] 10. The method of embodiment 1, wherein the metallocene complex is a complex compound having one or more cyclopentadienyl groups or derivative cyclopentadienyl groups bonded to the metal center.

[0215] 10. The method of embodiment 1, wherein the central metal is selected from a lanthanide element, scandium, yttrium, or any combination thereof.

[0216] 10. The method of claim 9, wherein the central metal is selected from samarium (Sm), neodymium (Nd), praseodymium (Pr), gadolinium (Gd), cerium (Ce), holmium (Ho), scandium (Sc), and yttrium (Y).

[0217] 10. The method of claim 1, further comprising converting the polymer into a tire.

[0218] One embodiment for the circular production of tires from a gaseous substrate is directed to a first gas fermentation process to produce at least one first product selected from butadiene, isoprene, conjugated dienes, or any combination thereof, and a second gas fermentation process to produce at least one second product selected from ethylene and olefins, or any combination thereof, wherein the at least one first product and the at least one second product are copolymerized to form a polymer, and the substrate is derived from the process comprising tires.

[0219] 10. The method of embodiment 1, wherein the substrate is derived from a process involving end-of-life tires.

[0220] One embodiment is directed to a method for the circular production of tires, the method comprising: 1) passing a gaseous substrate through a first bioreactor containing a culture of a recombinant C1-fixed microorganism capable of producing at least one first product selected from butadiene, isoprene, conjugated dienes, or any combination thereof in a culture medium, whereby the microorganism converts the gaseous substrate to the at least one first product, and recovering the at least one first product from the bioreactor; 2) passing the gaseous substrate through a second bioreactor containing a culture of a recombinant C1-fixed microorganism in a culture medium, whereby the microorganism converts the gaseous substrate to at least one second product selected from ethylene and olefins, or any combination thereof, whereby the microorganism converts the gaseous substrate to at least one second product, and recovering the at least one second product from the bioreactor; 3) polymerizing the at least one first product with at least one second product in the presence of a suitable polymerization catalyst to form a hybrid polymer; and 4) converting the hybrid polymer into a tire.

[0221]

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

[0222] 10. The method of embodiment 1, wherein the metallocene complex is a complex compound having one or more cyclopentadienyl groups or derivative cyclopentadienyl groups bonded to the metal center.

[0223] 10. The method of embodiment 1, wherein the central metal is selected from a lanthanide element, scandium, yttrium, or any combination thereof.

[0224] 10. The method of claim 9, wherein the central metal is selected from samarium (Sm), neodymium (Nd), praseodymium (Pr), gadolinium (Gd), cerium (Ce), holmium (Ho), scandium (Sc), and yttrium (Y).

[0225] 10. The method of claim 1, wherein the first bioreactor and the second bioreactor are run in parallel.

[0226] 10. The method of claim 1, wherein both the first bioreactor and the second bioreactor are operated continuously.

[0227] 10. The method of embodiment 1, wherein the substrate is derived from a process involving end-of-life tires.

[0228] 3. The method of claim 1, further comprising converting the isoprenoid into a product selected from synthetic rubber, a styrene-containing block polymer, a thermoplastic rubber, a pressure-sensitive or thermoset adhesive, a butyl rubber, a terpene selected from citral, linalool, ionone, myrcene, L-menthol, N,N-diethylnerylamine, geraniol, and nerolidol, a flavorant, a fragrance, a fuel additive, a plastic, and polyisoprene.

[0229] 10. The method of claim 1, further comprising converting the butadiene to a product selected from styrene butadiene rubber, synthetic rubber, tires, tire components, thermoplastic rubber, shoes, shoe soles, adhesives, sealants, asphalt, polymer-modified components, nylon, ABS resin, chloroprene / neoprene rubber, nitrile rubber, plastics, acrylic resins, acrylonitrile-butadiene-styrene resins, and synthetic elastomers.

[0230] One embodiment is directed to a method for chemical recycling, the method including providing to a pyrolysis, gasification, and / or partial oxidation process, a gas fermentation process, a pharmaceutical manufacturing process to produce products including butadiene, isoprenoids, ethylene, polyethylene terephthalate (PET), or any combination thereof, a synthetic rubber manufacturing process, a tire manufacturing process, a tire-using process, a process for collecting and shredding used tires, and returning to the pyrolysis, gasification, and / or partial oxidation process.

[0231] One embodiment is directed to a method for chemical recycling, the method including: 1) providing to a pyrolysis, gasification, and / or partial oxidation process; 2) providing to a gas fermentation process; 3) providing to a pharmaceutical manufacturing process to produce products including butadiene, isoprenoids, ethylene, polyethylene terephthalate (PET), or any combination thereof; 4) providing to a synthetic rubber manufacturing process; 5) providing to a tire manufacturing process; 6) providing to a tire-using process; 7) providing to a process for collecting and shredding used tires; and 8) providing back to pyrolysis, gasification, and / or partial oxidation process.

[0232] One embodiment is directed to a method for chemical recycling, the method comprising: 1) providing to a pyrolysis, gasification, and / or partial oxidation process; 2) providing to a gas fermentation process; 3) providing to a pharmaceutical manufacturing process to produce a product; 4) providing to a synthetic rubber manufacturing process; 5) providing to a tire manufacturing process; 6) providing to a tire use process; 7) providing to a process for collecting and shredding used tires; and 8) returning to the pyrolysis, gasification, and / or partial oxidation process.

[0233] Another embodiment is directed to a method for chemical recycling, the method including: 1) pyrolysis, gasification, and / or a partial oxidation process to produce an effluent; 2) passing the effluent through a gas fermentation process to produce a product; 3) passing the gas fermentation product through a chemical manufacturing process to produce a product; 4) passing the product through a synthetic rubber manufacturing process to produce synthetic rubber; 5) passing the synthetic rubber product through a tire manufacturing process to produce tires; 6) providing the tires to a tire-using process; 7) passing the used tires through a process for used tire collection and shredding; and 8) returning the used tires to the pyrolysis, gasification, and / or partial oxidation process.

[0234] One embodiment is directed to a genetically engineered microorganism capable of producing a commodity chemical, a tandem repeat protein product, a microbial biomass, a single cell protein (SCP), one or more intermediates, or any combination thereof.

[0235] 2. The microorganism of embodiment 1, wherein the microorganism produces a heterologous protein product, and wherein the microorganism comprises a heterologous nucleic acid encoding at least one protein having tandem repeats.

[0236] A microorganism described in an embodiment, wherein the microorganism comprises a genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one secreted chemical product from a gaseous substrate, the microorganism comprises a heterologous nucleic acid encoding at least one protein having tandem repeats and a heterologous nucleic acid encoding at least one secreted chemical product, and the microorganism is a C1-fixing bacterium.

[0237] A microorganism according to one embodiment, wherein the microorganism comprises a genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one secreted chemical product from a gaseous substrate, wherein the microorganism comprises a heterologous nucleic acid encoding at least one protein having one or more tandem repeats and a heterologous nucleic acid encoding at least one secreted chemical product, and wherein the microorganism is a C1-fixing bacterium.

[0238] A microorganism according to an embodiment, wherein the microorganism comprises a genetically engineered C1-fixing microorganism capable of co-producing a heterologous protein and a chemical product from a gaseous substrate, and the microorganism comprises a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeats and a heterologous nucleic acid encoding at least one chemical product.

[0239] 1. A microorganism according to one embodiment, wherein the microorganism comprises a genetically engineered C1-fixing microorganism capable of co-producing a heterologous protein and a chemical product from a gaseous substrate, wherein the microorganism comprises a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeats and a heterologous nucleic acid encoding at least one chemical product, and wherein the microorganism is capable of accumulating the at least one heterologous protein intracellularly and secreting the at least one chemical product from the cell.

[0240] 10. The microorganism of claim 1, wherein the microorganism comprises one or more heterologous enzymes from a genus selected from the group consisting of Bacillus, Clostridium, Cupriavidus, Escherichia, Gluconobacter, Hyphomicrobium, Lysinibacillus, Paenibacillus, Pseudomonas, Sedimenticola, Sporosarcina, Strreptomyces, Thermithiobacillus, Thermotoga, and Zea.

[0241] A microorganism according to an embodiment, wherein 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 two or more carbons from a gaseous substrate, wherein the microorganism comprises a heterologous nucleic acid encoding at least one protein having tandem repeats and a heterologous nucleic acid encoding at least one secreted chemical product.

[0242] A microorganism according to an embodiment, wherein 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 two or more carbons from a gaseous substrate, wherein the microorganism comprises a heterologous nucleic acid encoding a group of genes comprising at least one protein having tandem repeats and at least one secreted chemical product.

[0243] A microorganism according to one embodiment, a genetically engineered microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, wherein the microorganism comprises a heterologous nucleic acid encoding at least one protein having one or more tandem repeats and a heterologous nucleic acid encoding at least one chemical product, and the microorganism is a C1-fixing bacterium.

[0244]

[0013] In accordance with an embodiment of the present invention, the microorganism comprises a genetically engineered C1-fixing 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 repeats; b) a heterologous nucleic acid encoding at least one chemical product having two or more carbons, wherein the heterologous nucleic acid enables the microorganism to accumulate at least one heterologous protein within the cell and secrete at least one chemical product from the cell.

[0245] 10. The method of claim 1, wherein the method comprises culturing a genetically engineered C1-fixing microorganism in the presence of a gaseous substrate comprising one or more of CO, CO2, and H2, and wherein the culturing is a continuous fermentation process.

[0246] 10. The method of claim 1, wherein the culture is a continuous fermentation process.

[0247]

[0023] 3. The method of embodiment 1, wherein the gaseous substrate comprises a C1 carbon source comprising one or more of CO, CO2, and / or H2.

[0248] 10. The method of embodiment 1, wherein the gaseous substrate comprises synthesis gas or industrial waste gas.

[0249] 10. The method of claim 9, wherein the method comprises culturing an engineered C1 fixative to co-produce at least one heterologous protein having one or more tandem repeats and at least one chemical product, wherein the chemical product is one or more of ethylene, ethanol, acetone, isopropanol, or any combination thereof.

[0250] The microorganism of an embodiment, wherein the microorganism comprises a genetically engineered C1-fixing microorganism, and the at least one heterologous protein having one or more tandem repeats is selected from collagen, silk, elastin, keratin, resilin, titin, squid ring tooth (SRT) protein, saccharin, or any combination thereof.

[0251] 10. The microorganism of an embodiment, wherein 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.

[0252] Microorganisms include 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, Cupriavidus necator, Eubacterium limosum, Moorella thermautotrophica, Moorella thermoacetica, Oxobacter pfennigii, Ralstonia eutropha, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, and Thermoanaerobacter kiuvi.

[0253] 10. The microorganism of embodiment 1, wherein the microorganism is derived from a parent bacterium selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei.

[0254] 2. The microorganism of embodiment 1, wherein the microorganism is derived from a parent bacterium selected from the group consisting of Cupriavidus necator.

[0255] 2. The microorganism of embodiment 1, wherein the at least one heterologous protein having one or more tandem repeats is selected from a silk or SRT protein.

[0256] 10. The microorganism of embodiment 1, wherein the gas fermentation product is selected from an alcohol, an acid, a diacid, an alkene, a terpene, an isoprene, and an alkyne, or any combination thereof.

[0257] 10. The microorganism of embodiment 1, wherein the 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-propanediol, 1-propanol, 1-hexanol, 1-octanol, chorismic acid derived products, 3-hydroxybutyric acid, 1,3-butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, isoamyl alcohol, or monoethylene glycol.

[0258] 10. The microorganism of embodiment 1, wherein the 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-propanediol, 1-propanol, 1-hexanol, 1-octanol, chorismic acid derived products, 3-hydroxybutyric acid, 1,3-butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipate, 1,3-hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, isoamyl alcohol, monoethylene glycol, or a combination thereof.

[0259] 2. The microorganism of embodiment 1, wherein the microorganism further comprises a disruptive mutation in one or more genes.

[0260] 10. The microorganism of embodiment 1, wherein the genetically engineered C1-fixing microorganism further comprises a microbial biomass and at least one excipient.

[0261] 10. The microorganism of an embodiment, wherein the microorganism is a genetically engineered C1-fixing microorganism, wherein the microbial biomass is suitable as animal feed.

[0262] 10. The method of claim 1, wherein the animal feed is suitable for feeding one or more of beef cattle, dairy cattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, banteng, gayal, yaks, chickens, turkeys, ducks, geese, quails, guinea fowl, pigeons / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents.

[0263] A microorganism according to an embodiment, wherein the genetically engineered C1-fixing microorganism is suitable as a single cell protein (SCP).

[0264] In some aspects, the microbial biomass has a unit value. In one embodiment, the microbial biomass has a market value.

[0265] A microorganism according to an embodiment, wherein the genetically engineered C1-fixing microorganism is suitable as a cell-free protein synthesis (CFPS) platform.

[0266] 10. The microorganism of any one of embodiments 1 to 9, wherein the microorganism is a genetically engineered C1-fixing microorganism, wherein at least one secreted chemical product is native to the microorganism.

[0267] 2. The microorganism of claim 1, wherein the at least one heterologous protein is a squid ring tooth protein and the at least one chemical product is ethylene.

[0268] 2. The microorganism of embodiment 1, wherein the at least one heterologous protein is a silk protein and the at least one chemical product is ethylene.

[0269] 2. The microorganism of embodiment 1, wherein the at least one chemical product is ethylene.

[0270]

[0023] 3. The method of embodiment 1, wherein the substrate comprises one or more of CO, CO2, and H2.

[0271] 10. The method of embodiment 1, wherein at least a portion of the substrate is an industrial waste gas, an industrial off-gas, or a synthesis gas.

[0272]

[0013] In embodiments, the method can be performed using both anaerobic and aerobic gases to feed separate cultures (e.g., an anaerobic culture and an aerobic culture) in two or more different bioreactors that are both incorporated into the same process stream. [Example]

[0273] The following examples further illustrate the present disclosure but, of course, should not be construed as in any way limiting its scope.

[0274] Example 1: Production of tandem repeat proteins in the autotroph Clostridium autoethanogenum. 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 genes contain DNA encoding an N-terminal twin streptidase as a handle for protein detection via Western blot and / or affinity purification (Schmidt, Protein Expr Purif 92:54-61, 2013). These vectors contain pre-cloned Clostridial promoters and terminators. The promoter sequences are described in Karim et al. Synthetic Biology 2020;5(1):ysaa019. The resulting plasmids contain the ermB antibiotic selection marker. After transformation into Clostridium, sequence-verified strains were grown autotrophically in six-well plates.

[0275] Protein expression experiments were initiated in 6-well plates containing 3 mL of minimal medium containing yeast extract and a syngas mixture (55% CO, 5% H, 30% CO, and 10% N) at 200 kPa and grown at 37 °C until the strain reached a biomass concentration of 0.20–0.43 g DCW / L. Strains were then subcultured at 37 °C to 0.006–0.03 g DCW / L in 1 L Schott bottles containing 200 mL of minimal medium in the presence of a syngas mixture (55% CO, 5% H, 30% CO, and 10% N) at 150 kPa. Biomass concentrations were monitored until they reached 0.13–0.32 g DCW / L, and then the biomass was harvested for protein detection.

[0276] Tandem repeat protein production was assessed by Western blot analysis using an anti-Strep tag antibody. Cultures were lysed and clarified, and the clarified lysate and insoluble pellet (resuspended in 5 M urea) were analyzed separately for protein content. Samples were run on Tris-glycine SDS-PAGE, transferred to nitrocellulose membranes, and visualized by probing with an anti-Strep tag antibody conjugated to alkaline phosphatase. Proteins of the expected size were observed in the insoluble pellet for SRT008, SRT011, SRT012, and SS015. Additionally, SS015 was observed in the clarified lysate.

[0277] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0278] Example 2: Production of SRT008 and SRT012 from syngas fermentation in a batch CSTR. Tandem repeat protein-containing strains SRT008 and SRT012 (Table 2) were characterized in a CSTR in batch mode for protein and chemical production. Actively growing (early exponential) cultures harvested from Schott bottles were used to inoculate a 2 L CSTR using a synthetic gas blend (55% CO, 25% H, 30% CO, 10% N) at atmospheric pressure. A gas outage occurred during the run, causing culture perturbation and terminating SRT008 earlier than expected.

[0279] SRT008 achieved a peak biomass concentration of 2.5 g DCW / L (Figure 3B) and peak CO2 uptake of 1331 mmol / L / d (Figure 3C). In addition to a peak ethanol concentration of 26.88 g / L (Figures 3A and 3B), this strain produced a peak acetate titer of 4.14 mg / L and a peak butanediol titer of 4.73 mg / L (Figure 3B). SRT008 production was monitored via Western blot on days 0, 2.78, and 5.81, with the highest relative protein content on day 0.

[0280] SRTO12 achieved a peak biomass concentration of 1.31 g DCW / L (Figure 4B) and reached a peak CO uptake of 1537 mmol / L / d on day 2.73 before experiencing mechanical problems. In addition to a peak ethanol concentration of 9.57 g / L (Figures 4A and 4B), this strain produced a peak acetate titer of 8.27 mg / L and a peak butanediol titer of 0.57 mg / L (Figure 4B). SRT012 production was monitored via Western blot on days 0, 1.78, and 5.02, with the highest relative protein content on day 1.78 (Figure 4C). The final data point for protein content per biomass was obtained after gas shutoff.

[0281] Example 3: SRT012 production from syngas fermentation in a continuous CSTR. Under continuous CSTR conditions using strain SRT012, a syn-gas mixture (55% CO, 5% H, 30% CO, and 10% N), a 3 L reactor, and a cell recycling membrane (CRM) was used. A dilution (D) rate of 1.2 vessels per day (v / d) was initiated on day 5, then increased to 1.5 v / d on day 12.9, and finally reached a D of 2.5 v / d between days 12.9 and 60. Biomass concentration accumulated to a peak concentration of 20.85 g DCW / L (Figure 2B), and CO gas uptake peaked at 9,300 mmol / L / d (Figure 2C). This strain had a maximum ethanol concentration of 34.82 g / L and a maximum acetate concentration of 11.79 g / L (Figure 2B).

[0282] SRT008 protein was observed in all samples analyzed (Figure 2A). SRT008 production was assessed by Western blot analysis using an anti-Strep tag antibody. Samples were harvested from the CSTR and frozen for later analysis. After thawing, cells were lysed and the insoluble pellet was resuspended in 5 M urea. Samples were diluted with Laemmli sample buffer and run on a Tris-glycine SDS-PAGE protein gel. Samples were transferred to nitrocellulose membranes, stained with Ponceau S for total protein visualization, and then probed with an anti-Strep tag antibody conjugated to horseradish peroxidase for specific protein visualization. Specific protein content was measured and normalized to total protein content by densitometric analysis. Specific protein content normalized to total protein content is reported relative to day 5 (reactors are continuously run). The highest relative protein content was on day 0, with another peak occurring between days 45 and 55, approximately the same time a decline in ethanol production occurred and the cell recycling membrane was replaced.

[0283] Example 4: Production of SRT008 and SRT012 from high hydrogen syngas fermentation in a batch CSTR. Tandem repeat protein-containing strains SRT008 and SRT012 (Table 2) were characterized in a CSTR in batch mode for protein and chemical production. Actively growing (early exponential) cultures harvested from Schott bottles were used to inoculate a 2 L CSTR at atmospheric pressure using a synthetic gas blend (55% CO, 5% H, 30% CO, and 10% N). Cultures were grown in the reactor using a high-hydrogen gas blend (10% CO, 50% H, 30% CO, and 10% N).

[0284] SRT008 achieved a peak biomass concentration of 1.32 gDCW / L (Figure 5B), a peak CO uptake of 590 mmol / L / d, and a peak H2 uptake of 2060 mmol / L / d (Figure 5C). In addition to a peak ethanol concentration of 45.06 g / L (Figure 5B), this strain reached a peak acetate titer of 5.47 g / L (Figure 5B). As shown in all figures, there was an agitation event just before day 4, and the agitator was discontinued for approximately 45 minutes. The culture was affected but recovered shortly thereafter. SRT008 production was monitored via Western blot on days 0, 1.7, 2.7, 3.7, and 6.8, with peak protein content observed on day 0 (Figure 5A).

[0285] SRT012 achieved a peak biomass concentration of 1.75 gDCW / L (Figure 6B), a peak CO uptake of 535 mmol / L / d, and a maximum H uptake of 2074 mmol / L / d (Figure 6C). In addition to a peak ethanol concentration of 52.46 g / L (Figure 6B), this strain reached a peak acetate titer of 5.47 g / L (Figure 6B). SRT012 production was monitored via Western blot at days 0, 1.7, 3.7, 5.8, and 10.7, and peak protein content was measured at day 10.7 (Figure 6A).

[0286] Example 5: Continuous ethylene production from CO with H as energy source. A gene encoding an ethylene-forming enzyme was codon-adapted and synthesized for expression in Cupriavidus necator. The adapted gene, along with the constitutive promoter P10, was cloned into the broad-host-range expression vector pBBR1MCS2. The resulting product was used to transform E. coli, and positive clones identified by PCR were confirmed by DNA sequencing. The sequence-confirmed plasmid was then transformed into Cupriavidus necator PHB-4 via electroporation and selected on tryptic soy broth (TSB) agar plates containing 50 mg / L chloramphenicol. Transformants containing the pBBR1-Efe plasmid were confirmed via sequencing and single colonies were then grown in TSB at 30°C overnight and used to generate glycerol stocks for storage at -80°C. Strain recovery was performed by streaking onto TSB plates containing 50 mg / L chloramphenicol with 72-hour incubation at 30°C.

[0287] A single colony from a freshly streaked TSB plate was used to inoculate 3 mL of TSB containing 50 mg / L chloramphenicol in a 14 mL Falcon round-bottom polystyrene test tube with a snap cap. After overnight incubation at 30°C and 200 rpm in a Thermo MAXQ shaker, 1 mL of the culture was used to inoculate 100 mL of LB in a 200 mL Schott bottle. Cells were grown at 30°C and 200 rpm until an optical density of approximately 0.3–0.4 was reached.

[0288] 100 mL of the above culture was used to inoculate a 1.4 L Infors HT Multifors 2 CSTR containing 600 mL of 2x start-up medium. The reactor was incubated at 30°C and started with 250 rpm agitation and 150 nccm gas flow (3.14% O, 41% H, 3% CO, 52.86% N). Agitation and gas flow were increased to a maximum of 1450 rpm and 750 nccm as the culture grew. OD 600When the ΔΨ exceeded 0.5, the culture was continuously rotated using 4x medium containing 7 μL / h of Pluronic 31R1 antifoam. The supply oxygen rate was gradually increased to promote biomass production, with the balance removed from the nitrogen rate, and the outlet oxygen rate was constrained to remain below 4.5% as a safety measure.

[0289] Gas samples from the reactor were piped through 305 stainless steel to a flow selection valve controlled by a micro GC (manufacturer: Qmicro). The samples were then analyzed on a Rt-U SIP XP PLOT column under isothermal conditions (70 °C) via a thermal conductivity detector (TCD).

[0290] After the cultures were well established, the gas fraction was adjusted from O-limited to H-limited conditions and non-zero dissolved oxygen (DO) concentrations were observed. Ethylene production fluctuated as the system settled to a steady state and gas fractions were adjusted, but production was maintained over an 11-day period (Figure 7). During this period, the H fraction ranged from 11-18%, the O fraction from 5.5-6.6%, CO was held at 3%, and N was maintained as the balance. Upon returning to O-limited conditions, ethylene production ceased, demonstrating the importance of oxygen availability for ethylene production.

[0291] All references, including publications, patent applications, and patents, cited herein are incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The reference herein to any prior art is not, and should not be construed as, an acknowledgment that that prior art forms part of the common general knowledge in any country in the field of endeavor.

[0292] In the context of describing this disclosure (particularly in the context of the claims that follow), the use of the terms "a," "an," and "the" and similar referents shall be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" shall be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise noted. The term "consisting essentially of" limits the scope of a composition, process, or method to certain materials or steps, or to those that do not materially affect the basic and novel characteristics of the composition, process, or method. The use of alternatives (e.g., "or") should be understood to mean either one of the alternatives, both, or any combination thereof. As used herein, the term "about" means ±20% of the stated range, value, or structure, unless otherwise indicated.

[0293] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually recited herein. For example, any concentration range, percentage range, ratio range, integer range, size range, or thickness range should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as integer tenths and hundredths), unless otherwise indicated.

[0294] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better elucidate the invention and does not limit the scope of the invention unless otherwise asserted. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0295] Preferred embodiments of the present disclosure are described herein. Variations of those preferred embodiments may become apparent to those skilled in the art upon reading the above description. The inventors anticipate that those skilled in the art will adopt such variations as necessary, and the inventors intend for the present disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0296] Embodiment 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, wherein the microorganism comprises a heterologous nucleic acid encoding at least one protein having one or more tandem repeats and a heterologous nucleic acid encoding the at least one chemical product, and the microorganism is a C1-fixing bacterium.

[0297] Embodiment 2: The genetically engineered microorganism of embodiment 1, wherein the at least one heterologous protein having one or more tandem repeats is selected from collagen, silk, elastin, keratin, resilin, titin, squid ring tooth (SRT) protein, saccharin, or any combination thereof.

[0298] 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.

[0299] Embodiment 4: The genetically engineered microorganism of embodiment 2, wherein the at least one heterologous protein is a silk protein and the at least one chemical product is ethylene.

[0300] Embodiment 5: The genetically engineered microorganism of embodiment 1, wherein the at least one chemical product is ethylene.

[0301] Embodiment 6: The genetically engineered microorganism of embodiment 2, wherein the at least one heterologous protein having one or more tandem repeats is selected from a silk or SRT protein.

[0302] Embodiment 7: The genetically engineered microorganism of embodiment 1, wherein the 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-propanediol, 1-propanol, 1-hexanol, 1-octanol, chorismic acid-derived products, 3-hydroxybutyric acid, 1,3-butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, isoamyl alcohol, monoethylene glycol, or a combination thereof.

[0303] Embodiment 8: The genetically engineered microorganism of embodiment 1, further comprising a disruptive mutation in one or more genes.

[0304] Embodiment 9: The parent microorganism is Acetobacterium woodii, Alkalibaculum bacchii, Blautia product, 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, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Cupriavidus necator, and Thermoanaerobacter 2. The genetically engineered microorganism of embodiment 1 selected from the group consisting of:

[0305] Embodiment 10: A method for co-producing at least one heterologous protein having one or more tandem repeats and at least one chemical product by culturing the genetically engineered microorganism of Embodiment 1 in the presence of a gaseous substrate comprising one or more of CO, CO, and H, wherein the culturing is a continuous fermentation process.

[0306] Embodiment 11: The method of embodiment 10, wherein the gaseous substrate comprises a C1 carbon source comprising one or more of CO, CO2, and / or H2.

[0307] Embodiment 12: The method of embodiment 10, wherein the gaseous substrate comprises synthesis gas or industrial waste gas.

[0308] Embodiment 13: The method of embodiment 10, wherein the chemical product is one or more of ethylene, ethanol, acetone, isopropanol, or any combination thereof.

[0309] Embodiment 14: The genetically engineered microorganism of embodiment 1, further comprising a microbial biomass and at least one excipient.

[0310] Embodiment 15: The genetically engineered microorganism of embodiment 14, wherein the microbial biomass is suitable as animal feed.

[0311] Embodiment 16: The genetically engineered microorganism of embodiment 15, wherein the animal feed is suitable for feeding one or more of beef cattle, dairy cattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, banteng, gayal, yaks, chickens, turkeys, ducks, geese, quails, guinea pigs, squabs / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents.

[0312] Embodiment 17: The genetically engineered microorganism of embodiment 1, wherein the microorganism is suitable as a single cell protein (SCP).

[0313] Embodiment 18: The genetically engineered microorganism of embodiment 1, wherein the microorganism is suitable as a cell-free protein synthesis (CFPS) platform.

[0314] Embodiment 19: The genetically engineered microorganism of embodiment 1, wherein at least one chemical product is native to the microorganism.

[0315] Embodiment 20: A genetically engineered C1-fixing 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 repeats; b) a heterologous nucleic acid encoding at least one chemical product having two or more carbons, wherein the heterologous nucleic acid enables the microorganism to accumulate at least one heterologous protein within the cell and secrete 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, said microorganism comprising a heterologous nucleic acid encoding said at least one protein having one or more tandem repeats and a heterologous nucleic acid encoding said at least one chemical product, said microorganism being a C1-fixing bacterium.

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

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

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

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

7. 10. The genetically engineered microorganism of claim 1, wherein the 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-propanediol, 1-propanol, 1-hexanol, 1-octanol, chorismic acid derived products, 3-hydroxybutyric acid, 1,3-butanediol, 2-hydroxyisobutyrate or 2-hydroxyisobutyric acid, isobutylene, adipic acid, ketoadipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, isoamyl alcohol, monoethylene glycol, or combinations thereof.

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

9. The parent microorganism is Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Butyribacterium methylotrophicum, Clostridium. aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium 2. The genetically engineered microorganism of claim 1, wherein the microorganism is selected from the group consisting of: Clostridium magnum, Clostridium ragsdalei, Clostridium scatologenes, Cupriavidus necator, Eubacterium limosum, Moorella thermautotrophica, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Cupriavidus necator, and Thermoanaerobacter kiuvi.

10. CO, CO ２ , and H ２ 10. A method for co-producing at least one heterologous protein having one or more tandem repeats and at least one chemical product by culturing the genetically engineered microorganism of claim 1 in the presence of a gaseous substrate comprising one or more of:

11. The gaseous substrate is CO, CO ２ , and / or H ２ 11. The method of embodiment 10, comprising one or more C1 carbon sources comprising:

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

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

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

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

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

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

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

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

20. A genetically engineered C1-fixing microorganism capable of co-producing at least one heterologous protein and at least one chemical product from a gaseous substrate, said microorganism comprising: a) a heterologous nucleic acid encoding at least one heterologous protein having one or more tandem repeats; b) a heterologous nucleic acid encoding at least one chemical product having two or more carbons, wherein the heterologous nucleic acid enables the microorganism to accumulate the at least one heterologous protein within the cell and secrete the at least one chemical product from the cell.

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