Patent Application
20200239896
It has long been recognized that catalytic processes, such as the Fischer-Tropsch process, may be used to convert gases containing carbon dioxide (CO2), carbon monoxide (CO), and/or hydrogen (H2), such as industrial waste gas or syngas, into a variety of fuels and chemicals. Recently, however, gas fermentation has emerged as an alternative platform for the biological fixation of such gases. In particular, C1-fixing microorganisms have been demonstrated to convert gases containing CO2, CO, and/or H2 into products such as ethanol and 2,3-butanediol. Efficient production of such products may be limited, however, by slow microbial growth, limited gas uptake, sensitivity to toxins, or diversion of carbon substrates into undesired byproducts. Accordingly, there remains a need for genetically engineered microorganisms having improved characteristics.
The invention provides non-naturally occurring microorganisms comprising at least one disrupted gene. In the microorganisms of the invention, carbon flux is strategically diverted away from nonessential or undesirable products and towards products of interest. In certain embodiments, these disrupted genes divert carbon flux away from nonessential or undesirable metabolic nodes and through target metabolic nodes to improve production of products downstream of those target metabolic nodes.
The microorganisms of the invention are derived from parental bacteria such as Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium magnum, Clostridium ragsdalei, Clostridium scatologenes, Eubacterium limosum, Moorella thermautotrophica, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, or Thermoanaerobacter kiuvi. In a preferred embodiment, the parental bacterium is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a particularly preferred embodiment, the parental bacterium is Clostridium autoethanogenum.
In one embodiment, the invention provides a non-naturally occurring Wood-Ljungdahl bacterium comprising a heterologous thiolase and a disruptive mutation in one or more genes encoding, for example, one or more of NAD-dependent electron-bifurcating [FeFe]-hydrogenase, glutamate synthase, citramalate synthase, acetolactate decarboxylase, lactate dehydrogenase, acetate kinase, phosphate transacetylase, and aldehyde dehydrogenase, wherein the non-naturally occurring bacterium has improved carbon flux through acetoacetyl-CoA compared to a parental bacterium. Specifically, the expression of the one or more genes is decreased or eliminated compared to the parental bacterium.
In such an embodiment, the the non-naturally occurring bacterium may produce a product such as acetone, isopropanol, 3-hydroxyisovaleryl-CoA, 3-hydroxyisovalerate, isobutylene, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, isoprene, farnesene, 3-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxybutyrate, 3-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, or isoamyl alcohol.
For example, when the parental bacterium is Clostridium autoethanogenum, (a) the NAD-dependent electron-bifurcating [FeFe]-hydrogenase may be selected from the group consisting of CAETHG_1576, CAETHG_1578, CAETHG_3569, CAETHG_3570, and CAETHG_3571, (b) the glutamate synthase may be selected from the group consisting of CAETHG_0477, CAETHG_1580, CAETHG_3850, and CAETHG_3851, (c) the citramalate synthase may be CAETHG_2751, (d) the acetolactate decarboxylase may be CAETHG_2932, (e) the lactate dehydrogenase may be CAETHG_1147, (f) the acetate kinase may be CAETHG_3359, (g) the phosphate transacetylase may be CAETHG_3358, or (h) the aldehyde dehydrogenase may be selected from the group consisting of CAETHG_1819, CAETHG_3287, and CAETHG_1830.
In another embodiment, the invention provides a non-naturally occurring Wood-Ljungdahl bacterium comprising a disruptive mutation in one or more genes, wherein the non-naturally occurring bacterium has improved carbon flux through chorismate compared to a parental bacterium.
The one or more genes encode, for example, one or more of purine-nucleoside phosphorylase, lactate permease, cystathionine gamma-lyase, adenine phosphoribosyltransferase, 5′-nucleotidase/3′-nucleotidase/exopolyphosphatase, small conductance mechanosensitive channel, arginine deiminase, LL-diaminopimelate aminotransferase apoenzyme, and phosphopentomutase. Specifically, the expression of the one or more genes is decreased or eliminated compared to the parental bacterium.
In such an embodiment, the the non-naturally occurring bacterium may produce a product such as chorismate, para-hydroxybenzoic acid, salicylate, 2-aminobenzoate, dihydroxybenzoate, 4-hydroxycyclohexane carboxylic acid, and salts and ions thereof.
For example, when the parental bacterium is Clostridium autoethanogenum, the one or more genes may encode one or more of CAETHG_0160, CAETHG_0248, CAETHG_0498, CAETHG_1270, CAETHG_1371, CAETHG_2107, CAETHG_3021, CAETHG_3510, and CAETHG_3924; when the parental bacterium is Clostridium ljungdahlii, the one or more genes may encode one or more of CLJU_c20750, CLJU_c21610, CLJU_c24380, CLJU_c33720, CLJU_c34740, CLJU_c42810, CLJU_c09270, CLJU_c14280, and CLJU_c18150; and when the parental bacterium is Clostridium ragsdalei and the one or more genes may encode one or more of CLRAG_19250, CLRAG_31200, CLRAG_25120, CLRAG_24560, CLRAG_14800, CLRAG_25620, CLRAG_09600, or CLRAG_00520.
The invention also provides methods of producting products by culturing the microorganism of the invention in the presence of a substrate, such as a gaseous substrate comprising one or more of CO, CO2, and/or H2.
The term “non-naturally occurring” when used in reference to a microorganism is intended to mean that the microorganism has at least one genetic modification not found in a naturally occurring strain 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 terms “genetic modification,” “genetic alteration,” or “genetic engineering” broadly refer to manipulation of the genome or nucleic acids of a microorganism by the hand of man. Likewise, the terms “genetically modified,” “genetically altered,” or “genetically engineered” refers to a microorganism containing such a genetic modification, genetic alteration, or genetic engineering. These terms may be used to differentiate a lab-generated microorganism from a naturally-occurring microorganism. Methods of genetic modification of 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 methods, gene shuffling, and codon optimization.
“Recombinant” indicates that a nucleic acid, protein, or microorganism is the product of genetic modification, engineering, 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 microorganisms.
“Wild type” refers to the typical form of an organism, strain, gene, or characteristic as it occurs in nature, as distinguished from mutant or variant forms.
“Endogenous” refers to a nucleic acid or protein that is present or expressed in the wild-type or parental microorganism from which the microorganism of the invention is derived. For example, an endogenous gene is a gene that is natively present in the wild-type or parental microorganism from which the microorganism of the invention is derived. In one embodiment, the expression of an endogenous gene may be controlled by an exogenous regulatory element, such as an exogenous promoter.
“Exogenous” refers to a nucleic acid or protein that originates outside the microorganism of the invention. For example, an exogenous gene or enzyme may be artificially or recombinantly created and introduced to or expressed in the microorganism of the invention. An exogenous gene or enzyme may also be isolated from a heterologous microorganism and introduced to or expressed in the microorganism of the invention. Exogenous nucleic acids may be adapted to integrate into the genome of the microorganism of the invention or to remain in an extra-chromosomal state in the microorganism of the invention, for example, in a plasmid. “Heterologous” refers to a nucleic acid or protein that is derived from a different strain or species and introduced to or expressed in the microorganism of the invention.
The terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” “nucleic acid,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may 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 (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides or nucleotide analogs. If present, modifications to the nucleotide structure may 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.
As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene products.”
The terms “polypeptide”, “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, 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 and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
“Enzyme activity,” or simply “activity,” refers broadly to enzymatic activity, including, but not limited, to the activity of an enzyme, the amount of an enzyme, or the availability of an enzyme to catalyze a reaction. Accordingly, “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, “decreasing” 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.
“Mutated” refers to a nucleic acid or protein that has been modified in the microorganism of the invention compared to the wild-type or parental microorganism from which the microorganism of the invention 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.
“Disrupted gene” refers to a gene that has been modified in some way to reduce or eliminate expression of the gene, regulatory activity of the gene, or activity of an encoded protein or enzyme. The disruption may partially inactivate, fully inactivate, or delete the gene or enzyme. The disruption may be a knockout (KO) mutation that fully eliminates the expression or activity of a gene, protein, or enzyme. The disruption may also be a knock-down that reduces, but does not entirely eliminate, the expression or activity of a gene, protein, or enzyme. The disruption may be be anything that reduces, prevents, or blocks the biosynthesis of a product produced by an enzyme. The disruption may 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 nucleic acid which produces a protein that reduces or inhibits the activity of an enzyme, or the introduction of a nucleic acid (e.g., antisense RNA, RNAi, TALEN, siRNA, CRISPR, or CRISPRi) or protein which inhibits the expression of a protein or enzyme. The disruption may be introduced using any method known in the art. For the purposes of the present invention, disruptions are laboratory-generated, not naturally occurring.
“Codon optimization” refers to the mutation of a nucleic acid, such as a gene, for optimized or improved translation of the nucleic acid in a particular strain or species. Codon optimization may result in faster translation rates or higher translation accuracy. In a preferred embodiment, the genes of the invention are codon optimized for expression in Clostridium, particularly Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a further preferred embodiment, the genes of the invention are codon optimized for expression in Clostridium autoethanogenum LZ1561, which is deposited under DSMZ accession number DSM23693.
“Overexpressed” refers to an increase in expression of a nucleic acid or protein in the microorganism of the invention compared to the wild-type or parental microorganism from which the microorganism of the invention is derived. Overexpression may be achieved by any means known in the art, including modifying gene copy number, gene transcription rate, gene translation rate, or enzyme degradation rate.
The term “variants” includes nucleic acids and proteins whose sequence varies from the sequence of a reference nucleic acid and protein, such as a sequence of a reference nucleic acid and protein disclosed in the prior art or exemplified herein. The invention may be practiced using variant nucleic acids or proteins that perform substantially the same function as the reference nucleic acid or protein. For example, a variant protein may perform substantially the same function or catalyze substantially the same reaction as a reference protein. A variant gene may encode the same or substantially the same protein as a reference gene. A variant promoter may have substantially the same ability to promote the expression of one or more genes as a reference promoter.
Such nucleic acids or proteins may be referred to herein as “functionally equivalent variants.” By way of example, functionally equivalent variants of a nucleic acid may include allelic variants, fragments of a gene, mutated genes, polymorphisms, and the like. Homologous genes from other microorganisms are also examples of functionally equivalent variants. These include homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii, or Clostridium ljungdahlii, the details of which are publicly available on websites such as Genbank or NCBI. Functionally equivalent variants also include nucleic acids whose sequence varies as a result of codon optimization for a particular microorganism. A functionally equivalent variant of a nucleic acid will preferably have at least approximately 70%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 98%, or greater nucleic acid sequence identity (percent homology) with the referenced nucleic acid. A functionally equivalent variant of a protein will preferably have at least approximately 70%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 98%, or greater amino acid identity (percent homology) with the referenced protein. The functional equivalence of a variant nucleic acid or protein may be evaluated using any method known in the art.
“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which 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 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein 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.
“Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. 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 capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.
Nucleic acids may be delivered to a microorganism of the invention using any method known in the art. For example, nucleic acids may be delivered as naked nucleic acids or may be formulated with one or more agents, such as liposomes. The nucleic acids may be DNA, RNA, cDNA, or combinations thereof, as is appropriate. Restriction inhibitors may be used in certain embodiments. Additional vectors may include plasmids, viruses, bacteriophages, cosmids, and artificial chromosomes. In a preferred embodiment, nucleic acids are delivered to the microorganism of the invention using a plasmid. By way of example, transformation (including transduction or transfection) may be achieved by electroporation, ultrasonication, polyethylene glycol-mediated transformation, chemical or natural competence, protoplast transformation, prophage induction, or conjugation. In certain embodiments having active restriction enzyme systems, it may be necessary to methylate a nucleic acid before introduction of the nucleic acid into a microorganism.
Furthermore, nucleic acids may be designed to comprise a regulatory element, such as a promoter, 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.
A “microorganism” is a microscopic organism, especially a bacterium, archea, virus, or fungus. The microorganism of the invention is typically a bacterium. Herein, recitation of “microorganism” should be taken to encompass “bacterium.”
A “parental microorganism” is a microorganism used to generate a microorganism of the invention. The parental microorganism may 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). The microorganism of the invention may be modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parental microorganism. Similarly, the microorganism of the invention may be modified to contain one or more genes that were not contained by the parental microorganism. The microorganism of the invention may also be modified to not express or to express lower amounts of one or more enzymes that were expressed in the parental microorganism. In one embodiment, the parental microorganism is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the parental microorganism is Clostridium autoethanogenum LZ1561, which was deposited on Jun. 7, 2010 with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) located at InhoffenstraB 7B, D-38124 Braunschwieg, Germany on Jun. 7, 2010 under the terms of the Budapest Treaty and accorded accession number DSM23693. This strain is described in International Patent Application No. PCT/NZ2011/000144, which published as WO 2012/015317.
The term “derived from” indicates that a nucleic acid, protein, or microorganism is modified or adapted from a different (e.g., a parental or wild-type) nucleic acid, protein, or microorganism, so as to produce a new nucleic acid, protein, or microorganism. Such modifications or adaptations typically include insertion, deletion, mutation, or substitution of nucleic acids or genes. Generally, the microorganism of the invention is derived from a parental microorganism. In one embodiment, the microorganism of the invention is derived from Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the microorganism of the invention is derived from Clostridium autoethanogenum LZ1561, which is deposited under DSMZ accession number DSM23693.
The microorganism of the invention may be further classified based on functional characteristics. For example, the microorganism of the invention may be or may be derived from a C1-fixing microorganism, an anaerobe, an acetogen, an ethanologen, a carboxydotroph, and/or a methanotroph. Table 1 provides a representative list of microorganisms and identifies their functional characteristics.
Acetobacterium
woodii
Alkalibaculum
bacchii
Blautia
producta
Butyribacterium
methylotrophicum
Clostridium
aceticum
Clostridium
autoethanogenum
Clostridium
carboxidivorans
Clostridium
coskatii
Clostridium
drakei
Clostridium
formicoaceticum
Clostridium
ljungdahlii
Clostridium
magnum
Clostridium
ragsdalei
Clostridium
scatologenes
Eubacterium
limosum
Moorella
thermautotrophica
Moorella
thermoacetica (formerly
Clostridium
thermoaceticum)
Oxobacter
pfennigii
Sporomusa
ovata
Sporomusa
silvacetica
Sporomusa
sphaeroides
Thermoanaerobacter
kiuvi
1
Acetobacterium
woodi can produce ethanol from fructose, but not from gas.
2It has not been investigated whether Clostridiummagnum can grow on CO.
3One strain of Moorellathermoacetica, Moorella sp. HUC22-1, has been reported to produce ethanol from gas.
4It has not been investigated whether Sporomusaovata can grow on CO.
5It has not been investigated whether Sporomusasilvacetica can grow on CO.
6It has not been investigated whether Sporomusasphaeroides can grow on CO.
“Wood-Ljungdahl” refers to the Wood-Ljungdahl pathway of carbon fixation as described, e.g., by Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008. “Wood-Ljungdahl microorganism” refers, predictably, to a microorganism containing the Wood-Ljungdahl pathway. The microorganism of the invention is a Wood-Ljungdahl microorganism, usually a Wood-Ljungdahl bacterium. Generally, the microorganism of the invention contains a native Wood-Ljungdahl pathway. Herein, a Wood-Ljungdahl pathway may be a native, unmodified Wood-Ljungdahl pathway or it may be 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, CO2, and/or H2 to acetyl-CoA.
“C1” refers to a one-carbon molecule, for example, CO, CO2, CH4, or CH3OH. “C1-oxygenate” refers to a one-carbon molecule that also comprises at least one oxygen atom, for example, CO, CO2, or CH3OH. “C1-carbon source” refers a one carbon-molecule that serves as a partial or sole carbon source for the microorganism of the invention. For example, a C1-carbon source may comprise one or more of CO, CO2, CH4, CH3OH, or CH2O2. Preferably, the C1-carbon source comprises one or both of CO and CO2. A “C1-fixing microorganism” is a microorganism that has the ability to produce one or more products from a C1-carbon source. Typically, the microorganism of the invention is a C1-fixing bacterium. In a preferred embodiment, the microorganism of the invention is derived from a C1-fixing microorganism identified in Table 1.
An “anaerobe” is a microorganism that does not require oxygen for growth. An anaerobe may react negatively or even die if oxygen is present above a certain threshold. However, some anaerobes are capable of tolerating low levels of oxygen (e.g., 0.000001-5% oxygen). Typically, the microorganism of the invention is an anaerobe. In a preferred embodiment, the microorganism of the invention is derived from an anaerobe identified in Table 1.
An “acetogen” is a microorganism that produces or is capable of producing acetate (or acetic acid) as a product of anaerobic respiration. Typically, acetogens are obligately anaerobic bacteria that use the Wood-Ljungdahl pathway as their main mechanism for energy conservation and for synthesis of acetyl-CoA and acetyl-CoA-derived products, such as acetate (Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008). Acetogens use the acetyl-CoA pathway as a (1) mechanism for the reductive synthesis of acetyl-CoA from CO2, (2) terminal electron-accepting, energy conserving process, (3) mechanism for the fixation (assimilation) of CO2 in the synthesis of cell carbon (Drake, Acetogenic Prokaryotes, In: The Prokaryotes, 3rd edition, p. 354, New York, N.Y., 2006). All naturally occurring acetogens are C1-fixing, anaerobic, autotrophic, and non-methanotrophic. Typically, the microorganism of the invention is an acetogen. In a preferred embodiment, the microorganism of the invention is derived from an acetogen identified in Table 1.
An “ethanologen” is a microorganism that produces or is capable of producing ethanol. Typically, the microorganism of the invention is an ethanologen. In a preferred embodiment, the microorganism of the invention is derived from an ethanologen identified in Table 1.
An “autotroph” is a microorganism capable of growing in the absence of organic carbon. Instead, autotrophs use inorganic carbon sources, such as CO and/or CO2. Typically, the microorganism of the invention is an autotroph. In a preferred embodiment, the microorganism of the invention is derived from an autotroph identified in Table 1.
A “carboxydotroph” is a microorganism capable of utilizing CO as a sole source of carbon and energy. Typically, the microorganism of the invention is a carboxydotroph. In a preferred embodiment, the microorganism of the invention is derived from a carboxydotroph identified in Table 1.
A “methanotroph” is a microorganism capable of utilizing methane as a sole source of carbon and energy. In certain embodiments, the microorganism of the invention is a methanotroph or is derived from a methanotroph. In other embodiments, the microorganism of the invention is not a methanotroph or is not derived from a methanotroph.
More broadly, the microorganism of the invention may be derived from any genus or species identified in Table 1. For example, the microorganism may be a member of a genus selected from the group consisting of Acetobacterium, Alkalibaculum, Blautia, Butyribacterium, Clostridium, Eubacterium, Moorella, Oxobacter, Sporomusa, and Thermoanaerobacter. In particular, the microorganism may be derived from a parental bacterium selected from the group consisting of Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium magnum, Clostridium ragsdalei, Clostridium scatologenes, Eubacterium limosum, Moorella thermautotrophica, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, and Thermoanaerobacter kiuvi.
In a preferred embodiment, the microorganism of the invention is derived from the cluster of Clostridia comprising the species Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei. These species 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, WO 2008/028055 (Clostridium ragsdalei).
These three species have many similarities. In particular, these species are all C1-fixing, anaerobic, acetogenic, ethanologenic, and carboxydotrophic members of the genus Clostridium. These species have similar genotypes and phenotypes and modes of energy conservation and fermentative metabolism. Moreover, these species are clustered in clostridial rRNA homology group I with 16S rRNA DNA that is more than 99% identical, have a DNA G+C content of about 22-30 mol %, are gram-positive, have similar morphology and size (logarithmic growing cells between 0.5-0.7×3-5 μm), are mesophilic (grow optimally at 30-37° C.), have similar pH ranges of about 4-7.5 (with an optimal pH of about 5.5-6), lack cytochromes, and conserve energy via an Rnf complex. Also, reduction of carboxylic acids into their corresponding alcohols has been shown in these species (Perez, Biotechnol Bioeng, 110:1066-1077, 2012). Importantly, these species also all show strong autotrophic growth on CO-containing gases, produce ethanol and acetate (or acetic acid) as main fermentation products, and produce small amounts of 2,3-butanediol and lactic acid under certain conditions.
However, these three species also have a number of differences. These species were isolated from different sources: Clostridium autoethanogenum from rabbit gut, Clostridium ljungdahlii from chicken yard waste, and Clostridium ragsdalei from freshwater sediment. These species differ in 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). Moreover, these species differ in auxotrophy to certain vitamins (e.g., thiamine, biotin). These species have differences in nucleic and amino acid sequences of Wood-Ljungdahl pathway genes and proteins, although the general organization and number of these genes and proteins has been found to be the same in all species (Köpke, Curr Opin Biotechnol, 22: 320-325, 2011).
Thus, in summary, many of the characteristics of Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei are not specific to that species, but are rather general characteristics for this cluster of C1-fixing, anaerobic, acetogenic, ethanologenic, and carboxydotrophic members of the genus Clostridium. However, since these species are, in fact, distinct, the genetic modification or manipulation of one of these species may not have an identical effect in another of these species. For instance, differences in growth, performance, or product production may be observed.
The microorganism of the invention may also be derived from an isolate or mutant of Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. Isolates and mutants of Clostridium autoethanogenum include JAl-1 (DSM10061) (Abrini, Arch Microbiol, 161: 345-351, 1994), LBS1560 (DSM19630) (WO 2009/064200), and LZ1561 (DSM23693) (WO 2012/015317). Isolates and mutants of Clostridium ljungdahlii include ATCC 49587 (Tanner, Int J Syst Bacteriol, 43: 232-236, 1993), PETCT (DSM13528, ATCC 55383), ERI-2 (ATCC 55380) (U.S. Pat. No. 5,593,886), C-01 (ATCC 55988) (U.S. Pat. No. 6,368,819), 0-52 (ATCC 55989) (U.S. Pat. No. 6,368,819), and OTA-1 (Tirado-Acevedo, Production of bioethanol from synthesis gas using Clostridium ljungdahlii, PhD thesis, North Carolina State University, 2010). Isolates and mutants of Clostridium ragsdalei include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO 2008/028055).
“Substrate” refers to a carbon and/or energy source for the microorganism of the invention. Typically, the substrate is gaseous and comprises a C1-carbon source, for example, CO, CO2, and/or CH4. Preferably, the substrate comprises a C1-carbon source of CO or CO+CO2. The substrate may further comprise other non-carbon components, such as H2, N2, or electrons.
The substrate generally comprises at least some amount of CO, such as about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mol % CO. The substrate may comprise a range of CO, such as about 20-80, 30-70, or 40-60 mol % CO. Preferably, the substrate comprises about 40-70 mol % CO (e.g., steel mill or blast furnace gas), about 20-30 mol % CO (e.g., basic oxygen furnace gas), or about 15-45 mol % CO (e.g., syngas). In some embodiments, the substrate may comprise a relatively low amount of CO, such as about 1-10 or 1-20 mol % CO. The microorganism of the invention typically converts at least a portion of the CO in the substrate to a product. In some embodiments, the substrate comprises no or substantially no (<1 mol %) CO.
The substrate may comprise some amount of H2. For example, the substrate may comprise about 1, 2, 5, 10, 15, 20, or 30 mol % H2. In some embodiments, the substrate may comprise a relatively high amount of H2, such as about 60, 70, 80, or 90 mol % H2. In further embodiments, the substrate comprises no or substantially no (<1 mol %) H2.
The substrate may comprise some amount of CO2. For example, the substrate may comprise about 1-80 or 1-30 mol % CO2. In some embodiments, the substrate may comprise less than about 20, 15, 10, or 5 mol % CO2. In another embodiment, the substrate comprises no or substantially no (<1 mol %) CO2.
Although the substrate is typically gaseous, the substrate may also be provided in alternative forms. For example, the substrate may be dissolved in a liquid saturated with a CO-containing gas using a microbubble dispersion generator. By way of further example, the substrate may be adsorbed onto a solid support.
The substrate and/or C1-carbon source may be a waste gas obtained as a byproduct of an industrial process or from some other source, such as from automobile exhaust fumes or biomass gasification. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill manufacturing, non-ferrous products manufacturing, petroleum refining, coal gasification, electric power production, carbon black production, ammonia production, methanol production, and coke manufacturing. In these embodiments, the substrate and/or C1-carbon source may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method.
The substrate and/or C1-carbon source may be syngas, such as syngas obtained by gasification of coal or refinery residues, gasification of biomass or lignocellulosic material, or reforming of natural gas. In another embodiment, the syngas may be obtained from the gasification of municipal solid waste or industrial solid waste.
The composition of the substrate may have a significant impact on the efficiency and/or cost of the reaction. For example, the presence of oxygen (02) may 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 undesired impurities, such as toxins, undesired components, or dust particles, and/or increase the concentration of desirable components.
In certain embodiments, the fermentation is performed in the absence of carbohydrate substrates, such as sugar, starch, lignin, cellulose, or hemicellulose.
The microorganism of the invention may be cultured to produce one or more products. For instance, the microorganism of the invention may produce or may be engineered to produce ethanol (WO 2007/117157), acetate (WO 2007/117157), butanol (WO 2008/115080 and WO 2012/053905), 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), isoprene (WO 2013/180584), fatty acids (WO 2013/191567), 2-butanol (WO 2013/185123), 1,2-propanediol (WO 2014/036152), 1-propanol (WO 2014/0369152), chorismate-derived products (WO 2016/191625), 3-hydroxybutyrate (WO 2017/066498), and 1,3-butanediol (WO 2017/0066498). In addition to one or more target products, the microorganism of the invention may also produce ethanol, acetate, and/or 2,3-butanediol. In certain embodiments, microbial biomass itself may be considered a product.
A “native product” is a product produced by a genetically unmodified microorganism. For example, ethanol, acetate, and 2,3-butanediol are native products of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei. A “non-native product” is a product that is produced by a genetically modified microorganism, but is not produced by a genetically unmodified microorganism from which the genetically modified microorganism is derived.
Herein, reference to an acid (e.g., acetic acid or 2-hydroxyisobutyric acid) should be taken to also include the corresponding salt (e.g., acetate or 2-hydroxyisobutyrate).
“Selectivity” refers to the ratio of the production of a target product to the production of all fermentation products produced by a microorganism. The microorganism of the invention may be engineered to produce products at a certain selectivity or at a minimum selectivity. In one embodiment, a target product account for at least about 5%, 10%, 15%, 20%, 30%, 50%, or 75% of all fermentation products produced by the microorganism of the invention. In one embodiment, the target product accounts for at least 10% of all fermentation products produced by the microorganism of the invention, such that the microorganism of the invention has a selectivity for the target product of at least 10%. In another embodiment, the target product accounts for at least 30% of all fermentation products produced by the microorganism of the invention, such that the microorganism of the invention has a selectivity for the target product of at least 30%.
“Increasing the efficiency,” “increased efficiency,” and the like include, but are not limited to, increasing growth rate, product production rate or volume, product volume per volume of substrate consumed, or product selectivity. Efficiency may be measured relative to the performance of parental microorganism from which the microorganism of the invention is derived.
Typically, the culture is performed in a bioreactor. The term “bioreactor” includes a culture/fermentation device consisting of one or more vessels, towers, 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 other device suitable for gas-liquid contact. In some embodiments, the bioreactor may comprise a first growth reactor and a second culture/fermentation reactor. The substrate may be provided to one or both of these reactors. As used herein, the terms “culture” and “fermentation” are used interchangeably. These terms encompass both the growth phase and product biosynthesis phase of the culture/fermentation process.
The culture is generally maintained in an aqueous culture medium that contains nutrients, vitamins, and/or minerals sufficient to permit growth of the microorganism. Preferably the aqueous culture medium is an anaerobic microbial growth medium, such as a minimal anaerobic microbial growth medium. Suitable media are well known in the art.
The culture/fermentation should desirably be carried out under appropriate conditions for production of the target product. Typically, the culture/fermentation is performed under anaerobic conditions. Reaction conditions to consider include pressure (or partial pressure), temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas substrate concentrations to ensure that gas in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition. In particular, the rate of introduction of the substrate may be controlled to ensure that the concentration of gas in the liquid phase does not become limiting, since products may be consumed by the culture under gas-limited conditions.
Operating a bioreactor at elevated pressures allows for an increased rate of gas mass transfer from the gas phase to the liquid phase. Accordingly, it is generally preferable to perform the culture/fermentation at pressures higher than atmospheric pressure. Also, since a given gas conversion rate is, in part, a function of the substrate retention time and retention time dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the volume of the bioreactor required and, consequently, the capital cost of the culture/fermentation equipment. This, in turn, means that the retention time, defined as the liquid volume in the bioreactor divided by the input gas flow rate, can be reduced when bioreactors are maintained at elevated pressure rather than atmospheric pressure. The optimum reaction conditions will depend partly on the particular microorganism used. However, in general, it is preferable to operate the fermentation at a pressure higher than atmospheric pressure. Also, since a given gas conversion rate is in part a function of substrate retention time and achieving a desired retention time in turn dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the volume of the bioreactor required, and consequently the capital cost of the fermentation equipment.
In certain embodiments, the fermentation is performed in the absence of light or in the presence of an amount of light insufficient to meet the energetic requirements of photosynthetic microorganisms. In certain embodiments, the microorganism of the invention is a non-photosynthetic microorganism.
Target products may be separated or purified from a 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, target products are recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering one or more target products from the broth. Alcohols and/or acetone may be recovered, for example, by distillation. Acids may be recovered, for example, by adsorption on activated charcoal. Separated microbial cells are preferably returned to the bioreactor. The cell-free permeate remaining after target products have been removed is also preferably returned to the bioreactor. Additional nutrients (such as B vitamins) may be added to the cell-free permeate to replenish the medium before it is returned to the bioreactor.
The microorganism of the invention contains at least one disrupted gene. In some embodiments, the microorganism of the invention contains more than one disrupted genes, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, or 200 disrupted genes. For example, the disrupted gene may be selected from Table 2. Although representative accession numbers are provided for C. autoethanogenum, C. ljungdahlii, and C. ragsdalei, a person of ordinary skill in the art would be capable of readily identifying homologs in other Wood-Ljungdahl microorganisms.
Clostridium
Clostridium
Clostridium
autoethanogenum
ljungdahlii
ragsdalei
The inventors have further identified key metabolic pathways and key metabolic nodes in Wood-Ljungdahl microorganisms (
The invention finally provides methods of producting products by culturing the microorganism of the invention in the presence of a substrate, such as a gaseous substrate comprising one or more of CO, CO2, and/or H2. Possible combinations of disrupted genes for optimizing production of particular products are described in Examples 2-19.
As described elsewhere in this application, such products may include native or non-native products of Wood-Ljungdahl microorganisms. For example, such products include, but are not limited to acetyl-CoA, ethanol, acetate, butanol, butyrate, butyryl-CoA, 2,3-butanediol, lactate, butene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate (3-HP), isoprene, farnesene, fatty acids (fatty acid ethyl esters, fatty acid butyl esters), 2-butanol, 1,2-propanediol, 1-propanol, chorismate-derived products, 3-hydroxybutyrate, 1,3-butanediol, C6-C8 alcohols (hexanol, heptanol, octanol), caproate, octanoate, isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), acetoacetyl-CoA, 3-hydroxybutyrate-CoA (3-HB-CoA), malonyl-CoA, pyruvate, dehydroshikimate, chorismate, para-hydroxybenzoic acid, salicylate, 2-aminobenzoate, 2,3-dihydroxybenzoate, 2-hydroxycyclohexane carboxylic acid, citramalate, ketobutyrate, acetolactate, acetoin, valine, leucine, and isoleucine.
The following examples further illustrate the invention but, of course, should not be construed to limit its scope in any way.
This example describes metabolic modeling in Wood-Ljungdahl microorganisms.
A genome-scale metabolic model of Clostridium autoethanogenum like the one described by Marcellin, Green Chem, 18: 3020-3028, 2016 was utilized. This model was used to simulate the design, construction, in silico growth and screening of strains with disruptive gene mutations to predict those that would produce higher yields of native compounds. In addition, new genome-scale models were built for a number non-native compound-producing strains. For these, heterologous genes and metabolic reactions were added to the wild type Clostridium autoethanogenum model structure to represent the incorporation of the non-native compound production pathway. Although the model used for the experimental work described herein is based on Clostridium autoethanogenum, the results can reasonably be expected to apply to other Wood-Ljungdahl microorganisms as well, given similarities in metabolism.
For each chemical production strain, millions of mutant strains incorporating different combinations of disruptive gene mutations were built in silico. Boolean gene-protein-reaction associations were used to determine which metabolic reactions were inactivated upon disruption of a gene (Thiele, Nature Protocols, 5: 93-121, 2010). The design, construction and screening of mutant strains was carried out using cameo version 0.11.2 (Sonnenschein, Biosustain/Cameo: 0.11.0, doi: 10.5281/zenodo.835730, 2017) and evolutionary algorithms implemented by inspyred version 1.0.1.
The growth of these mutant strains was simulated using two constraint-based computational modeling techniques: flux balance analysis (FBA) and linear minimization of metabolic adjustment (LMOMA). These growth simulation techniques are used to capture two likely metabolic phenotypes, following genetic perturbation (Maia, Proceedings of the Genetic and Evolutionary Computation Conference Companion on—GECCO '17, New York, N.Y., ACM Press, 1661-1668, 2017). An experimental metabolic flux profile was constructed and used as the reference state for LMOMA simulations. Growth simulations were run using scripts from cobrapy version 0.8.2 (Ebrahim., COBRApy: COnstraints-Based Reconstruction and Analysis for Python, BMC Syst Biol, 7: 74, 2013), with optlang version 1.2.3 (Jensen, Optlang: An Algebraic Modeling Language for Mathematical Optimization,” The Journal of Open Source Software, 2, doi:10.21105/joss.00139, 2017) as the solver interface and Gurobi Optimizer version 7.0.2 as the optimization solver.
Growth rates and key metabolic fluxes including those for fermentation products were recorded and used to screen strains. For each strain simulation, the biomass-product coupled yield (BPCY) and the and the carbon molar yield were calculated. These yields were used to determine the fitness score.
In addition, flux variability analysis (FVA) was carried out to determine whether the mutant strain requires production of the compound of interest for growth to occur (growth-coupled strain designs). If the minimum boundary flux of the compound of interest was greater than zero during growth, the strain was classified as growth-coupling. These growth-coupled strain designs should allow greater fermentation stability during continuous fermentation. This minimum flux was converted to carbon yield (FVA minimum yield) and used to compare the level of growth coupling between strains.
This example describes disruptions for improved production of acetate in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Acetate is a native product of Wood-Ljungdahl microorganisms.
This example describes disruptions for improved production of ethanol in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Ethanol is a native product of Wood-Ljungdahl microorganisms.
This example describes disruptions for improved production of acetone in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of acetone in Wood-Ljungdahl microorganisms is described, e.g., in WO 2012/115527. The following pathway was used to model acetone production herein: 2.0 Acetyl-CoA-->CoA+Acetoacetyl-CoA; Acetate+Acetoacetyl-CoA-->Acetyl-CoA+Acetoacetate; Acetoacetate-->CO2+Acetone; Acetone-->Acetone_ext.
This example describes disruptions for improved production of isopropanol in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of isopropanol in Wood-Ljungdahl microorganisms is described, e.g., in WO 2012/115527. The following pathway was used to model isopropanol production herein: 2.0 Acetyl-CoA-->CoA+Acetoacetyl-CoA; Acetate+Acetoacetyl-CoA-->Acetyl-CoA+Acetoacetate; Acetoacetate-->CO2+Acetone; Isopropanol-->Isopropanol_ext.
This example describes disruptions for improved production of lactate in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Lactate is a native product of Wood-Ljungdahl microorganisms.
This example describes disruptions for improved production of 1,3-butanediol in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of 1,3-butanediol in Wood-Ljungdahl microorganisms is described, e.g., in WO 2017/0066498. The following pathway was used to model 1,3-butanediol production herein: 2.0 Acetyl-CoA-->CoA+Acetoacetyl-CoA; NADPH+H++Acetoacetyl-CoA-->NADP+(R)-3-Hydroxybutyryl-CoA; Phosphate+(R)-3-Hydroxybutyryl-CoA-->CoA+(R)-3-hydroxybutyryl-Phosphate; ADP+(R)-3-hydroxybutyryl-Phosphate-->ATP+(R)-3-Hydroxybutyrate; (R)-3-Hydroxybutyrate+Reducedferredoxin-->Oxidizedferredoxin+(R)-3-hydroxybutyraldehyde; NADPH+H++(R)-3-hydroxybutyraldehyde-->NADP+13BDO; NADH+H++(R)-3-hydroxybutyraldehyde-->NAD+13BDO; 13BDO-->13BDO_ext.
This example describes disruptions for improved production of 2,3-butanediol in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. 2,3-butanediol is a native product of at least some Wood-Ljungdahl microorganisms.
This example describes disruptions for improved production of 2-butanol in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of 2-butanol in Wood-Ljungdahl microorganisms is described, e.g., in WO 2013/185123. The following pathway was used to model 2-butanol production herein: NADH+H++(R)-Acetoin-->NAD+meso-2,3-Butanediol; meso-2,3-Butanediol-->H2O+MEK; MEK+NADPH+H+-->2-butanol+NADP; 2-butanol-->2-butanol ext.
This example describes disruptions for improved production of 2-hydroxyisobutyric acid in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of 2-hydroxyisobutyric acid in Wood-Ljungdahl microorganisms is described, e.g., in WO 2017/0066498.
The following pathway was used to model 2-hydroxyisobutyric acid production in rows 1-40 below: 2.0 Acetyl-CoA-->CoA+Acetoacetyl-CoA; NADH+H++Acetoacetyl-CoA-->NAD+(S)-3-Hydroxybutyryl-CoA; (S)-3-Hydroxybutyryl-CoA-->2HIB-CoA; 2HIB-CoA+H2O-->2hib+CoA; 2hib-->2hib_ext.
The following pathway was used to model 2-hydroxyisobutyric acid production in rows 41-93 below: 2.0 Acetyl-CoA-->CoA+Acetoacetyl-CoA; NADH+H++Acetoacetyl-CoA-->NAD+(S)-3-Hydroxybutyryl-CoA; (S)-3-Hydroxybutyryl-CoA-->2HIB-CoA; 2HIB-CoA+ADP+Phosphate-->2hib+ATP+CoA; 2hib-->2hib_ext.
The following pathway was used to model 2-hydroxyisobutyric acid production in rows 94-103 below: 2.0 Acetyl-CoA-->CoA+Acetoacetyl-CoA; NADH+H++Acetoacetyl-CoA-->NAD+(S)-3-Hydroxybutyryl-CoA; (S)-3-Hydroxybutyryl-CoA-->2HIB-CoA; 2HIB-CoA+Acetate-->2hib+Acetyl-CoA; 2hib-->2hib_ext.
This example describes disruptions for improved production of 3-hydroxybutyrate in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of 3-hydroxybutyrate in Wood-Ljungdahl microorganisms is described, e.g., in WO 2017/066498. The following pathway was used to model 3-hydroxybutyrate production herein: 3-Hydroxybutyrate-->3-Hydroxybutyrate_ext; Acetate+Acetoacetyl-CoA-->Acetyl-CoA+Acetoacetate; NADH+H++Acetoacetate-->NAD+3-Hydroxybutyrate; 2.0 Acetyl-CoA-->CoA+Acetoacetyl-CoA.
This example describes disruptions for improved production of methyl ethyl ketone (MEK), i.e., 2-butanone, in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of MEK in Wood-Ljungdahl microorganisms is described, e.g., in WO 2012/024522 and WO 2013/185123. The following pathway was used to model methyl ethyl ketone production herein: NADH+H++(R)-Acetoin-->NAD+meso-2,3-Butanediol; meso-2,3-Butanediol-->H2O+MEK; H++MEK-->H+_ext+MEK_ext.
This example describes disruptions for improved production of acetolactate in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Acetolactate is a native product of at least some Wood-Ljungdahl microorganisms.
This example describes disruptions for improved production of isoprene in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of isoprene in Wood-Ljungdahl microorganisms is described, e.g., in WO 2013/180584. The following pathway was used to model isoprene production herein: 2.0 Acetyl-CoA-->CoA+Acetoacetyl-CoA; 2.0 NADH+2.0H++(S)-3-Hydroxy-3-methylglutaryl-CoA-->2.0 NAD+CoA+(R)-Mevalonate; ATP+(R)-Mevalonate-->ADP+(R)-5-Phosphomevalonate; ATP+(R)-5-Phosphomevalonate-->ADP+(R)-5-Diphosphomevalonate; ATP+(R)-5-Diphosphomevalonate-->ADP+Phosphate+C02+Isopentenyldiphosphate; Isopentenyldiphosphate-->DMAPP; DMAPP-->PPi+Isoprene; H2O+Acetyl-CoA+Acetoacetyl-CoA-->CoA+(S)-3-Hydroxy-3-methylglutaryl-CoA; Isoprene-->Isoprene_ext. These disruptions would also be useful for improving production of IPP/DMAPP and/or products downstream of IPP/DMAPP, such as famesene and other terpenoids.
This example describes disruptions for improved production of adipic acid in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of adipic acid in Wood-Ljungdahl microorganisms is described, e.g., in WO 2017/0066498. The following pathway was used to model adipic acid production herein: ATP+CoA+Succinate-->ADP+Phosphate+Succinyl-CoA; NAD+CoA+2-Oxoglutarate-->NADH+C02+Succinyl-CoA; Acetyl-CoA+Succinyl-CoA-->CoA+3-Oxo-adipyl-CoA; NADH+H++3-Oxo-adipyl-CoA-->NAD+3-Hydroxyadipyl-CoA; 3-Hydroxyadipyl-CoA-->2,3-Dehydroadipyl-CoA; NADH+H++2,3-Dehydroadipyl-CoA-->NAD+Adipyl-CoA; Phosphate+Adipyl-CoA-->CoA+Adipyl-P; ADP+Adipyl-P-->ATP+Adipate; Adipate-->Adipate_ext.
This example describes disruptions for improved production of 2-aminobenzoate in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of 2-aminobenzoate in Wood-Ljungdahl microorganisms is described, e.g., in WO 2016/191625. The following pathway was used to model 2-aminobenzoate production herein: NH3+Chorismate=>H2O+Pyruvate+H++2-aminobenzoate.
This example describes disruptions for improved production of salicylate in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of salicylate in Wood-Ljungdahl microorganisms is described, e.g., in WO 2016/191625. The following pathway was used to model salicylate production herein: Chorismate-->Isochorismate; Isochorismate-->Salicylate+Pyruvate.
This example describes disruptions for improved production of chorismate in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of chorismate in Wood-Ljungdahl microorganisms is described, e.g., in WO 2016/191625. Chorismate is a native product of at least some Wood-Ljungdahl microorganisms. These same disruptions would be expected to similarly increase flux through dehydroshikimate, a metabolic node just upstream of chorismate.
This example describes disruptions for improved production of famesene in Wood-Ljungdahl microorganisms. Metabolic modeling experiments were performed as described in Example 1. Production of famesene in Wood-Ljungdahl microorganisms is described, e.g., in WO 2013/180584. The following pathway was used to model famesene production herein: 2.0 Acetyl-CoA-->CoA+Acetoacetyl-CoA; H2O+Acetyl-CoA+Acetoacetyl-CoA-->CoA+(S)-3-Hydroxy-3-methylglutaryl-CoA; 2.0 NADH+2.0H++(S)-3-Hydroxy-3-methylglutaryl-CoA-->2.0 NAD+CoA+(R)-Mevalonate; ATP+(R)-Mevalonate-->ADP+(R)-5-Phosphomevalonate; ATP+(R)-5-Diphosphomevalonate-->ADP+Phosphate+CO2+Isopentenyldiphosphate; ATP+(R)-5-Diphosphomevalonate-->ADP+Phosphate+CO2+Isopentenyldiphosphate; Isopentenyldiphosphate-->DMAPP; 2.0 Isopentenyldiphosphate+DMAPP-->PPi+trans,trans-famesyl-diphosphate; trans,trans-famesyl-diphosphate-->Famesene+PPi; Famesene-->Famesene_ext.
This example describes gene disruption targets common across different product pathways. Optimizations were run using an evolutionary algorithm on 444 pathways. Each strain design was scored based on the achieved yield (non-growth coupled designs) and biomass-product coupled yield (growth coupled designs). Each gene was ranked based on how often it appeared in strain designs. 19 genes were commonly identified for disruption in optimized strains.
This example describes gene disruption targets to increase target compound production during autotrophic growth. This strategy involves eliminating or decreasing the production of other fermentation byproducts, making the target compound a required growth byproduct. Metabolic modeling experiments were performed as described in Example 1.
Modeling evidence demonstrates that this strategy is appropriate for target compounds whose production imposes minimal ATP burden. This strategy is not well suited for products with significant ATP burden during autotrophic growth. This is because this strategy decreases cellular ATP yields through the elimination of substrate level phosphorylation catalysed by acetate kinase.
In particular, production of products such as acetone, isopropanol, 1,3-butanediol, 3-hydroxybutyrate, 2-hydroxyisobutyrate, 3-hydroxyisovalerate, and adipic acid can be improved by introducing a disruptive mutation into genes encoding acetate kinase and/or phosphate transacetylase, and optionally further introducing a disruptive mutation into one or more genes encoding acetolactate decarboxylase, lactate dehydrogenase, aldehyde dehydrogenase, or citramalate synthase.
Each model was assessed using flux variability analysis to determine the minimum required flux towards the target compound during normal growth. Then, the proposed set of disruptive gene mutations was applied to each model. Flux variability analysis was carried out again to identify any existence of coupling between compound production and growth. Simulations were carried out using cobrapy version 0.13.4.
This example describes increasing target compound production during autotrophic growth on gas mixes with a low proportion of CO by decreasing required acetate co-production. Metabolic modeling experiments were performed as described in Example 1.
The strategy involves adjusting the redox cofactor balance so there is excess NADPH. To maintain redox homeostasis, the cell must make products whose production pathway requires NADPH. As acetate production does not fulfil this, the cell will be required to make other products to achieve maximum growth rates.
Modeling evidence demonstrates that this strategy is appropriate for target compounds with an ATP burden that requires the co-production of acetate. This strategy is also appropriate for strains that produce ethanol as a primary product. This strategy is predicted to work on low CO gases, where the cell can utilise the hydrogenase enzyme to reduce ferredoxin and NAD(P)+. In some cases, the maximum possible yield of the target compound will decrease, as this strategy reduces the efficiency of the energy metabolism of the cell.
In particular, production of products such as ethanol, acetone, isopropanol, 1,3-butanediol, 2-butanol, 2-hydroxyisobutyrate, 3-hydroxyisovalerate, adipic acid, methyl ethyl ketone, isoprene, salicylate, chorismate, and farnesene can be improved by introducing a disruptive mutation into a gene encoding NAD-dependent electron-bifurcating [FeFe]-hydrogenase (e.g., Hyd), and optionally further introducing a disruptive mutation into one or more genes encoding glutamate synthase, citramalate synthase, acetolactate decarboxylase, or lactate dehydrogenase.
Each model was assessed using flux variability analysis to determine the minimum required flux to acetate at high growth rates. Then, the proposed set of disruptive gene mutations was applied to each model. The NAD-dependent hydrogenase (Hyd) was removed from the stoichiometric matrix to represent the knock out of this enzyme. Flux through the glutamate synthase reaction was decreased by 30% to represent a disruption of this enzyme. Flux variability analysis was carried out again to determine the minimum acetate production requirement to achieve maximum growth. Simulations were carried out using cobrapy version 0.13.4
This example describes increasing flux through acetoacetyl-CoA, a central metabolic node. Increasing flux through this node will increase production of downstream products such as acetone, isopropanol, 3-hydroxyisovaleryl-CoA, 3-hydroxyisovalerate, isobutylene, isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), isoprene, terpenoids such as farnesene, 3-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxybutyrate, 3-hydroxybutyrylaldehyde, 1,3-butanediol, 2-hydroxyisobutyryl-CoA, 2-hydroxyisobutyrate, butyryl-CoA, butyrate, butanol, caproate, hexanol, octanoate, octanol, 1,3-hexanediol, 2-buten-1-ol, isovaleryl-CoA, isovalerate, or isoamyl alcohol. Metabolic modeling experiments were performed as described in Example 1.
Most Wood-Ljungdahl microorganisms are not natively capable of converting acetyl-CoA to acetoacetyl-CoA, such that this step may require the introduction of a heterologous enzyme, such as a thiolase (i.e., acetyl-CoA acetyltransferase) (EC 2.3.1.9). The thiolase may be, for example, ThlA from Clostridium acetobutylicum (WP_010966157.1), PhaA from Cupriavidus necator (WP_013956452.1), BktB from Cupriavidus necator (WP_011615089.1), AtoB from Escherichia coli (NP_416728.1), or a similar.
In particular, flux through acetoacetyl-CoA can be improved by introducing a disruptive mutation into one or more genes encoding one or more, two or more, three or more, four or more, or five or more of NAD-dependent electron-bifurcating [FeFe]-hydrogenase (e.g., Hyd), glutamate synthase, citramalate synthase, acetolactate decarboxylase, lactate dehydrogenase, acetate kinase, phosphate transacetylase, or aldehyde dehydrogenase.
All references, including publications, patent applications, and patents, cited herein are hereby 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 to any prior art in this specification is not, and should not be taken as, an acknowledgement that that prior art forms part of the common general knowledge in the field of endeavour in any country.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to 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 the specified 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 the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the term “about” means±20% of the indicated range, value, or structure, unless otherwise indicated.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, any concentration range, percentage range, ratio range, integer range, size range, or thickness range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
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., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention 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 invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This application is the U.S. national phase of International Patent Application No. PCT/US2018/053587 filed on Sep. 28, 2018, which claims the benefit of U.S. Provisional Patent Application No. 62/565,000 filed Sep. 28, 2017, both of which are incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/053587 | 9/28/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62565000 | Sep 2017 | US |
