WO2014089025A1 - Increased yields of biosynthesized products - Google Patents

Increased yields of biosynthesized products Download PDF

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Publication number
WO2014089025A1
WO2014089025A1 PCT/US2013/072781 US2013072781W WO2014089025A1 WO 2014089025 A1 WO2014089025 A1 WO 2014089025A1 US 2013072781 W US2013072781 W US 2013072781W WO 2014089025 A1 WO2014089025 A1 WO 2014089025A1
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reductase
butadiene
nitrite
coa
microbial organism
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PCT/US2013/072781
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French (fr)
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Mark J. Burk
Anthony P. Burgard
Steven J. VAN DIEN
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Genomatica, Inc.
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Publication of WO2014089025A1 publication Critical patent/WO2014089025A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes

Definitions

  • the present invention relates generally to biosynthetic processes, and more specifically to increasing yields of biosynthesized products under anaerobic culture conditions.
  • the invention provides a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or pathway for reducing nitrate (N03-) or nitrite (N02-) and a metabolic modification attenuating the amount or activity of a
  • composition including a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or pathway for reducing nitrate (N03-) or nitrite (N02-) and a medium containing nitrate.
  • Figure 1 shows a schematic depicting a process for anaerobic production of butadiene with nitrate.
  • Figure 2 shows nitrate respiration and butadiene energetic.
  • Figure 3 shows the energetics associated with anaerobic butadiene production in the presence of nitrate.
  • Figure 4 shows exemplary pathways for production of butadiene from acetyl-CoA, glutaconyl-CoA, glutaryl-CoA, 3-aminobutyryl-CoA or 4-hydroxybutyryl-CoA via crotyl alcohol.
  • Enzymes for transformation of the identified substrates to products include: A. acetyl- CoA:acetyl-CoA acyltransferase, B. acetoacetyl-CoA reductase, C. 3-hydroxybutyryl-CoA dehydratase, D. crotonyl-CoA reductase (aldehyde forming), E.
  • crotonaldehyde reductase (alcohol forming)
  • F. crotyl alcohol kinase G. 2-butenyl-4-phosphate kinase, H. butadiene synthase, I. crotonyl-CoA hydrolase, synthetase, transferase, J. crotonate reductase, K. crotonyl- CoA reductase (alcohol forming),
  • L. glutaconyl-CoA decarboxylase M., glutaryl-CoA dehydrogenase, N. 3-aminobutyryl-CoA deaminase, O. 4-hydroxybutyryl-CoA dehydratase, P. crotyl alcohol diphosphokinase.
  • Figure 5 shows exemplary pathways for production of butadiene from erythrose-4- phosphate. Enzymes for transformation of the identified substrates to products include: A.
  • Erythrose-4-phosphate reductase B. Erythritol-4-phospate cytidylyltransferase, C. 4-(cytidine 5'- diphospho)-erythritol kinase, D. Erythritol 2,4-cyclodiphosphate synthase, E. l-Hydroxy-2- butenyl 4-diphosphate synthase, F. l-Hydroxy-2-butenyl 4-diphosphate reductase, G. Butenyl 4- diphosphate isomerase, H. Butadiene synthase I. Erythrose-4-phosphate kinase, J. Erythrose reductase, K. Erythritol kinase.
  • Figure 6 shows an exemplary pathway for production of butadiene from malonyl- CoA plus acetyl-CoA.
  • Enzymes for transformation of the identified substrates to products include: A. malonyl-CoA:acetyl-CoA acyltransferase, B. 3 -oxoglutaryl-Co A reductase (ketone - reducing), C. 3-hydroxyglutaryl-CoA reductase (aldehyde forming), D. 3-hydroxy-5- oxopentanoate reductase, E. 3,5-dihydroxypentanoate kinase, F. 3H5PP kinase, G. 3H5PDP decarboxylase, H.
  • butenyl 4-diphosphate isomerase I. butadiene synthase, J. 3 -hydroxy glutaryl- CoA reductase (alcohol forming), K. 3 -oxoglutaryl-Co A reductase (aldehyde forming), L. 3,5- dioxopentanoate reductase (ketone reducing), M. 3,5-dioxopentanoate reductase (aldehyde reducing), N. 5-hydroxy-3-oxopentanoate reductase, O. 3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming).
  • 3H5PP 3-Hydroxy-5- phosphonatooxypentanoate
  • 3H5PDP 3-Hydroxy-5- [hydroxy(phosphonooxy)phosphoryl]oxy pentanoate.
  • Figure 7 shows the energetics associate with oxygen respiration for the biosynthesis of 1,4-butanediol.
  • Figure 8 shows exemplary biochemical pathways to 1,4-butanediol from succinate.
  • Enzymes for transformation of the identified substrates to products include: 1) CoA-independent succinic semialdehyde dehydrogenase; 2) Succinyl-CoA synthetase; 3) CoA-dependent succinic semialdehyde dehydrogenase; 4) 4-hydroxybutanoate dehydrogenase; 5) CoA-independent aldehyde dehydrogenase; 6) 4-hydroxybutanoate: acetate CoA transferase; 7) CoA-dependent aldehyde dehydrogenase; 8) alcohol dehydrogenase
  • This invention is directed to the use of nitrate as an alternative electron acceptor for simulated microaerobic respiration (or micro-respiric growth). Since it can be added in a solution, use of nitrate is cheaper and easier for precise control than aeration.
  • the invention provides, for example, a method for anaerobic production of reduced biochemical products with high energetic demands, using controlled addition of nitrate as an electron acceptor.
  • the invention is applicable for the production of a large number of biosynthesized chemicals that are industrially useful. Particular examples of such biosynthesized chemicals include butadiene and 1 ,4-butanediol (BDO) production in microorganisms.
  • oxygen-reactive products such as butadiene or other olefins
  • Higher product yields can be obtained, for example, as a result of reduced product loss from reaction with oxygen, and are obtained under safer conditions.
  • the methods and compositions described herein minimize fouling of fermentation equipment that typically occurs with olefins such as butadiene when oxygen is present, thus providing additional economic benefits.
  • microorganism can use alternative molecules as terminal electron acceptors, such as nitrate (NO3 " ) and sulfate (SO4 “2 ), reducing them to nitrite (NO2 ) and sulfite (SO3 "2 ), respectively.
  • the nitrate/nitrite couple is about half that of the O 2 /H 2 O couple, and can result in the translocation of up to about two protons (Unden et al, Antonie Van Leeuwenhoek, 66:3-23 (1994)).
  • the nitrite formed from this process can also be used as a terminal electron acceptor, via the cytoplasmic nitrate reductase (nirBD) or the membrane-associated nitrite reductase (nrfABCDEFG), resulting in the formation of NH 4 + .
  • This ammonium can also be used as a nitrogen source for cell component biosynthesis.
  • nitrite reduction via nirBD does not result in proton translocation and is undesirable in some instances.
  • the nirBD genes are known to be repressed by nitrate, even when nitrite is present in excess (Cole and Brown, F EMS Microbiol. Lett., 7:65-72 (1980)).
  • the nirBD genes can also be deleted to assure that basal reduction of nitrite in the cytoplasm does not occur.
  • nitrate respiration is well studied in microorganisms, including bacteria such as E. coli and B. subtilis
  • industrial fermentation processes have generally not utilized this approach.
  • the denitrification process has been employed for the treatment of municipal wastewater, in which nitrate is a common contaminant.
  • nitrate in the wastewater is reduced to NH4 + or N 2 , usually by a microbial community (Metcalf E.,
  • the invention differs from the above reports in several ways: (1) The controlled addition of nitrate (or nitrite) is used to improve an inherently anaerobic process, not to replace an aerobic process; (2) nitrate (or nitrite) will be added in limited quantities, not in excess; and (3) addition of nitrate (or nitrite) will be used in a controlled manner to regulate the relative rates of respiration and fermentation and to govern the amount of biomass required for optimum yield and productivity of the desired product.
  • Figure 1 is a schematic depicting the general process of culturing or fermenting microorganisms in, for example, a nitrate containing media, isolating the broth or a crude fraction containing the biosynthesized butadiene and purifying, partially or completely, the biosynthesized butadiene produced using micro-respiric growth of the invention.
  • non-naturally occurring when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species.
  • Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material.
  • modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species.
  • Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon.
  • Exemplary metabolic polypeptides include enzymes or proteins within a butadiene biosynthetic pathway or a denitrification enzyme or pathway.
  • a metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof. Exemplary metabolic modifications are disclosed herein.
  • butadiene having the molecular formula C4H6 and a molecular mass of 54.09 g/mol (see Figures 2-4) (IUPAC name Buta-l,3-diene) is used interchangeably throughout with 1,3 -butadiene, biethylene, erythrene, divinyl, vinylethylene.
  • Butadiene is a colorless, non corrosive liquefied gas with a mild aromatic or gasoline-like odor.
  • Butadiene is both explosive and flammable because of its low flash point.
  • isolated refers an organism or biosynthetic product that is substantially free of at least one component as the referenced microbial organism is found in nature.
  • the term includes a microbial organism or biosynthetic product that is removed from some or all components as it is found in its natural environment.
  • the term also includes a microbial organism or biosynthetic product of the invention that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism or biosynthetic product is partly or completely separated from other substances as it is found in nature or as it is grown, synthesized, stored or subsisted in non-naturally occurring environments.
  • isolated microbial organisms or biosynthetic products include partially pure microbes or products, substantially pure microbes or products and microbes cultured in a medium that is non-naturally occurring or products produced in a medium that is non-naturally occurring.
  • microbial As used herein, the terms "microbial,” “microbial organism” or “microorganism” are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.
  • CoA or "coenzyme A” refers to an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system.
  • Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.
  • anaerobic when used in reference to a culture or growth condition is intended to mean that the conditions are substantially anaerobic.
  • substantially anaerobic refers to an amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media.
  • the term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.
  • Exogenous as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism.
  • the molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism.
  • the source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas
  • homologous refers to a molecule or activity derived from the host microbial organism.
  • exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.
  • exogenous nucleic acid refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid.
  • a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein.
  • two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism
  • the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids.
  • exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids.
  • the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism
  • the term "gene disruption,” or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive or attenuated.
  • the genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate or attenuate the encoded gene product.
  • One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the non-naturally occurring microorganisms of the invention.
  • the phenotypic effect of a gene disruption can be a null mutation, which can arise from many types of mutations including inactivating point mutations, entire gene deletions, and deletions of chromosomal segments or entire chromosomes.
  • Specific enzyme inhibitors, such as antibiotics, can also produce null mutant phenotype, therefore being equivalent to gene disruption.
  • the term "growth-coupled" when used in reference to the production of a biosynthesized product is intended to mean that the biosynthesis of the referenced biochemical product is produced during the growth phase of a microorganism.
  • the growth-coupled production can be obligatory, meaning that the biosynthesis of the referenced biochemical is an obligatory product produced during the growth phase of a microorganism.
  • the term "attenuate,” or grammatical equivalents thereof, is intended to mean to weaken, reduce or diminish the activity or amount of an enzyme or protein.
  • Attenuation of the activity or amount of an enzyme or protein can mimic complete disruption if the attenuation causes the activity or amount to fall below a critical level required for a given pathway to function. However, the attenuation of the activity or amount of an enzyme or protein that mimics complete disruption for one pathway, can still be sufficient for a separate pathway to continue to function. For example, attenuation of an endogenous enzyme or protein can be sufficient to mimic the complete disruption of the same enzyme or protein for production of a fatty alcohol, fatty aldehyde or fatty acid product of the invention, but the remaining activity or amount of enzyme or protein can still be sufficient to maintain other pathways, such as a pathway that is critical for the host microbial organism to survive, reproduce or grow.
  • Attenuation of an enzyme or protein can also be weakening, reducing or diminishing the activity or amount of the enzyme or protein in an amount that is sufficient to increase yield of a fatty alcohol, fatty aldehyde or fatty acid product of the invention, but does not necessarily mimic complete disruption of the enzyme or protein.
  • the non-naturally occurring microbal organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration.
  • stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.
  • a particularly useful stable genetic alteration is a gene deletion.
  • the use of a gene deletion to introduce a stable genetic alteration is particularly useful to reduce the likelihood of a reversion to a phenotype prior to the genetic alteration.
  • stable growth-coupled production of a biochemical can be achieved, for example, by deletion of a gene encoding an enzyme catalyzing one or more reactions within a set of metabolic modifications.
  • the stability of growth-coupled production of a biochemical can be further enhanced through multiple deletions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity.
  • An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms.
  • mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides.
  • Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor.
  • Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable.
  • Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.
  • Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species.
  • a specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase.
  • a second example is the separation of mycoplasma 5 '-3' exonuclease and Drosophila DNA polymerase III activity.
  • the DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.
  • paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions.
  • Paralogs can originate or derive from, for example, the same species or from a different species.
  • microsomal epoxide hydrolase epoxide hydrolase I
  • soluble epoxide hydrolase epoxide hydrolase II
  • Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor.
  • Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.
  • a nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species.
  • a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein.
  • a nonorthologous gene includes, for example, a paralog or an unrelated gene.
  • Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score.
  • Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related.
  • Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept- 16- 1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.
  • the term "denitrification enzyme” or a "denitrification pathway” is intended to mean the loss of nitrate from an environment by an enzyme or enzymatic pathway having reducing activity.
  • the term includes one or more of the following enzymatic reduction steps: nitrate (N0 3 ⁇ ) reduction to nitrite (N0 2 ⁇ ); nitrite reduction to ammonia (NH 4 ); nitrite reduction to nitric oxide (NO); nitric oxide reduction to nitrous oxide (N 2 0); and nitrous oxide reduction to nitrogen gas (N 2 ), and the use of formate as an electron donor with nitrate being the electron acceptor.
  • An exemplary denitrification pathway includes reduction from nitrate to nitrite to ammonia using the denitrification enzymes nitrate reductase (narGHJI, narZYWB, and/or napFDAGHBC-ccmABCDEFGH) and membrane-associated, ammonia forming, nitrite reductase (nrfABCDEFG), respectively.
  • nitrate reductase narGHJI, narZYWB, and/or napFDAGHBC-ccmABCDEFGH
  • nrfABCDEFG membrane-associated, ammonia forming, nitrite reductase
  • Another exemplary denitrification pathway includes reduction from nitrate to nitrite to nitric oxide to nitrous oxide to nitrogen using the
  • denitrification enzymes nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase (Nor), and nitrous oxide reductase (Nos), respectively.
  • a denitrification enzyme refers to one enzymatic reduction step whereas a pathway includes two or more enzymatic reduction steps.
  • denitrification pathway includes part of a denitrification pathway as it is found or utilized naturally in a microorganism.
  • denitrification enzyme also is intended to include the upstream action of formate dehydrogenase to allow the use of formate as electron donor when nitrate is an electron acceptor.
  • fermentation refers to catabolic reactions producing ATP in which organic compounds serve as both the primary electron donor and ultimate electron acceptor.
  • the process of fermentation can be carried out under anaerobic, substantially anaerobic or microaerobic culture conditions of microorganisms including bacteria and fungi.
  • reducing equivalents is intended to mean a molecule that is capable of undergoing reduction or oxidation.
  • CO and/or H 2 can provide reducing equivalents by means of carbon monoxide dehydrogenase and hydrogenase enzymes, respectively.
  • Reducing equivalents also can come in the form of, for example, NADH, NADPH, FADH, reduced quinones, reduced cytochromes, reduced ferredoxins, reduced flavodoxins and thioredoxins.
  • the reducing equivalents can serve as cofactors for the for various enzymes disclosed herein as well as, for example, malate dehydrogenase, fumarate reductase, alpha-ketoglutarate: ferredoxin oxidoreductase (alternatively known as 2-oxoglutarate:ferredoxin oxidoreductase, alpha-ketoglutarate synthase, or 2-oxoglutarate synthase), pyruvate: ferredoxin oxidoreductase and isocitrate dehydrogenase.
  • the electrons from these reducing equivalents can alternatively pass through an ion-gradient producing electron transport chain where they are passed to an acceptor such as oxygen, nitrate, oxidized metal ions, protons, or an electrode.
  • the ion-gradient can then be used for ATP generation via an ATP synthase or similar enzyme.
  • the invention provides a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or denitrification pathway for reducing nitrate (NO 3 ) or nitrite (N0 2 ⁇ ) and a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase, wherein the non-naturally occurring microbial organism has at least one exogenous nucleic acid encoding an enzyme or protein within the butadiene
  • the biosynthetic pathway at least one exogenous nucleic acid encoding a denitrification enzyme or at least one exogenous nucleic acid encoding an enzyme or protein within the denitrification pathway.
  • the referenced enzymes or proteins are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired chemical product such as butadiene or a desired chemical product that results from an enzymatic reduction reaction of a denitrification reaction and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein.
  • the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a butadiene pathway, an electron transport pathway or denitrification enzyme or pathway pathway, such as that shown in Figures 2, 4-6, 7 or 8.
  • Metabolic pathways of microbial organisms can be engineered to biosynthesize chemical products such as butadiene.
  • the engineered pathways also can be engineered to generate excess reducing equivalents that can be harnessed for the production of cellular energy in the form of ATP, for example.
  • Butadiene biosynthetic pathways of the invention produce, for example, one or more excess NADH equivalents (reducing equivalents).
  • electrons from one reducing equivalents can be passed to 1 ⁇ 4 nitrate to form 3 ⁇ 4 water, 1 ⁇ 4 NH 4 + and concomitantly form a proton gradient.
  • ATP is synthesized by coupling the excess reducing equivalents produced from a butadiene biosynthetic pathway with enzymes involved in denitrification, thus allowing nitrate (NO 3 ) or nitrite (N0 2 ⁇ ) to be an electron acceptor under anaerobic conditions.
  • nitrate NO 3
  • nitrite N0 2 ⁇
  • excess reducing equivalents in the form of NADH are oxidized by a proton-pumping NADH dehydrogenase (Nuo) to produce a proton gradient across the membrane.
  • the proton gradient can then generate ATP via a proton/ ATPase complex (see, for example, Atp in Figure 7).
  • the electrons can be passed via an electron carrier to a nitrate reductase for generation of additional protons with further enhancement of the proton gradient.
  • Figure 2 depicts electrons being transferred to nitrate for its reduction to nitrite by nitrate reductase (Nar) and the generation of additional protons.
  • the nitrite can be further transported across a membrane, such as a bacterial periplasmic membrane, and reduced to ammonia (NH 4 ) by nitrite reductase (Nrf), for example.
  • the Nrf enzyme complex includes the NrfA, NrfB, NrfC and NrfD subunits.
  • NrfA is linked to the menaquinol pool in the cytoplasmic membrane through a soluble electron carrier, NrfB, that in turn interacts with a membrane integral quinol dehydrogenase, NrfCD.
  • NrfCD membrane integral quinol dehydrogenase
  • NrfA is associated with the cytoplasmic membrane with its cytochrome components facing or free in the periplasm; acting as a terminal electron acceptor of an electron transport chain beginning with membrane-associated formate -oxidizing enzymes it generates a proton gradient. This proton gradient is the driving force for the generation of ATP.
  • a similar electron transport occurs using an ubiquinol pool as shown in Figure 2.
  • Some, many or all of these enzymes can be utilized for nitrate and/or nitrite reduction by, for example, introducing encoding nucleic acids or modifying encoding nucleic acids to attenuate gene product activity in a host production organism to generate the desired combination of denitrification enzyme, enzymes or pathway.
  • polypeptides involved in regulatory, transport, and enzymatic functions related to the reduction of nitrate to nitrite to ammonium include, for example, three nitrate reductases, two nitrite reductases, three nitrate/nitrite transporters, two two-component regulatory systems, two formate dehydrogenases, and two NADH dehydrogenases.
  • the three nitrate reductases catalyzing the reduction of nitrate into nitrite include the cytoplasmic, membrane-associated enzymes NarG and NarZ and a periplasmic nitrate reductase (Nap).
  • Nitrate and nitrite are transported in and out of the cytoplasm by two nitrate (NarK and NarU) and three nitrite (NarK, NarU, and NirC) transporters.
  • Nitrite extrusion in the presence of nitrate mainly takes place through NarK, but nitrite uptake can be supported at similar rates by either NarK or NirC.
  • Nitrate uptake can be equivalently supported by either NarK or NarU.
  • the expression of nitrate- and nitrite-regulated genes is mediated by two environmental signals (the absence of oxygen and the presence of nitrate/nitrite ions in the culture medium), several global regulators (Fnr, Fis, Ihf, and H-Ns, and Cra), and by the homologous two-component regulatory systems NarX/NarL and NarQ/NarP. Formate and NADH are among the electron donors for nitrate and nitrite reduction.
  • Formate dehydrogenases (FdhN and FdhO) and NADH dehydrogenases deliver the electrons from formate and NADH to the quinone pools, which, in turn, pass the electrons to the nitrate and nitrite reductases. This conserves cellular energy and generates ATP via the proton- translocating ATPase.
  • NirB is locted in the cytoplasm and contributes little to the production of a proton gradient for generation of ATP. Accordingly, the activity of NirB (or other components of the complex, NirC and/or NirD) can be attenuated as either as being unnecessary or superfluous, or as a metabolic strategy to channel nitrite to the periplasmic space for production of
  • ammonia/ammonium as a beneficial nitrogen source for the microorganisms of the invention.
  • ammonia/ammonium as a beneficial nitrogen source for the microorganisms of the invention.
  • those skilled in the art will understand what additional gene attenuation strategies can be utilized to further the production of a proton gradient or a beneficial nitrogen source from the above denitrification enzymes and pathways.
  • the invention provides a non-naturally occurring microbial organism having a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase.
  • the metabolic modification includes attenuating the amount or activity of a gene product encoded by nirB, nirC or nirD.
  • a denitrification enzyme or denitrification pathway of the invention can include, for example, one or more formate dehydrogenases, nitrate reductases, NO-forming nitrite reductases, nitric oxide reductases or nitrous oxide reductases. Any of the previously described polypeptides having regulatory, transport or enzymatic functions also can be utilized as a denitrification enzyme or utilized in a denitrification pathway of the invention to couple with reducing equivalents for the production of ATP.
  • the desired activity can be introduced into a non-naturally occurring microbial organism of the invention by recombinant expression of an encoding nucleic acid
  • a host organism can be selected to have one or more endogenous activities or both recombinant expression or endogenous activities can be utilized to achieve any desired combination of enzymatic activities. Therefore, for the denitrification activities of the invention, for example, one or more denitrification enzymes or pathways can be introduced, utilized through endogenous activities or any combination thereof.
  • reducing equivalents can be harnessed for production of a proton gradient by passing electrons from a reducing equivalent to nitrate.
  • Nitrate (NO 3 ) can be reduced by the action of nitrate reductase to generate nitrite (N0 2 ⁇ ) and a proton gradient.
  • Subsequent steps also can be included such as the conversion of nitrite (N0 2 ⁇ ) to ammonia (NH 4 ), catalyzed by nitrite reductase for the production of a microbial nitrogen source.
  • One or more steps in a denitrification pathway from nitrate to gaseous nitrogen (N 2 ) can alternatively or additionally be included.
  • steps can initiate by the generation of nitrite from nitrate as described above.
  • Production of nitrogen gas can proceed via the gaseous intermediates nitric oxide (NO), produced from nitrite by NO-forming nitrite reductase, and nitrous oxide (N 2 0), produced from nitric oxide by nitric oxide reductase.
  • NO gaseous intermediates nitric oxide
  • N 2 0 nitrous oxide
  • Formate dehydrogenase also can be utilized to generate a proton gradient from reducing equivalents through the use of formate as electron donor when nitrate is an electron acceptor. Given the teachings and guidance provided herein, any of the above steps can occur alone or in combination.
  • nitrate reductases include members of the Nar complex.
  • Members of this enzyme complex include the following for Nar complex 1 : respiratory nitrate reductase 1, alpha chain (NarG) (1.7.99.4); respiratory nitrate reductase 1, beta chain (NarH) (1.7.99.4); respiratory nitrate reductase 1, gamma chain (Narl) (1.7.99.4); respiratory nitrate reductase 1, delta chain (NarJ) (1.7.99.4).
  • the following members belong to Nar complex 2: respiratory nitrate reductase 2, alpha chain (NarZ) (1.7.99.4);
  • An alternative gamma chain and a delta chains for complex 2 include: NarV respiratory nitrate reductase, gamma subunit; respiratory nitrate reductase 2, delta chain (NarW) (1.7.99.4) nitrite is converted to either ammonia or gaseous nitrogen.
  • Nitrite reductases catalyze the reduction of nitrite to ammonia or gaseous nitrogen.
  • the conversion of nitrite to ammonia is catalyzed by the NirBD enzyme.
  • the conversion of nitrite to gaseous nitrogen can proceed via two intermediate gaseous forms of nitrogen, nitric oxide (NO) and nitrous oxide (N 2 0).
  • NO-forming nitrite reductase (NirK or NirS) catalyzes the conversion of nitrite to nitric oxide.
  • Nitric oxide reductase (Nor) catalyzes the conversion of nitric oxide to nitrous oxide.
  • Nitrous oxide reductase catalyzes the conversion of nitrous oxide to nitrogen. Conversion of nitrite to gaseous nitrogen can be energetically less favorable than the conversion of nitrite to ammonia because of a smaller redox potential for the former as compared to the latter.
  • Homologues, orthologs, paralogs and/or nonorthologous gene displacements of the above proton gradient and/or denitrification enzymes of the invention such as Nuo, Nar, Nrf, Nir, Nor and Nos polypeptides and their encoding nucleic acids can be obtained from a variety of different bacterial, fungal and other microorganism species that are capable of carrying out anerobic respiration using nitrate as an electron acceptor. Exemplary members for nucleic acids encoding these polypeptides are described further below in Example I.
  • any of the denitrification enzymes or denitrification pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of Figures 2 and 7, can be utilized to generate a non-naturally occurring microbial organism that produces any denitrification pathway intermediate or product, as desired. It is further understood that a non-naturally occurring microbial organism that produces a
  • the denitrification enzyme product or a denitrification pathway intermediate can be utilized to produce the intermediate as a desired product for facilitation of micro-respiric growth and proton gradient generation.
  • the invention provides for at least the following reduction steps employing nitrate or nitrite as an electron acceptor: Nitrate (NO 3 ) can be reduced to nitrite (N0 2 ), nitrite can be reduced to ammonia (NH 4 ), nitrite can be reduced to nitric oxide (NO), nitric oxide can be reduced to nitrous oxide (N 2 0) , nitrous oxide can be reduced to nitrogen (N 2 ).
  • the invention provides a non-naturally occurring microbial organism, including a microbial organism having a butadiene pathway and a denitrification enzyme or pathway.
  • the butadiene biosynthetic pathway includes at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase, a butadiene synthase, a crotyl alcohol kin
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl- CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase ( Figure 4, steps A-H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) ( Figure 4, steps A-C, K, F, G, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase ( Figure 4, steps A-C, K, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase, ( Figure 4, steps A-C, I, J, E, F, G, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3- hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase ( Figure 4, steps A-C, I, J, E, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl- CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase and a crotyl alcohol diphosphokinase ( Figure 4, steps A-E, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotonyl-CoA reductase (aldehyde forming), a
  • crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase ( Figure 4, steps L, D-H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) ( Figure 4, steps L, K, F, G, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 4, steps L, K, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 4, steps L, I, J, E, F, G, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase ( Figure 4, steps L, I, J, E, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl- CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene a glutaconyl-CoA decarboxylase and a crotyl alcohol
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase ( Figure 4, steps M, D-H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotyl alcohol kinase, a 2-butenyl-4- phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) ( Figure 4, steps M, K, F, G, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a butadiene synthase, a crotonyl- CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase ( Figure 4, steps M, K, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 4, steps M, I, J, E, F, G, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase ( Figure 4, steps M, I, J, E, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl- CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a glutaryl-CoA dehydrogenase and a crotyl alcohol diphosphokinase (Figure 4, steps M, C, D, E, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 4, steps N, D-H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) ( Figure 4, steps N, K, F, G, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase ( Figure 4, steps N, K, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a
  • crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 4, steps N, I, J, E, F, G, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl- CoA deaminase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl- CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a 3-aminobutyryl-CoA deaminase and a crotyl alcohol diphosphokinase ( Figure 4, steps N, C, D, E, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 4, steps O, D-H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) ( Figure 4, steps O, K, F, G, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase ( Figure 4, steps O, K, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a
  • crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase ( Figure 4, steps O, I, J, E, F, G, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl- CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase ( Figure 4, steps O, I, J, E, P, H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a 4-hydroxybutyryl-CoA dehydratase and a crotyl alcohol diphosphokinase (Figure 4, steps L, C, D, E, P, H).
  • the invention provides a non-naturally occurring microbial organism, including a microbial organism having a butadiene pathway and a denitrification enzyme or pathway.
  • the butadiene biosynthetic pathway includes at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythrose-4-phosphate reductase, an erythritol-4- phospate cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4- cyclodiphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate synthase, a l-hydroxy-2- butenyl 4-diphosphate reductase, a butenyl 4-diphosphate
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythrose-4-phosphate reductase, an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate synthase, a l-hydroxy-2- butenyl 4-diphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate reductase and a butadiene synthase ( Figure 5, steps A-F, and H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythrose-4-phosphate reductase, an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate synthase, a 1- hydroxy-2-butenyl 4-diphosphate reductase, a butenyl 4-diphosphate isomerase and butadiene synthase ( Figure 5, steps A-H).
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythritol-4-phospate cytidylyltransferase, a 4- (cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate synthase, a 1- hydroxy-2 -butenyl 4-diphosphate synthase, a l-hydroxy-2 -butenyl 4-diphosphate reductase, a butadiene synthase, an erythrose-4-phosphate kinase, an erythrose reductase and a erythritol kinase ( Figure 5, steps I, J, K
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythritol-4-phospate
  • cytidylyltransferase a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4- cyclodiphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate synthase, a l-hydroxy-2- butenyl 4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a butadiene synthase, an erythrose-4-phosphate kinase, an erythrose reductase and an erythritol kinase ( Figure 5, steps I, J, K, B-H).
  • the invention provides a non-naturally occurring microbial organism, including a microbial organism having a butadiene pathway and a denitrification enzyme or pathway.
  • the butadiene biosynthetic pathway includes at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, an 3- oxoglutaryl-CoA reductase (ketone -reducing), a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a 3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate kinase, a 3- hydroxy-5-phosphonatooxypentanoate kinase, a 3-hydroxy-5-
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone- reducing), a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a 3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4- diphosphate isomerase and a butadiene synthase
  • the non- naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5- phosphonatooxypentanoate kinase, a 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene synthase, an 3- oxoglutaryl-CoA reductase (aldehyde forming), a 3,5-dioxopentanoate reductase (aldehyde reducing) and a 5-hydroxy-3-oxopent
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, a 3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate kinase, a 3-Hydroxy-5-phosphonatooxypentanoate kinase, a 3- Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4- diphosphate isomerase, a butadiene synthase, an 3-oxoglutaryl-CoA reductase (alde
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a 3-hydroxy-5- [hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene synthase, a 5-hydroxy-3-oxopentanoate reductase and a 3-oxo-glutaryl- CoA reductase (CoA reducing
  • the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing), a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a 3- hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4- diphosphate isomerase, a butadiene synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol forming).
  • the invention provides a non-naturally occurring microbial organism having a butadiene pathway and a denitrification enzyme or pathway.
  • the butadiene biosynthetic pathway includes at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of acetyl-CoA to acetoacetyl-CoA, acetoacetyl-CoA to 3-hydroxybutyryl-CoA, 3-hydroxybutyryl- CoA to crotonyl-CoA, crotonyl-CoA to crotonaldehyde, crotonaldehyde to crotyl alcohol, crotyl alcohol to 2-betenyl-phosphate, 2-betenyl-phosphate to 2-butenyl-4-diphosphate, 2-butenyl-4- diphosphate to butadiene, erythrose-4-phosphate to erythritol-4-phosphate
  • the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a butadiene pathway, such as that shown in Figures 4-6.
  • the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce an intermediate of a butadiene pathway.
  • exemplary butadiene pathways are disclosed herein (see above and figures).
  • the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a butadiene pathway enzyme, where the microbial organism produces a butadiene pathway intermediate, for example, acetoacetyl-CoA,
  • such a microbial organism that produces an intermediate can be used in combination with another microbial organism
  • the invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product.
  • reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.
  • the intermediates crotanate; 3,5-dioxopentanoate, 5-hydroxy-3- oxopentanoate, 3 -hydroxy-5 -oxopentanoate, 3-oxoglutaryl-CoA and 3-hydroxyglutaryl-CoA, as well as other intermediates are carboxylic acids, which can occur in various ionized forms, including fully protonated, partially protonated, and fully deprotonated forms. Accordingly, the suffix "-ate,” or the acid form, can be used interchangeably to describe both the free acid form as well as any deprotonated form, in particular since the ionized form is known to depend on the pH in which the compound is found.
  • carboxylate products or intermediates includes ester forms of carboxylate products or pathway intermediates, such as O-carboxylate and S-carboxylate esters.
  • O- and S-carboxylates can include lower alkyl, that is CI to C6, branched or straight chain carboxylates.
  • O- or S-carboxylates include, without limitation, methyl, ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl O- or S-carboxylates, any of which can further possess an unsaturation, providing for example, propenyl, butenyl, pentyl, and hexenyl O- or S-carboxylates.
  • O-carboxylates can be the product of a biosynthetic pathway.
  • Exemplary O-carboxylates accessed via biosynthetic pathways can include, without limitation: methyl crotanate; methy-3,5-dioxopentanoate; methyl-5-hydroxy-3- oxopentanoate; methyl-3 -hydroxy-5 -oxopentanoate; 3-oxoglutaryl-CoA, methyl ester; 3- hydroxyglutaryl-CoA, methyl ester; ethyl crotanate; ethyl-3,5-dioxopentanoate; ethyl-5 -hydroxy- 3-xopentanoate; ethyl-3-hydroxy-5-oxopentanoate; 3-oxoglutaryl-CoA, ethyl ester; 3- hydroxyglutaryl-CoA, ethyl ester; n-propyl crotanate; n-propyl-3,5-dioxopentanoate; n-propyl
  • O- carboxylates can include medium to long chain groups, that is C7-C22, O-carboxylate esters derived from fatty alcohols, such heptyl, octyl, nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl alcohols, any one of which can be optionally branched and/or contain unsaturations.
  • O-carboxylate esters derived from fatty alcohols, such heptyl, octyl, nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl, stearyl
  • O- carboxylate esters can also be accessed via a biochemical or chemical process, such as esterification of a free carboxylic acid product or transesterification of an O- or S-carboxylate.
  • S-carboxylates are exemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters, and various aryl and heteroaryl thioesters.
  • the non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more of the denitrification pathways or one or more of the butadiene biosynthetic pathways.
  • nucleic acids for some or all of a particular denitrification pathways or one or more of the butadiene biosynthetic pathway can be expressed.
  • nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression.
  • an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve reduction of nitrate or one or more of nitrogen containing compounds within a denitrification pathway or to achieve butadiene biosynthesis.
  • a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired denitrification or biosynthetic pathway or a desired denitrification pathway or biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as reduction of nitrate or downstream product for the generation of a proton gradient and/or a desired product such as butadiene.
  • Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, fungus or any of a variety of other microorganisms applicable or suitable to fermentation processes.
  • Exemplary bacteria include any species selected from the order Enterobacteriales, family Enterobacteriaceae, including the genera Escherichia and Klebsiella; the order Aeromonadales, family Succinivibrionaceae, including the genus Anaerobio spirillum; the order Pasteur ellales, family Pasteur ellaceae, including the genera Actinobacillus and Mannheimia; the order Rhizobiales, family
  • Bradyrhizobiaceae including the genus Rhizobium
  • the order Bacillales family Bacillaceae, including the genus Bacillus
  • the order Actinomycetales families Corynebacteriaceae and Streptomycetaceae, including the genus Corynebacterium and the genus Streptomyces, respectively
  • order Rhodospirillales family Acetobacteraceae, including the genus
  • Gluconobacter the order Sphingomonadales, family Sphingomonadaceae, including the genus Zymomonas; the order Lactobacillales, families Lactobacillaceae and Streptococcaceae, including the genus Lactobacillus and the genus Lactococcus, respectively; the order
  • Clostridiales family Clostridiaceae, genus Clostridium; and the order Pseudomonadales , family Pseudomonadaceae, including the genus Pseudomonas.
  • Non-limiting species of host bacteria include Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens,
  • Actinobacillus succinogenes Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Cory neb acterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.
  • exemplary species of fungi species include any species selected from the order Eurotiales, family Trichocomaceae, including the genus Aspergillus; and the order Mucorales, family Mucoraceae, including the genus Rhizopus.
  • Further exemplary species of yeast or fungi include genera selected from the order Sordariaceae, family Sordariales including the genus Neurospora, and the order Nectriaceae, family Hypocreales including the genus Fusarium.
  • Exemplary species of Neurospora include N. africana, N. bonaerensis, N.
  • mesoamericanum F. moniliforme, F. napiforme, F. nygamai, F. oxysporum, F. proliferatum, F. pseudocircinatum, F. pseudograminearum, F. sacchari, F. semitectum, F. solani, F.
  • Non-limiting species of host yeast or fungi include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and the like.
  • E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering.
  • Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product.
  • the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed denitrification or butadiene pathway- encoding nucleic acid and up to all encoding nucleic acids for one or more denitrification or butadiene biosynthetic pathways.
  • denitrification or butadiene biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid.
  • exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins.
  • exogenous expression of all enzymes or proteins in a pathway for production of denitrification product or butadiene can be included.
  • a non-naturally occurring microbial organism of the invention can have one, two, three, four or more enzymes and up to all nucleic acids encoding the enzymes or proteins constituting a denitrification pathway or butadiene biosynthetic pathway disclosed herein.
  • the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize reduction of a substrate in a denitrification pathway or butadiene biosynthesis or that confer other useful functions onto the host microbial organism.
  • One such other functionality can include, for example, augmentation of the synthesis of one or more of the denitrification pathway or butadiene pathway precursors such as nitrate, nitrite or crotyl alcohol, for example.
  • a host microbial organism is selected such that it produces the precursor of a denitrification or butadiene biosynthetic pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism.
  • formate for denitrification and acetyl-CoA are produced naturally in a host organism such as E. coli.
  • a host organism can be engineered to increase production of a precursor, as disclosed herein.
  • a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a denitrification or butadiene pathway.
  • a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to reduce nitrate or other products within a denitrification pathway of the invention or to synthesize butadiene.
  • it can be useful to increase the synthesis or accumulation of a denitrification pathway product or butadiene pathway product to, for example, drive reduction reactions or butadiene pathway reactions toward enhancement of a proton gradient or butadiene production.
  • Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described denitrification or butadiene pathway enzymes or proteins.
  • Overexpression of the enzyme or enzymes and/or protein or proteins of the denitrification or butadiene pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, reducing nitrate, nitrite or other nitrogen containing compounds or producing butadiene, through overexpression of one, two, three, four, five or up to all enzymes in the referenced pathway, that is, up to all nucleic acids encoding denitrification and/or butadiene biosynthetic pathway enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the denitrification activity or butadiene biosynthetic pathway.
  • exogenous expression of the encoding nucleic acids is employed.
  • Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user.
  • endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element.
  • an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time.
  • an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.
  • any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention.
  • the nucleic acids can be introduced so as to confer, for example, a denitrification or butadiene biosynthetic pathway onto the microbial organism.
  • encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer denitrification or butadiene biosynthetic capability.
  • a non-naturally occurring microbial organism having a denitrification or butadienebiosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the
  • nitrate reductase and nitrite reductase and the like.
  • any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention.
  • any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, nitrate reductase, nitrite reductase and NO-forming nitrite reductase, nitric oxide reductase and/or nitrous oxide reductase, and so forth, as desired, and including butadiene biosynthetic pathway combinations so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.
  • any combination of two, three, four or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.
  • the non- naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and/or with other microbial organisms and methods well known in the art to achieve enhanced product yield from increased ATP synbthesis and enhanced product biosynthesis by other routes.
  • a host organism can be selected based on desired characteristics for introduction of one or more gene disruptions to increase generation of a proton gradient and/or production of biosynthesized butadiene.
  • a genetic modification is to be introduced into a host organism to disrupt a gene, any homologs, orthologs or paralogs that catalyze similar, yet non-identical metabolic reactions can similarly be disrupted to ensure that a desired metabolic reaction is sufficiently disrupted. Because certain differences exist among metabolic networks between different organisms, those skilled in the art will understand that the actual genes disrupted in a given organism may differ between organisms.
  • the methods of the invention can be applied to any suitable host microorganism to identify the cognate metabolic alterations needed to construct an organism in a species of interest that will increase proton gradient and butadiene biosynthesis.
  • the increased production couples biosynthesis of butadiene to growth of the organism, and can obligatorily couple production of butadiene to growth of the organism if desired and as disclosed herein.
  • Sources of encoding nucleic acids for a denitrification enzyme, denitrification pathway enzymes and/or a butadiene pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction.
  • species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human.
  • An exemplary species for such sources include, for example, Escherichia coli and all other microbial species having endogenous denitrification pathways as well as other exemplary species disclosed herein or available as source organisms for
  • genes encoding the requisite denitrification pathway enzyme or enzymes and/or a butadiene biosynthetic activity for one or more genes in related or distant species including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art.
  • the metabolic alterations allowing biosynthesis of denitrification pathway enzyme or enzymes and/or butadiene described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.
  • denitrification pathway activities and/or butadiene biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non- identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ.
  • teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will carry out denitrification of nitrate and/or nitrite to enhance proton gradient production and synthesize butadiene.
  • Methods for constructing and testing the expression levels of a non-naturally occurring denitrification exhibiting and butadiene-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).
  • Exogenous nucleic acid sequences involved in a pathway for conferring denitrification pathway activity and for the production of butadiene can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation.
  • some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al, J. Biol. Chem.
  • genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells.
  • a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells.
  • An expression vector or vectors can be constructed to include one or more denitrification and/or butadiene biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism.
  • Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
  • both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • the transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art.
  • Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA
  • PCR polymerase chain reaction
  • immunoblotting for expression of gene products
  • the invention also provides a composition comprising a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or pathway for reducing nitrate (NO 3 ) or nitrite (N0 2 ⁇ ) and a medium comprising nitrate.
  • the medium can alternatively contain nitrite or both nitrate and nitrite.
  • the medium can alternatively or additionally contain elevated levels of ammonia or ammonium as a terminal product during culture of the non-naturally occurring organisms of the invention.
  • nitrate and/or nitrite instead of oxygen to provide for respiration simulates a microaerobic environment and is beneficial because it is much easier to control relative to oxygen.
  • the use of nitrate and/or nitrite does not require high agitation rates and therefore allows the use of anaerobic fermentation conditions and vessels that can be much larger than fermenters for aerobic processes and are significantly less expensive than aerobic fermenters.
  • the non-naturally occurring microorganisms of the invention can be in medium containing from about 0.1 mg/L to about 10 g/L or more nitrate and/or nitrite.
  • concentrations within this range include, for example, 0.1 mg/L, 0.2 mg/L, 0.3 mg/L, 0.4 mg/L, 0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1.0 g/L, 1.5 g/L, 2.0 g/L, 2.5 g/L, 3.0 g/L, 3.5 g/L, 4.0 g/L, 4.5 g/L, 5.0 g/L, 5.5 g/L, 6.0 g/L, 6.5 g/L, 7.0 g/L, 7.5 g/L, 8.0 g/L, 8.5 g/L, 9.0 g/L, 9.5 g/L, 10 g/L or more.
  • any concentration in between the above exemplary concentrations also can be used in the invention.
  • the concentration of nitrate and/or nitrite can be maintained at or near the Km of the nitrate reductase or nitrite reductase that are employed.
  • Such values can be in the nanomolar or micromolar range including, for example, 10 uM, 20 uM, 30 uM, 40 uM, 50 uM, 60 uM, 70 uM, 80 uM, 90 uM, 0.1 ⁇ , 0.2 ⁇ , 0.3 ⁇ , 0.4 ⁇ , 0.5 ⁇ , 0.6 ⁇ , 0.7 ⁇ , 0.8 ⁇ , 0.9 ⁇ , 1.0 ⁇ , 2.0 ⁇ , 3.0 ⁇ , 4.0 ⁇ , 5.0 ⁇ , 6.0 ⁇ , 7.0 ⁇ , 8.0 ⁇ , 9.0 ⁇ , 10 ⁇ or more.
  • Ammonia and ammonium can be elevated in the medium in amounts up to 1.0 M or more than its concentration as it is found in a comparable culture of a microbial organism that does not couple a denitrification pathway for increased production of a proton gradient and ATP synthesis as described herein.
  • the elevated amounts can include, for example, elevated amounts up to about 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M or more compared to a microbial organism that does not couple a denitrification pathway for increased production of a proton gradient and ATP synthesis as described herein.
  • the total concentrations of ammonia that can be formed using the culture and/or fermentation conditions of the invention include, for example, about 250 mM to 2.0 M, particularly about 250 mM to 1.0 M, and more particularly, about 400 mM to 800 mM.
  • the invention provides a non-naturally occurring microbial organism as described previously in a medium containing a nitrogen compound for accepting electrons under micro-respiric conditions such as nitrate and/or nitrite or in a medium contain a downstream or final product of one or more electron transport steps of a denitrification pathway as described herein. Accordingly, the invention additionally provides a non-naturally occurring microbial organism wherein the denitrification enzyme or pathway includes formate dehydrogenase, nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase or nitrous oxide reductase.
  • the non-naturally occurring microbial organism in nitrate containing medium can reduce nitrate (NO3 ) to nitrite (N0 2 ⁇ ), ammonia (N3 ⁇ 4), nitric oxide (NO), nitrous oxide (N 2 0) or nitrogen (N 2 ).
  • the non-naturally occurring microbial organism of can further include a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase.
  • the nitrite reductase can be a NADH-dependent nitrite reductase, including for example a NADH-dependent nitrite reductase encoded by nirB, nirC or nirD. Additionally, the butadiene biosynthetic pathway of the non-naturally occurring organisms of the invention can produce excess reducing equivalents, including NADH reducing equivalent.
  • An exemplary butadiene biosynthetic pathway of a non-naturally occurring microbial organism in nitrate containing medium can include an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase and a butadiene synthase.
  • the invention further provides a non- naturally occurring microbial organism in nitrate containing medium, for example, wherein the microbial organism is Escherichia.
  • the invention further provides a composition comprising bioderived butadiene in a nitrate or ammonium (NH4+) containing medium.
  • the medium alternatively or additionally can contain any of the nitrogen compounds exemplified previously as an intermediate or final product in the denitrification pathways of the invention.
  • the microorganism of the invention can be used to generate bioderived butadiene.
  • the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in butadiene or any butadiene pathway intermediate.
  • the various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources.”
  • Uptake sources can provide isotopic enrichment for any atom present in the product butadiene or butadiene pathway intermediate, or for side products generated in reactions diverging away from a butadiene pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.
  • the uptake sources can be selected to alter the carbon-12, carbon- 13, and carbon- 14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen- 16, oxygen- 17, and oxygen- 18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can be selected to alter the nitrogen- 14 and nitrogen- 15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31 , phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios.
  • the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources.
  • An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom.
  • An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction.
  • Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio.
  • a target atom isotopic ratio of an uptake source can be achieved by selecting a desired origin of the uptake source as found in nature.
  • a natural source can be a biobased derived from or synthesized by a biological organism or a source such as petroleum-based products or the atmosphere.
  • a source of carbon for example, can be selected from a fossil fuel-derived carbon source, which can be relatively depleted of carbon- 14, or an environmental or atmospheric carbon source, such as C0 2 , which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart.
  • the unstable carbon isotope carbon-14 or radiocarbon makes up for roughly 1 in 10 12 carbon atoms in the earth's atmosphere and has a half-life of about 5700 years.
  • the stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen ( 14 N).
  • Fossil fuels contain no carbon-14, as it decayed long ago. Burning of fossil fuels lowers the atmospheric carbon-14 fraction, the so-called "Suess effect”.
  • Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR).
  • AMS accelerated mass spectrometry
  • SIRMS Stable Isotope Ratio Mass Spectrometry
  • SNIF-NMR Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance
  • mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC), high performance liquid
  • ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product's biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective April 1, 2011). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein.
  • the biobased content of a compound is estimated by the ratio of carbon- 14 ( 14 C) to carbon- 12 ( 12 C).
  • An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available.
  • the Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933 ⁇ 0.001 (the weighted mean). The isotopic ratio of HOx II is -17.8 per mille.
  • ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)).
  • a Fm 0% represents the entire lack of carbon-14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source.
  • a Fm 100%, after correction for the post-1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a "modern" source includes biobased sources.
  • the percent modern carbon (pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon-14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a "pre-bomb" standard, and because nearly all new biobased products are produced in a post-bomb
  • polypropylene terephthalate (PPT) polymers derived from renewable 1,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30% (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al, supra, 2000).
  • polybutylene terephthalate polymer derived from both renewable 1 ,4-butanediol and renewable terephthalic acid resulted in bio-based content exceeding 90%> (Colonna et al, supra, 2011).
  • the present invention provides butadiene or a butadiene pathway intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source.
  • the butadiene or a butadiene pathway intermediate can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or as much as 100%.
  • the uptake source is C0 2 .
  • the present invention provides butadiene or a butadiene pathway intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects petroleum-based carbon uptake source.
  • the butadiene or a butadiene pathway intermediate can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%>, less than 45%, less than 40%>, less than 35%, less than 30%>, less than 25%, less than 20%), less than 15%, less than 10%, less than 5%, less than 2% or less than 1%.
  • the present invention provides butadiene or a butadiene pathway intermediate that has a carbon- 12, carbon-13, and carbon- 14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source.
  • a combination of uptake sources is one way by which the carbon- 12, carbon-13, and carbon- 14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.
  • the present invention relates to the biologically produced butadiene or butadiene pathway intermediate as disclosed herein, and to the products derived therefrom, wherein the butadiene or a butadiene pathway intermediate has a carbon- 12, carbon-13, and carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment.
  • the invention provides bioderived butadiene or a bioderived butadiene intermediate having a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment, or any of the other ratios disclosed herein.
  • a product can have a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived butadiene or a bioderived butadiene pathway intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product.
  • Methods of chemically modifying a bioderived product of butadiene, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein.
  • the invention further provides products made from bioderived butadiene such as synthetic rubber, tires and the like having a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment, wherein the synthetic rubber, tires, and the like are generated directly from or in combination with bioderived butadiene or a bioderived butadiene pathway intermediate as disclosed herein.
  • bioderived butadiene such as synthetic rubber, tires and the like having a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment
  • butadiene is a chemical used in commercial and industrial applications. Non-limiting examples of such applications include production of synthetic rubber or elastomers and all products made from such rubber and elastomers such as tires and the like. Moreover, butadiene is also used as a raw material in the production of a wide range of products including tires, styrene-butadiene rubber (SBR), polybutadiene rubber (PBR), polychloroprene (Neoprene), nitrile rubber (NR) and the like.
  • SBR styrene-butadiene rubber
  • PBR polybutadiene rubber
  • Neoprene polychloroprene
  • NR nitrile rubber
  • the invention provides biobased synthetic rubber, other elastomers and products derived therefrom comprising one or more bioderived butadiene or bioderived butadiene pathway intermediate produced by a non- naturally occurring microorganism of the invention or produced using a method disclosed herein.
  • bioderived means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism.
  • a biological organism in particular the microbial organisms of the invention disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source.
  • the biological organism can utilize atmospheric carbon.
  • biobased means a product as described above that is composed, in whole or in part, of a bioderived compound of the invention.
  • a biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock.
  • the invention provides synthetic rubber and all products made from such rubber comounds such as tires and the like comprising bioderived butadiene or bioderived butadiene pathway intermediate, wherein the bioderived butadiene or bioderived butadiene pathway intermediate includes all or part of the butadiene or butadiene pathway intermediate used in the production of synthetic rubber and all products made from such rubber comounds such as tires and the like.
  • the final synthetic rubber, elastomer or products made therefrom can contain the bioderived butadiene, butadiene pathway intermediate, or a portion thereof that is the result of the manufacturing of synthetic rubber, elastomer or products made therefrom.
  • Such manufacturing can include chemically reacting the bioderived butadiene or bioderived butadiene pathway intermediate (e.g. chemical conversion, chemical functionalization, chemical coupling, oxidation, reduction, polymerization, copolymerization and the like) into the final synthetic rubber, elastomer or products made therefrom.
  • bioderived butadiene or bioderived butadiene pathway intermediate e.g. chemical conversion, chemical functionalization, chemical coupling, oxidation, reduction, polymerization, copolymerization and the like
  • the invention provides a biobased synthetic rubber, elastomer or products made therefrom comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived butadiene or bioderived butadiene pathway intermediate as disclosed herein.
  • the invention provides a composition having a bioderived butadiene or butadiene pathway intermediate disclosed herein and a compound other than the bioderived butadiene or butadiene pathway intermediate.
  • the invention provides a biobased synthetic rubber, elastomer or products made thererfrom such as tires and the like wherein the butadiene or butadiene pathway intermediate used in its production is a combination of bioderived and petroleum derived butadiene or butadiene pathway intermediate.
  • a biobased synthetic rubber, elastomer or products made therefrom such as tires and the like can be produced using 50%> bioderived butadiene and 50%> petroleum derived butadiene or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of
  • bioderived/petroleum derived precursors so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing synthetic rubber, elastomer or products made therefrom such as rubber using the bioderived butadiene or bioderived butadiene pathway intermediate of the invention are well known in the art.
  • the invention provides a process for producing a biobased synthetic rubber, elastomer or products made therefrom such as tires disclosed herein by chemically reacting the bioderived butadiene with itself or another compound in a synthetic rubber producing reaction. It is understood that such process are well known in the art.
  • a compound other than the bioderived butadiene in a composition of the invention is a trace amount of a cellular portion of a non-naturally occurring microbial organism having a butadiene pathway of the invention disclosed here.
  • a cellular portion of a microbial organism includes without limitation proteins, polypeptides, peptides, amino acids, nucleic acids, polynucleotides, components of the cell wall or a cellular membrane including, for example, peptidoglycans, glycoproteins, and polysaccharides, or any other cellular component.
  • a "trace amount” as used herein refers to the presence of a compound or material in the composition, but in a quantity approaching a detectable limit. Such trace amounts can be so small as to not be accurately measured.
  • the invention provides a molded product obtained by molding a biobased synthetic rubber or product made therefrom disclosed herein.
  • molded products may be produced into any number of industrially desirable forms including, for example, a pellet.
  • the invention additionally provides culture medium comprising bioderived butadiene, wherein said bioderived butadiene has a carbon- 12, carbon- 13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source.
  • the culture medium can contain nitrate, nitrite and/or ammonium as described previously.
  • the culture medium can be separated from a non-naturally occurring microbial organism having a butadiene pathway.
  • the bioderived butadiene can have a carbon- 12, carbon- 13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source.
  • the bioderived butadiene can have an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.
  • the invention also provides a composition including bioderived butadiene and a compound other than bioderived butadiene.
  • the compound other than bioderived butadiene can be a trace amount of a cellular portion of a non-naturally occurring microbial organism having a butadiene pathway.
  • biobased synthetic rubber or product made therefrom having any of the bioderived butadiene described above.
  • the biobased synthetic rubber or product made therefrom can have at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50%) bioderived butadiene.
  • the invention additionally provides methods of utilizing the microbial organisms of the invention containing a denitrification pathway for making a desired product, including butadiene.
  • the invention provides a method of making bioderived butadiene.
  • the method can include the steps of culturing a non-naturally occurring microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway for reducing nitrate (NO3 ) or nitrite (N0 2 ⁇ ) in a medium containing a sufficient amount of nitrate to produce a proton gradient under anaerobic conditions for a sufficient period of time to produce the bioderived butadiene.
  • NO3 nitrate
  • N0 2 ⁇ nitrite
  • the denitrification enzyme or pathway can comprise formate dehydrogenase, nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase and/or nitrous oxide reductase.
  • the nitrate (NO3) can be reduced to nitrite (N0 2 ⁇ ), ammonia (NH 4 ), nitric oxide (NO), nitrous oxide (N 2 0) or nitrogen (N 2 ).
  • the microbial organism can further comprise a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase.
  • the nitrite reductase can be a NADH-dependent nitrite reductase.
  • the metabolic modification of the nitrite reductase can comprise attenuating the amount or activity of, for example, nirB, nirC or nirD.
  • the butadiene biosynthetic pathway in which a microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway is utilized, can produce excess reducing equivalents.
  • the reducing equivalent can be NADH.
  • the method of the invention can utilize a microbial organism where the butadiene biosynthetic pathway comprises an acetyl- CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase.
  • the butadiene biosynthetic pathway comprises an acetyl- CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotony
  • any of a number of host microbial organisms can be used in a method of the invention, including but not limited to Escherichia species.
  • the sufficient amount of nitrate can comprise, for example, 0.1 mg/L to 10 g/L nitrate, including intermediate concentrations as disclosed herein.
  • the anaerobic conditions can comprise an amount of oxygen that is less than about 10% of saturation for dissolved oxygen in liquid media.
  • the invention further provides a method of producing bioderived butadiene by growing a non-naturally occurring microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway for reducing nitrate ( ⁇ 0 3 ⁇ ) or nitrite (N0 2 ⁇ ) in fermentation broth containing nitrate under fermentation conditions for a sufficient period of time to produce said bioderived butadiene.
  • the denitrification enzyme or pathway can comprise formate dehydrogenase, nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase and/or nitrous oxide reductase.
  • the nitrate (NO3) can be reduced to nitrite (N0 2 ⁇ ), ammonia (NH 4 ), nitric oxide (NO), nitrous oxide (N 2 0) or nitrogen (N 2 ).
  • the microbial organism can further comprise a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase.
  • the nitrite reductase can be a NADH-dependent nitrite reductase.
  • the metabolic modification of the nitrite reductase can comprise attenuating the amount or activity of, for example, nirB, nirC or nirD.
  • the butadiene biosynthetic pathway in which a microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway is utilized, can produce excess reducing equivalents.
  • the reducing equivalent can be NADH.
  • the method of the invention can utilize a microbial organism where the butadiene biosynthetic pathway comprises an acetyl- CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase.
  • the butadiene biosynthetic pathway comprises an acetyl- CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotony
  • any of a number of host microbial organisms can be used in a method of the invention, including but not limited to Escherichia species.
  • the sufficient amount of nitrate can comprise, for example, 0.1 mg/L to 10 g/L nitrate, including intermediate concentrations as disclosed herein.
  • the anaerobic conditions can comprise an amount of oxygen that is less than about 10% of saturation for dissolved oxygen in liquid media.
  • the method can further comprise purifying the bioderived biobutadiene.
  • the bioderived biobutadiene can be purified by compression and cryoscopic distillation, as disclosed herein.
  • Suitable purification and/or assays to test for the production of a desired product such as butadiene can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant.
  • HPLC High Performance Liquid Chromatography
  • GC-MS Gas Chromatography-Mass Spectroscopy
  • LC-MS Liquid Chromatography-Mass Spectroscopy
  • Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al, Biotechnol. Bioeng., 90:775-779 (2005)), or other suitable assay and detection methods well known in the art.
  • the individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art. For typical Assay Methods, see Manual on Hydrocarbon Analysis (ASTM Manula Series, A.W. Drews, ed., 6th edition, 1998, American Society for Testing and Materials, Baltimore,
  • a desired product such as butadien can be separated from other components in the culture using a variety of methods well known in the art.
  • separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.
  • any of the non-naturally occurring microbial organisms described herein can be cultured under conditions to utilize a denitrification pathway to produce and/or secrete the biosynthetic products of the invention.
  • the denitrification pathway utilizing and butadiene producers can be cultured for the biosynthetic production of butadiene.
  • the invention provides culture medium having the butadiene or butadiene pathway intermediate described herein.
  • the culture mediums can also be separated from the non-naturally occurring microbial organisms of the invention that produced the butadiene or butadiene pathway intermediate.
  • Methods for separating a microbial organism from culture medium are well known in the art. Exemplary methods include filtration, flocculation, precipitation, centrifugation, sedimentation, and the like.
  • the recombinant strains are cultured under conditions suitable for utilizing a denitrification enzyme or pathway, as disclosed herein, and in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap.
  • microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration.
  • Exemplary anaerobic conditions have been described previously and are well-known in the art.
  • Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719, filed August 10, 2007.
  • Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. Fermentations can also be conducted in two phases, if desired. The first phase can be aerobic to allow for high growth and therefore high productivity, followed by an anaerobic phase of high butadiene yields.
  • the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH.
  • the growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.
  • the growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism.
  • Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch.
  • Other sources of carbohydrate include, for example, renewable feedstocks and biomass.
  • Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks.
  • Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
  • carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
  • renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of a desired product such as butadiene.
  • the microbial organisms of the invention also can be modified for growth on syngas as its source of carbon.
  • one or more proteins or enzymes are expressed in the denitrification pathway utilizing and butadiene producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.
  • Synthesis gas also known as syngas or producer gas
  • syngas is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues.
  • Syngas is a mixture primarily of H 2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H 2 and CO, syngas can also include C0 2 and other gases in smaller quantities.
  • synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C0 2 .
  • the Wood-Ljungdahl pathway catalyzes the conversion of CO and H 2 to acetyl-CoA and other products such as acetate.
  • Organisms capable of utilizing CO and syngas also generally have the capability of utilizing C0 2 and C0 2 /H 2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway.
  • H 2 -dependent conversion of C0 2 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved.
  • non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize C0 2 and H 2 mixtures as well for the production of acetyl-CoA and other desired products.
  • the Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch.
  • the methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA.
  • the reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase,
  • methyltetrahydrofolatexorrinoid protein methyltransferase for example, AcsE
  • corrinoid iron- sulfur protein for example, nickel-protein assembly protein
  • nickel-protein assembly protein for example, AcsF
  • ferredoxin for example, ferredoxin
  • acetyl-CoA synthase carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC).
  • the non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a denitrification and/or butadiene pathway enzyme or protein in sufficient amounts to produce utilize a denitrification pathway and produce butadiene. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce butadiene and utilize a denitrification pathway.
  • the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of butadiene resulting in intracellular concentrations between about 0.001-2000 mM or more.
  • the intracellular concentration of butadiene is between about 3-1500 mM, particularly between about 5-1250 mM and more particularly between about 8-1000 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, 100 mM, 200 mM, 500 mM, 800 mM, or more.
  • Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms of the invention.
  • culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions.
  • Exemplary anaerobic conditions have been described previously and are well known in the art.
  • Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication
  • Exemplary fermentation processes include, but are not limited to, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation; and continuous fermentation and continuous separation.
  • the production organism is grown in a suitably sized bioreactor sparged with an appropriate gas.
  • the culture is sparged with an inert gas or combination of gases, for example, nitrogen, N 2 /C0 2 mixture, argon, helium, and the like.
  • additional carbon source(s) and/or other nutrients are fed into the bioreactor at a rate approximately balancing consumption of the carbon source and/or nutrients.
  • the temperature of the bioreactor is maintained at a desired temperature, generally in the range of 22-37 degrees C, but the temperature can be maintained at a higher or lower temperature depending on the the growth characteristics of the production organism and/or desired conditions for the fermentation process. Growth continues for a desired period of time to achieve desired characteristics of the culture in the fermentor, for example, cell density, product concentration, and the like. In a batch fermentation process, the time period for the fermentation is generally in the range of several hours to several days, for example, 8 to 24 hours, or 1, 2, 3, 4 or 5 days, or up to a week, depending on the desired culture conditions.
  • the pH can be controlled or not, as desired, in which case a culture in which pH is not controlled will typically decrease to pH 3-6 by the end of the run.
  • the fermenter contents can be passed through a cell separation unit, for example, a centrifuge, filtration unit, and the like, to remove cells and cell debris.
  • a cell separation unit for example, a centrifuge, filtration unit, and the like.
  • the cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product.
  • the fermentation broth can be transferred to a product separations unit. Isolation of product occurs by standard separations procedures employed in the art to separate a desired product from dilute aqueous solutions.
  • Such methods include, but are not limited to, liquid-liquid extraction using a water immiscible organic solvent (e.g, toluene or other suitable solvents) to provide an organic solution of the product, if appropriate, standard distillation methods, and the like, depending on the chemical characteristics of the product of the
  • the production organism is generally first grown up in batch mode in order to achieve a desired cell density.
  • feed medium of the same composition is supplied continuously at a desired rate, and fermentation liquid is withdrawn at the same rate.
  • the product concentration in the bioreactor generally remains constant, as well as the cell density.
  • the temperature of the fermentor is maintained at a desired temperature, as discussed above.
  • the bioreactor is operated continuously for extended periods of time, generally at least one week to several weeks and up to one month, or longer, as appropriate and desired.
  • the fermentation liquid and/or culture is monitored periodically, including sampling up to every day, as desired, to assure consistency of product concentration and/or cell density.
  • fermenter contents are constantly removed as new feed medium is supplied.
  • the exit stream, containing cells, medium, and product are generally subjected to a continuous product separations procedure, with or without removing cells and cell debris, as desired.
  • Continuous separations methods employed in the art can be used to separate the product from dilute aqueous solutions, including but not limited to continuous liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene), standard continuous distillation methods, and the like, or other methods well known in the art.
  • growth condition for achieving biosynthesis of butadiene in a denitfication utilizing microbial organism can include the addition of an osmoprotectant to the culturing conditions.
  • the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant.
  • an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress.
  • Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose.
  • Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2- methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine.
  • the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used.
  • the amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about lOmM, no more than about 50mM, no more than about lOOmM or no more than about 500mM.
  • anaerobic conditions are particularly useful in methods using a denitrification pathway utilizing microbial organism since anaerobic conditions allow the use of nitrate instead of oxygen for respiration.
  • the use of nitrate can be useful since it can be controlled more easily relative to oxygen.
  • the methods of the invention utilizing a denitrification pathway and anaerobic conditions do not require high agitation rates and hence allow the use of anaerobic fermentation conditions and vessels.
  • Such anaerobic fermentation conditions and vessels can be much larger than fermenters for aerobic processes and are significantly less expensive than aerobic fermenters.
  • nitrate instead of oxygen minimizes undesired oxidative side reactions for a product such as butadiene that is highly reactive in the presence of oxygen, including reducing the potential risk of explosion in reaction vessels containing products that are highly reactive in the presence of oxygen.
  • Methods for purifying butadiene are well known to those skilled in the art. Accordingly, such methods can be used to separate or purify the bioderived butadiene form culture media, fermentation broth and the like.
  • compression, cryoscopic distillation or both compression and cryoscopic distillation can be used to separate and/or purify bioderived butadiene from culture media or fermentation broth.
  • Compression can be used to convert a material or mixtures of materials from a gaseous to its liquid phase. This method is applicable for purifying the bioderived butadiene of the invention because bioderived butadiene is found in its gaseous form once released by the non-naturally occurring microbial organisms of the invention.
  • cryoscopic distillation is a distillative freezing process in which a mixture of two or more components to be separated are vaporized under sufficiently reduced pressure and only one component freezes.
  • a first step partial vaporization of a mixture under reduced pressure results in crystallization of a major component of the mixture to be purified.
  • a pure product of the major component is obtained by repeating this operation to completely eliminate the liquid phase.
  • the low pressure vapor is transformed into a condensed mass by slightly lowering the temperature without pressurizing it. Distillation procedures and the recovery process of butadiene are described in Chemico-Biological
  • the invention provides bioderived butadiene separated or purified by compression, cryoscopic distillation or by both compression and cryoscopic distillation.
  • the method includes butadiene containing media or fermentation broth obtained following culturing under anaerobic conditions in the presence of nitrate or other nitrogen containing compound described herein for achieving micro-respiric growth.
  • This Example describes exemplary denitrification enzymes and denitrification pathways that can be included a butadiene producing microbial for growing in the presence of nitrate.
  • Nitrate respiration has been extensively studied (Kraft et al, Journal of
  • NADH dehydrogenase NADH -> NAD
  • NADH ubiquinone oxidoreductase I (NDH-1) is an NADH dehydrogenase that catalyzes the transfer of electrons from NADH to the quinone pool in the cytoplasmic membrane and is able to generate a proton electrochemical gradient.
  • Nitrate reductase Nitrate -> Nitrite
  • E. coli contains three nitrate reductases. Two of them, nitrate reductase A (NRA) and nitrate reductase Z (NRZ), are membrane bound and biochemically similar. The third nitrate reductase, Nap, is located in the periplasm. Nitrate reductase A is expressed when levels of nitrate in the environment are high, Nap is expressed when they are low, while NRZ expression is not dependent on nitrate levels or anaerobiosis, but induced during stationary phase.
  • NUA nitrate reductase A
  • NRZ nitrate reductase Z
  • Nitrate reductase A functions anaerobically as a terminal electron acceptor. It accepts electrons from the quinone pool and in so doing expels two protons from the cell, thereby adding to the proton motive force. Formate dehydrogenase N and nitrate reductase A form a respiratory chain, where a redox loop of quinone molecules couples electron transfer from formate in the periplasm to nitrate in the cytoplasm.
  • Nitrate reductase A is a heterotrimer composed of the ⁇ -, ⁇ - and ⁇ chains.
  • a fourth polypeptide, encoded by the narJ gene, is required for the incorporation of the molybdenum cofactor into NarG, the a subunit. If it is coexpressed with the private chaperone NarJ, the NarGH complex alone is soluble and active with artificial electron donors such as benzyl viologen. NarGH becomes localized to the cytoplasmic side of the inner membrane by interaction with Narl.
  • nitrate reductase Z expression is not dependent on nitrate levels or anaerobiosis. During entry into stationary phase, transcription of the narZYWV operon is induced, and induction is mainly dependent on the alternative sigma factor RpoS.
  • RpoS alternative sigma factor
  • nitrate reductase Z is a heterotrimer composed of the ⁇ -, ⁇ - and ⁇ chains.
  • a fourth polypeptide, encoded by the narW gene is required for the incorporation of the molybdenum cofactor into NarZ, the a subunit.
  • Nap Periplasmic nitrate reductase
  • Nap The physiological role of Nap is that of mediating anaerobic respiration at the expense of low concentrations of nitrate. Owing to the periplasmic location of Nap, the cost of pumping nitrate into the cell is avoided. In addition, Nap has a significantly higher affinity for nitrate than nitrate reductase A and is thus able to exploit the low concentrations of nitrate occuring in the natural environment of E. coli. Notably, several pathogenic bacterial species, such as Haemophilus influenzae, only contain orthologs of the periplasmic nitrate reductase. During glucose fermentation in the absence of menaquinone, a very low level of Nap activity appears to substitute for the redox-balancing role of fumarate reductase, which is dependent on menaquinone.
  • the nap operon encodes seven proteins.
  • the catalytic portion of the protein consisting of the periplasmic NapA and NapB polypeptides, receives electrons via the membrane-bound cytochrome NapC from NapGH or directly from the quinone pool.
  • the NapD polypeptide is required for enzyme activity and is thought to be involved in the post-translational assembly of the molybdoprotein NapA.
  • NapF, NapG and NapH are predicted to encode iron- sulfur proteins and are not required for Nap activity; they do, however, contribute to the maximum rate of nitrate reduction.
  • NapG and NapH facilitate electron transfer from ubiquinol via NapC to NapAB.
  • NrfABCD is a respiratory enzyme which couples to the formate oxidising enzymes via menaquinone in order to generate electron potential.
  • nrfA encodes a cytochrome c nitrite reductase (cyt c552). It is a novel pentahaem enzyme that forms a redox complex with NrfB - also a pentahaem protein - to catalyse the reduction of nitrite to ammonia.
  • nrfC and nrfD are predicted to encode electron transfer proteins that couple electron transport from the menaquinol pool in the membrane to the NrfAB complex in the periplasm.
  • NrfA is linked to the menaquinol pool in the cytoplasmic membrane through the electron carrier NrfB which in turn interacts with the membrane integral proteins NrfC and NrfD. Under physiological conditions it is likely that the periplasmic subunit NrfA is tightly associated with the membrane assoicated subunit NrfB.
  • NrfA can also reduce and thereby detoxify nitric oxide (NO); mutant strains lacking NrfA activity have increased sensitivity to NO.
  • NrfA Nitrite reduction and synthesis of the NrfA protein are repressed by oxygen.
  • nitrite reduction by the Nrf pathway is induced by nitrite and repressed by nitrate.
  • the Fnr transciptional regulator is essential for NrfA synthesis and nitrite reduction by formate.
  • the nrf operon is further regulated by the NarQP and NarXL two-component systems in response to levels of nitrite and nitrate in the environment.
  • NrfE, NrfF and NrfG are presumed to part of a heme lyase that adds heme groups to apocytrochrome c552 (NrfA)
  • Nitrate/nitrite Transport Enzymes [00163] Nitrate/nitrite transporter:
  • NarU and NarK are the two nitrate transporters in E. coli. Both function as nitrate/nitrite antiporters in anaerobic nitrate respiration. NarK is a member of the major facilitator superfamily (MFS) of transporters, and probably also functions as a proton/nitrite antiporter. NarK is thought to act as a nitrate/nitrite antiporter based on physiological studies of narK mutants as well as mutants expressing narK but lacking the other nitrate/nitrite transporters, NarU and NirC.
  • MFS major facilitator superfamily
  • Nitrate transport experiments in whole cells demonstrated that nitrate uptake is not drastically affected by mutations in narK (likely due to the presence of NarU), but the mutations instead affected nitrate utilization due to a decreased ability to excrete nitrite.
  • narK is regulated by nitrate and oxygen via the regulatory proteins NarL, NarX, NarQ, FNR, IHF, and Fis.
  • the nitrate-induced expression of narK is partially dependent upon ModE-molybdate as a transcriptional activator as well as the catalyzed product of MoeA. and iron.
  • NarU is a member of the major facilitator superfamily (MFS) of transporters, and is highly similar to NarK.
  • narU is the first gene of the narUZYWV operon. The cloned narU gene was shown to be able to complement a narK mutation based on assays of nitrite concentrations in the external medium. However, narU was not able to complement a narK nirC mutation for nitrite uptake.
  • a strain expressing narU without narK had a selective advantage over a strain expressing narK without narU during slow growth or starvation, but the opposite was true during fast growth.
  • Membrane topology predictions using experimentally determined C terminus locations indicate that NarU has 12 transmembrane helices and the C-terminus is located in the cytoplasm.
  • dissimilatory nitrite reductase enzymes There are two types of dissimilatory nitrite reductase enzymes. These two types catalyzes precisely the same reaction, but differ in their cofactors. One type contains a cytochrome cdl, while the second type contains copper.
  • One exemplary copper-containing dissimilatory nitrite reductase can be found in the halophilic archaeon Haloarcula marismortui. The enzyme contains two subunits, whose apparent molecular masses are 46 and 42 kDa.
  • nirK 150 YP 137309.1 151) GL55379459 152) Haloarcula marismortui
  • NirS genes contain a cytochrome cdl .
  • NirS is a homodimer, and each subunit contains one c-type heme and one dl-type heme.
  • Nitric oxide reductase NO -» N 2 0
  • Nitric oxide reductase is an integral membrane component of the anaerobic respiratory chain, which results in denitrification.
  • the enzyme catalyzes the reduction of nitric oxide to nitrous oxide, a reaction that involves the formation of an N-N bond, in essence a reversal of nitrogen fixation. NO reduction is coupled to electron transport phosphorylation.
  • the enzyme consists of a cytochrome c and cytochrome b subunits, encoded by the norC and norB, respectively.
  • the two genes are contiguous and transcribed as a single 2.0-kb transcript in Pseudomonas stutzeri.
  • Both proteins consist of a single subunit that contains both high- spin and low- spin heme b, no heme c, and one non-heme iron. While this single subunit is homologous to the catalytic subunits of common NO reductases, which are heterodimers, it possesses an N-terminal extension of approximately 280 amino acids. Mutation of either norB or norZ havs no obvious phenotype, but the simultaneous inactivation of both genes is lethal to the cells under anaerobic growth conditions, indicating that they are isofunctional and instrumental in denitrification. The enzyme encoded by the norZ gene is monomeric and was shown to accept electrons from a quinol. While it is located in the membrane, the enzyme's active site faces the periplasm. 165) norZ 166) AAZ64403.1 167) GL72122217 168) Ralstonia eutropha
  • the nitrous oxide reductase of Pseudomonas stutzeri is a multicopper enzyme composed of two identical subunits, encoded by the nosZ gene.
  • the enzyme contains 8 copper ions.
  • Two conserved domains corresponding to the binuclear centers CuA, the entry site for electrons, and CuZ, the catalytic site, have been identified in NosZ.
  • Hydrazine hydrolase is a unique enzyme that was predicted based on the proposed anammox (anaerobic ammonia oxidation) pathway.
  • anammox anaerobic ammonia oxidation pathway.
  • kuste2854 through kuste2861 encode a ⁇ -propeller complex, similar to nitrous oxide reductase.
  • anammoxosome identified products of three of these genes, namley kuste2859, kuste2860 and kuste2861, as very abundant members of the anammoxosome proteome. Antibodies produced against two of these peptides verified the association between these proteins and
  • a multiheme protein having hydrazine-oxidizing activity was purified from the anammox bacterium planctomycete KSU-1.
  • the protein which is homodimeric, contains 8 heme c molecules per monomer, one of which is a specialized heme with absorbance at at 472 nm (known as cytochrome P472).
  • the purified protein catalyzed the oxidation of hydrazine in vitro, using cytochrome c as an electron acceptor, and could not accept hydroxylamine as a substrate, although it was a competitive inhibitor for hydrazine.
  • Two similar genes, hzoA and hzoB encode the enzyme, with only two amino acid residues different between the polypeptides.
  • Kustdl340 A similar gene, Kustdl340, has been identified in the sequenced metagenome of Candidatus Kuenenia stuttgartiensis. The product of that gene was found to be highly abundant in the anammoxosome (Karlsson et al, FEMS Microbiol. Lett., 297(l):87-94 (2009); Strous et al, Nature, 440(7085):790-794 (2006)).
  • NADH dehydrogenase non-proton pumping
  • NADH:ubiquinone oxidoreductase II is a type IIA NADH dehydrogenase that catalyzes the transfer of electrons from NADH to the quinone pool in the cytoplasmic membrane. It is thus part of the aerobic respiratory chain of the cell, and its primary function may be maintenance of the [NADH]/[NAD ] balance of the cell.
  • NDH-2 is one of two distinct NADH dehydrogenases in E. coli. In contrast to NDH-1 (encoded by the nuo genes), NDH-2 utilizes NADH exclusively, and electron flow from NADH to ubiquinone does not generate an electrochemical gradient.
  • NirB cytoplasmic nitrite reductase
  • the prosthetic groups of nitrite reductase are FAD, an iron-sulfur cluster and siroheme.
  • the product of the cysG gene is necessary for the synthesis of the siroheme prosthetic group of NirB. The reaction is active only during anaerobic growth and fulfills a dissimilatory rather than
  • Nitrite transporter Nitrite (periplasm) Nitrite (cytosol)
  • NirC is a nitrite transporter which is a member of the FNT family of formate and nitrite transporters.
  • the nirC gene is located in the nir operon which codes for a NADH- dependent nitrite reductase.
  • NirC functions to import nitrite as a substrate for this enzyme complex.
  • the nir operon is anaerobically expressed and is repressed by oxygen.
  • Strains expressing nirC but lacking the two other nitrate/nitrite transporters (NarK and NarU) had higher activity of nitrite uptake and reduction than a strain expressing only narK.
  • NirC is able to reimport nitrite that is extruded by NarK for further reduction to ammonia. 237) nirC 238) YP_026212.2 239) GL90111575 240) Escherichia coli K-12
  • SIMPHENY is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system.
  • This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.
  • This computational approach is consistent with biological realities because biological systems are flexible and can reach the same result in many different ways.
  • the electrons are passed to 1 ⁇ 2 0 2 to form water and generate ATP.
  • About 1.5-2.0 ATP's can be synthesized per NADH equivalent depending upon the assumed proton translocation stoichiometry of ATP synthase (3 vs. 4 H+/ATP) as illustrated in Figure 7.
  • Figure 8 shows reactions that can participate in the synthesis of BDO from succinate, a central intermediate of the TCA cycle which is a fermentation product of wild-type E. coli.
  • the two-step reduction of succinate to 4-hydroxybutanoate (4-HB) is known to occur in nature, and the genes responsible have been found in several bacterial species (Sohling and Gottschalk, J. Bacteriol, 178-871-880 (1996)).
  • the subsequent two-step reduction of 4-HB to BDO has been reported in Clostridium acetobutylicum (Jewell et al, Curr. Microbiol, 13:215-219 (2007)).
  • the analogous step in the second part of the pathway is the conversion of 4-HB to 4-hydroxybutanal. This step can occur via the CoA intermediate, 4-hydroxybutyryl-CoA using a CoA transferase, converting acetyl-CoA to acetate in the process (Sholing and Gottschalk, J. Bacteriol., 178:871- 880 (1996)), and ATP is required to regenerate acetyl-CoA. If both acid-to-aldehyde conversions occur by way of the CoA derivatives, 2 additional ATP molecules are needed for each BDO produced.
  • the only products are BDO, biomass, and C0 2 .
  • the yield drops and either acetate or ethanol is made as a byproduct. Controlled nitrate addition can therefore provide the same or similar yield as oxygen, the exact amount depending on whether further reduction of nitrite to ammonia occurs.
  • One objective for the above BDO process is to couple growth to BDO production, so that some carbon and energy must be directed to biomass as well as BDO.
  • a final BDO titer of 100 g/L and a batch time of 33.3 hours were assumed, which corresponds to a volumetric productivity of 3 g/L*hr (neglecting reactor cleaning and filling times) and a cell growth rate of 0.094 hr-1 if the inoculum is 1/20 the final cell density.
  • the glucose specific uptake rate or the BDO/cell growth ratio can be increased beyond the values specified in Table 2 via strain engineering, higher BDO titers and/or volumetric productivities will be achieved. Finally, the BDO/glucose yield is decreased at low specific glucose uptake rates because a greater percentage of the incoming substrate is needed to satisfy the non-growth associated ATP maintenance requirement.
  • Table 2 The minimum specific glucose uptake rates required to enable 100 g/L of BDO production at a 3 g/L/hr volumetric productivity were calculated assuming various final cell densities for the five growth scenarios.

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Abstract

The invention provides a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or pathway for reducing nitrate (N03-) or nitrite (N02-) and a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase. Also provided is a composition including a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or pathway for reducing nitrate (N03-) or nitrite (N02-) and a medium containing nitrate. Methods of producting bioderived butadiene are also provided.

Description

INCREASED YIELDS OF BIO SYNTHESIZED PRODUCTS
[0001] This application claims the benefit of priority of United States Provisional application serial No. 61/733,400, filed December 4, 2012, the entire contents of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to biosynthetic processes, and more specifically to increasing yields of biosynthesized products under anaerobic culture conditions.
[0003] Industrial fermentations for the production of reduced chemicals, such as alcohols and organic acids, are generally performed under anaerobic conditions. First, anaerobic growth facilitates the production of these compounds because the NAD(P)H produced during glycolysis has no outlet by respiration. Second, aerobic fermentations tend to be more expensive, due to the capital and energy requirements to achieve high agitation rates. However, strains engineered for non-native products can have increased energy requirements, so that exclusive synthesis of the product may no longer be energetically feasible under anaerobic conditions. Therefore, the cell can produce a product of interest along with other undesired products, such as ethanol and acetate by-products, in order to balance the energy requirements. Production of such by-products can complicate downstream separations, or the cell may favor secretion of these native fermentation products over the product of interest. One possible solution to this problem is to grow the culture microaerobically by adding a small amount of oxygen to allow enough respiration to produce the right amount of ATP required for product synthesis and optimal growth. However, control of dissolved oxygen on industrial scale is difficult, and costly capital and energy infrastructure required for aeration will be needed.
[0004] Thus, there exists a need for an efficient system for microaerobic respiration for the production of biochemically synthesized chemicals. The invention satisfies this need and provides related advantages as well. SUMMARY OF INVENTION
[0005] The invention provides a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or pathway for reducing nitrate (N03-) or nitrite (N02-) and a metabolic modification attenuating the amount or activity of a
cytoplasmic nitrite reductase. Also provided is a composition including a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or pathway for reducing nitrate (N03-) or nitrite (N02-) and a medium containing nitrate.
Methods of producting bioderived butadiene are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] Figure 1 shows a schematic depicting a process for anaerobic production of butadiene with nitrate.
[0007] Figure 2 shows nitrate respiration and butadiene energetic.
[0008] Figure 3 shows the energetics associated with anaerobic butadiene production in the presence of nitrate.
[0009] Figure 4 shows exemplary pathways for production of butadiene from acetyl-CoA, glutaconyl-CoA, glutaryl-CoA, 3-aminobutyryl-CoA or 4-hydroxybutyryl-CoA via crotyl alcohol. Enzymes for transformation of the identified substrates to products include: A. acetyl- CoA:acetyl-CoA acyltransferase, B. acetoacetyl-CoA reductase, C. 3-hydroxybutyryl-CoA dehydratase, D. crotonyl-CoA reductase (aldehyde forming), E. crotonaldehyde reductase (alcohol forming), F. crotyl alcohol kinase, G. 2-butenyl-4-phosphate kinase, H. butadiene synthase, I. crotonyl-CoA hydrolase, synthetase, transferase, J. crotonate reductase, K. crotonyl- CoA reductase (alcohol forming), L. glutaconyl-CoA decarboxylase, M., glutaryl-CoA dehydrogenase, N. 3-aminobutyryl-CoA deaminase, O. 4-hydroxybutyryl-CoA dehydratase, P. crotyl alcohol diphosphokinase.
[0010] Figure 5 shows exemplary pathways for production of butadiene from erythrose-4- phosphate. Enzymes for transformation of the identified substrates to products include: A.
Erythrose-4-phosphate reductase, B. Erythritol-4-phospate cytidylyltransferase, C. 4-(cytidine 5'- diphospho)-erythritol kinase, D. Erythritol 2,4-cyclodiphosphate synthase, E. l-Hydroxy-2- butenyl 4-diphosphate synthase, F. l-Hydroxy-2-butenyl 4-diphosphate reductase, G. Butenyl 4- diphosphate isomerase, H. Butadiene synthase I. Erythrose-4-phosphate kinase, J. Erythrose reductase, K. Erythritol kinase.
[0011] Figure 6 shows an exemplary pathway for production of butadiene from malonyl- CoA plus acetyl-CoA. Enzymes for transformation of the identified substrates to products include: A. malonyl-CoA:acetyl-CoA acyltransferase, B. 3 -oxoglutaryl-Co A reductase (ketone - reducing), C. 3-hydroxyglutaryl-CoA reductase (aldehyde forming), D. 3-hydroxy-5- oxopentanoate reductase, E. 3,5-dihydroxypentanoate kinase, F. 3H5PP kinase, G. 3H5PDP decarboxylase, H. butenyl 4-diphosphate isomerase, I. butadiene synthase, J. 3 -hydroxy glutaryl- CoA reductase (alcohol forming), K. 3 -oxoglutaryl-Co A reductase (aldehyde forming), L. 3,5- dioxopentanoate reductase (ketone reducing), M. 3,5-dioxopentanoate reductase (aldehyde reducing), N. 5-hydroxy-3-oxopentanoate reductase, O. 3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming). Compound abbreviations include: 3H5PP = 3-Hydroxy-5- phosphonatooxypentanoate and 3H5PDP = 3-Hydroxy-5- [hydroxy(phosphonooxy)phosphoryl]oxy pentanoate.
[0012] Figure 7 shows the energetics associate with oxygen respiration for the biosynthesis of 1,4-butanediol.
[0013] Figure 8 shows exemplary biochemical pathways to 1,4-butanediol from succinate. Enzymes for transformation of the identified substrates to products include: 1) CoA-independent succinic semialdehyde dehydrogenase; 2) Succinyl-CoA synthetase; 3) CoA-dependent succinic semialdehyde dehydrogenase; 4) 4-hydroxybutanoate dehydrogenase; 5) CoA-independent aldehyde dehydrogenase; 6) 4-hydroxybutanoate: acetate CoA transferase; 7) CoA-dependent aldehyde dehydrogenase; 8) alcohol dehydrogenase
DETAILED DESCRIPTION OF THE INVENTION
[0014] This invention is directed to the use of nitrate as an alternative electron acceptor for simulated microaerobic respiration (or micro-respiric growth). Since it can be added in a solution, use of nitrate is cheaper and easier for precise control than aeration. [0015] The invention provides, for example, a method for anaerobic production of reduced biochemical products with high energetic demands, using controlled addition of nitrate as an electron acceptor. The invention is applicable for the production of a large number of biosynthesized chemicals that are industrially useful. Particular examples of such biosynthesized chemicals include butadiene and 1 ,4-butanediol (BDO) production in microorganisms. Due to high cellular energy (ATP) requirements, butadiene and BDO are challenging to produce fermentatively. The use of nitrate instead of oxygen to provide a limited amount of respiration simulates a microaerobic environment and is beneficial because it is much easier to control relative to oxygen. In addition, the method of the invention does not require high agitation rates and, therefore, allows the use of anaerobic fermentation conditions and vessels that can be much larger compared to fermenters used in aerobic processes and are significantly less expensive than aerobic fermenters. The use of non-oxygen electron acceptors in anerobic anoxic fermentation in the methods and compositions described herein provides benefits over micro-aerobic
fermentation when oxygen-reactive products, such as butadiene or other olefins, are produced. Higher product yields can be obtained, for example, as a result of reduced product loss from reaction with oxygen, and are obtained under safer conditions. In addition, the methods and compositions described herein minimize fouling of fermentation equipment that typically occurs with olefins such as butadiene when oxygen is present, thus providing additional economic benefits.
[0016] During aerobic growth, NADH generated during glycolysis and the TCA cycle is oxidized back to NAD+ through the electron transport chain. During each electron transfer step in this chain, protons are pumped across the cell membrane to drive the proton motive force, resulting in ATP production. For the chain to function, an external terminal electron acceptor should be present. Certain microorganisms, including E. coli, for example, preferentially uses oxygen as the terminal electron acceptor for the electron transport chain, via a cytochrome oxidase, which translocates two protons in the process. In the absence of oxygen,
microorganism can use alternative molecules as terminal electron acceptors, such as nitrate (NO3") and sulfate (SO4"2), reducing them to nitrite (NO2 ) and sulfite (SO3"2), respectively. The nitrate/nitrite couple is about half that of the O2/H2O couple, and can result in the translocation of up to about two protons (Unden et al, Antonie Van Leeuwenhoek, 66:3-23 (1994)). In
Escherichia coli, this coupling occurs by the Nar systems (Nitrate reductase A: narGHIJ; Nitrate reductase Z: narZYWV) which are well known to those skilled in the art. The nar operon is activated in the presence of nitrate and is repressed in the presence of oxygen (Unden et al., Antonie Van Leeuwenhoek, 66:3-23 (1994)). The periplasmic nitrate reductase {napFDAGHBC) can also be used to convert nitrate to nitrite. The nitrite formed from this process can also be used as a terminal electron acceptor, via the cytoplasmic nitrate reductase (nirBD) or the membrane-associated nitrite reductase (nrfABCDEFG), resulting in the formation of NH4 +. This ammonium can also be used as a nitrogen source for cell component biosynthesis. Note that nitrite reduction via nirBD does not result in proton translocation and is undesirable in some instances. The nirBD genes are known to be repressed by nitrate, even when nitrite is present in excess (Cole and Brown, F EMS Microbiol. Lett., 7:65-72 (1980)). The nirBD genes can also be deleted to assure that basal reduction of nitrite in the cytoplasm does not occur.
[0017] Although the pathway for nitrate respiration is well studied in microorganisms, including bacteria such as E. coli and B. subtilis, industrial fermentation processes have generally not utilized this approach. The denitrification process has been employed for the treatment of municipal wastewater, in which nitrate is a common contaminant. In this process, nitrate in the wastewater is reduced to NH4+ or N2, usually by a microbial community (Metcalf E.,
Wastewater engineering treatment, disposal, and reuse, McGraw Hill, New York (1991)). There are several other reports worth mentioning. Production of rhamnolipids in Pseudomonas aeruginosa in an anoxic environment containing nitrate (Chayabutra et al., Bioitechnol. Bioeng. 72:25-33 (2001)) has been reported to lower the need for high agitation rates required for aerobic growth in the presence of these surfactants. Anoxic production of amino acids by
Cory neb acterium glutamicum, a strict aerobe, also has been demonstrated using nitrate as the terminal electron acceptor (Takeno et al., Appl. Microbiol. BiotechnoL, 74(5): 1173-1182 (2007)). Yields obtained were 20-40% lower compared to those obtained with aeration. Finally, the engineering of nitrate respiration in fungus Fusarium oxysporum has been reported (Panagiotou et al, Metab. Eng., 8:474-482 (2007)).
[0018] The invention differs from the above reports in several ways: (1) The controlled addition of nitrate (or nitrite) is used to improve an inherently anaerobic process, not to replace an aerobic process; (2) nitrate (or nitrite) will be added in limited quantities, not in excess; and (3) addition of nitrate (or nitrite) will be used in a controlled manner to regulate the relative rates of respiration and fermentation and to govern the amount of biomass required for optimum yield and productivity of the desired product. Figure 1 is a schematic depicting the general process of culturing or fermenting microorganisms in, for example, a nitrate containing media, isolating the broth or a crude fraction containing the biosynthesized butadiene and purifying, partially or completely, the biosynthesized butadiene produced using micro-respiric growth of the invention.
[0019] As used herein, the term "non-naturally occurring" when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a butadiene biosynthetic pathway or a denitrification enzyme or pathway.
[0020] A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof. Exemplary metabolic modifications are disclosed herein.
[0021] As used herein, the term "butadiene," having the molecular formula C4H6 and a molecular mass of 54.09 g/mol (see Figures 2-4) (IUPAC name Buta-l,3-diene) is used interchangeably throughout with 1,3 -butadiene, biethylene, erythrene, divinyl, vinylethylene. Butadiene is a colorless, non corrosive liquefied gas with a mild aromatic or gasoline-like odor. Butadiene is both explosive and flammable because of its low flash point.
[0022] When used in reference to a microbial organism or product of the invention,
"isolated" refers an organism or biosynthetic product that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism or biosynthetic product that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism or biosynthetic product of the invention that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism or biosynthetic product is partly or completely separated from other substances as it is found in nature or as it is grown, synthesized, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms or biosynthetic products include partially pure microbes or products, substantially pure microbes or products and microbes cultured in a medium that is non-naturally occurring or products produced in a medium that is non-naturally occurring..
[0023] As used herein, the terms "microbial," "microbial organism" or "microorganism" are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.
[0024] The term "CoA" or "coenzyme A" refers to an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.
[0025] As used herein, the term "anaerobic" when used in reference to a culture or growth condition is intended to mean that the conditions are substantially anaerobic. Substantially anaerobic refers to an amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.
[0026] "Exogenous" as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term "endogenous" refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species whereas
"homologous" refers to a molecule or activity derived from the host microbial organism.
Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.
[0027] It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism
[0028] As used herein, the term "gene disruption," or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive or attenuated. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate or attenuate the encoded gene product. One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the non-naturally occurring microorganisms of the invention. The phenotypic effect of a gene disruption can be a null mutation, which can arise from many types of mutations including inactivating point mutations, entire gene deletions, and deletions of chromosomal segments or entire chromosomes. Specific enzyme inhibitors, such as antibiotics, can also produce null mutant phenotype, therefore being equivalent to gene disruption.
[0029] As used herein, the term "growth-coupled" when used in reference to the production of a biosynthesized product is intended to mean that the biosynthesis of the referenced biochemical product is produced during the growth phase of a microorganism. In a particular embodiment, the growth-coupled production can be obligatory, meaning that the biosynthesis of the referenced biochemical is an obligatory product produced during the growth phase of a microorganism.
[0030] As used herein, the term "attenuate," or grammatical equivalents thereof, is intended to mean to weaken, reduce or diminish the activity or amount of an enzyme or protein.
Attenuation of the activity or amount of an enzyme or protein can mimic complete disruption if the attenuation causes the activity or amount to fall below a critical level required for a given pathway to function. However, the attenuation of the activity or amount of an enzyme or protein that mimics complete disruption for one pathway, can still be sufficient for a separate pathway to continue to function. For example, attenuation of an endogenous enzyme or protein can be sufficient to mimic the complete disruption of the same enzyme or protein for production of a fatty alcohol, fatty aldehyde or fatty acid product of the invention, but the remaining activity or amount of enzyme or protein can still be sufficient to maintain other pathways, such as a pathway that is critical for the host microbial organism to survive, reproduce or grow.
Attenuation of an enzyme or protein can also be weakening, reducing or diminishing the activity or amount of the enzyme or protein in an amount that is sufficient to increase yield of a fatty alcohol, fatty aldehyde or fatty acid product of the invention, but does not necessarily mimic complete disruption of the enzyme or protein.
[0031] The non-naturally occurring microbal organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.
[0032] In the case of gene disruptions, a particularly useful stable genetic alteration is a gene deletion. The use of a gene deletion to introduce a stable genetic alteration is particularly useful to reduce the likelihood of a reversion to a phenotype prior to the genetic alteration. For example, stable growth-coupled production of a biochemical can be achieved, for example, by deletion of a gene encoding an enzyme catalyzing one or more reactions within a set of metabolic modifications. The stability of growth-coupled production of a biochemical can be further enhanced through multiple deletions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity.
[0033] Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.
[0034] An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.
[0035] Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5 '-3' exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.
[0036] In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co- evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.
[0037] A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.
[0038] Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having butadiene or denitrification biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. Similarly for a gene disruption, evolutionally related genes can also be disrupted or deleted in a host microbial organism to reduce or eliminate functional redundancy of enzymatic activities targeted for disruption.
[0039] Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related.
Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.
[0040] Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept- 16- 1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.
[0041] As used herein, the term "denitrification enzyme" or a "denitrification pathway" is intended to mean the loss of nitrate from an environment by an enzyme or enzymatic pathway having reducing activity. The term includes one or more of the following enzymatic reduction steps: nitrate (N03 ~) reduction to nitrite (N02 ~); nitrite reduction to ammonia (NH4); nitrite reduction to nitric oxide (NO); nitric oxide reduction to nitrous oxide (N20); and nitrous oxide reduction to nitrogen gas (N2), and the use of formate as an electron donor with nitrate being the electron acceptor. An exemplary denitrification pathway includes reduction from nitrate to nitrite to ammonia using the denitrification enzymes nitrate reductase (narGHJI, narZYWB, and/or napFDAGHBC-ccmABCDEFGH) and membrane-associated, ammonia forming, nitrite reductase (nrfABCDEFG), respectively. Another exemplary denitrification pathway includes reduction from nitrate to nitrite to nitric oxide to nitrous oxide to nitrogen using the
denitrification enzymes nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase (Nor), and nitrous oxide reductase (Nos), respectively. A denitrification enzyme refers to one enzymatic reduction step whereas a pathway includes two or more enzymatic reduction steps. Thus, the term denitrification pathway includes part of a denitrification pathway as it is found or utilized naturally in a microorganism. The term denitrification enzyme also is intended to include the upstream action of formate dehydrogenase to allow the use of formate as electron donor when nitrate is an electron acceptor.
[0042] As used herein, the term "fermentation" refers to catabolic reactions producing ATP in which organic compounds serve as both the primary electron donor and ultimate electron acceptor. The process of fermentation can be carried out under anaerobic, substantially anaerobic or microaerobic culture conditions of microorganisms including bacteria and fungi.
[0043] As used herein, the term "reducing equivalents" is intended to mean a molecule that is capable of undergoing reduction or oxidation. For example, CO and/or H2 can provide reducing equivalents by means of carbon monoxide dehydrogenase and hydrogenase enzymes, respectively. Reducing equivalents also can come in the form of, for example, NADH, NADPH, FADH, reduced quinones, reduced cytochromes, reduced ferredoxins, reduced flavodoxins and thioredoxins. The reducing equivalents, particularly NADH, NADPH, and reduced ferredoxin, can serve as cofactors for the for various enzymes disclosed herein as well as, for example, malate dehydrogenase, fumarate reductase, alpha-ketoglutarate: ferredoxin oxidoreductase (alternatively known as 2-oxoglutarate:ferredoxin oxidoreductase, alpha-ketoglutarate synthase, or 2-oxoglutarate synthase), pyruvate: ferredoxin oxidoreductase and isocitrate dehydrogenase. The electrons from these reducing equivalents can alternatively pass through an ion-gradient producing electron transport chain where they are passed to an acceptor such as oxygen, nitrate, oxidized metal ions, protons, or an electrode. The ion-gradient can then be used for ATP generation via an ATP synthase or similar enzyme.
[0044] The invention provides a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or denitrification pathway for reducing nitrate (NO3 ) or nitrite (N02 ~) and a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase, wherein the non-naturally occurring microbial organism has at least one exogenous nucleic acid encoding an enzyme or protein within the butadiene
biosynthetic pathway, at least one exogenous nucleic acid encoding a denitrification enzyme or at least one exogenous nucleic acid encoding an enzyme or protein within the denitrification pathway. One skilled in the art will understand that the referenced enzymes or proteins are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired chemical product such as butadiene or a desired chemical product that results from an enzymatic reduction reaction of a denitrification reaction and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a butadiene pathway, an electron transport pathway or denitrification enzyme or pathway pathway, such as that shown in Figures 2, 4-6, 7 or 8.
[0045] Metabolic pathways of microbial organisms can be engineered to biosynthesize chemical products such as butadiene. The engineered pathways also can be engineered to generate excess reducing equivalents that can be harnessed for the production of cellular energy in the form of ATP, for example. Butadiene biosynthetic pathways of the invention produce, for example, one or more excess NADH equivalents (reducing equivalents). As shown in Figure 2, electrons from one reducing equivalents can be passed to ¼ nitrate to form ¾ water, ¼ NH4 + and concomitantly form a proton gradient. Using the micro-respiric conditions of the invention, about 1.1-1.5 ATP ' s can be synthesized per NADH equivalent depending upon the assumed proton translocation stoichiometry of ATP synthase (3 vs. 4 H /ATP). This additional ATP yield can support an anaerobic production phase for biosynthesized butadiene and other chemicals. Figure 3 exemplifies the biochemical considerations and stoichiometries used to calculate the above ATP yield for butadiene biosynthesis coupled to a denitrification enzyme or enzyme or pathway for enhancement of a proton gradient.
[0046] ATP is synthesized by coupling the excess reducing equivalents produced from a butadiene biosynthetic pathway with enzymes involved in denitrification, thus allowing nitrate (NO3 ) or nitrite (N02 ~) to be an electron acceptor under anaerobic conditions. Referring to Figure 2, for example, excess reducing equivalents in the form of NADH are oxidized by a proton-pumping NADH dehydrogenase (Nuo) to produce a proton gradient across the membrane. The proton gradient can then generate ATP via a proton/ ATPase complex (see, for example, Atp in Figure 7). Additionally, the electrons can be passed via an electron carrier to a nitrate reductase for generation of additional protons with further enhancement of the proton gradient. Figure 2 depicts electrons being transferred to nitrate for its reduction to nitrite by nitrate reductase (Nar) and the generation of additional protons. The nitrite can be further transported across a membrane, such as a bacterial periplasmic membrane, and reduced to ammonia (NH4) by nitrite reductase (Nrf), for example.
[0047] Referring to the denitrification pathway exemplified in Figure 2, electrons are transferred to Nar or Nrf by electron carriers. In this specific example, the Nrf enzyme complex includes the NrfA, NrfB, NrfC and NrfD subunits. NrfA is linked to the menaquinol pool in the cytoplasmic membrane through a soluble electron carrier, NrfB, that in turn interacts with a membrane integral quinol dehydrogenase, NrfCD. The ubiquinol and menaquinol pools shown in Figure 2 can be used interchangeably as electron carriers by the enzymes shown therein. NrfA is associated with the cytoplasmic membrane with its cytochrome components facing or free in the periplasm; acting as a terminal electron acceptor of an electron transport chain beginning with membrane-associated formate -oxidizing enzymes it generates a proton gradient. This proton gradient is the driving force for the generation of ATP. A similar electron transport occurs using an ubiquinol pool as shown in Figure 2.
[0048] There are a number of denitrification enzymes and/or pathways that can be harnessed for the production of additional ATP using nitrate, nitrite or other nitrogen containing
compounds as an electron acceptor. Some, many or all of these enzymes can be utilized for nitrate and/or nitrite reduction by, for example, introducing encoding nucleic acids or modifying encoding nucleic acids to attenuate gene product activity in a host production organism to generate the desired combination of denitrification enzyme, enzymes or pathway.
[0049] For example, polypeptides involved in regulatory, transport, and enzymatic functions related to the reduction of nitrate to nitrite to ammonium include, for example, three nitrate reductases, two nitrite reductases, three nitrate/nitrite transporters, two two-component regulatory systems, two formate dehydrogenases, and two NADH dehydrogenases. The three nitrate reductases catalyzing the reduction of nitrate into nitrite include the cytoplasmic, membrane-associated enzymes NarG and NarZ and a periplasmic nitrate reductase (Nap). The reduction of nitrite to ammonia is catalyzed by the cytoplasmic, NADH-dependent nitrite reductase NirBD and the periplasmic, cytocrome c nitrite reductase Nrf. Nitrate and nitrite are transported in and out of the cytoplasm by two nitrate (NarK and NarU) and three nitrite (NarK, NarU, and NirC) transporters. Nitrite extrusion in the presence of nitrate mainly takes place through NarK, but nitrite uptake can be supported at similar rates by either NarK or NirC.
Nitrate uptake can be equivalently supported by either NarK or NarU. The expression of nitrate- and nitrite-regulated genes is mediated by two environmental signals (the absence of oxygen and the presence of nitrate/nitrite ions in the culture medium), several global regulators (Fnr, Fis, Ihf, and H-Ns, and Cra), and by the homologous two-component regulatory systems NarX/NarL and NarQ/NarP. Formate and NADH are among the electron donors for nitrate and nitrite reduction. Formate dehydrogenases (FdhN and FdhO) and NADH dehydrogenases deliver the electrons from formate and NADH to the quinone pools, which, in turn, pass the electrons to the nitrate and nitrite reductases. This conserves cellular energy and generates ATP via the proton- translocating ATPase. [0050] NirB is locted in the cytoplasm and contributes little to the production of a proton gradient for generation of ATP. Accordingly, the activity of NirB (or other components of the complex, NirC and/or NirD) can be attenuated as either as being unnecessary or superfluous, or as a metabolic strategy to channel nitrite to the periplasmic space for production of
ammonia/ammonium as a beneficial nitrogen source for the microorganisms of the invention. Using the teachings and guidance provided herein, those skilled in the art will understand what additional gene attenuation strategies can be utilized to further the production of a proton gradient or a beneficial nitrogen source from the above denitrification enzymes and pathways.
[0051] Thus, the invention provides a non-naturally occurring microbial organism having a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase. The metabolic modification includes attenuating the amount or activity of a gene product encoded by nirB, nirC or nirD.
[0052] A denitrification enzyme or denitrification pathway of the invention can include, for example, one or more formate dehydrogenases, nitrate reductases, NO-forming nitrite reductases, nitric oxide reductases or nitrous oxide reductases. Any of the previously described polypeptides having regulatory, transport or enzymatic functions also can be utilized as a denitrification enzyme or utilized in a denitrification pathway of the invention to couple with reducing equivalents for the production of ATP.
[0053] As described further below with respect to engineering a dentitrafication enzyme, denitrification pathway and/or a butadiene pathway of the invention, the desired activity can be introduced into a non-naturally occurring microbial organism of the invention by recombinant expression of an encoding nucleic acid, a host organism can be selected to have one or more endogenous activities or both recombinant expression or endogenous activities can be utilized to achieve any desired combination of enzymatic activities. Therefore, for the denitrification activities of the invention, for example, one or more denitrification enzymes or pathways can be introduced, utilized through endogenous activities or any combination thereof.
[0054] Thus, for example, reducing equivalents can be harnessed for production of a proton gradient by passing electrons from a reducing equivalent to nitrate. Nitrate (NO3 ) can be reduced by the action of nitrate reductase to generate nitrite (N02 ~) and a proton gradient. Subsequent steps also can be included such as the conversion of nitrite (N02 ~) to ammonia (NH4), catalyzed by nitrite reductase for the production of a microbial nitrogen source. One or more steps in a denitrification pathway from nitrate to gaseous nitrogen (N2) can alternatively or additionally be included. These steps can initiate by the generation of nitrite from nitrate as described above. Production of nitrogen gas can proceed via the gaseous intermediates nitric oxide (NO), produced from nitrite by NO-forming nitrite reductase, and nitrous oxide (N20), produced from nitric oxide by nitric oxide reductase. The final step to nitrogen can be performed by a nitrous oxide reductase. Formate dehydrogenase also can be utilized to generate a proton gradient from reducing equivalents through the use of formate as electron donor when nitrate is an electron acceptor. Given the teachings and guidance provided herein, any of the above steps can occur alone or in combination.
[0055] By way of exemplification, and as previously described, nitrate reductases include members of the Nar complex. Members of this enzyme complex include the following for Nar complex 1 : respiratory nitrate reductase 1, alpha chain (NarG) (1.7.99.4); respiratory nitrate reductase 1, beta chain (NarH) (1.7.99.4); respiratory nitrate reductase 1, gamma chain (Narl) (1.7.99.4); respiratory nitrate reductase 1, delta chain (NarJ) (1.7.99.4). The following members belong to Nar complex 2: respiratory nitrate reductase 2, alpha chain (NarZ) (1.7.99.4);
respiratory nitrate reductase 2, beta chain (NarY) (1.7.99.4); respiratory nitrate reductase 2, gamma chain (NarV) (1.7.99.4). An alternative gamma chain and a delta chains for complex 2 include: NarV respiratory nitrate reductase, gamma subunit; respiratory nitrate reductase 2, delta chain (NarW) (1.7.99.4) nitrite is converted to either ammonia or gaseous nitrogen.
[0056] Nitrite reductases catalyze the reduction of nitrite to ammonia or gaseous nitrogen. The conversion of nitrite to ammonia is catalyzed by the NirBD enzyme. The conversion of nitrite to gaseous nitrogen can proceed via two intermediate gaseous forms of nitrogen, nitric oxide (NO) and nitrous oxide (N20). NO-forming nitrite reductase (NirK or NirS) catalyzes the conversion of nitrite to nitric oxide. Nitric oxide reductase (Nor) catalyzes the conversion of nitric oxide to nitrous oxide. Nor consists of two subunits, namely a large subunit NorB
(1.7.99.7) and the small subunit NorC (1.7.99.7). Nitrous oxide reductase (Nos) catalyzes the conversion of nitrous oxide to nitrogen. Conversion of nitrite to gaseous nitrogen can be energetically less favorable than the conversion of nitrite to ammonia because of a smaller redox potential for the former as compared to the latter.
[0057] Homologues, orthologs, paralogs and/or nonorthologous gene displacements of the above proton gradient and/or denitrification enzymes of the invention such as Nuo, Nar, Nrf, Nir, Nor and Nos polypeptides and their encoding nucleic acids can be obtained from a variety of different bacterial, fungal and other microorganism species that are capable of carrying out anerobic respiration using nitrate as an electron acceptor. Exemplary members for nucleic acids encoding these polypeptides are described further below in Example I.
[0058] It is understood that any of the denitrification enzymes or denitrification pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of Figures 2 and 7, can be utilized to generate a non-naturally occurring microbial organism that produces any denitrification pathway intermediate or product, as desired. It is further understood that a non-naturally occurring microbial organism that produces a
denitrification enzyme product or a denitrification pathway intermediate can be utilized to produce the intermediate as a desired product for facilitation of micro-respiric growth and proton gradient generation. Thus, the invention provides for at least the following reduction steps employing nitrate or nitrite as an electron acceptor: Nitrate (NO3 ) can be reduced to nitrite (N02), nitrite can be reduced to ammonia (NH4), nitrite can be reduced to nitric oxide (NO), nitric oxide can be reduced to nitrous oxide (N20) , nitrous oxide can be reduced to nitrogen (N2).
[0059] In some embodiments, the invention provides a non-naturally occurring microbial organism, including a microbial organism having a butadiene pathway and a denitrification enzyme or pathway. The butadiene biosynthetic pathway includes at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase, a crotonate reductase, a crotonyl-CoA reductase (alcohol forming), a glutaconyl- Co A decarboxylase, a glutaryl-CoA dehydrogenase, an 3-aminobutyryl-CoA deaminase, a 4- hydroxybutyryl-CoA dehydratase or a crotyl alcohol diphosphokinase (Figure 4). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl- CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 4, steps A-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) (Figure 4, steps A-C, K, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 4, steps A-C, K, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase, (Figure 4, steps A-C, I, J, E, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3- hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase (Figure 4, steps A-C, I, J, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl- CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase and a crotyl alcohol diphosphokinase (Figure 4, steps A-E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotonyl-CoA reductase (aldehyde forming), a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 4, steps L, D-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) (Figure 4, steps L, K, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 4, steps L, K, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 4, steps L, I, J, E, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase (Figure 4, steps L, I, J, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl- CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene a glutaconyl-CoA decarboxylase and a crotyl alcohol
diphosphokinase (Figure 4, steps L, C, D, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 4, steps M, D-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotyl alcohol kinase, a 2-butenyl-4- phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) (Figure 4, steps M, K, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a butadiene synthase, a crotonyl- CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 4, steps M, K, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 4, steps M, I, J, E, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase (Figure 4, steps M, I, J, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl- CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a glutaryl-CoA dehydrogenase and a crotyl alcohol diphosphokinase (Figure 4, steps M, C, D, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 4, steps N, D-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) (Figure 4, steps N, K, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 4, steps N, K, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 4, steps N, I, J, E, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl- CoA deaminase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl- CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol
diphosphokinase (Figure 4, steps N, I, J, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a 3-aminobutyryl-CoA deaminase and a crotyl alcohol diphosphokinase (Figure 4, steps N, C, D, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 4, steps O, D-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) (Figure 4, steps O, K, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 4, steps O, K, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 4, steps O, I, J, E, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl- CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase (Figure 4, steps O, I, J, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a 4-hydroxybutyryl-CoA dehydratase and a crotyl alcohol diphosphokinase (Figure 4, steps L, C, D, E, P, H).
[0060] In some embodiments, the invention provides a non-naturally occurring microbial organism, including a microbial organism having a butadiene pathway and a denitrification enzyme or pathway. The butadiene biosynthetic pathway includes at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythrose-4-phosphate reductase, an erythritol-4- phospate cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4- cyclodiphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate synthase, a l-hydroxy-2- butenyl 4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a butadiene synthase, an erythrose-4-phosphate kinase, an erythrose reductase or an erythritol kinase (Figure 5). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythrose-4-phosphate reductase, an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate synthase, a l-hydroxy-2- butenyl 4-diphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate reductase and a butadiene synthase (Figure 5, steps A-F, and H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythrose-4-phosphate reductase, an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate synthase, a 1- hydroxy-2-butenyl 4-diphosphate reductase, a butenyl 4-diphosphate isomerase and butadiene synthase (Figure 5, steps A-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythritol-4-phospate cytidylyltransferase, a 4- (cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate synthase, a 1- hydroxy-2 -butenyl 4-diphosphate synthase, a l-hydroxy-2 -butenyl 4-diphosphate reductase, a butadiene synthase, an erythrose-4-phosphate kinase, an erythrose reductase and a erythritol kinase (Figure 5, steps I, J, K, B-F, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4- cyclodiphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate synthase, a l-hydroxy-2- butenyl 4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a butadiene synthase, an erythrose-4-phosphate kinase, an erythrose reductase and an erythritol kinase (Figure 5, steps I, J, K, B-H).
[0061] In some embodiments, the invention provides a non-naturally occurring microbial organism, including a microbial organism having a butadiene pathway and a denitrification enzyme or pathway. The butadiene biosynthetic pathway includes at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, an 3- oxoglutaryl-CoA reductase (ketone -reducing), a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a 3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate kinase, a 3- hydroxy-5-phosphonatooxypentanoate kinase, a 3-hydroxy-5-
[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene synthase, a 3-hydroxyglutaryl-CoA reductase (alcohol forming), an 3- oxoglutaryl-CoA reductase (aldehyde forming), a 3,5-dioxopentanoate reductase (ketone reducing), a 3,5-dioxopentanoate reductase (aldehyde reducing), a 5-hydroxy-3-oxopentanoate reductase or an 3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming) (Figure 6). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone- reducing), a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a 3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4- diphosphate isomerase and a butadiene synthase (Figure 6, steps A-I). In one aspect, the non- naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5- phosphonatooxypentanoate kinase, a 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene synthase, an 3- oxoglutaryl-CoA reductase (aldehyde forming), a 3,5-dioxopentanoate reductase (aldehyde reducing) and a 5-hydroxy-3-oxopentanoate reductase. (Figure 6, steps A, K, M, N, E, F, G, H, I). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, a 3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate kinase, a 3-Hydroxy-5-phosphonatooxypentanoate kinase, a 3- Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4- diphosphate isomerase, a butadiene synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming) and a 3,5-dioxopentanoate reductase (ketone reducing). (Figure 6, steps A, K, L, D, E, F, G, H, I). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a 3-hydroxy-5- [hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene synthase, a 5-hydroxy-3-oxopentanoate reductase and a 3-oxo-glutaryl- CoA reductase (CoA reducing and alcohol forming). (Figure 6, steps A, O, N, E, F, G, H, I). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing), a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a 3- hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4- diphosphate isomerase, a butadiene synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol forming). (Figure 6, steps A, B, J, E, F, G, H, I).
[0062] In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a butadiene pathway and a denitrification enzyme or pathway. The butadiene biosynthetic pathway includes at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of acetyl-CoA to acetoacetyl-CoA, acetoacetyl-CoA to 3-hydroxybutyryl-CoA, 3-hydroxybutyryl- CoA to crotonyl-CoA, crotonyl-CoA to crotonaldehyde, crotonaldehyde to crotyl alcohol, crotyl alcohol to 2-betenyl-phosphate, 2-betenyl-phosphate to 2-butenyl-4-diphosphate, 2-butenyl-4- diphosphate to butadiene, erythrose-4-phosphate to erythritol-4-phosphate, erythritol-4- phosphate to 4-(cytidine 5'-diphospho)-erythritol, 4-(cytidine 5'-diphospho)-erythritol to 2- phospho-4-(cytidine 5'-diphospho)-erythritol, 2-phospho-4-(cytidine 5'-diphospho)-erythritol to erythritol-2,4-cyclodiphosphate, erythritol-2,4-cyclodiphosphate to l-hydroxy-2-butenyl 4- diphosphate, l-hydroxy-2 -butenyl 4-diphosphate to butenyl 4-diphosphate, butenyl 4- diphosphate to 2-butenyl 4-diphosphate, l-hydroxy-2-butenyl 4-diphosphate to 2-butenyl 4- diphosphate, 2-butenyl 4-diphosphate to butadiene, malonyl-CoA and acetyl-CoA to 3- oxoglutaryl-CoA, 3-oxoglutaryl-CoA to 3-hydroxyglutaryl-CoA to 3 -hydroxy-5 -oxopentanoate, 3 -hydroxy-5 -oxopentanoate to 3,5-dihydroxy pentanoate, 3,5-dihydroxy pentanoate to 3- hydroxy-5-phosphonatooxypentanoate, 3 -hydroxy-5 -phosphonatooxypentanoate to 3 -hydroxy-5 - [hydroxy(phosphonooxy)phosphoryl]oxy pentanoate, 3-hydroxy-5-
[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate to butenyl 4-biphosphate, glutaconyl-CoA to crotonyl-CoA, glutaryl-CoA to crotonyl-CoA, 3-aminobutyryl-CoA to crotonyl-CoA, 4- hydroxybutyryl-CoA to crotonyl-CoA, crotonyl-CoA to crotonate, crotonate to crotonaldehyde, crotonyl-CoA to crotyl alcohol, crotyl alcohol to 2-butenyl-4-diphosphate, erythrose-4-phosphate to erythrose, erythrose to erythritol, erythritol to erythritol-4-phosphate, 3-oxoglutaryl-CoA to 3,5-dioxopentanoate, 3,5-dioxopentanoate to 5 -hydroxy-3 -oxopentanoate, 5-hydroxy-3- oxopentanoate to 3,5-dihydroxypentanoate, 3-oxoglutaryl-CoA to 5 -hydroxy-3 -oxopentanoate, 3,5-dioxopentanoate to 3 -hydroxy-5 -oxopentanoate and 3-hydroxyglutaryl-CoA to 3,5- dihydroxypentanoate. One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a butadiene pathway, such as that shown in Figures 4-6.
[0063] The biosynthesis of butadiene by metabolically engineered microorganisms employing a variety of different pathways other than those described above can be found described in, for example, US Publication No. 2011-0300597, US Publication No. 2012- 0021478, published January 26, 2012 and US Publication No. 2012-0225466, published September 6, 2012, which are incorporated herein by reference.
[0064] While generally described herein as a microbial organism that contains a butadiene pathway, it is understood that the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce an intermediate of a butadiene pathway. Such exemplary butadiene pathways are disclosed herein (see above and figures). Therefore, in addition to a microbial organism containing a butadiene pathway that produces butadiene, the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a butadiene pathway enzyme, where the microbial organism produces a butadiene pathway intermediate, for example, acetoacetyl-CoA,
3- hydroxybutyryl-CoA, crotonyl-CoA, crotonaldehyde, crotyl alcohol, 2-betenyl-phosphate, 2- butenyl-4-diphosphate, erythritol-4-phosphate, 4-(cytidine 5'-diphospho)-erythritol, 2-phospho-
4- (cytidine 5'-diphospho)-erythritol, erythritol-2,4-cyclodiphosphate, l-hydroxy-2-butenyl 4- diphosphate, butenyl 4-diphosphate, 2-butenyl 4-diphosphate, 3-oxoglutaryl-CoA, 3- hydroxyglutaryl-CoA, 3-hydroxy-5-oxopentanoate, 3,5-dihydroxy pentanoate, 3-hydroxy-5- phosphonatooxypentanoate, 3 -hydroxy-5 - [hydroxy(phosphonooxy)phosphoryl]oxy pentanoate, crotonate, erythrose, erythritol, 3,5-dioxopentanoate or 5-hydroxy-3-oxopentanoate.
[0065] It is understood that any of the pathways disclosed herein, as described in the
Examples and exemplified in the Figures, including the pathways of Figures 2-4, can be utilized to generate a non-naturally occurring microbial organism that produces any pathway
intermediate or product, as desired. As disclosed herein, such a microbial organism that produces an intermediate can be used in combination with another microbial organism
expressing downstream pathway enzymes to produce a desired product. However, it is understood that a non-naturally occurring microbial organism that produces a butadiene pathway intermediate can be utilized to produce the intermediate as a desired product.
[0066] The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.
[0067] As disclosed herein, the intermediates crotanate; 3,5-dioxopentanoate, 5-hydroxy-3- oxopentanoate, 3 -hydroxy-5 -oxopentanoate, 3-oxoglutaryl-CoA and 3-hydroxyglutaryl-CoA, as well as other intermediates, are carboxylic acids, which can occur in various ionized forms, including fully protonated, partially protonated, and fully deprotonated forms. Accordingly, the suffix "-ate," or the acid form, can be used interchangeably to describe both the free acid form as well as any deprotonated form, in particular since the ionized form is known to depend on the pH in which the compound is found. It is understood that carboxylate products or intermediates includes ester forms of carboxylate products or pathway intermediates, such as O-carboxylate and S-carboxylate esters. O- and S-carboxylates can include lower alkyl, that is CI to C6, branched or straight chain carboxylates. Some such O- or S-carboxylates include, without limitation, methyl, ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl O- or S-carboxylates, any of which can further possess an unsaturation, providing for example, propenyl, butenyl, pentyl, and hexenyl O- or S-carboxylates. O-carboxylates can be the product of a biosynthetic pathway. Exemplary O-carboxylates accessed via biosynthetic pathways can include, without limitation: methyl crotanate; methy-3,5-dioxopentanoate; methyl-5-hydroxy-3- oxopentanoate; methyl-3 -hydroxy-5 -oxopentanoate; 3-oxoglutaryl-CoA, methyl ester; 3- hydroxyglutaryl-CoA, methyl ester; ethyl crotanate; ethyl-3,5-dioxopentanoate; ethyl-5 -hydroxy- 3-xopentanoate; ethyl-3-hydroxy-5-oxopentanoate; 3-oxoglutaryl-CoA, ethyl ester; 3- hydroxyglutaryl-CoA, ethyl ester; n-propyl crotanate; n-propyl-3,5-dioxopentanoate; n-propyl-5- hydroxy-3 -oxopentanoate; n-propyl-3 -hydroxy-5 -oxopentanoate; 3-oxoglutaryl-CoA, n-propyl ester; and 3-hydroxyglutaryl-CoA, n-propyl ester. Other biosynthetically accessible O- carboxylates can include medium to long chain groups, that is C7-C22, O-carboxylate esters derived from fatty alcohols, such heptyl, octyl, nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl alcohols, any one of which can be optionally branched and/or contain unsaturations. O- carboxylate esters can also be accessed via a biochemical or chemical process, such as esterification of a free carboxylic acid product or transesterification of an O- or S-carboxylate. S-carboxylates are exemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters, and various aryl and heteroaryl thioesters. [0068] The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more of the denitrification pathways or one or more of the butadiene biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular denitrification pathways or one or more of the butadiene biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve reduction of nitrate or one or more of nitrogen containing compounds within a denitrification pathway or to achieve butadiene biosynthesis. Thus, a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired denitrification or biosynthetic pathway or a desired denitrification pathway or biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as reduction of nitrate or downstream product for the generation of a proton gradient and/or a desired product such as butadiene.
[0069] Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, fungus or any of a variety of other microorganisms applicable or suitable to fermentation processes. Exemplary bacteria include any species selected from the order Enterobacteriales, family Enterobacteriaceae, including the genera Escherichia and Klebsiella; the order Aeromonadales, family Succinivibrionaceae, including the genus Anaerobio spirillum; the order Pasteur ellales, family Pasteur ellaceae, including the genera Actinobacillus and Mannheimia; the order Rhizobiales, family
Bradyrhizobiaceae, including the genus Rhizobium; the order Bacillales, family Bacillaceae, including the genus Bacillus; the order Actinomycetales, families Corynebacteriaceae and Streptomycetaceae, including the genus Corynebacterium and the genus Streptomyces, respectively; order Rhodospirillales, family Acetobacteraceae, including the genus
Gluconobacter; the order Sphingomonadales, family Sphingomonadaceae, including the genus Zymomonas; the order Lactobacillales, families Lactobacillaceae and Streptococcaceae, including the genus Lactobacillus and the genus Lactococcus, respectively; the order
Clostridiales, family Clostridiaceae, genus Clostridium; and the order Pseudomonadales , family Pseudomonadaceae, including the genus Pseudomonas. Non-limiting species of host bacteria include Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens,
Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Cory neb acterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.
[0070] Similarly, exemplary species of fungi species include any species selected from the order Eurotiales, family Trichocomaceae, including the genus Aspergillus; and the order Mucorales, family Mucoraceae, including the genus Rhizopus. Further exemplary species of yeast or fungi include genera selected from the order Sordariaceae, family Sordariales including the genus Neurospora, and the order Nectriaceae, family Hypocreales including the genus Fusarium. Exemplary species of Neurospora include N. africana, N. bonaerensis, N.
brevispora, N. caffera, N. calospora, N. cerealis, N. crassa, N. cratophora, N. dictyophora, N. discrete, N. dodgei, N. himalayensis, N. hippopotami, N. indica, N. intermedia, N. inversa, N. kobi, N. lineolata, N. longispora, N. novoguineensis, N. pannonica, N. pseudocalospora, N. pseudoreticulata, N. reticulate, N. sitophila andN. tetrasperma. Exemplary species of Fusarium include F. acaciae-mearnsii, F. aquaeductuum, F. aquaeductuum var. media, F. asiaticum, F. boothii, F. cerealis, F. chlamydosporum, F. circinatum, F. coeruleum, F. culmorum, F. dimerum, F. graminearum, F. incarnatum, F. konzum, F. lumulosporum, F. meridionale, F.
mesoamericanum, F. moniliforme, F. napiforme, F. nygamai, F. oxysporum, F. proliferatum, F. pseudocircinatum, F. pseudograminearum, F. sacchari, F. semitectum, F. solani, F.
sporotrichoides, F. subglutinans, F. tabacinum, F. thapsinum, F. verticillioides. Non-limiting species of host yeast or fungi include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and the like. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product.
[0071] Depending on the denitrification or butadiene biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed denitrification or butadiene pathway- encoding nucleic acid and up to all encoding nucleic acids for one or more denitrification or butadiene biosynthetic pathways. For example, denitrification or butadiene biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a denitrification or butadiene pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of denitrification product or butadiene can be included.
[0072] Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the denitrification pathway or portion thereof or the butadiene pathway
deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have one, two, three, four or more enzymes and up to all nucleic acids encoding the enzymes or proteins constituting a denitrification pathway or butadiene biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize reduction of a substrate in a denitrification pathway or butadiene biosynthesis or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the denitrification pathway or butadiene pathway precursors such as nitrate, nitrite or crotyl alcohol, for example.
[0073] Generally, a host microbial organism is selected such that it produces the precursor of a denitrification or butadiene biosynthetic pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. For example, formate for denitrification and acetyl-CoA are produced naturally in a host organism such as E. coli. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a denitrification or butadiene pathway.
[0074] In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to reduce nitrate or other products within a denitrification pathway of the invention or to synthesize butadiene. In this specific embodiment it can be useful to increase the synthesis or accumulation of a denitrification pathway product or butadiene pathway product to, for example, drive reduction reactions or butadiene pathway reactions toward enhancement of a proton gradient or butadiene production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described denitrification or butadiene pathway enzymes or proteins. Overexpression of the enzyme or enzymes and/or protein or proteins of the denitrification or butadiene pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, reducing nitrate, nitrite or other nitrogen containing compounds or producing butadiene, through overexpression of one, two, three, four, five or up to all enzymes in the referenced pathway, that is, up to all nucleic acids encoding denitrification and/or butadiene biosynthetic pathway enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the denitrification activity or butadiene biosynthetic pathway.
[0075] In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.
[0076] It is understood that, in methods of the invention, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, a denitrification or butadiene biosynthetic pathway onto the microbial organism.
Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer denitrification or butadiene biosynthetic capability. For example, a non-naturally occurring microbial organism having a denitrification or butadienebiosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the
combination of nitrate reductase and nitrite reductase, and the like. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, nitrate reductase, nitrite reductase and NO-forming nitrite reductase, nitric oxide reductase and/or nitrous oxide reductase, and so forth, as desired, and including butadiene biosynthetic pathway combinations so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. Similarly, any combination of two, three, four or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. [0077] In addition to the reduction of nitrate and other nitrogen containing compounds for generation a proton gradient and to the biosynthesis of butadiene as described herein, the non- naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and/or with other microbial organisms and methods well known in the art to achieve enhanced product yield from increased ATP synbthesis and enhanced product biosynthesis by other routes.
[0078] Similarly, it is understood by those skilled in the art that a host organism can be selected based on desired characteristics for introduction of one or more gene disruptions to increase generation of a proton gradient and/or production of biosynthesized butadiene. Thus, it is understood that, if a genetic modification is to be introduced into a host organism to disrupt a gene, any homologs, orthologs or paralogs that catalyze similar, yet non-identical metabolic reactions can similarly be disrupted to ensure that a desired metabolic reaction is sufficiently disrupted. Because certain differences exist among metabolic networks between different organisms, those skilled in the art will understand that the actual genes disrupted in a given organism may differ between organisms. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the methods of the invention can be applied to any suitable host microorganism to identify the cognate metabolic alterations needed to construct an organism in a species of interest that will increase proton gradient and butadiene biosynthesis. In a particular embodiment, the increased production couples biosynthesis of butadiene to growth of the organism, and can obligatorily couple production of butadiene to growth of the organism if desired and as disclosed herein.
[0079] Sources of encoding nucleic acids for a denitrification enzyme, denitrification pathway enzymes and/or a butadiene pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. An exemplary species for such sources include, for example, Escherichia coli and all other microbial species having endogenous denitrification pathways as well as other exemplary species disclosed herein or available as source organisms for
corresponding genes. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite denitrification pathway enzyme or enzymes and/or a butadiene biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of denitrification pathway enzyme or enzymes and/or butadiene described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.
[0080] In some instances, such as when an alternative denitrification pathway or butadiene biosynthetic pathway exists in an unrelated species, denitrification pathway activities and/or butadiene biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non- identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will carry out denitrification of nitrate and/or nitrite to enhance proton gradient production and synthesize butadiene.
[0081] Methods for constructing and testing the expression levels of a non-naturally occurring denitrification exhibiting and butadiene-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). [0082] Exogenous nucleic acid sequences involved in a pathway for conferring denitrification pathway activity and for the production of butadiene can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E.
Figure imgf000041_0001
or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al, J. Biol. Chem.
280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.
[0083] An expression vector or vectors can be constructed to include one or more denitrification and/or butadiene biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism.
Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.
[0084] The invention also provides a composition comprising a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or pathway for reducing nitrate (NO3 ) or nitrite (N02 ~) and a medium comprising nitrate. The medium can alternatively contain nitrite or both nitrate and nitrite. In addition, the medium can alternatively or additionally contain elevated levels of ammonia or ammonium as a terminal product during culture of the non-naturally occurring organisms of the invention.
[0085] As described previously, the use of nitrate and/or nitrite instead of oxygen to provide for respiration simulates a microaerobic environment and is beneficial because it is much easier to control relative to oxygen. In addition, the use of nitrate and/or nitrite does not require high agitation rates and therefore allows the use of anaerobic fermentation conditions and vessels that can be much larger than fermenters for aerobic processes and are significantly less expensive than aerobic fermenters. The non-naturally occurring microorganisms of the invention can be in medium containing from about 0.1 mg/L to about 10 g/L or more nitrate and/or nitrite.
Exemplary concentrations within this range include, for example, 0.1 mg/L, 0.2 mg/L, 0.3 mg/L, 0.4 mg/L, 0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1.0 g/L, 1.5 g/L, 2.0 g/L, 2.5 g/L, 3.0 g/L, 3.5 g/L, 4.0 g/L, 4.5 g/L, 5.0 g/L, 5.5 g/L, 6.0 g/L, 6.5 g/L, 7.0 g/L, 7.5 g/L, 8.0 g/L, 8.5 g/L, 9.0 g/L, 9.5 g/L, 10 g/L or more. However, any concentration in between the above exemplary concentrations also can be used in the invention. In general, the concentration of nitrate and/or nitrite can be maintained at or near the Km of the nitrate reductase or nitrite reductase that are employed. Such values can be in the nanomolar or micromolar range including, for example, 10 uM, 20 uM, 30 uM, 40 uM, 50 uM, 60 uM, 70 uM, 80 uM, 90 uM, 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, 1.0 μΜ, 2.0 μΜ, 3.0 μΜ, 4.0 μΜ, 5.0 μΜ, 6.0 μΜ, 7.0 μΜ, 8.0 μΜ, 9.0 μΜ, 10 μΜ or more.
[0086] Ammonia and ammonium can be elevated in the medium in amounts up to 1.0 M or more than its concentration as it is found in a comparable culture of a microbial organism that does not couple a denitrification pathway for increased production of a proton gradient and ATP synthesis as described herein. Accordingly, the elevated amounts can include, for example, elevated amounts up to about 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M or more compared to a microbial organism that does not couple a denitrification pathway for increased production of a proton gradient and ATP synthesis as described herein. The total concentrations of ammonia that can be formed using the culture and/or fermentation conditions of the invention include, for example, about 250 mM to 2.0 M, particularly about 250 mM to 1.0 M, and more particularly, about 400 mM to 800 mM.
[0087] Therefore, the invention provides a non-naturally occurring microbial organism as described previously in a medium containing a nitrogen compound for accepting electrons under micro-respiric conditions such as nitrate and/or nitrite or in a medium contain a downstream or final product of one or more electron transport steps of a denitrification pathway as described herein. Accordingly, the invention additionally provides a non-naturally occurring microbial organism wherein the denitrification enzyme or pathway includes formate dehydrogenase, nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase or nitrous oxide reductase.
[0088] The non-naturally occurring microbial organism in nitrate containing medium, for example, can reduce nitrate (NO3 ) to nitrite (N02 ~), ammonia (N¾), nitric oxide (NO), nitrous oxide (N20) or nitrogen (N2). The non-naturally occurring microbial organism of can further include a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase. The nitrite reductase can be a NADH-dependent nitrite reductase, including for example a NADH-dependent nitrite reductase encoded by nirB, nirC or nirD. Additionally, the butadiene biosynthetic pathway of the non-naturally occurring organisms of the invention can produce excess reducing equivalents, including NADH reducing equivalent. An exemplary butadiene biosynthetic pathway of a non-naturally occurring microbial organism in nitrate containing medium, for example, can include an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase and a butadiene synthase. The invention further provides a non- naturally occurring microbial organism in nitrate containing medium, for example, wherein the microbial organism is Escherichia.
[0089] The invention further provides a composition comprising bioderived butadiene in a nitrate or ammonium (NH4+) containing medium. The medium alternatively or additionally can contain any of the nitrogen compounds exemplified previously as an intermediate or final product in the denitrification pathways of the invention.
[0090] The microorganism of the invention can be used to generate bioderived butadiene. In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in butadiene or any butadiene pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." Uptake sources can provide isotopic enrichment for any atom present in the product butadiene or butadiene pathway intermediate, or for side products generated in reactions diverging away from a butadiene pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.
[0091] In some embodiments, the uptake sources can be selected to alter the carbon-12, carbon- 13, and carbon- 14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen- 16, oxygen- 17, and oxygen- 18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can be selected to alter the nitrogen- 14 and nitrogen- 15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31 , phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios.
[0092] In some embodiments, the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources. An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom. An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio. In some embodiments, a target atom isotopic ratio of an uptake source can be achieved by selecting a desired origin of the uptake source as found in nature. For example, as discussed herein, a natural source can be a biobased derived from or synthesized by a biological organism or a source such as petroleum-based products or the atmosphere. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel-derived carbon source, which can be relatively depleted of carbon- 14, or an environmental or atmospheric carbon source, such as C02, which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart.
[0093] The unstable carbon isotope carbon-14 or radiocarbon makes up for roughly 1 in 1012 carbon atoms in the earth's atmosphere and has a half-life of about 5700 years. The stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen (14N). Fossil fuels contain no carbon-14, as it decayed long ago. Burning of fossil fuels lowers the atmospheric carbon-14 fraction, the so-called "Suess effect".
[0094] Methods of determining the isotopic ratios of atoms in a compound are well known to those skilled in the art. Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC), high performance liquid
chromatography (HPLC) and/or gas chromatography, and the like. [0095] In the case of carbon, ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product's biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective April 1, 2011). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein.
[0096] The biobased content of a compound is estimated by the ratio of carbon- 14 (14C) to carbon- 12 (12C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm = (S-B)/(M-B), where B, S and M represent the 14C/12C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the 14C/12C ratio of a sample from "Modern." Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard reference materials (SRM) 4990b) normalized to 513CVPDB=-19 per mil (Olsson, The use of Oxalic acid as a Standard, in, Radiocarbon Variations and Absolute Chronology, Nobel Symposium, 12th Proc, John Wiley & Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to 513CVPDB=-19 per mil. This is equivalent to an absolute (AD 1950) 14C/12C ratio of 1.176 ± 0.010 x 10"12 (Karlen et al., Arkiv Geqfysik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one istope with respect to another, for example, the preferential uptake in biological systems of C12 over C13 over C14, and these corrections are reflected as a Fm corrected for δ13.
[0097] An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available. The Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). The isotopic ratio of HOx II is -17.8 per mille. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm = 0% represents the entire lack of carbon-14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source. A Fm = 100%, after correction for the post-1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a "modern" source includes biobased sources.
[0098] As described in ASTM D6866, the percent modern carbon (pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon-14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a "pre-bomb" standard, and because nearly all new biobased products are produced in a post-bomb
environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old.
[0099] ASTM D6866 quantifies the biobased content relative to the material's total organic content and does not consider the inorganic carbon and other non-carbon containing substances present. For example, a product that is 50%> starch-based material and 50%> water would be considered to have a Biobased Content = 100% (50%> organic content that is 100%) biobased) based on ASTM D6866. In another example, a product that is 50% starch-based material, 25% petroleum-based, and 25% water would have a Biobased Content = 66.7%> (75% organic content but only 50%> of the product is biobased). In another example, a product that is 50%> organic carbon and is a petroleum-based product would be considered to have a Biobased Content = 0%> (50%) organic carbon but from fossil sources). Thus, based on the well known methods and known standards for determining the biobased content of a compound or material, one skilled in the art can readily determine the biobased content and/or prepared downstream products that utilize of the invention having a desired biobased content.
[00100] Applications of carbon-14 dating techniques to quantify bio-based content of materials are known in the art (Currie et al., Nuclear Instruments and Methods in Physics Research B, 172:281-287 (2000)). For example, carbon- 14 dating has been used to quantify bio- based content in terephthalate-containing materials (Colonna et al., Green Chemistry, 13:2543- 2548 (2011)). Notably, polypropylene terephthalate (PPT) polymers derived from renewable 1,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30% (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al, supra, 2000). In contrast, polybutylene terephthalate polymer derived from both renewable 1 ,4-butanediol and renewable terephthalic acid resulted in bio-based content exceeding 90%> (Colonna et al, supra, 2011).
[00101] Accordingly, in some embodiments, the present invention provides butadiene or a butadiene pathway intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source. For example, in some aspects the butadiene or a butadiene pathway intermediate can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or as much as 100%. In some such embodiments, the uptake source is C02. In some embodiments, the present invention provides butadiene or a butadiene pathway intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the butadiene or a butadiene pathway intermediate can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%>, less than 45%, less than 40%>, less than 35%, less than 30%>, less than 25%, less than 20%), less than 15%, less than 10%, less than 5%, less than 2% or less than 1%. In some embodiments, the present invention provides butadiene or a butadiene pathway intermediate that has a carbon- 12, carbon-13, and carbon- 14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake sources is one way by which the carbon- 12, carbon-13, and carbon- 14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.
[00102] Further, the present invention relates to the biologically produced butadiene or butadiene pathway intermediate as disclosed herein, and to the products derived therefrom, wherein the butadiene or a butadiene pathway intermediate has a carbon- 12, carbon-13, and carbon- 14 isotope ratio of about the same value as the C02 that occurs in the environment. For example, in some aspects the invention provides bioderived butadiene or a bioderived butadiene intermediate having a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C02 that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C02 that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived butadiene or a bioderived butadiene pathway intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product. Methods of chemically modifying a bioderived product of butadiene, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein. The invention further provides products made from bioderived butadiene such as synthetic rubber, tires and the like having a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C02 that occurs in the environment, wherein the synthetic rubber, tires, and the like are generated directly from or in combination with bioderived butadiene or a bioderived butadiene pathway intermediate as disclosed herein.
[00103] Butadiene is a chemical used in commercial and industrial applications. Non-limiting examples of such applications include production of synthetic rubber or elastomers and all products made from such rubber and elastomers such as tires and the like. Moreover, butadiene is also used as a raw material in the production of a wide range of products including tires, styrene-butadiene rubber (SBR), polybutadiene rubber (PBR), polychloroprene (Neoprene), nitrile rubber (NR) and the like. Accordingly, in some embodiments, the invention provides biobased synthetic rubber, other elastomers and products derived therefrom comprising one or more bioderived butadiene or bioderived butadiene pathway intermediate produced by a non- naturally occurring microorganism of the invention or produced using a method disclosed herein.
[00104] As used herein, the term "bioderived" means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism. Such a biological organism, in particular the microbial organisms of the invention disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source. Alternatively, the biological organism can utilize atmospheric carbon. As used herein, the term "biobased" means a product as described above that is composed, in whole or in part, of a bioderived compound of the invention. A biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock.
[00105] In some embodiments, the invention provides synthetic rubber and all products made from such rubber comounds such as tires and the like comprising bioderived butadiene or bioderived butadiene pathway intermediate, wherein the bioderived butadiene or bioderived butadiene pathway intermediate includes all or part of the butadiene or butadiene pathway intermediate used in the production of synthetic rubber and all products made from such rubber comounds such as tires and the like. For example, the final synthetic rubber, elastomer or products made therefrom can contain the bioderived butadiene, butadiene pathway intermediate, or a portion thereof that is the result of the manufacturing of synthetic rubber, elastomer or products made therefrom. Such manufacturing can include chemically reacting the bioderived butadiene or bioderived butadiene pathway intermediate (e.g. chemical conversion, chemical functionalization, chemical coupling, oxidation, reduction, polymerization, copolymerization and the like) into the final synthetic rubber, elastomer or products made therefrom. Thus, in some aspects, the invention provides a biobased synthetic rubber, elastomer or products made therefrom comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived butadiene or bioderived butadiene pathway intermediate as disclosed herein.
[00106] Additionally, in some embodiments, the invention provides a composition having a bioderived butadiene or butadiene pathway intermediate disclosed herein and a compound other than the bioderived butadiene or butadiene pathway intermediate. For example, in some aspects, the invention provides a biobased synthetic rubber, elastomer or products made thererfrom such as tires and the like wherein the butadiene or butadiene pathway intermediate used in its production is a combination of bioderived and petroleum derived butadiene or butadiene pathway intermediate. For example, a biobased synthetic rubber, elastomer or products made therefrom such as tires and the like can be produced using 50%> bioderived butadiene and 50%> petroleum derived butadiene or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of
bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing synthetic rubber, elastomer or products made therefrom such as rubber using the bioderived butadiene or bioderived butadiene pathway intermediate of the invention are well known in the art.
[00107] In some aspects, the invention provides a process for producing a biobased synthetic rubber, elastomer or products made therefrom such as tires disclosed herein by chemically reacting the bioderived butadiene with itself or another compound in a synthetic rubber producing reaction. It is understood that such process are well known in the art.
[00108] In another aspect, a compound other than the bioderived butadiene in a composition of the invention is a trace amount of a cellular portion of a non-naturally occurring microbial organism having a butadiene pathway of the invention disclosed here. A cellular portion of a microbial organism includes without limitation proteins, polypeptides, peptides, amino acids, nucleic acids, polynucleotides, components of the cell wall or a cellular membrane including, for example, peptidoglycans, glycoproteins, and polysaccharides, or any other cellular component. A "trace amount" as used herein refers to the presence of a compound or material in the composition, but in a quantity approaching a detectable limit. Such trace amounts can be so small as to not be accurately measured.
[00109] In some embodiments, the invention provides a molded product obtained by molding a biobased synthetic rubber or product made therefrom disclosed herein. Such molded products may be produced into any number of industrially desirable forms including, for example, a pellet.
[00110] Thus, the invention additionally provides culture medium comprising bioderived butadiene, wherein said bioderived butadiene has a carbon- 12, carbon- 13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. The culture medium can contain nitrate, nitrite and/or ammonium as described previously. The culture medium can be separated from a non-naturally occurring microbial organism having a butadiene pathway. The bioderived butadiene can have a carbon- 12, carbon- 13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. The bioderived butadiene can have an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.
[00111] The invention also provides a composition including bioderived butadiene and a compound other than bioderived butadiene. The compound other than bioderived butadiene can be a trace amount of a cellular portion of a non-naturally occurring microbial organism having a butadiene pathway.
[00112] Further provided is a biobased synthetic rubber or product made therefrom having any of the bioderived butadiene described above. The biobased synthetic rubber or product made therefrom can have at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50%) bioderived butadiene. Further provided is a molded product obtained by molding a biobased synthetic rubber or product made therefrom and a process for producing a biobased synthetic rubber or product made therefrom that includes chemically reacting the bioderived of the invention with itself or another compound in a synthetic rubber producing reaction.
[00113] The invention additionally provides methods of utilizing the microbial organisms of the invention containing a denitrification pathway for making a desired product, including butadiene. In particular, the invention provides a method of making bioderived butadiene. The method can include the steps of culturing a non-naturally occurring microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway for reducing nitrate (NO3 ) or nitrite (N02 ~) in a medium containing a sufficient amount of nitrate to produce a proton gradient under anaerobic conditions for a sufficient period of time to produce the bioderived butadiene. In such a method, the denitrification enzyme or pathway can comprise formate dehydrogenase, nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase and/or nitrous oxide reductase. In a particular embodiment, the nitrate (NO3) can be reduced to nitrite (N02 ~), ammonia (NH4), nitric oxide (NO), nitrous oxide (N20) or nitrogen (N2).
[00114] In methods of the invention utilizing a microbial organism containing a denitrification pathway, the microbial organism can further comprise a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase. In an embodiment of the invention, the nitrite reductase can be a NADH-dependent nitrite reductase. In a particular embodiment, the metabolic modification of the nitrite reductase can comprise attenuating the amount or activity of, for example, nirB, nirC or nirD.
[00115] In another embodiment of the invention, in which a microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway is utilized, the butadiene biosynthetic pathway can produce excess reducing equivalents. For example, the reducing equivalent can be NADH. In a furthe embodiment, the method of the invention can utilize a microbial organism where the butadiene biosynthetic pathway comprises an acetyl- CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase. As disclosed herein, any of a number of host microbial organisms can be used in a method of the invention, including but not limited to Escherichia species. In a particular embodiment, the sufficient amount of nitrate can comprise, for example, 0.1 mg/L to 10 g/L nitrate, including intermediate concentrations as disclosed herein. In another embodiment, the anaerobic conditions can comprise an amount of oxygen that is less than about 10% of saturation for dissolved oxygen in liquid media.
[00116] The invention further provides a method of producing bioderived butadiene by growing a non-naturally occurring microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway for reducing nitrate (Ν03 ~) or nitrite (N02 ~) in fermentation broth containing nitrate under fermentation conditions for a sufficient period of time to produce said bioderived butadiene. In one embodiment of the method, the denitrification enzyme or pathway can comprise formate dehydrogenase, nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase and/or nitrous oxide reductase. In a particular embodiment, the nitrate (NO3) can be reduced to nitrite (N02 ~), ammonia (NH4), nitric oxide (NO), nitrous oxide (N20) or nitrogen (N2).
[00117] In methods of the invention utilizing a microbial organism containing a denitrification pathway, the microbial organism can further comprise a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase. In an embodiment of the invention, the nitrite reductase can be a NADH-dependent nitrite reductase. In a particular embodiment, the metabolic modification of the nitrite reductase can comprise attenuating the amount or activity of, for example, nirB, nirC or nirD.
[00118] In another embodiment of the invention, in which a microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway is utilized, the butadiene biosynthetic pathway can produce excess reducing equivalents. For example, the reducing equivalent can be NADH. In a furthe embodiment, the method of the invention can utilize a microbial organism where the butadiene biosynthetic pathway comprises an acetyl- CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase. As disclosed herein, any of a number of host microbial organisms can be used in a method of the invention, including but not limited to Escherichia species. In a particular embodiment, the sufficient amount of nitrate can comprise, for example, 0.1 mg/L to 10 g/L nitrate, including intermediate concentrations as disclosed herein. In another embodiment, the anaerobic conditions can comprise an amount of oxygen that is less than about 10% of saturation for dissolved oxygen in liquid media.
[00119] In an additional embodiment of the invention, in which a microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway is utilized, the method can further comprise purifying the bioderived biobutadiene. In a particular embodiment, the bioderived biobutadiene can be purified by compression and cryoscopic distillation, as disclosed herein.
[00120] Suitable purification and/or assays to test for the production of a desired product such as butadiene can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al, Biotechnol. Bioeng., 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art. For typical Assay Methods, see Manual on Hydrocarbon Analysis (ASTM Manula Series, A.W. Drews, ed., 6th edition, 1998, American Society for Testing and Materials, Baltimore,
Maryland).
[00121] A desired product such as butadien can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.
[00122] Any of the non-naturally occurring microbial organisms described herein can be cultured under conditions to utilize a denitrification pathway to produce and/or secrete the biosynthetic products of the invention. For example, the denitrification pathway utilizing and butadiene producers can be cultured for the biosynthetic production of butadiene. Accordingly, in some embodiments, the invention provides culture medium having the butadiene or butadiene pathway intermediate described herein. In some aspects, the culture mediums can also be separated from the non-naturally occurring microbial organisms of the invention that produced the butadiene or butadiene pathway intermediate. Methods for separating a microbial organism from culture medium are well known in the art. Exemplary methods include filtration, flocculation, precipitation, centrifugation, sedimentation, and the like.
[00123] For the production of a desired product such as butadiene in a microbial organism having a denitrification pathway, the recombinant strains are cultured under conditions suitable for utilizing a denitrification enzyme or pathway, as disclosed herein, and in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. Fermentations can also be conducted in two phases, if desired. The first phase can be aerobic to allow for high growth and therefore high productivity, followed by an anaerobic phase of high butadiene yields.
[00124] If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.
[00125] The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of a desired product such as butadiene. [00126] In addition to renewable feedstocks such as those exemplified above, the microbial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the denitrification pathway utilizing and butadiene producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.
[00127] Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H2 and CO, syngas can also include C02 and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C02.
[00128] The Wood-Ljungdahl pathway catalyzes the conversion of CO and H2 to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing C02 and C02/H2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H2-dependent conversion of C02 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of C02 and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:
2 C02 + 4 H2 + n ADP + n Pi→ CH3COOH + 2 H20 + n ATP
Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize C02 and H2 mixtures as well for the production of acetyl-CoA and other desired products.
[00129] The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase,
methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: methyltetrahydrofolatexorrinoid protein methyltransferase (for example, AcsE), corrinoid iron- sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a denitrification and/or butadiene pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability.
[00130] The non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a denitrification and/or butadiene pathway enzyme or protein in sufficient amounts to produce utilize a denitrification pathway and produce butadiene. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce butadiene and utilize a denitrification pathway.
[00131] Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of butadiene resulting in intracellular concentrations between about 0.001-2000 mM or more. Generally, the intracellular concentration of butadiene is between about 3-1500 mM, particularly between about 5-1250 mM and more particularly between about 8-1000 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, 100 mM, 200 mM, 500 mM, 800 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms of the invention. [00132] In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication
2009/0047719, filed August 10, 2007. Any of these conditions can be employed with the non- naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the denitrification pathway utilizing and butadiene producers can synthesize butadiene at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, butadiene producing microbial organisms can produce butadiene intracellularly and/or secrete the product into the culture medium.
[00133] Exemplary fermentation processes include, but are not limited to, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation; and continuous fermentation and continuous separation. In an exemplary batch fermentation protocol, the production organism is grown in a suitably sized bioreactor sparged with an appropriate gas. Under anaerobic conditions, the culture is sparged with an inert gas or combination of gases, for example, nitrogen, N2/C02 mixture, argon, helium, and the like. As the cells grow and utilize the carbon source, additional carbon source(s) and/or other nutrients are fed into the bioreactor at a rate approximately balancing consumption of the carbon source and/or nutrients. The temperature of the bioreactor is maintained at a desired temperature, generally in the range of 22-37 degrees C, but the temperature can be maintained at a higher or lower temperature depending on the the growth characteristics of the production organism and/or desired conditions for the fermentation process. Growth continues for a desired period of time to achieve desired characteristics of the culture in the fermentor, for example, cell density, product concentration, and the like. In a batch fermentation process, the time period for the fermentation is generally in the range of several hours to several days, for example, 8 to 24 hours, or 1, 2, 3, 4 or 5 days, or up to a week, depending on the desired culture conditions. The pH can be controlled or not, as desired, in which case a culture in which pH is not controlled will typically decrease to pH 3-6 by the end of the run. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit, for example, a centrifuge, filtration unit, and the like, to remove cells and cell debris. In the case where the desired product is expressed intracellularly, the cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. The fermentation broth can be transferred to a product separations unit. Isolation of product occurs by standard separations procedures employed in the art to separate a desired product from dilute aqueous solutions. Such methods include, but are not limited to, liquid-liquid extraction using a water immiscible organic solvent (e.g, toluene or other suitable solvents) to provide an organic solution of the product, if appropriate, standard distillation methods, and the like, depending on the chemical characteristics of the product of the
fermenation process.
[00134] In an exemplary fully continuous fermentation protocol, the production organism is generally first grown up in batch mode in order to achieve a desired cell density. When the carbon source and/or other nutrients are exhausted, feed medium of the same composition is supplied continuously at a desired rate, and fermentation liquid is withdrawn at the same rate. Under such conditions, the product concentration in the bioreactor generally remains constant, as well as the cell density. The temperature of the fermentor is maintained at a desired temperature, as discussed above. During the continuous fermentation phase, it is generally desirable to maintain a suitable pH range for optimized production. The pH can be monitored and maintained using routine methods, including the addition of suitable acids or bases to maintain a desired pH range. The bioreactor is operated continuously for extended periods of time, generally at least one week to several weeks and up to one month, or longer, as appropriate and desired. The fermentation liquid and/or culture is monitored periodically, including sampling up to every day, as desired, to assure consistency of product concentration and/or cell density. In continuous mode, fermenter contents are constantly removed as new feed medium is supplied. The exit stream, containing cells, medium, and product, are generally subjected to a continuous product separations procedure, with or without removing cells and cell debris, as desired.
Continuous separations methods employed in the art can be used to separate the product from dilute aqueous solutions, including but not limited to continuous liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene), standard continuous distillation methods, and the like, or other methods well known in the art. [00135] In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of butadiene in a denitfication utilizing microbial organism can include the addition of an osmoprotectant to the culturing conditions. In certain
embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2- methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about lOmM, no more than about 50mM, no more than about lOOmM or no more than about 500mM.
[00136] As described herein, anaerobic conditions are particularly useful in methods using a denitrification pathway utilizing microbial organism since anaerobic conditions allow the use of nitrate instead of oxygen for respiration. The use of nitrate can be useful since it can be controlled more easily relative to oxygen. In addition, the methods of the invention utilizing a denitrification pathway and anaerobic conditions do not require high agitation rates and hence allow the use of anaerobic fermentation conditions and vessels. Such anaerobic fermentation conditions and vessels can be much larger than fermenters for aerobic processes and are significantly less expensive than aerobic fermenters. In addition, use of nitrate instead of oxygen minimizes undesired oxidative side reactions for a product such as butadiene that is highly reactive in the presence of oxygen, including reducing the potential risk of explosion in reaction vessels containing products that are highly reactive in the presence of oxygen. [00137] Methods for purifying butadiene are well known to those skilled in the art. Accordingly, such methods can be used to separate or purify the bioderived butadiene form culture media, fermentation broth and the like.
[00138] For example, compression, cryoscopic distillation or both compression and cryoscopic distillation can be used to separate and/or purify bioderived butadiene from culture media or fermentation broth. Compression can be used to convert a material or mixtures of materials from a gaseous to its liquid phase. This method is applicable for purifying the bioderived butadiene of the invention because bioderived butadiene is found in its gaseous form once released by the non-naturally occurring microbial organisms of the invention. The process of using compression for the purpose of butadiene purification is decribed in Chemico-Biological Interactions 166: 10-14 (2007); Patent US 4,433,558; Patent US 5,859,304; Patent Application Publication US 2004/0045804; Patent Application Publication US 2004/0267078 and
international patent application WO 2012/145096.
[00139] Briefly, cryoscopic distillation is a distillative freezing process in which a mixture of two or more components to be separated are vaporized under sufficiently reduced pressure and only one component freezes. For example, in a first step, partial vaporization of a mixture under reduced pressure results in crystallization of a major component of the mixture to be purified. A pure product of the major component is obtained by repeating this operation to completely eliminate the liquid phase. In a second step the low pressure vapor is transformed into a condensed mass by slightly lowering the temperature without pressurizing it. Distillation procedures and the recovery process of butadiene are described in Chemico-Biological
Interactions, 166: 10-14 (2007); Patent US 4,433,558; Patent US 5,859,304; Patent Application Publication US 2004/0045804; Patent Application Publication US 2004/0267078 and
international patent application WO 2012/145096. Other methods well known in the art can similarly be used to separate and/or purify butadiene from a culture media and/or fermentation broth.
[00140] Accordingly, the invention provides bioderived butadiene separated or purified by compression, cryoscopic distillation or by both compression and cryoscopic distillation. The method includes butadiene containing media or fermentation broth obtained following culturing under anaerobic conditions in the presence of nitrate or other nitrogen containing compound described herein for achieving micro-respiric growth.
[00141] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.
EXAMPLE I
Anaerobic Production of Butadiene in the Presence of Nitrate
[00142] This Example describes exemplary denitrification enzymes and denitrification pathways that can be included a butadiene producing microbial for growing in the presence of nitrate.
[00143] Nitrate respiration has been extensively studied (Kraft et al, Journal of
Biotechnology, 155: 104- 117 (2011); Takeno et al, Appl. Microbiol. Biotechnol, 75: 1173-1182 (2007); Prohl et al, Arch. Microbiol, 170: 1-7 (1998); Wang and Gunsales, Journal of
Bacteriology, 180(20):5813-5822 (2000); Einsle, Methods in Enzymology, 496:399-422 (2011); Gonzalez, WO 2008/147470 A2). In the following sections, genes are described that encode enzymes with activities imparting nitrate respiration capabilities.
[00144] Complex I Enzyme
[00145] NADH dehydrogenase: NADH -> NAD
[00146] NADH ubiquinone oxidoreductase I (NDH-1) is an NADH dehydrogenase that catalyzes the transfer of electrons from NADH to the quinone pool in the cytoplasmic membrane and is able to generate a proton electrochemical gradient.
1) nuoA 2) NP 416791.3 3) GI 49176207 4) Escherichia coli K-12 MG1655
5) nuoB 6) NP 416790.1 7) GI 16130222 8) Escherichia coli K-12 MG1655
9) nuoC 10) NP 416789.2 11) GI 145698291 12) Escherichia coli K-12 MG1655
13) nuoE 14) P 416788.1 15) GI 16130220 16) Escherichia coli K-12 MG1655
17) nuoF 18)NP 416787.1 19) GI 16130219 20) Escherichia coli K-12 MG1655
21) nuoG 22) NP 416786.4 23) GI 145698290 24) Escherichia coli K-12 MG1655 25) nuoH 26) NP 416785.1 27) GI 16130217 28) Escherichia coli K-12 MG1655
29) nuoI 30)NP 416784.1 31) GI 16130216 32) Escherichia coli K-12 MG1655
33) nuoJ 34) NP 416783.1 35) GI 16130215 36) Escherichia coli K-12 MG1655
37) nuoK 38)NP 416782.1 39) GI 16130214 40) Escherichia coli K-12 MG1655
41) nuoL 42)NP 416781.1 43) GI 16130213 44) Escherichia coli K-12 MG1655
45) nuoM 46)NP 416780.1 47) GI 16130212 48) Escherichia coli K-12 MG1655
49) nuoN 50)NP 416779.2 51) GI 145698289 52) Escherichia coli K-12 MG1655
[00147] Nitrate to Nitrite Enzymes
[00148] Nitrate reductase: Nitrate -> Nitrite
[00149] E. coli contains three nitrate reductases. Two of them, nitrate reductase A (NRA) and nitrate reductase Z (NRZ), are membrane bound and biochemically similar. The third nitrate reductase, Nap, is located in the periplasm. Nitrate reductase A is expressed when levels of nitrate in the environment are high, Nap is expressed when they are low, while NRZ expression is not dependent on nitrate levels or anaerobiosis, but induced during stationary phase.
[00150] Nitrate reductase A functions anaerobically as a terminal electron acceptor. It accepts electrons from the quinone pool and in so doing expels two protons from the cell, thereby adding to the proton motive force. Formate dehydrogenase N and nitrate reductase A form a respiratory chain, where a redox loop of quinone molecules couples electron transfer from formate in the periplasm to nitrate in the cytoplasm.
[00151] Nitrate reductase A is a heterotrimer composed of the α-, β- and γ chains. A fourth polypeptide, encoded by the narJ gene, is required for the incorporation of the molybdenum cofactor into NarG, the a subunit. If it is coexpressed with the private chaperone NarJ, the NarGH complex alone is soluble and active with artificial electron donors such as benzyl viologen. NarGH becomes localized to the cytoplasmic side of the inner membrane by interaction with Narl.
53) narG 54) NP 415742.1 55) GI: 16129187 56) Escherichia coli K-12 MG1655
57) narH 58)NP 415743.1 59) GI: 16129188 60) Escherichia coli K-12 MG1655
61) narJ 62) NP 415744.1 63) GI: 16129189 64) Escherichia coli K-12 MG1655
65) narl 66) NP 415745.1 67) GI: 16129190 68) Escherichia coli K-12 MG1655 [00152] Unlike nitrate reductase A, nitrate reductase Z expression is not dependent on nitrate levels or anaerobiosis. During entry into stationary phase, transcription of the narZYWV operon is induced, and induction is mainly dependent on the alternative sigma factor RpoS. By homology whith nitrate reductase A, nitrate reductase Z is a heterotrimer composed of the α-, β- and γ chains. A fourth polypeptide, encoded by the narW gene, is required for the incorporation of the molybdenum cofactor into NarZ, the a subunit.
Figure imgf000065_0001
[00153] Periplasmic nitrate reductase (Nap) is induced by anaerobiosis through the mediation of the transcription factor Fnr and low concentrations of nitrate through the mediation of NarP. Nap is not itself a coupling site for generating proton motive force; acting as a terminal electron acceptor, it does support anaerobic respiration of various carbon sources Λ
[00154] The physiological role of Nap is that of mediating anaerobic respiration at the expense of low concentrations of nitrate. Owing to the periplasmic location of Nap, the cost of pumping nitrate into the cell is avoided. In addition, Nap has a significantly higher affinity for nitrate than nitrate reductase A and is thus able to exploit the low concentrations of nitrate occuring in the natural environment of E. coli. Notably, several pathogenic bacterial species, such as Haemophilus influenzae, only contain orthologs of the periplasmic nitrate reductase. During glucose fermentation in the absence of menaquinone, a very low level of Nap activity appears to substitute for the redox-balancing role of fumarate reductase, which is dependent on menaquinone.
[00155] The nap operon encodes seven proteins. The catalytic portion of the protein, consisting of the periplasmic NapA and NapB polypeptides, receives electrons via the membrane-bound cytochrome NapC from NapGH or directly from the quinone pool. The NapD polypeptide is required for enzyme activity and is thought to be involved in the post-translational assembly of the molybdoprotein NapA. NapF, NapG and NapH are predicted to encode iron- sulfur proteins and are not required for Nap activity; they do, however, contribute to the maximum rate of nitrate reduction. NapG and NapH facilitate electron transfer from ubiquinol via NapC to NapAB.
Figure imgf000066_0001
[00156] Nitrite to Ammonia Enzymes
[00157] Membrane-associated, ammonia forming, nitrite reductase: Nitrite Ammonia
[00158] In E. coli K-12, the nrfABCD genes encode a membrane associated nitrite reductase complex responsible for the 6 electron reduction of nitrite to ammonia. NrfABCD is a respiratory enzyme which couples to the formate oxidising enzymes via menaquinone in order to generate electron potential.
[00159] nrfA encodes a cytochrome c nitrite reductase (cyt c552). It is a novel pentahaem enzyme that forms a redox complex with NrfB - also a pentahaem protein - to catalyse the reduction of nitrite to ammonia. nrfC and nrfD are predicted to encode electron transfer proteins that couple electron transport from the menaquinol pool in the membrane to the NrfAB complex in the periplasm. NrfA is linked to the menaquinol pool in the cytoplasmic membrane through the electron carrier NrfB which in turn interacts with the membrane integral proteins NrfC and NrfD. Under physiological conditions it is likely that the periplasmic subunit NrfA is tightly associated with the membrane assoicated subunit NrfB.
[00160] NrfA can also reduce and thereby detoxify nitric oxide (NO); mutant strains lacking NrfA activity have increased sensitivity to NO.
[00161] Nitrite reduction and synthesis of the NrfA protein are repressed by oxygen. During anaerobic growth nitrite reduction by the Nrf pathway is induced by nitrite and repressed by nitrate. The Fnr transciptional regulator is essential for NrfA synthesis and nitrite reduction by formate. The nrf operon is further regulated by the NarQP and NarXL two-component systems in response to levels of nitrite and nitrate in the environment. NrfE, NrfF and NrfG are presumed to part of a heme lyase that adds heme groups to apocytrochrome c552 (NrfA)
Figure imgf000067_0001
[00162] Nitrate/nitrite Transport Enzymes [00163] Nitrate/nitrite transporter:
Nitrite (periplasm) Nitrite (cytosol)
Nitrite (cytosol) + Nitrate (periplasm) Nitrite (periplasm) + Nitrate (cytosol)
[00164] NarU and NarK are the two nitrate transporters in E. coli. Both function as nitrate/nitrite antiporters in anaerobic nitrate respiration. NarK is a member of the major facilitator superfamily (MFS) of transporters, and probably also functions as a proton/nitrite antiporter. NarK is thought to act as a nitrate/nitrite antiporter based on physiological studies of narK mutants as well as mutants expressing narK but lacking the other nitrate/nitrite transporters, NarU and NirC. Nitrate transport experiments in whole cells demonstrated that nitrate uptake is not drastically affected by mutations in narK (likely due to the presence of NarU), but the mutations instead affected nitrate utilization due to a decreased ability to excrete nitrite.
Transport experiments in proteoliposomes using a nitrite fluorophore indicated that NarK mediates electrogenic nitrite extrusion with a Km of approx 300 μΜ. Competition experiment using chemostat cultures show narK+ narU strains out-compete narK narU+ strains during exponential growth, but the opposite is true during stationary phase.
[00165] Expression of narK is regulated by nitrate and oxygen via the regulatory proteins NarL, NarX, NarQ, FNR, IHF, and Fis. The nitrate-induced expression of narK is partially dependent upon ModE-molybdate as a transcriptional activator as well as the catalyzed product of MoeA. and iron.
[00166] NarU is a member of the major facilitator superfamily (MFS) of transporters, and is highly similar to NarK. narU is the first gene of the narUZYWV operon. The cloned narU gene was shown to be able to complement a narK mutation based on assays of nitrite concentrations in the external medium. However, narU was not able to complement a narK nirC mutation for nitrite uptake. A strain expressing narU without narK had a selective advantage over a strain expressing narK without narU during slow growth or starvation, but the opposite was true during fast growth. Membrane topology predictions using experimentally determined C terminus locations indicate that NarU has 12 transmembrane helices and the C-terminus is located in the cytoplasm.
Figure imgf000068_0001
[00167] Nitrite to N2 Enzymes
[00168] NO-forming nitrite reductase: Nitrite -> NO
[00169] There are two types of dissimilatory nitrite reductase enzymes. These two types catalyzes precisely the same reaction, but differ in their cofactors. One type contains a cytochrome cdl, while the second type contains copper. One exemplary copper-containing dissimilatory nitrite reductase can be found in the halophilic archaeon Haloarcula marismortui. The enzyme contains two subunits, whose apparent molecular masses are 46 and 42 kDa.
However, the two subunits are encoded by the same nirK gene, but the smaller subunit is missing 16 amino acid residues from the N-terminal region. 149) nirK 150) YP 137309.1 151) GL55379459 152) Haloarcula marismortui
[00170] NirS genes contain a cytochrome cdl . NirS is a homodimer, and each subunit contains one c-type heme and one dl-type heme.
Figure imgf000069_0001
[00171] Nitric oxide reductase: NO -» N20
[00172] Nitric oxide reductase is an integral membrane component of the anaerobic respiratory chain, which results in denitrification. The enzyme catalyzes the reduction of nitric oxide to nitrous oxide, a reaction that involves the formation of an N-N bond, in essence a reversal of nitrogen fixation. NO reduction is coupled to electron transport phosphorylation.The enzyme consists of a cytochrome c and cytochrome b subunits, encoded by the norC and norB, respectively. The two genes are contiguous and transcribed as a single 2.0-kb transcript in Pseudomonas stutzeri.
Figure imgf000069_0002
[00173] While most organisms have a dimeric nitric oxide reductase encoded by the norB and norC genes (see nitric oxide reductase from Pseudomonas stutzeri), a few microorganisms posses a monomeric type.The bacterium Ralstonia eutropha HI 6 contains two independent nitric oxide reductases. One is encoded by the chromosomal norZ gene, while the other is encoded by the megaplasmid pHGl -located norB gene. The two genes share 90% amino acid sequence identity, and are integral membrane proteins composed of 14 membrane-spanning helices. Both proteins consist of a single subunit that contains both high- spin and low- spin heme b, no heme c, and one non-heme iron. While this single subunit is homologous to the catalytic subunits of common NO reductases, which are heterodimers, it possesses an N-terminal extension of approximately 280 amino acids. Mutation of either norB or norZ havs no obvious phenotype, but the simultaneous inactivation of both genes is lethal to the cells under anaerobic growth conditions, indicating that they are isofunctional and instrumental in denitrification. The enzyme encoded by the norZ gene is monomeric and was shown to accept electrons from a quinol. While it is located in the membrane, the enzyme's active site faces the periplasm. 165) norZ 166) AAZ64403.1 167) GL72122217 168) Ralstonia eutropha
169) norB 170) NP 942879.1 171) GL38637905 172) Ralstonia eutropha
[00174] Nitrous oxide reductase
[00175] The nitrous oxide reductase of Pseudomonas stutzeri is a multicopper enzyme composed of two identical subunits, encoded by the nosZ gene. The enzyme contains 8 copper ions. Two conserved domains corresponding to the binuclear centers CuA, the entry site for electrons, and CuZ, the catalytic site, have been identified in NosZ.
Figure imgf000070_0001
[00176] Hydrazine hydrolase: NO + NH4+ -> N2H4
[00177] Hydrazine hydrolase is a unique enzyme that was predicted based on the proposed anammox (anaerobic ammonia oxidation) pathway. When the metagenome of Candidatus Kuenenia stuttgartiensis became available, a cluster of eight genes was recognized as potentially encoding the enzyme. These genes, kuste2854 through kuste2861, encode a β-propeller complex, similar to nitrous oxide reductase. A later study which identified proteins within the
anammoxosome identified products of three of these genes, namley kuste2859, kuste2860 and kuste2861, as very abundant members of the anammoxosome proteome. Antibodies produced against two of these peptides verified the association between these proteins and
anammoxosomes (Karlsson et al, FEMS Microbiol. Lett., 297(l):87-94 (2009); Straus et al, Nature, 440(7085):790-794 (2006)).
177) Kuste2861 178) CAJ73613.1 179) GL91200564 180) Candidatus Kuenenia stuttgartiensis
181) Kuste2860 182) CAJ73612.1 183) GL91200563 184) Candidatus Kuenenia stuttgartiensis
185) Kuste2859 186) CAJ73611.1 187) GL91200562 188) Candidatus Kuenenia stuttgartiensis
189) Kuste2858 190) CAJ73610.1 191) GL91200561 192) Candidatus Kuenenia stuttgartiensis
193) Kuste2857 194) CAJ73609.1 195) GL91200560 196) Candidatus Kuenenia stuttgartiensis
197) Kuste2856 198) CAJ73608.1 199) GL91200559 200) Candidatus Kuenenia stuttgartiensis
201) Kuste2855 202) CAJ73607.1 203) GL91200558 204) Candidatus Kuenenia stuttgartiensis 205) Kuste2854 206) CAJ73606.1 207) GL91200557 208) Candidatus Kuenenia stuttgartiensis
[00178] Hydrazine oxidoreductase
[00179] A multiheme protein having hydrazine-oxidizing activity was purified from the anammox bacterium planctomycete KSU-1. The protein, which is homodimeric, contains 8 heme c molecules per monomer, one of which is a specialized heme with absorbance at at 472 nm (known as cytochrome P472). The purified protein catalyzed the oxidation of hydrazine in vitro, using cytochrome c as an electron acceptor, and could not accept hydroxylamine as a substrate, although it was a competitive inhibitor for hydrazine. Two similar genes, hzoA and hzoB, encode the enzyme, with only two amino acid residues different between the polypeptides.
Transcription analysis showed that while both genes were transcribed, the hzoB transcript is present at a 5 -fold higher level, indicating that hydrazine oxidoreductase B is the dominant form.
[00180] A similar gene, Kustdl340, has been identified in the sequenced metagenome of Candidatus Kuenenia stuttgartiensis. The product of that gene was found to be highly abundant in the anammoxosome (Karlsson et al, FEMS Microbiol. Lett., 297(l):87-94 (2009); Strous et al, Nature, 440(7085):790-794 (2006)).
Figure imgf000071_0001
[00181] Undesirable activities particularly if E. coli is the chosen host organism
[00182] It can be desirable to delete or attenuate the following activities to maximize the ATP yield of nitrate respiration.
[00183] NADH dehydrogenase (non-proton pumping): NADH -> NAD
[00184] NADH:ubiquinone oxidoreductase II (NDH-2) is a type IIA NADH dehydrogenase that catalyzes the transfer of electrons from NADH to the quinone pool in the cytoplasmic membrane. It is thus part of the aerobic respiratory chain of the cell, and its primary function may be maintenance of the [NADH]/[NAD ] balance of the cell. NDH-2 is one of two distinct NADH dehydrogenases in E. coli. In contrast to NDH-1 (encoded by the nuo genes), NDH-2 utilizes NADH exclusively, and electron flow from NADH to ubiquinone does not generate an electrochemical gradient.
221) ndh 222) NP_415627.1 223) GI: 16129072 224) Escherichia coli K-12
MG1655
[00185] Cytoplasmic, ammonia forming, nitrite reductase: Nitrite Ammonia
[00186] At high concentrations of nitrate, the cytoplasmic nitrite reductase (NirB) is made almost exclusively. NirB is located in the cytoplasm and does not generate a proton gradient. Its probable metabolic role is to detoxify nitrite. NirB catalyzes the six-electron reduction of nitrite to ammonia and also catalyzes the two-electron reduction of hydroxylamine to ammonia. The prosthetic groups of nitrite reductase are FAD, an iron-sulfur cluster and siroheme. The product of the cysG gene is necessary for the synthesis of the siroheme prosthetic group of NirB. The reaction is active only during anaerobic growth and fulfills a dissimilatory rather than
assimilatory role.
Figure imgf000072_0001
[00187] Nitrite transporter (nirC): Nitrite (periplasm) Nitrite (cytosol)
[00188] NirC is a nitrite transporter which is a member of the FNT family of formate and nitrite transporters. The nirC gene is located in the nir operon which codes for a NADH- dependent nitrite reductase. NirC functions to import nitrite as a substrate for this enzyme complex. The nir operon is anaerobically expressed and is repressed by oxygen. Strains expressing nirC but lacking the two other nitrate/nitrite transporters (NarK and NarU) had higher activity of nitrite uptake and reduction than a strain expressing only narK. NirC is able to reimport nitrite that is extruded by NarK for further reduction to ammonia. 237) nirC 238) YP_026212.2 239) GL90111575 240) Escherichia coli K-12
MG1655
EXAMPLE II
Anaerobic Production of 1,4-Butanediol in the Presence of Nitrate
[00189] This Example shows the production of bioderived 1 ,4-butanediol from
microorganisms grown in the presence of nitrate.
[00190] To exemplify the principle of micro-respiric growth for chemical production, the expression of a recombinant pathway to produce 1 ,4-butanediol (BDO) in E. coli. This process was simulated by adding the proposed reactions to the current genome-scale E. coli model (Version 1.4) in SIMPHENY. This computational method and system is described in, for example, U.S. Patent Nos. 7,856,317 and 8,301,430. SIMPHENY is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components. This computational approach is consistent with biological realities because biological systems are flexible and can reach the same result in many different ways.
[00191] Maximum theoretical yields were calculated assuming different scenarios: 1.) oxygen available to meet demand, 2.) strictly anaerobic conditions allowing either acetate or ethanol secretion, 3.) reduction of nitrate to nitrite, and 4.) the 2-step reduction of nitrate to ammonia. Next, maximum yield was calculated in each case for various scenarios assuming different levels of cell growth. A non-growth associated maintenance energy of 7.6 mmol/gDCW-hr was assumed in all cases. [00192] For comparison with the micro-respiric conditions below, Figure 7 depicts oxygen respiration and the energetic for BDO biosynthesis. Briefly, the BDO pathway generates 1 excess NADH equivalent using the pathways exemplified in Figure 8 and described further below. The electrons are passed to ½ 02 to form water and generate ATP. About 1.5-2.0 ATP's can be synthesized per NADH equivalent depending upon the assumed proton translocation stoichiometry of ATP synthase (3 vs. 4 H+/ATP) as illustrated in Figure 7.
[00193] Figure 8 shows reactions that can participate in the synthesis of BDO from succinate, a central intermediate of the TCA cycle which is a fermentation product of wild-type E. coli. The two-step reduction of succinate to 4-hydroxybutanoate (4-HB) is known to occur in nature, and the genes responsible have been found in several bacterial species (Sohling and Gottschalk, J. Bacteriol, 178-871-880 (1996)). The subsequent two-step reduction of 4-HB to BDO has been reported in Clostridium acetobutylicum (Jewell et al, Curr. Microbiol, 13:215-219 (2007)). The direct conversion of succinate to succinic semialdehyde can be thermodynamically challenging, so an alternative pathway proceeding through succinyl-CoA also is available (Lutke-Eversloh and Steinbuchel, F EMS Microbiol. Lett., 181 :63-71 (1999)). The genes encoding these steps have been cloned and overexpressed for synthesis of 4-HB as an intermediate to the copolymer polyhydroxyalkanoate (Song et al, Wei Sheng Wu Xue Bao, 45(3):382-386 (2005); Valentin et al, Eur. J. Biochem., 227:43-60 (1995)). An additional ATP is required, which serves to overcome the thermodynamic barrier between succinate and succinic semialdehyde. The analogous step in the second part of the pathway is the conversion of 4-HB to 4-hydroxybutanal. This step can occur via the CoA intermediate, 4-hydroxybutyryl-CoA using a CoA transferase, converting acetyl-CoA to acetate in the process (Sholing and Gottschalk, J. Bacteriol., 178:871- 880 (1996)), and ATP is required to regenerate acetyl-CoA. If both acid-to-aldehyde conversions occur by way of the CoA derivatives, 2 additional ATP molecules are needed for each BDO produced. This makes the net ATP generation from glucose to BDO negative. Therefore, additional ATP is necessary to make this conversion. In the absence of respiration, this can occur by diverting some glucose to other products that have a net positive ATP gain, such as acetate. However, the production of unwanted byproducts can be prevented by adding a controlled amount of external electron acceptor. [00194] The maximum theoretical yields for each of the above described scenarios assuming that the production of BDO must be energetically neutral are shown in Table 1. The maximum theoretical yield without assuming energetic neutrality is 1.091 mol/mol glucose for all cases. The highest yield is obtained when oxygen uptake is allowed. The model represents the case where just enough oxygen is taken up to allow the proper amount of ATP generation through respiration. In this scenario, the only products are BDO, biomass, and C02. In the anaerobic case, the yield drops and either acetate or ethanol is made as a byproduct. Controlled nitrate addition can therefore provide the same or similar yield as oxygen, the exact amount depending on whether further reduction of nitrite to ammonia occurs.
Table 1. The maximum theoretical yields of 1 ,4-butanediol (BDO) for five sets of environmental conditions: 1) aerobic respiration, 2) anaerobic fermentation with acetate co-production, 3) anaerobic fermentation with ethanol co-production, 4) nitrate respiration leading to nitrite formation, and 5) nitrate respiration leading to ammonia formation. Negative values indicate metabolites taken up; positive values indicate metabolites secreted. Molar units are assumed along with pathway energetic neutrality.
Figure imgf000075_0001
[00195] One objective for the above BDO process is to couple growth to BDO production, so that some carbon and energy must be directed to biomass as well as BDO. For this scenario, a final BDO titer of 100 g/L and a batch time of 33.3 hours were assumed, which corresponds to a volumetric productivity of 3 g/L*hr (neglecting reactor cleaning and filling times) and a cell growth rate of 0.094 hr-1 if the inoculum is 1/20 the final cell density. These values were chosen as a realistic example; the calculations could be repeated in SimPheny using different parameters to obtain qualitatively similar results. For all calculations, the growth and specific BDO production rates were fixed and a non-growth associated ATP maintenance requirement of 7.6 mmol/gDW/hr was assumed as suggested in ref. (Varma and Palsson, Appl. Environ. Microbiol, 60:3724-3731 (1994)). The specific glucose uptake rate was then minimized subject to these constraints. The results are shown in Table 2. In general, the ability to achieve high cell densities lessens the extent to which metabolic engineering will be required to alter the BDO/growth ratio and also lowers the required specific uptake rate of glucose. On the other hand, if the glucose specific uptake rate or the BDO/cell growth ratio can be increased beyond the values specified in Table 2 via strain engineering, higher BDO titers and/or volumetric productivities will be achieved. Finally, the BDO/glucose yield is decreased at low specific glucose uptake rates because a greater percentage of the incoming substrate is needed to satisfy the non-growth associated ATP maintenance requirement.
Table 2: The minimum specific glucose uptake rates required to enable 100 g/L of BDO production at a 3 g/L/hr volumetric productivity were calculated assuming various final cell densities for the five growth scenarios.
Figure imgf000077_0001
[00196] Throughout this application various publications have been referenced. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

Claims

CLAIMS What is claimed is:
1. A non-naturally occurring microbial organism comprising a butadiene
biosynthetic pathway, a denitrification enzyme or pathway for reducing nitrate (NO3 ) or nitrite (NO2 ) and a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase.
2. The non-naturally occurring microbial organism of claim 1 , wherein said denitrification enzyme or pathway comprises formate dehydrogenase, nitrate reductase, NO- forming nitrite reductase, nitric oxide reductase or nitrous oxide reductase.
3. The non-naturally occurring microbial organism of claim 1, wherein said nitrate (NO3") is reduced to nitrite (N02 ~), ammonia (NH4), nitric oxide (NO), nitrous oxide (N20) or nitrogen (N2).
4. The non-naturally occurring microbial organism of claim 1 , wherein said nitrite reductase is a NADH-dependent nitrite reductase.
5. The non-naturally occurring microbial organism of claim 1 , wherein said metabolic modification of said nitrite reductase comprises attenuating the amount or activity of nirB, nirC or nirD.
6. The non-naturally occurring microbial organism of claim 1 , wherein said butadiene biosynthetic pathway produces excess reducing equivalents.
7. The non-naturally occurring microbial organism of claim 6, wherein said reducing equivalent is NADH.
8. The non-naturally occurring microbial organism of claim 1 , wherein said butadiene biosynthetic pathway comprises an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase and a butadiene synthase.
9. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism is Escherichia.
10. A composition comprising a non-naturally occurring microbial organism having a butadiene biosynthetic pathway, a denitrification enzyme or pathway for reducing nitrate (NO3 ) or nitrite (N02 ~) and a medium comprising nitrate.
11. The non-naturally occurring microbial organism of claim 10, wherein said denitrification enzyme or pathway comprises formate dehydrogenase, nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase or nitrous oxide reductase.
12. The non-naturally occurring microbial organism of claim 10, wherein said nitrate (NO3 ") is reduced to nitrite (N02 ~), ammonia (NH4), nitric oxide (NO), nitrous oxide (N20) or nitrogen (N2).
13. The non-naturally occurring microbial organism of claim 10, further comprising a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase.
14. The non-naturally occurring microbial organism of claim 13, wherein said nitrite reductase is a NADH-dependent nitrite reductase.
15. The non-naturally occurring microbial organism of claim 13, wherein said metabolic modification of said nitrite reductase comprises attenuating the amount or activity of nirB, nirC or nirD.
16. The non-naturally occurring microbial organism of claim 10, wherein said butadiene biosynthetic pathway produces excess reducing equivalents.
17. The non-naturally occurring microbial organism of claim 16, wherein said reducing equivalent is NADH.
18. The non-naturally occurring microbial organism of claim 10, wherein said butadiene biosynthetic pathway comprises an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase and a butadiene synthase.
19. The non-naturally occurring microbial organism of claim 10, wherein said microbial organism is Escherichia.
20. A composition comprising bioderived butadiene in a nitrate or ammonium (NH4 ) containing medium.
21. A method of making bioderived butadiene, comprising culturing a non-naturally occurring microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway for reducing nitrate (NO3 ) or nitrite (N02 ~) in a medium containing a sufficient amount of nitrate to produce a proton gradient under anaerobic conditions for a sufficient period of time to produce said bioderived butadiene.
22. The method of claim 21, wherein said denitrification enzyme or pathway comprises formate dehydrogenase, nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase or nitrous oxide reductase.
23. The method of claim 21 , wherein said nitrate (NO3) is reduced to nitrite (N02 ~), ammonia (NH4), nitric oxide (NO), nitrous oxide (N20) or nitrogen (N2).
24. The method of claim 21, further comprising a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase.
25. The method of claim 24, wherein said nitrite reductase is a NADH-dependent nitrite reductase.
26. The method of claim 24, wherein said metabolic modification of said nitrite reductase comprises attenuating the amount or activity of nirB, nirC or nirD.
27. The method of claim 21, wherein said butadiene biosynthetic pathway produces excess reducing equivalents.
28. The method of claim 27, wherein said reducing equivalent is NADH.
29. The method of claim 21, wherein said butadiene biosynthetic pathway comprises an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl- CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase.
30. The method of claim 21 , wherein said microbial organism is Escherichia.
31. The method of claim 21 , wherein said sufficient amount of nitrate comprises from about 1 to 10 g/L.
32. The method of claim 21, wherein said anaerobic conditions comprise an amount of oxygen that is less than about 10% of saturation for dissolved oxygen in liquid media.
33. A method of producing bioderived butadiene comprising growing a non-naturally occurring microbial organism having a butadiene biosynthetic pathway and a denitrification enzyme or pathway for reducing nitrate (N03 ~) or nitrite (N02 ~) in fermentation broth containing nitrate under fermentation conditions for a sufficient period of time to produce said bioderived butadiene.
34. The method of claim 33, wherein said denitrification enzyme or pathway comprises formate dehydrogenase, nitrate reductase, nitrite reductase, NO-forming nitrite reductase, nitric oxide reductase or nitrous oxide reductase.
35. The method of claim 33, wherein said nitrate (N03 ~) is reduced to nitrite (N02 ~), ammonia (NH4), nitric oxide (NO), nitrous oxide (N20) or nitrogen (N2).
36. The method of claim 33, further comprising a metabolic modification attenuating the amount or activity of a cytoplasmic nitrite reductase.
37. The method of claim 36, wherein said nitrite reductase is a NADH-dependent nitrite reductase.
38. The method of claim 36, wherein said metabolic modification of said nitrite reductase comprises attenuating the amount or activity of nirB, nirC or nirD.
39. The method of claim 33, wherein said butadiene biosynthetic pathway produces excess reducing equivalents.
40. The method of claim 39, wherein said reducing equivalent is NADH.
41. The method of claim 33, wherein said butadiene biosynthetic pathway comprises an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl- CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase.
42. The method of claim 33, wherein said microbial organism is Escherichia.
43. The method of claim 33, wherein said fermentation broth containing nitrate under fermentation conditions comprises from about 1 to 10 g/L nitrate.
44. The method of claim 33, wherein said anaerobic conditions comprise an amount of oxygen that is less than about 10% of saturation for dissolved oxygen in liquid media.
45. The method of claim 33, further comprising purifying said bioderived biobutadiene.
46. The method of claim 33, wherein said bioderived biobutadiene is purified by compression and cryoscopic distillation.
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WO2021257618A1 (en) * 2020-06-15 2021-12-23 Florida Food Products, LLC Continuous fermentation process for meat curing agents
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