MODIFIED ORGANISMS FOR ETHYLENE, ETHANE, AND METHANE BIOGENESIS AND METHODS FOR USE THEREOF

The present disclosure provides non-naturally occurring microbial organisms capable of producing ethylene, ethane, and/or methane, as well was methods for producing ethylene, ethane, and/or methane using the same.

BACKGROUND

Nitrogenases are an ancient group of enzymes, existing approximately 3.2 billion years ago, before the evolution of oxygenic photosynthesis and subsequent widespread oxygenation (1, 2), Their essential function is reduction of dinitrogen gas into ammonia, contributing over half of the annual global nitrogen fixation required for the synthesis of nucleic and amino acids by all life on earth (3). Ancestors to nitrogenase in anaerobic prokaryotes also gave rise to distinct nitrogenase-like reductases for bacterial photosynthesis and archaeal methanogenesis cofactor metabolism (4, 5, 6, 7). These include the dark operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide α oxidoreductase (COR) of bacteriochlorophyll biosynthesis, and Ni2+-sirohydrochlorin a,c-diamide reductive cyclase for biosynthesis of the archaeal methyl coenzyme-M reductase cofactor F430 (4, 5, 6, 7). However, the evolutionary history of nitrogen fixation revealed overlooked nitrogen fixation-like (NFL) sequences in the genomes of anaerobic bacteria with entirely unknown function. Some were surprisingly associated with sulfur metabolism and transport genes (8, 9). This suggested that certain members of the nitrogenase family potentially have a role in sulfur metabolism.

Previous production of ethylene gas (>1 μmol/h/g dry cell weight) was observed from photosynthetic Alphaproteobacteria such asRhodospirillum rubrumandRhodopseudomonas palustriswhen growing anaerobically under the low sulfate concentrations (<200 μM) commonly encountered in their freshwater and soil habitats (seeFIGS.5A-C) (10). The precursor of ethylene, (2-methylthio)ethanol (MT-EtOH), and the pathway for its production was documented (P2017-343-096 Novel Microbial Process to Synthesize Ethylene) (10). This volatile organic sulfur compound (VOSC) was produced from byproducts of S-adenosyl-L-methionine (SAM) utilization to regenerate methionine (FIG.1A; DHAP shunt) (10). SAM is a key cellular cofactor synthesized directly from methionine and is required by all organisms for diverse processes including DNA, RNA, and protein methylations, polyamine and neurotransmitter synthesis, quorum sensing, and 5′-deoxyadenosyl radical generation by radical SAM enzymes (11). However, the enzymes responsible for the liberation of sulfur from MT-EtOH for methionine regeneration and concomitant ethylene formation were unresolved (10). These enzymes are thus disclosed to be a reductase of the nitrogenase-like family of enzymes, specifically a methylthio-alkane reductase (Mar) composed of components MarB, MarH, MarD, and MarK (MarBHDK) (seeFIG.1A).

There is a clear need for methods of producing the industrial precursor compounds ethylene, ethane, and methane, and microorganisms for the same. In particular, known ethylene producing enzyme systems require oxygen (aminocyclopropanecarboxylate oxidase and 2-oxoglutatate dioxygenase), forming a flammable ethylene-oxygen gas mixture. In addition methane and ethane when mixed with air are also explosive and flammable. Therefore, a microorganism and enzyme system to produce significant levels of ethylene, ethane, or methane in the absence of oxygen would have great utility.

SUMMARY

The present disclosure provides non-naturally occurring microbial organisms which are capable of producing ethylene, ethane, methane, or combinations thereof.

In one aspect, a non-naturally occurring microbial organism is provided comprising a nucleic acid encoding one or more genes of a methylthio-alkane reductase complex and one or more genes of a methionine salvage pathway.

In another aspect, a non-naturally occurring microbial organism is provided, wherein the organism is an anaerobic organism which produces ethylene, ethane, and/or methane using a methylthio-alkane reductase complex and a methionine salvage pathway, and wherein the organism has been optimized for producing ethylene, ethane, and/or methane with one or more non-naturally occurring genes.

In another aspect, a method of producing ethylene, ethane, and/or methane is provided, the method comprising:

culturing a population of the non-naturally occurring microbial organism described herein in a culture medium comprising one or more carbon sources; and

A bioreactor is further provided comprising the non-naturally occurring microbial organism described herein.

A vector is also provided comprising: one or more exogenous nucleic acid molecules encoding one or more genes of a methylthio-alkane reductase complex and one to or more genes of a methionine salvage pathway.

DETAILED DESCRIPTION

Methane is used for the production of energy, hydrogen gas, synthesis gas, and methanol used in the manufacturing of various organic chemicals. Methane is the second most used energy source next to electricity. Ethylene is used in a variety of industrial processes, including the production of polyethylene for plastic bags, polystyrene for packaging and insulation, and ethylene oxide for detergents. In addition, ethylene may be converted to C5-C10 gasoline-like molecules. Ethylene is thus thought to be the most widely used chemical on earth (over 175 million tons in 2018) and the demands and market for this feedstock are steadily increasing, with nearly a $300 billion annual market. Thus, there is considerable interest in developing new and innovative ways to produce these key industrial precursor compounds (ethylene, ethane, methane) with bio-based methods as a potential way to supplement chemical-based processes.

For anaerobic ethylene production by microorganisms, the novel and widespread bacterial carbon and sulfur salvage pathway, the DHAP Shunt (FIG.1A), converts the ubiquitous S-adenosyl-L-methionine byproduct, MTA, into adenine, DHAP, and the volatile organic sulfur compound, (2-methylthio)ethanol (MT-EtOH). This includes freshwater and soil bacteria such asRhodospirillum rubrumandRhodopseudomonas palustris,extra-intestinal pathogenicEscherichia coli,and pathogenicBacillusspecies (10, 25, 26, 67). It was demonstrated that the Alphaproteobacteria,R. rubrumandR. palustris,were able to further utilize MT-EtOH as a sole sulfitr source for growth and synthesis of sulfur-containing amino acids (e.g. methionine), producing stoichiometric amounts of ethylene gas in the process (10). This process was strictly anaerobic and clearly enzymatic in nature (10). This was the first reported solely anaerobic route to ethylene, and involves a novel cooperation of genes and enzymes (MarBHDK). It was subsequently found that the enzyme system producing ethylene from MT-EtOH (MarBHDK) was a member of the nitrogenase family of enzymes from a novel and distinct Glade (FIG.4andFIG.16). This strictly anaerobic methylthio-alkane reductase system not only could product ethylene form MT-EtOH, but it could also produce ethane from ethylmethylsulfide (CH3—S—CH2—CH3) and methane from dimethylsulfide (CH3—S—CH3). This was verified in alphaproteobacteria, includingRhodopseudomonas plaustris, Rhodospirillum rubrum,andBlastochloris viridis.A search of the available database for other organisms that possess the same set of discovered genes encoding nitrogenase-like methylthio-alkane reductase enzymes for reactions for ethylene, ethane, and methane formation indicated that this enzyme was prevalent in genomes from multiple phyla of industrially relevant Proteobacteria and Firmicutes. It was also found that these genes were detected in anoxic high carbon ecosystems including wetland soils and animal rumen. Notably, expressed proteins for methylthio-alkane reductase were recovered in situ, supporting the ability to use a functional screen to potentially recover catalytically active enzymes from the environment.

Disclosed herein is an exclusively anaerobic enzyme system and associated pathways that couples sulfur metabolism to ethylene and methane production in the purple non-sulfur alpha-proteobacteria.Rhodospirillum rubrum, Rhodopseudomonas palustris,andBlastochloris viridis(FIGS.1A-1C). Genes for this anaerobic enzyme system are widely distributed amongst bacteria (FIG.17), and this pathway reveals a possible route by which ethylene and methane, both of which are frequently observed in anoxic environments, can be produced by indigenous microbes.

Disclosed herein are methods for the development of a potential industrially compatible process to biologically produce ethylene and methane in high yields. Disclosed herein is a method to fully characterize the anaerobic ethylenelethane/methane producing enzyme system and determine how the genes are regulated at the molecular level. Computational modeling of the chemical reactions performed by the relevant enzymes are initiated to learn the mechanisms by which these enzymes catalyze the reactions involved in ethylene biosynthesis. In addition, since ethylene/ethane/methane synthesis from the respective precursor compound is an inducible process, further studies probe the molecular regulation of the genes involved during photosynthetic metabolism using a variety of “omics” tools. These biochemical and molecular studies are invaluable for optimizing ethylene/ethane/methane production and creating bacterial strains that over-produce ethylene/ethane/methane under controlled conditions.

Also disclosed herein is a method to maximize ethylene and methane production with different feedstocks; e.g., lignocellulose digests as well as inorganic carbon sources (FIGS.20A-20D). Rps. palustris, as well as cellulolytic and acetogenic bacteria such asRuminiclostridium josuiandClostridium ljungdhaliispecies all contain the genes for the ethylene/ethane/methane producing enzyme system MarBHDK (FIG.17), and each of these organisms has the capacity to grow on cellulosic digests as well as inorganic carbon sources (CO2). Conditions are optimized for each of these growth conditions.

Further disclosed are metagenomics and bioinformatic/computational approaches to discover more effective enzymes of uncultured organisms from anaerobic environments. Analysis of existing genome and metagenome databases allow identification of potential gene sequences for ethylene/ethane/methane producing enzymes systems that have specific or enhanced catalytic properties. Such sequences, homologous to known genes, may then be screened for their effectiveness in catalyzing key reactions of ethylene/ethane/methane synthesis. This leverages over 4 billion years of evolution to obtain the most efficient enzymes. In addition, a functional genomics approach may be established to isolate relevant genes from the metagenome without previous knowledge of sequences; e.g., by complementing specific mutant host organisms with environmental DNA (68). These metagenomics approaches, plus a full battery of other synthetic biology and “omics” approaches is utilized to optimize ethylene/ethane/methane formation.

Definitions

The term “culture”, “cultivate”, and “ferment” are used interchangeably and refer to the intentional growth, propagation, proliferation, and/or enablement of metabolism, catabolism, and/or anabolism of one or more cells (e.g. a microbial organism). The combination of both growth and propagation may be termed proliferation, Examples include production by an organism of ethylene, ethane, or methane. Culture does not refer to the growth or propagation of microorganisms in nature or otherwise without human intervention.

The term “growth” means an increase in cell size, total cellular contents, and/or cell mass or weight of a cell (e.g. a microbial organism).

A “growth media” or “growth medium” as used herein can be a solid, powder, or liquid mixture which comprises all or substantially all of the nutrients necessary to support the growth of microbial organisms; various nutrient compositions are preferably prepared when particular microbial species are being assayed. Amino acids, carbohydrates, minerals, vitamins and other elements known to those skilled in the art to be necessary for the growth of microbial organisms are provided in the medium. In one embodiment, the growth medium is liquid.

The term “propagation” refers to an increase in cell number via cell division.

The term “promoter” or “regulatory element” refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of origin in the microbial organism used, for example, promoters derived from viruses or from other organisms can be used in the compositions or methods described herein,

A polynucleotide sequence is “heterologous” to a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified by human action from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from naturally occurring allelic variants.

The term “recombinant” refers to a human manipulated nucleic acid (e.g. polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g. polynucleotide), or if in reference to a protein (i.e, a “recombinant protein”), a protein encoded by a recombinant nucleic acid (e.g. polynucleotide). In embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al.,Molecular Cloning—A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may comprise nucleic acids (e.g. polynucleotides) combined in such a way that the nucleic acids (e.g. polynucleotides) are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second nucleic acid (e.g. polynucleotide).

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together. The term “nucleic acid” includes single-, double-, or multiple-stranded DNA, RNA and analogs (derivatives) thereof. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids and polynucleotides are polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. In certain embodiments, the nucleic acids herein contain phosphodiester bonds. In other embodiments, nucleic acid analogs are included that may have alternate backbones. The term encompasses nucleic acids containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. A particular nucleic acid sequence also encompasses “splice variants.” Similarly, a particular protein encoded by a nucleic acid encompasses any protein encoded by a splice variant of that nucleic acid. “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. An example of splice variants is discussed in Leicher, et al.,J. Biol. Chem.273 (52):35095-35101 (1998).

The term “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively. In some embodiments, an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al.,Molecular Cloning—A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In some embodiments, an expression cassette comprising a terminator (or termination sequence) operably linked to a second nucleic acid (e.g. polynucleotide) may include a terminator that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation. In some embodiments, the expression cassette comprises a promoter operably linked to a second nucleic acid (e.g. polynucleotide) and a terminator operably linked to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation. In some embodiments, the expression cassette comprises an endogenous promoter. In some embodiments, the expression cassette comprises an endogenous terminator. In some embodiments, the expression cassette comprises a synthetic (or non-natural) promoter. In some embodiments, the expression cassette comprises a synthetic (or non-natural) terminator.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

The phrase “codon optimized” as it refers to genes or coding regions of nucleic acid molecules for the transformation of various hosts, refers to the alteration of codons in the gene or coding regions of polynucleic acid molecules to reflect the typical codon usage of a selected organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that selected organism.

The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence with a higher affinity, e.g., under more stringent conditions, than to other nucleotide sequences (e.g., total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen,Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH The Tmis the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology,ed. Ausubel, et al. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Polypeptides which are “substantially similar” share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

The term “modulator” refers to a composition that increases or decreases the level of a target molecule or the level of activity or function of a target molecule or the physical state of the target of the molecule. In embodiments a modulator is a recombinant nucleic acid that is capable of increasing or decreasing the amount of a protein in a cell or the level of activity of a protein in a cell or transcription of a second nucleic acid in a cell. In embodiments, a modulator increases or decreases the level of activity of a protein or the amount of the protein in a cell. The term “modulate” is used in accordance with its plain and ordinary meaning and refers to the act of changing or varying one or more properties. “Modulation” refers to the process of changing or varying one or more properties. For example, as applied to the effects of a modulator on a target protein, to modulate means to change by increasing or decreasing a property or function of the target molecule or the amount of the target molecule. In embodiments, a recombinant nucleic acid that modulates the level of activity of a protein may increase the activity or amount of the protein relative the absence of the recombinant nucleic acid. In embodiments, an increase in the activity or amount of a protein may include overexpression of the protein. “Overexpression” is used in accordance with its plain and ordinary meaning and refers to an increased level of expression of a protein relative to a control (e.g. cell or expression system not including a recombinant nucleic acid that contributes to the overexpression of a protein). In embodiments, a decrease in the activity or amount of a protein may include a mutation (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid; all/any of which may be in the coding region for a protein or in an operably linked region (e.g, promoter)) of the protein. The term “increased” refers to a detectable increase compared to a control.

A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g. enhancers and coding sequences) do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. In embodiments, a promoter is operably linked with a coding sequence when it is capable of affecting (e.g. modulating relative to the absence of the promoter) the expression of a protein from that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

Transformation” refers to the transfer of a nucleic acid molecule into a host organism (e.g. a microbial organism). In embodiments, the nucleic acid molecule may be a plasmid that replicates autonomously or it may integrate into the genome of the host organism (e.g. a microbial organism). Host organisms containing the transformed nucleic acid molecule may be referred to as “transgenic” or “recombinant” or “transformed” organisms (e.g. microbial organisms). A “genetically modified” organism (e.g. genetically modified microbial organism) is an organism (e.g. microbial organism) that includes a nucleic acid that has been modified by human intervention. Examples of a nucleic acid that has been modified by human intervention include, but are not limited to, insertions, deletions, mutations, expression nucleic acid constructs (e.g. over-expression or expression from a non-natural promoter or control sequence or an operably linked promoter and gene nucleic acid distinct from a naturally occurring promoter and gene nucleic acid in an organism), extra-chromosomal nucleic acids, and genomically contained modified nucleic acids. Genetically modified organisms may be made by rational modification of a nucleic acid or may be made by use of a mutagen or mutagenesis protocol that results in a mutation that was not identified (e.g. intended or targeted) prior to the use of the mutagen or mutagenesis protocol (e.g. UV exposure, EMS exposure, mutagen exposure, random genomic mutagenesis, transformation of a library of different nucleic acid constructs). Genetically modified organisms that include a modification (e.g. modification, insertion, deletion, mutation) not previously known or intended prior to making of the genetically modified organism may be identified through screening a plurality of organism including one or more genetically modified organisms by using a selection criteria that identifies the genetically modified organism of interest. In embodiments, a genetically modified organism includes a recombinant nucleic acid.

As used herein, the term “episome” or “episomally” is intended to refer to an extrachromosomal DNA moiety or plasmid that can replicate autonomously in a host cell when physically separated from the chromosomal DNA of the host cell.

Methods for synthesizing sequences and bringing sequences together are well established and known to those of skill in the art. For example, in vitro mutagenesis and selection, site-directed mutagenesis, error prone PCR (Melnikov et al., Nucleic Acids Research, 27 (4)1056-1062 (Feb. 15, 1999)), “gene shuffling” or other means can be employed to obtain mutations of naturally occurring genes.

Compositions

Microbial Organisms

The present disclosure provides non-naturally occurring microbial organisms which are capable of producing ethylene, ethane, methane, or combinations thereof. some aspects, the microbial organism has been genetically modified with one or more genes directed to the production of ethylene, ethane, methane, or combinations thereof. In other aspects, the microbial organism may naturally produce ethylene, ethane, methane, or combinations thereof, but has been optimized for said production by the introduction of one or more non-naturally occurring genes.

Thus, in one aspect, a non-naturally occurring microbial organism is provided comprising a nucleic acid encoding one or more genes of a methylthio-alkane reductase complex and one or more genes of a methionine salvage pathway.

In some embodiments, the organism can produce ethylene, ethane, methane, or combinations thereof, In some embodiments, the organism produces ethylene, In some embodiments, the organism produces ethane. In some embodiments, the organism produces methane.

In another aspect, a non-naturally occurring microbial organism is provided, wherein the organism is an anaerobic organism which produces ethylene, ethane, and/or methane using a methylthio-alkane reductase complex and a methionine salvage pathway, and wherein the organism has been optimized for producing ethylene, ethane, and/or methane with one or more non-naturally occurring genes. In some embodiments, the one or more non-naturally occurring genes comprise one or more genes of a SAM hydrolase. In some embodiments, the one or more non-naturally occurring genes comprise one or more genes of a methanethiol methylase (mddik), a methionine gamma lyase (mgt), or combinations thereof.

In some embodiments, the one or more genes of a methylthio-alkane reductase complex may comprise marB, marH, marD, marK, or combinations thereof.

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise marB. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the nucleic acid sequence of SEQ ID NO: 1 (marB).

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 1 (marB). In some embodiments, the one or more genese of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID No: 1.

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the peptide sequence of SEQ ID NO: 2 (MarB).

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the peptide sequence of SEQ ID NO: 2. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein of SEQ ID NO: 2. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

In some embodiments, the one or more genes of a methyltbio-alkane reductase complex comprise marH. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the nucleic acid sequence of SEQ ID NO: 3 (marH).

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the one or more genese of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 3.

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise one or more marll genes associated with an accession number found in Table 1 below:

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the peptide sequence of SEQ ID NO: 4 (MarH).

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the peptide sequence of SEQ ID NO: 4. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein of SEQ ID NO: 4. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise marD. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the nucleic acid sequence of SEQ ID NO: 5 (marD).

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 5.

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise one or more marD genes associated with an accession number found in Table 2 below:

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the peptide sequence of SEQ ID NO: 6 (MarD).

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the peptide sequence of SEQ ID NO: 6. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein of SEQ ID NO: 6. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

In some embodiments, the one or more genes of a methyltbio-alkane reductase complex comprise marK. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the nucleic acid sequence of SEQ ID NO: 7 (marK).

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 7.

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise one or more marK genes associated with an accession number found in Table 3 below:

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the peptide sequence of SEQ ID NO: 8 (MarK).

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the peptide sequence of SEQ ID NO: 8. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein of SEQ ID NO: 8. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

The art is familiar with the methods and techniques used to identify other methylthio-alkane reductase genes and nucleotide sequences.

Methionine Salvage Pathways

In some embodiments, the one or more genes of a methionine salvage pathway comprise one or more genes of a dihydroxyacetone phosphate (DHAP) shunt pathway. In some embodiments, the one or more genes of a DHAP shunt pathway comprise 5′-methylthioadenosine phosphorylase (mtnP), methylthioadenosine nucleosidase (mtn1), 5-methylthioribose kinase (mtnK), 5-methylthioribose-1-phosphate isomerase (mtnA), 5-methylthioribulose-1-phosphate aldolase (ald2), or combinations thereof.

In some embodiments, the one or more genes of a methionine salvage pathway comprises mtnP. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid). In some embodiments, the one or more genes of a methionine salvage pathway comprises an mtnP gene associated with an accession number found in Table 4 below:

The art, is familiar with the methods and techniques used to identify other 5′-methylthioadenosine phosphorylase genes and nucleotide sequences.

In some embodiments, the one or more genes of a methionine salvage pathway comprises mtnK. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid). In some embodiments, the one or more genes of a methionine salvage pathway comprises an mtnK gene associated with an accession number found in Table 5 below:

In some embodiments, the one or more genes of a methionine salvage pathway comprises mtnA. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid). In some embodiments, the one or more genes of a methionine salvage pathway comprises an mtnA gene associated with an accession number found in Table 6 below:

In some embodiments, the one or more genes of a methionine salvage pathway comprises ald2. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid). In some embodiments, the one or more genes of a methionine salvage pathway comprises an ald2 gene associated with an accession number found in Table 7 below:

Additional Genes

In some embodiments, the nucleic acid may encode one or more genes of a SAM hydrolase. In some embodiments, the one or more genes of a SAM hydrolase may be a non-naturally occurring, or exogenous, gene. In some embodiments, the SAM hydrolase may be derived from a coliphage virus. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

The art is familiar with the methods and techniques used to identify other SAM hydrolase genes and nucleotide sequences.

In some embodiments, the nucleic acid may encode one or more genes of a methanethiol methylase (mddA), a methionine gamma lyase (mgl), or combinations thereof. In some embodiments, the one or more genes of mddA, mgi, or combinations thereof, may be a non-naturally occurring, or exogenous, gene. In some embodiments, the one or more genes of mddA and/or mgl are derived fromRhodopseudomonal palsutris.In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

The art is familiar with the methods and techniques used to identify other methanethiol methylase and/or methionine gamma lyase genes and nucleotide sequences.

In some embodiments, the nucleic acid may be codon optimized. In some embodiments, the one or more may be optionally and independently linked to a control element. In some embodiments, the control element comprises a promoter.

Vectors

In another aspect, vectors are provided comprising one or more exogenous nucleic acid molecules encoding one or more genes of a methylthio-alkane reductase complex and one or more genes of a methionine salvage pathway. Vectors are also provided for use in the methods disclosed herein. For example, one or more of the vectors disclosed herein can be used to transform a microbial organism. Microbial organisms are also described transformed with or comprising one or more of the vectors described herein.

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex may comprise marB, marH, marD, marK, or combinations thereof.

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise marB. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the nucleic acid sequence of SEQ ID NO: 1 (marB).

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 1 (marB). In some embodiments, the one or more genese of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 1.

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the peptide sequence of SEQ ID NO: 2 (MarB).

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the peptide sequence of SEQ ID NO: 2. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein of SEQ ID NO: 2. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise marH. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the nucleic acid sequence of SEQ ID NO: 3 (marH).

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the one or more genese of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 3.

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise one or more marH genes associated with an accession number found in Table 1.

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the peptide sequence of SEQ ID NO: 4 (MarH).

In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the peptide sequence of SEQ ID NO: 4. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein of SEQ ID NO: 4. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise marD. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the nucleic acid sequence of SEQ ID NO: 5 (marD).

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID No: 5.

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise one or more marD genes associated with an accession number found in Table 2.

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the peptide sequence of SEQ ID NO: 6 (MarD).

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the peptide sequence of SEQ ID NO: 6. In some embodiments, the one or more genes of a inethylthio-alkane reductase complex comprise a gene encoding a protein of SEQ ID NO: 6. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise marK. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence haying at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the nucleic acid sequence of SEQ ID NO: 7 (marK).

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 7.

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise one or more marK genes associated with an accession number found in Table 3.

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein haying at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the peptide sequence of SEQ ID NO: 8 (MarK).

In some embodiments of the vectors described herein, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the peptide sequence of SEQ ID NO: 8. In some embodiments, the one or more genes of a methylthio-alkane reductase complex comprise a gene encoding a protein of SEQ ID NO: 8. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

In some embodiments of the vectors described herein, the one or more genes of a methionine salvage pathway comprise one or more genes of a dihydroxyacetone phosphate (DHAP) shunt pathway. In some embodiments, the one or more genes of a DHAP shunt pathway comprise 5′-methylthioadenosine phosphorylase (mtnP), 5-methylthioribose kinase (mtnK), 5-methylthioribose-1-phosphate isomerase (mtnA), 5-methylthioribulose-1-phosphate aldolase (ald2), or combinations thereof.

In some embodiments of the vectors described herein, the one or more genes of a methionine salvage pathway comprises mtnP. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid). In some embodiments, the one or more genes of a methionine salvage pathway comprises an mtnP gene associated with an accession number found in Table 4.

In some embodiments of the vectors described herein, the one or more genes of a methionine salvage pathway comprises mtnl. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

In some embodiments of the vectors described herein, the one or more genes of a methionine salvage pathway comprises mtnK. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid). In some embodiments, the one or more genes of a methionine salvage pathway comprises an mtnK, gene associated with an accession number found in Table 5.

In some embodiments of the vectors described herein, the one or more genes of a methionine salvage pathway comprises mtnA. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid). In some embodiments, the one or more genes of a methionine salvage pathway comprises an mtnA gene associated with an accession number found in Table 6.

In some embodiments of the vectors described herein, the one or more genes of a methionine salvage pathway comprises ald2. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid). In some embodiments, the one or more genes of a methionine salvage pathway comprises an ald2 gene associated with an accession number found in Table 7.

In some embodiments of the vectors described herein, the exogenous nucleic acid molecules may further encode one or more genes of a SAM hydrolase. In some embodiments, the one or more genes of a SAM hydrolase may be a non-naturally occurring, or exogenous, gene. In some embodiments, the SAM hydrolase may be derived from a coliphage virus. In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).

In some embodiments of the vectors described herein, the exogenous nucleic acid molecules may encode one or more genes of a methanethiol methylase (mddA), a methionine gamma lyase (mgl), or combinations thereof. In some embodiments, the one or more genes of mddA, mgl, or combinations thereof, may be a non-naturally occurring, or exogenous, gene. In some embodiments, the one or more genes of mddA and/or mgl are derived fromRhodopseudomonal palsutris.In some embodiments, the gene is a wildtype version of the gene or encodes a wildtype form of the associated protein. In some embodiments, the gene is a mutant form of the gene or may encode a mutant form of the associated protein (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid),

In some embodiments the one or more exogenous nucleic acid molecules are integrated into a gene expression cassette. In some embodiments, the gene expression cassette comprises one or more control elements. In some embodiments, the one or more exogenous nucleic acid molecules disclosed herein are operably linked to a control element. In some embodiments, the control element is a promoter. In some embodiments, the promoter may be constitutively active or inducibly active. In some embodiments, the promoter is constitutively active regardless of sulfate concentration, i.e., sulfate limitation is not required in order to induce expression of the gens found in the one or more exogenous nucleic acid molecules.

In some embodiments, the promoter comprises a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the sequence of SEQ ID NO: 9:

In some embodiments, the promoter comprises a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the promoter comprises a nucleic acid sequence of SEQ ID NO: 9.

In some embodiments, the promoter comprises a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more identity to the sequence of SEQ ID NO: 10:

In some embodiments, the promoter comprises a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the promoter comprises a nucleic acid sequence of SEQ ID NO: 10.

In another aspect, a non-naturally occurring organism is provided comprising a vector described herein.

Methods of Use

In another aspect, methods of producing ethylene, ethane, and/or methane are provided comprising:

culturing a population of the non-naturally occurring microbial organism described herein in a culture medium comprising one or more carbon sources; and

In some embodiments, the methods described herein may be used in the production of ethylene. In some embodiments, the methods described herein may be used in the production of ethane. In some embodiments, the methods described herein may be used in the production of methane.

The term “carbon source” means a carbon source that a microbial organism described herein will metabolize to derive energy (e.g. monosaccharides, oligosaccharides, polysaccharides, alkanes, fatty acids, esters of fatty acids, monoglycerides, acetate, carbon dioxide, methanol, formaldehyde, formate or carbon-containing amines). The term “carbon source” refers to a carbon containing composition (e.g. compound, mixture of compounds) that an organism may metabolize for use by the organism or that may be used for organism viability. A “majority carbon source” refers to a carbon containing composition that accounts for greater than 50% of the available carbon sources for an organism (e.g. in a media, in a growth media, in a defined media for the organism, or in a defined media for producing ethylene, ethane, and/or methane by an organism) at a specified time (e.g. media when starting a culture, media in a bioreactor when growing the organism, or media when producing ethylene, ethane, and/or methane from the organism). In embodiments, an organism may be cultured using a medium comprising a majority carbon source selected from the group consisting of glucose, glycerol, xylose, fructose, mannose, ribose, sucrose, and lignocellusic biomass. In embodiments, an organism may be cultured using a medium comprising one or more carbon sources selected from the group consisting of glucose, fructose, sucrose, lactose, galactose, xylose, mannose, rhamnose, arabinose, glycerol, acetate, depolymerized sugar beet pulp, black liquor, corn starch, depolymerized cellulosic material, corn stover, sugar beet pulp, switchgrass, milk whey, molasses, potato, rice, sorghum, sugar cane, wheat, and mixtures thereof (e.g. mixtures of glycerol and glucose, mixtures of glucose and xylose, mixtures of fructose and glucose, mixtures of sucrose and depolymerized sugar beet pulp, black liquor, corn starch, depolymerized cellulosic material, corn stover, sugar beet pulp, switchgrass, milk whey, molasses, potato, rice, sorghum, sugar cane, and/or wheat). In some embodiments, an organism is cultured using a medium comprising one or more carbon sources selected from the group consisting of depolymerized sugar beet pulp, black liquor, corn starch, depolymerized cellulosic material, corn stover, sugar beet pulp, switchgrass, milk whey, molasses, potato, rice, sorghum, sugar cane, thick cane juice, sugar beet juice, and wheat. In some embodiments, an organism is cultured using a medium comprising lignocellulosic biomass. In some embodiments, carbon sources may be monosaccharides (e.g., glucose, fructose), disaccharides (e.g., lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch, cellulose or mixtures thereof), sugar alcohols (e.g., glycerol) or mixtures from renewable feedstocks (e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses, or barley malt). Additionally, carbon sources may include alkanes, thtty acids, esters of fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, various commercial sources of fatty acids including vegetable oils (e.g., soybean oil) or animal fats. In some embodiments, the culture medium may contain, in addition to the primary (or majority) carbon source, one or more secondary carbon sources. In some embodiments, the secondary carbon source comprises lignin or lignin derived aromatic compounds. In some embodiments, the secondary carbon source comprises lignin breakdown products.

In some embodiments, the one or more carbon sources may comprise biomass, for example lignocellulosic biomass. The term “biomass” refers to material produced by growth and/or propagation of cells. “Lignocellulosic biomass” is used according to it plain and ordinary meaning and refers to plant dry matter comprising carbohydrate (e.g. cellulose or hemicellulose) and polymer (e.g. lignin). Lignocellulosic biomass may include agricultural residues (e.g. corn stover or sugarcane bagasse), energy crops (e.g. poplar trees, willow,Miscanthus purpureum, Pennisetum purpureum,elephant grass, maize, Sudan grass, millet, white sweet clover, rapeseed, giant miscanthus, switchgrass, jatropha,Miscanthus giganteus,or sugarcane), wood residues (e.g. sawmill or papermill discard), or municipal paper waste.

In some embodiments, the one or more carbon sources may be selected from one or more in combination of: carbon dioxide and carbon monoxide, mono and disaccharide sugars, organic acids (for example, malate, succinate, pyruvate, and fumarate), volatile fatty acids (for example, formate, acetate, propionate, and butyrate), alcohols (for example, ethanol and glycerol), and cellulosic plant biomass including but not limited to corn stover, miscanthus, switchgrass.

A “growth media” or “growth medium” as used herein can be a solid, powder, or liquid mixture which comprises all or substantially all of the nutrients necessary to support the growth of an organism; various nutrient compositions are preferably prepared when particular species are being assayed. Amino acids, carbohydrates, minerals, vitamins and other elements known to those skilled in the art to be necessary for the growth of microbial organisms are provided in the medium. In one embodiment, the growth medium is liquid. In one embodiment, the growth medium is a production medium (for example, medium optionally containing higher concentrations of glucose and/or altered concentrations of nitrogen).

In some embodiments, the growth media is sufficiently deficient in or absent of sulfate.

In another aspect, a bioreactor is provided comprising a non-naturally occurring organism as described herein. Such bioreactors may be used in the methods described herein.

EMBODIMENTS

Further embodiments of the present disclosure are provided as follows:Embodiment 1: a non-naturally occurring microbial organism comprising a nucleic acid encoding one or more genes of a methylthio-alkane reductase complex and one or more genes of a methionine salvage pathway.Embodiments 2: a non-naturally occurring microbial organism of embodiment 1, wherein the organism produces ethylene, ethane, methane, or combinations thereof.Embodiment 3: the non-naturally occurring microbial organism of embodiment 2, wherein the organism produces ethylene.Embodiment 4: the non-naturally occurring microbial organism of embodiment 2, wherein the organism produces ethane.Embodiment 5: the non-naturally occurring microbial organism of embodiment 2, wherein the organism produces methane.Embodiment 6: the non-naturally occurring microbial organism of any one of embodiments 1-5, wherein the one or more genes of a methylthio-alkane reductase complex comprise marB, marD, marK, or combinations thereof.Embodiment 7: the non-naturally occurring microbial organism of any one of embodiments 1-6, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 1.Embodiment 8: the non-naturally occurring microbial organism of embodiment 7, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 1.Embodiment 9: the non-naturally occurring microbial organism of any one of embodiments 1-8, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 3.Embodiment 10: the non-naturally occurring microbial organism of embodiment 9, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 3.Embodiment 11: the non-naturally occurring microbial organism of any one of embodiments 1-10, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 5.Embodiment 12: the non-naturally occurring microbial organism of embodiment 11, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 5.Embodiment 13: the non-naturally occurring microbial organism of any one of embodiments 1-12, wherein the one or more genes of a methylthio-alkane reductase comprise a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 7.Embodiment 14: the non-naturally occurring microbial organism of embodiment 13, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 7.Embodiment 15: the non-naturally occurring organism of any one of embodiments 1-14, wherein the one or more genes of a methionine salvage pathway comprise one or more genes of a dihydroxyacetone phosphate (DHAP) shunt pathway.Embodiment 16: the non-naturally occurring organism of embodiment 15, wherein the one or more genes of a DHAP shunt pathway comprise 5′-methylthioadenosine phosphorylase (mtnP), methylthioadenosine nucleosidase (mtn1), 5-methylthioribose kinase (mtnK), 5-methylthioribose-1-phosphate isomerase (mtnA), 5-methylthioribulose-1-phosphate aldolase (ald2), or combinations thereof.Embodiment 17: the non-naturally occurring organism of embodiment 16, wherein the one or more genes of a DHAP shunt pathway comprise mtnP.Embodiment 18: the non-naturally occurring organism of embodiment 16, wherein the one or more genes of a DHAP shunt pathway comprise intni and mtnK.Embodiment 19: the non-naturally occurring organism of any one of embodiments 16-18, wherein the one or more genes of a DHAP shunt pathway comprise mtnA.Embodiment 20: the non-naturally occurring organism of any one of embodiments 16-19, wherein the one or more genes of a DHAP shunt pathway comprise ald2.Embodiment 21: the non-naturally occurring microbial organism of any one of embodiments 1-20, wherein the nucleic acid further encodes one or more genes of a SAM hydrolase.Embodiment 22: the non-naturally occurring microbial organism of any one of embodiments 1-10, wherein the nucleic acid further encodes one or more genes of a methanethiol methylase (mddA), a methionine gamma lyase, or combinations thereof.Embodiment 23: the non-naturally occurring microbial organism of any one of embodiments 1-22, wherein the nucleic acid is codon optimized.Embodiment 24: the non-naturally occurring microbial organism of any one of embodiments 1-23, wherein the nucleic acid is integrated into the genome of the organism.Embodiment 25: the non-naturally occurring microbial organism of any one of embodiments 1-23, wherein the nucleic acid is episomally integrated into a plasmid.Embodiment 26: a non-naturally occurring microbial organism, wherein the organism is an anaerobic organism which produces ethylene, ethane, and/or methane using a methylthio-alkane reductase complex and a methionine salvage pathway, and wherein the organism has been optimized for producing ethylene, ethane, and/or methane with one or more non-naturally occurring genes.Embodiment 27: the non-naturally occurring microbial organism of embodiment 26, wherein the one or more non-naturally occurring genes comprise one or more genes of a SAM hydrolase.Embodiment 28: the non-naturally occurring microbial organism of embodiment 26, wherein the one or more non-naturally occurring genes comprise one or more genes of a methanethiol methylase (mddA), a methionine gamma lyase (mgl), or combinations thereof.Embodiment 29: the non-naturally occurring microbial organism of any one of embodiments 26-28, wherein the one or more non-naturally occurring genes are integrated into the genome of the organism.Embodiment 30: the non-naturally occurring microbial organism of any one of embodiments 26-28, wherein the one or more non-naturally occurring genes are episomally expressed from a plasmic.Embodiment 31: the non-naturally occurring microbial organism of any one of embodiments 26-30, wherein the one or more non-naturally occurring genes are codon optimized.Embodiment 32: a method of producing ethylene, ethane, and/or methane comprising:culturing a population of the non-naturally occurring microbial organism of any one of embodiments 1-31 in a culture medium comprising one or more carbon sources; andrecovering the ethylene, ethane, and/or methane.Embodiment 33: the method of embodiment 32, wherein the one or more carbon sources comprise carbon dioxide, carbon monoxide, an organic acid, a volatile fatty acid, an alcohol, cellulosic plant mass, or combinations thereof.Embodiment 34: the method of embodiment 32 or 33, wherein the one or more carbon sources comprise carbon dioxide, carbon monoxide, malate, succinate, pyruvate, fumarate, formate, acetate, propionate, butyrate, ethanol, glycerol, corn stover, miscanthus, or switchgrass.Embodiment 35: the method of any one of embodiments 32-34, wherein the one or more carbon sources comprise corn stover.Embodiment 36: the method of embodiment 32, wherein the one or more carbon sources comprise lignoceliulosic biomass.Embodiment 3: the method of any one of embodiments 32-36, wherein the population is cultured in the absence of sulfate.Embodiment 38: a bioreactor comprising the non-naturally occurring microbial organism of any one of embodiments 1-31.

Embodiment 39: a vector comprising: one or more exogenous nucleic acid molecules encoding one or more genes of a methylthio-alkane reductase complex and one or more genes of a methionine salvage pathway.Embodiment 40: the vector of embodiment 39, wherein the one or more genes of a methylthio-alkane reductase complex comprise marB, marH, marD, marK, or combinations thereof.Embodiment 41: the vector of embodiment 39 or embodiment 40, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 1.Embodiment 42: the vector of embodiment 41, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 1.Embodiment 43: the vector of any one of embodiments 39-42, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 3.Embodiment 44: the vector of embodiment 43, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 3.Embodiment 45: the vector of any one of embodiments 39-44, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 5.Embodiment 46: the vector of embodiment 43, wherein the one or more genes of a methylthio-alkane reductase complex comprise a nucleic acid sequence of SEQ ID NO: 5.Embodiment 47: the vector of any one of embodiments 39-46, wherein the one or more genes of a methylthio-alkane reductase comprise a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 7.Embodiment 48: the vector of embodiment 47, wherein the one or more genes of a methylthio-alkane reductase comprise a nucleic acid sequence of SEQ ID NO: 7.Embodiment 49: the vector of any one of embodiments 39-48, wherein the one or more genes of a methionine salvage pathway comprise one or more genes of a dihydroxyacetone phosphate (DHAP) shunt pathway.Embodiment 50: the vector of embodiment 49, wherein the one or more genes of a DHAP shunt pathway comprise 5′-methylthioadenosine phosphorylase (mtnP), 5-methylthioribose kinase (mtnK) 5-methylthioribose-1-phosphate isomerase (mtnA), 5-methylthioribulose-1-phosphate aldolase (ald2), alcohol dehydrogenase (adh), or combinations thereof.Embodiment 51: the vector of embodiment 50, wherein the one or more genes of a DHAP shunt pathway comprise mtnP.Embodiment 52: the vector of embodiment 50, wherein the one or more genes of a DHAP shunt pathway comprise mtn1 and mtnK.Embodiment 53: the vector of any one of embodiments 50-52, wherein the one or more genes of a DHAP shunt pathway comprise mtnA.Embodiment 54: the vector of any one of embodiments 50-53, wherein the one or more genes of a DHAP shunt pathway comprise ald2.Embodiment 55: the vector of any one of embodiments 39-54, wherein the one or more exogenous nucleic acid molecules further encode one or more genes of a SAM hydrolase.Embodiment 56: the vector of any one of embodiments 39-55, wherein the one or more exogenous nucleic acid molecules further encode one or more genes of a methanethiol methylase (mddA), a methionine gamma lyase (mgl), or combinations thereof.Embodiment 57: the vector of any one of embodiments 39-56, wherein the one or more genes are integrated into a gene expression cassette.Embodiment 58: the vector of embodiment 57, wherein the gene expression cassette comprises a promoter.Embodiment 59: the vector of embodiment 58, wherein the promoter comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 9.Embodiment 60: the vector of embodiment 59, wherein the promoter comprises a nucleic acid sequence of SEQ ID NO: 9.Embodiment 61: the vector of embodiment 58, wherein the promoter comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 10.Embodiment 62: the vector of embodiment 61, wherein the promoter comprises a nucleic acid sequence of SEQ ID NO: 10.Embodiment 63: the vector of any one of embodiments 39-62, wherein the one or more genes have been codon optimized.Embodiment 64: a non-naturally occurring organism comprising a vector of any one of embodiments 39-63.

EXAMPLES

Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.

A Nitrogenase-Like Enzyme System Catalyzes Methionine, Ethylene, Ethane, and Methane Biogenesis

R. rubrumwas grown under conditions for ethylene induction (50 μM limiting sulfate or 1 mM MT-EtOH as sole S-source) and ethylene repression (1 mM sulfate) (FIGS.5A-5C) (10). Proteomics differential abundance analysis identified multiple proteins that increased over 20-fold in proteomes from induced versus repressed cells (FIG.1B). Among these were enzymes involved in cysteine and methionine metabolism: homoserine/serine: O-acetyltransferase (CysE), O-acetyl-L-homoserine sulfhydrylase, cystathionine beta-synthase, and cystathionine gamma-lyase (FIGS.1A-1C, reactions 2, 3, 6, 7, respectively).

Several proteins previously identified as NFL sequences of unknown function (8,9) showed some of the highest increases in abundance under ethylene inducing conditions (FIG.1B, Rru_A0772-Rru_A0773 and Rru_A0793-Rru_A0796, seeFIG.6for gene organization). In addition, there was also a large increase in abundance of proteins likely involved in iron-sulfur cluster metabolism; NifS cysteine desulfurase and a putative Fe4—S4scaffold protein (FIG.1B, Rru_A1068-Rru_A1069). This appears analogous to theAzotobacter vinelandiiNifUS system for synthesis of nitrogenase-destined iron-sulfur clusters from cysteine (12). However, the precise iron-sulfur cluster assembly pathway inR. rubrumis unknown. The involvement of the nitrogenase-like system in ethylene production was further bolstered by theR. rubrumtransposon mutant strain WRdht-66B3, possessing an inactivated gene encoding a putative nitrogenase reductase-like iron protein (Rru_A0795;FIG.1B). This and other mutants identified in a random mutagenesis screen were unable to grow anaerobically in the presence of MT-EtOH as sole S-source but could still grow utilizing sulfate, indicatirig a defect in the ethylene-producing pathway (FIGS.7A-7D). Consistent with the Tn5 mutagenesis results, specific deletion of NFL gene cluster Rru_A0793-Rru_A0796 renderedR rubrumincapable of growth or production of ethylene above basal levels with MT-EtOH as sole S-source (FIGS.2A-BandFIG.8C). This result confirmed that the putative nitrogenase-like system encoded by NFL gene duster Rru_A0793-Rru_A0796 was essential for assimilating sulfur from MT-EtOH to produce ethylene and methionine.

Other biologically relevant volatile organic sulfur compounds (VOSCs) were then tested for utilization by this putative nitrogenase-like enzyme system (FIG.2A-BandFIG.9A). In addition to MT-EtOH, VOSC utilization with concomitant hydrocarbon production was specific to dimethyl sulfide (DMS), the most abundant environmental VOSC, and ethyl methyl sulfide (EMS) (FIG.2A-B). Analogous to MT-EtOH (10), use of DMS or EMS resulted in methane or ethane production, respectively, in a 1 to 1 stoichiometry (FIG.3A-B). Specific deletion of the other two NFL genes, Rru_A0772-Rru_A0773, did not affect growth or hydrocarbon production (FIG.2A-BandFIG.8B). Thus, we designateR. rubrumgenes Rru_A0793-Rru_A0796, previously identified as NFL genes nflBHDK of unknown function (8, 9), asmethylthio-alkane reductase genes, marBHDK. This is based on corresponding amino acid similarity toR. rubrummolybdenum nitrogenase gene products NifB (synthesis of the NifB-cofactor precursor to the nitrogenase catalytic cofactor), NifH (nitrogenase-reductase iron protein), NifD (nitrogenase catalytic subunit α), and NifK (nitrogenase catalytic subunit β) (FIG.10-FIG.13). NFL genes Rru_A0772-Rru_A0773 remain designated nflDK genes of unknown function (8, 9).

When allR. rubrumNFL genes were deleted (strain Δ0772:3/Δ0793:6) and specific gene combinations were re-introduced via expression from a plasmid, expression of marBHDK was necessary and sufficient to restore growth and hydrocarbon metabolism from VOSCs (FIG.2B-CandFIG.9B-C). The NFL genes of unknown function, nflDK, could not replace marDK in complementing for growth. Upon feeding cells expressing marBH and nflDK with VOSCs, ethylene and ethane production was poorly catalyzed at 3- to 4-fold above basal levels and no methane enhancement was observed (FIG.2B-CandFIG.9B-C). This revealed thatR. rubrumNflDK could only weakly catalyze methylthio-alkane reduction, indicating a different primary function. Given nflDK is expressed not just in the presence of MT-EtOH but also in response to general sulfate limitation (FIG.1B-C), NflDK may catalyze sulfur liberation from alternate albeit unknown compounds. Alternately, given gene proximity and amino acid similarity (40%) to MarDK, NflDK may serve as accessory proteins for MarDK assembly analogous to NifEN (14). NifEN arose evolutionarily by gene duplication of NifDK and contains considerable sequence homology (˜40%) to NifDK, including P-cluster and FeMo-cofactor coordination sites (8, 9, 12). While NifEN does not have nitrogenase and hydrogen formation activity, it still retains acetylene and azide reduction capabilities (66). TheR. rubrumNflDK, group IV nitrogenase-like proteins of unknown function (Rru_A0772-Rru_A0773 gene products) share 40% sequence identity with MarDK and are evolutionarily closer to MarDK than NifDK (FIG.4). Coordinately, the nfIDK genes are located near marBHDK analogous to the association of nifEN with nigBHDK (8,9). However, unlike NifEN, NflDK is entirely dispensable, and homologous nflDK sequences are not observed to be present and associated with marBRDK gene clusters in several other organisms (FIG.18).

These results demonstrated the requirement of the MarBHDK nitrogenase-like system for the anaerobic assimilation of sulfur from common environmental VOSCs such as DMS and MT-EtOH in order to support growth and methionine metabolism. Moreover, these observations revealed a previously unknown mechanism for the bacterial production of methane and ethylene.

The link between VOSC utilization and methionine synthesis via the marBHDK gene products was characterized by feeding experiments with (2-[methyl-C14]thio)ethanol. This enabled detection of the methylthio-moiety of MT-EtOH. Upon feeding the wild type strain, MT-EtOH was consumed. Labeled methanethiol (C14H3—SH) and methionine (methyl-C14) were concomitantly produced and observed at low levels (˜2% of MT-ETOH concentration) until MT-EtOH was depleted (FIG.2D). These low levels, like previously observed for methanethiol metabolism from 5′-methylthioadenosine inR. rubrum(12), are likely due to the flux of methanethiol to methionine and subsequent utilization thereof for protein synthesis and SAM-dependent processes (11). This is substantiated by C14incorporation from MT-EtOH into insoluble cell material (FIGS.14A-14B). Conversely, in the marBHDK deletion strain there was no detectable metabolism of MT-EtOH, and hence, no methanethiol or methionine produced (FIG.2EandFIGS.14A-14B). Given that ethylene, ethane, and methane are produced from MT-EtOH, EMS, and DMS, respectively, the observed methanethiol is consistent with C—S single bond reduction and methylthio-release from these substrates by the methylthio-alkane reductase (FIG.1A, reaction 1 andFIG.2F). Each process is thermodynamically favored for the substrates and products observed (FIG.2FandFIGS.15A-15B). The methanethiol along with O-acetyl-homoserine then serve as substrates for O-acetylhomoserine sulfhydrylase, which catalyzes the synthesis of methionine (FIG.1A, reaction 3) (13). This defines an anaerobic methylthio-alkane reductase methionine synthesis pathway and establishes the role of a nitrogenase-like enzyme system in sulfur metabolism (FIG.1A).

Native Expression of Methylthio-Alkane Reductase is Regulated by Sulfur Response

SalR—Sulfur metabolism evidently is the primary function of these nitrogenase-like methylthio-alkane reductases, as opposed to nitrogen fixation by nitrogenase.R. rubrumpossesses molybdenum nitrogenase (NifHDK), which is the default nitrogenase, and iron only nitrogenase (AnfHDGK) nitrogenase, which is synthesized in the absence of molybdenum (9). In in vivo activity assays, theR. rubrummolybdenum nitrogenase could not perform methylthio-alkane reduction, even under maximally inducing conditions, and vice versa (FIG.3D; glutamate as N-source and 50 μM sulfate). Indeed, nitrogenase and methylthio-alkane reductase activities were independent, separately regulated, and both systems could be expressed simultaneously (FIG.3D).R. rubrumnitrogenase gene expression (nifHDK) is regulated by the transcriptional regulator NifA in response to nitrogen availability (14). Methylthio-alkane reductase activity in the presence of 1 mM MT-EtOH or DMS was regulated by sulfate availability, with an EC50˜150 μM sulfate for 50% repression of activity (FIG.3C). Our random mutagenesis screen identified the specific regulatory gene in the vicinity of marBHDK (Rru_A0785;FIG.1B,FIG.6, andFIGS.7A-7D). We designate this LysR family regulator as SalR (sulfursalvageregulator). Inactivation of salR rendered strains incapable of growth or hydrocarbon production utilizing MT-EtOH, DMS and EMS as sole S-source (FIG.2A-BandFIG.8E; strain 0785::Tn5). Transcriptomics and differential expression analysis of the parent (WRdht) and salR deletion strain (0785::Tn5) growing under marBHDK inducing and repressing conditions revealed that marBHDK and the rest of the methylthio-alkane reductase methionine synthesis pathway are under transcriptional control of SalR (FIG.1C). Thus, when sufficient sulfur is available (>150 μM), expression appears repressed, but when sulfate becomes limiting, marBHDK and O-acetylhomoserine sulfhydrylase gene transcription is specifically upregulated via SalR to utilize VOSCs for methionine metabolism (FIG.1A; reactions 1 and 3). Therefore, as shown inFIG.2B, expression of marBHDK from a non-natural gene promoter DNA sequence enables synthesis of MarBHDK and concomitant ethylenelethanelmethane production without the native regulation imposed by sulfate-sensitive SalR.

Organisms With Methylthio-Alkane Reductase are Widespread in Nature Including Industrially Relevant Acetogenic and Lignocellulosic Clostridia

The nitrogenase superfamily is composed of the bona fide nitrogenase sequences (groups I-III) and nitrogen fixation-like sequences (NFL; groups IV-VI) (FIG.4) (9). Phylogenetic analysis places methylthio-alkane reductase homologues in their own clade within group IV, which we denote as group IVC (FIG.4andFIG.16). In contrast, theR. rubrumNflD protein resides in a separate clade with other NflD sequences of unknown function (FIG.4), consistent with the poor methylthio-alkane reductase activity exhibited by NflDK (FIG.2B). Bacteria possessing MarBHDK sequence homologs of this previously uncharacterized group IV-C clade include members of the Fibrobacter and Bacteriodetes phyla, Rhodospirillales and Rhizobiales within the Proteobacteria phylum, and Selenomonadales andClostridiumspecies within the Firmicutes phylum (FIG.17). To verify the phylogeny results for the Proteobacteria,Rhodopseudomonas palustrisandBlastochloris viridiswere tested, which possess group IV-C marBHDK homologues. Also tested was closely related speciesRhodobacter capsulatus,which possesses nitrogenase and nflBHDK but no marBHDK (FIG.4,FIG.16, andFIG.18; Rp, Bv, Rc). BothR. palustrisandB. viridiswere able to grow with MT-EtOH, EMS, or DMS as sole sulfur source and correspondingly produced ethylene, ethane, or methane (FIG.2AandFIGS.19A-19C), demonstrating that methylthio-alkane reductase homologues from these organisms catalyze the same process. Conversely,R. capsulatuscould not utilize any of these VOSCs as sole sulfur source for growth (FIG.2AandFIGS.19A-19C), likeR. rubrumexpressing NflDK but not MarDK (FIGS.2B-C), indicating that group IV NFL proteins of unknown function catalyze processes distinct from methylthio-alkane reductase.

Amino Acid Sequence Comparison of Nitrogenase and Methylthio-Alkane Reductase Proteins Indicate a Distinct Function for Each Group

Nitrogenase functions via a coordinated transfer of electrons through a network of highly modified iron and sulfur metal clusters. The minimal molybdenum nitrogenase system requires gene products NifBHDKEN; the vanadium (Vnf) and iron (Anf) nitrogenases have similar requirements (8, 9). The NifH homodimer possesses a single Fe4—S4cluster at the homodimer interface. The NifDK heterotetramer contains Fe8—S7P-clusters coordinated at each of the two NifDK subunit interfaces, and each NifD subunit contains the characteristic catalytic FeMo-cofactor [Fe7—S9—C—Mo-homocitrate] (12). In the Vnf and Anf nitrogenase systems Mo is replaced with V or Fe, respectively. Initially, electrons are donated to the NifH Fe4—S4cluster from a reducing agent such as a ferredoxin or flavodoxin (61). When NifH is in complex with NifDK, these electrons are transferred in an ATP binding and hydrolysis dependent manner to the P-cluster of NifDK. NifH also has roles in P-duster assembly from two Fe4—S4clusters on the apo-NifDK heterotetramer and synthesis of FeMo-cofactor when in complex with NifDK-like FeMo-cofactor assembly proteins, NifEN (12). P-cluster electrons are then passed to the FeMo-cofactor catalytic cluster and ultimately to FeMo-cofactor-bound dinitrogen for stepwise reduction to ammonia (17, 62).

MarH: MarH contains the same NifH conserved residues for MgATP hydrolysis and Fe4—S4cluster coordination that enables transfer of electrons from the NifH Fe4—S4cluster to the NifDK P-cluster (FIG.12). The NifH conserved Arg-100 (V. vinelandiinumbering) is also conserved in MarH. This residue is modifiable by ADP-ribosylation to prevent NifH from complexing from NifDK. As nitrogenase activity is an ATP intensive process, this post translational modification effectively inactivates nitrogenase to prevent unnecessary ATP consumption when energy supply is insufficient or diazotrophy is not required (e.g. ammonium available as N-source). ForR. rubrumnitrogenase, ADP-ribosylation is catalyzed by dinitrogenase reductase ADP-ribosyltransferase (DRAT) and removed by dinitrogenase reductase activating glycohydrolase (DRAG). An analogous system appears to exist inA. vinelandii(63).

MarDK: MarD and MarK each possess the triad of cysteines conserved in the molybdenum nitrogenase subunits NifD and NifK for P-cluster coordination (FIG.10andFIG.11). One or more of these conserved cysteines are absent in the bacteriochlorophyll oxidoreductase (ChlLNB and BchXYZ) and reductive cyclase F430 synthesis (GbfCD) systems, which complex a catalytic Fe4—S4cluster instead (64, 65). MarD also has a conserved cysteine for coordinating a catalytic metallocofactor as in NifD for the FeMo-cofactor (Cys-275 inA. vinelandii). In contrast, however, the conserved NifD His-442 residue (A. vinelandiinumbering) responsible for coordinating FeMo-cofactor homocitrate and molybdenum is replaced with a Gly-Asp-Glu motif in MarD and there are no homocitrate synthase genes associated with marBHDK gene clusters (FIG.10) (9,15,16). In addition, the conserved NifD Glu-191 and His-195 residues involved in coordinating nitrogen intermediates bound to the FeMo-cofactor are replaced in MarD with aromatic residues Trp and Phe (9, 17).

MarB: NifB is a radical SAM enzyme responsible for carbide insertion and formation of the 8Fe—9S—C NifB-cofactor, the precursor to FeMo-cofactor (12). MarB possesses all of the identified motifs conserved across NifB enzymes associated with bona fide nitrogenases (FIG.13). For nitrogenase, NifB-cofactor maturation to FeMo-cofactor requires NifH and NifEN for addition of molybdenum and homocitrate (12).

Together, this indicates that methylthio-alkane reductase proceeds via a mechanism, similar but distinct to that of nitrogenase to convert MT-EtOH to ethylene, ethylmethylsulfide to ethane, and dimethylsulfide to methane (17). Methane release from DMS by the methylthio-alkane reductases is separate and distinct from the other known non-archaeal methanogenic processes, including photosynthesis-linked methane production by cyanobacteria (18), methane release from methylphosphonates by marine bacteria (19), and direct reduction of carbon dioxide to methane by iron-only nitrogenase (AnfDHGK) (20). In waterlogged soils, strictly anaerobic microbial processes produce ethylene that can accumulate to levels inhibitory to plant root growth, causing crop damage (21, 22). Early attempts at identifying ethylene-producing organisms surprisingly isolated oxygen-dependent soil bacteria and fungi (23, 24). The organisms and methylthio-alkane reductases identified here function a,naerobically and could contribute to this soil-ethylene paradox (10). This anaerobic ethylene process is distinct from the oxygen-dependent reactions catalyzed by aminocyclopropanecarboxylate oxidase and 2-oxoglutate dioxygenase in plants, fungi, and certain bacteria.

Non-Natural Pathways for Optimized Microbial Ethylene and Methane Production

The ethylene precursor, 5′-methylthioadenosine (MTA) is a routine byproduct of highly regulated processes such as quorum sensing, polyamine production, etc. These are highly regulated processes, making the native production of MTA for subsequent ethylene production rate limiting. The coliphage SAM hydrolase (MTA-forming) is a viral enzyme that directly converts SAM to MTA (FIG.20D) (69, 70). When this non-naturally occurring gene element is synthesized inRhodospirillum rubruinandRhodopseudomonas palustrisfor ethylene biogas production vial the DHAP shunt MarBHDK system (FIG.20C), ethylene production is enhanced 20-50 fold above the native amount produced by the organism in the absence of SAM hydrolase (FIG.20D).

The methane precursor, dimethylsulfide, is the most abundant organic sulfur compound in the environment. It is produced by marine bacteria from dimethylsulfinypropionate and by terrestrial bacteria from methanethiol (71, 72). A non-natural methionine salvage pathway fromRhodopseudomonal palsutrisfor the conversion of methionine to dimethylsulfide is constructed using methionine gamma lyase (mgl) and methanethiol methyltransferase (mddA) (FIG.20B) (72). This directly converts methionine to dimethylsulfide for methane production by methylthio-alkane reductase (MarBHDK) (FIG.20C) in photosynthetic bacteria (e.g.Rhodospirillum rubrum) or lignocellulose degrading bacteria (e.g.Clostridium cellulolyticum).

Materials and Methods

Fine chemicals: Dimethyl sulfide, methanethiol, L-methionine, 5′-methylthioadenosine, and S-methyl-t-cysteine were from Sigma; ethyl methyl sulfide, (2-methylthio)ethanol, (2-methylthio)acetate, and (3-methylthio)propanol were from Alfa Aesar. All media components were of ultrapure grade from Sigma or J. T. Baker, For targeted metabolite detection, (2-[methyl-C14]thio)ethanol was synthesized from [methyl-C14]-S-adenosylmethionine (Perkin Elmer). Labeled S-adenosylmethionine was acid hydrolyzed in 0.01 N H2SO4under reflux at 100° C. for 30 min to form [methyl-C14]-5′-methylthioadenosine. (2-[methyl-C14]thio)ethanol was subsequently formed enzymatically in a reaction containing 50 mM potassium phosphate pH 7.8, 5 mM MgCl2, 0.2 mM NADH, 60 μM substrate, and 2 μM each of purifiedR. rubrum5′-methylthioadenosine phosphorylase (10),Bacillus subtilis5-methylthioribose-1-phosphate isomerase (29),E. coli5-methylthioribulose-1-phosphate aldolase (25), andS. cerevisiaealcohol dehydrogenase (Sigma) at 30° C. for 2 h. Enzymes were synthesized and purified as previously described (10). Complete conversion was monitored by reverse phase HPLC with an inline scintillation detector as previously described (10), followed by enzyme removal via Amicon (Millipore) centrifugal concentration device.

Bacterial strains and growth conditions:R. rubrumATCC 11170 wild type strain (SmR; NC_007643.1; American Type Culture Collection), Rru_A1998 deletion strain WR (ΔrlpA::GmR) in which the MTA-isoprenoid shunt is inactivated, and Rru_A1998/Rru_A0359 deletion strain WRdht (ΔrlpA::GmR/Δald2) in which the MTA-isoprenoid and DHAP shunts are inactivated were as previously described (10, 30).Rhodobacter capsulatusSB1003 (NC_014034.1, American Type Culture Collection) (31),Rhodopseudomonas palustrisCGA010 (32), andBlastochloris viridisDSM133 (NZ_AP014854.2, University of Leibnitz DSMZ) (33) wild type strains were also as previously described.Rhodopseudomonal palustrisCGA010 (Caroline Harwood, University of Washington) is a derivative of CGA009 (SmR; NC_005296.1, American Type Culture Collection) in which a frame shift mutation is corrected. Anaerobic growth ofR. rubrumandR. capsulatuswas performed in static anaerobic culture tubes and serum bottles at 30° C. with 2000 lux incandescent illumination. Cultures were composed of sulfur-free Ormerod's malate (30 mM) minimal medium supplemented with the indicated sulfur source under a 95:5 mixture of N2LH2gaseous headspace as previously described (34, 35). Anaerobic growth ofR. palustriswas similarly performed by replacing malate with 0.5% (v/v) ethanol and 0.2% (w/v) sodium bicarbonate and adding 2 μg/ml para-aminobenzoic acid. All anaerobic manipulations were performed using an anaerobic chamber under 5% hydrogen and 95% nitrogen (Coy Laboratories).

Proteomics analysis: To optimize ethylene induction, and by inference of the remaining steps of the pathway in metabolizing MT-EtOH to methionine, the growth ofR. rubrumstrain WR (ΔrlpA::GmR) was measured spectrophotometrically by optical density at 660 nm (O.D.660nm) and the specific rate of ethylene production (μmol/h/g dry cell weight) was independently measured by gas chromatography (see GC analysis below) at regular intervals for a given sulfate or MT-EtOH concentration (FIGS.5A-5C). Cells were grown anaerobically, photoheterotrophically in anaerobic culture tubes containing 20 ml of sulfur-free malate minimal medium supplemented with 25, 50, 100, 1000 μM ammonium sulfate or 200-1000 μM MT-EtOH. For limiting sulfate, maximum ethylene specific rate was observed under 50 μM sulfate at an O.D.660nmof 0.6-0.75. For 200-1000 μM MT-EtOH, maximum ethylene specific rate was also observed in the same O.D.660nmrange. Subsequently,R. rubrumstrain WR was grown in triplicate (biological replicates) anaerobically, photoheterotrophically in rectangular flasks containing 0.5 L sulfur-free malate minimal medium supplemented with 50 μM or 1000 μM ammonium sulfate or 1000 μM MT-EtOH to an O.D.660nmof ˜0.60. Cultures were harvested anaerobically by centrifugation at 3000×g for 5 min and remaining media was thoroughly removed by decanting. Cell pellets were aliquoted in 0.4-0.6 g fractions and flash frozen in liquid N2.

Each cell pellet was lysed by 4% sodium deoxycholate in 100 mM ammonium bicarbonate with the application of sonication (20% amplitude, 10 s pulse, 10 s rest, 2 min total puke time). Crude protein extract was precleared via centrifugation, reduced with 10 mM dithiothreitol, alkylated with 30 mM iodoacetamide, and then collected on top of a 10 kDa cutoff spin column filter (VIVASPIN 500, Sartorius). Collected proteins were digested to peptides with two sequential aliquots of sequencing-grade trypsin (Sigma) at a 1:75 enzyme:protein ratio (w/w), initially overnight at room temperature followed by additional 3 h at room temperature. Peptides were collected by centrifugation and acidified to 1% formic acid followed by extraction with ethyl acetate to remove sodium deoxycholate. The peptide containing aqueous phase was recovered and concentrated. Concentrated peptides were measured using the bicinchoninic acid assay (Pierce).

Each peptide mixture was analyzed on a two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) platform using a Q Exactive Plus (QE+) mass spectrometer (Thermo Fisher Scientific) equipped with an Ultimate 3000 RS system (Thermo Fisher Scientific). 9 μg of each peptide sample was loaded via autosampler onto a triphasic pre-column (5 cm C18 reversed phase (RP), 5 cm strong cation exchange, and 5 cm C18 RP). Bound peptides were then washed and separated over three successive salt cuts of ammonium acetate (35 mM, 50 mM and 500 mM), each followed by an RP-LC elution via an in-house pulled nano-electrospray emitter (75 μm ID) packed with 30 cm of C18 RP. Mass spectra were acquired on QE+ in a data-dependent mode with full scan at 70K resolution, followed by HCD fragmentation of the top 15 most abundant ions at 15K resolution.

Acquired MS/MS spectra were matched with theoretical tryptic peptides generated from a concatenatedRhodaspirillum rubrumproteome FASTA database with contaminants and decoy sequences using MyriMatch v. 2.2 (37). Peptide spectral matches were filtered to achieve peptide false-discovery rates (FDR) <1% and assembled to their respective proteins using IDPicker v. 3.1 (38). Peptide abundance intensities were derived in IDPicker by extracting precursor intensities from chromatograms with lower and upper retention time of 90 s and tnass tolerance of 5 ppm. Protein abundances were calculated by summing up intensities of all identified peptides and normalized by their protein lengths respectively. Protein intensities were further log2 transformed and median centered using InfernoRDN version 1.1 (39), to approximate a normal distribution and reduce technical variance for further pairwise comparison. Student's T-test was then performed for every pair condition using Perseus platform (40) for two different thresholds (Benjamini-Hochberg FDR adjusted p-value <0.05 and fold change >2, or Benjamini-Hochberg FDR adjusted p-value <0.01 and fold change >4; two-sided).

Transcriptomies analysis:R. rubrumstrain WRdht (ΔrlpA/Δald2) and 0785::Tn5 (ΔrlpA/Δald2/0785::Tn5) were grown in triplicate (biological replicates) photoheterotrophically in anaerobic culture tubes containing 20 ml sulfur-free malate minimal medium supplemented with 50 μM (“Lo”) or 1000 μM (“Hi”) sulfate. When cells reached an O.D.660nmof 0.65-0.8, cells were harvested and stabilized by RNA protect reagent (Qiagen). RNA was isolated using the RNeasy protect kit (Qiagen) and quantified by UV absorbance. RNA-seq library construction and sequencing were performed at The Genomics and Microarray Shared Resource at University of Colorado Denver Cancer Center, Denver, CO, USA. Library preparation and rRNA depletion were performed using to the Zymo-Seq Ribo Free Total RNA Library Kit Cat No. R3000 with input of 250 ng and libraries were sequenced on the Illumina NovaSeq 6000 using 2×150 paired end reads. Raw RNA-seq data were trimmed using sickle (github.com/najoshi/sickle) (41). Prior genomic sequencing ofR. rubrumstrain WRdht confirmed the rlpA and ald2 deletions and >99% nucleotide identity to theR. rubrumATCC11170 genome. Mapping of transcriptomic reads to the reference was conducted using Bowtie2 (v2.3.5.1) with the options—very-sensitive and—score-min L,0, −0.1 (42). Differential expression analysis was performed using DESEq2 (v 1.22.2) (fitType=local, test=Wald) (43). Comparison of transcriptomes from the parent strain (WRdht) grown under 50 μM versus 1000 μM sulfate indicated all genes that were transcriptionally regulated >1.5-fold in response to sulfate availability (two-sided Wald Chi-square test, BH-FDR adjusted p<0.002 as implemented by DESeq2 (43)). Corresponding comparison for the SalR deletion strain (0785::Tn5) indicated which of these genes were no longer regulated in response to sulfate availability. Comparison of the SalR deletion strain to the parent strain under 1000 μM sulfate indicated which of these genes were potentially transcriptionally activated or repressed by SalR.

Transposon-insertion isolates ofR. rubrumwere individually picked into 96-well flat-bottom tissue culture plates containing 200 μl of sulfur-free Ormerod's malate minimal medium supplemented with 100 μM ammonium sulfate and 25 μg/ml kanamycin. Inoculated plates were incubated in an anaerobic chamber for 2 h, sealed with thermal adhesive film to prevent evaporation, and further sealed in thermal-seal bags (Kapak, ProAmpac) to maintain anaerobic conditions. Isolates were grown anaerobically at 30° C. under 2000 lux incandescent illumination to late log phase. Cultures were briefly exposed to air atmosphere, quickly transferred by 96-pin transfer device to new anaerobic 96-well plates containing 200 μl of anaerobic sulfur-free Ormerod's malate minimal medium supplemented with 1 mM ammonium sulfate or 1 mM MT-EtOH, and then incubated and sealed in an anaerobic chamber as before. Isolates were again grown anaerobically under illumination to screen for mutants incapable of growth on MT-EtOH but still able to grow on sulfate as sole S-source. 11,250 mutants were screened to ensure each gene received a transposon insertion at least once (FIGS.7A-7D). Putative ethylene pathway mutants were verified by confirmatory growth experiment in anaerobic culture tubes. The false discovery rate was 80% due to the sensitive nature of growingR. rubrumin 96-well plates with MT-EtOH as sole S-source. Validated ethylene pathway mutants were sequenced to determine the location of the Tn5 insertion as previously described (44,).

Gene deletion and complementation studies: Nonpolar gene cluster deletions of Rru_A1066-Rru_A1069, Rru_A0772-Rru_A0773, and Rru_A0793-Rru_A0796 in theR. rubrumwild type strain were performed by homologous recombination using previously described methods (10). Briefly, DNA fragments were amplified by PCR using primers listed in Table A below, digested with the indicated restriction enzyme following manufacturer's protocols (New England Biolabs), and ligated into pK18mobSacBgm (10) using T4 DNA ligase (New England Biolabs). Sequence verified plasmids were transformed intoE. coliStellar strain (TaKaRa Bio) and mobilized intoR. rubrumwild type by triparental conjugation with helper strainE. coliJM109/pRK2013 (American Type Culture Collection) (45), similar to methods used for the transposon mutagenesis. Transconjugants were selected on 16% PYE agar plates with 25 μg/ml kanamycin and 50 μg/ml streptomycin under aerobic growth at 30° C. First and second homologous recombination events were selected by 10% (w/v) sucrose sensitivity and kanamycin resistance of the isolates, and second recombinants possessing the proper gene deletion were sequence verified.

Gene complementation of theR. rubrumNFL gene deletion strain Δ0772:3/Δ0793:6 was performed in trans by NFL genes expressed from complementation plasmid pMTAP (70). Genes were amplified by PCR using primers listed in Table A, digested with the indicated restriction enzyme, and ligated into pMTAP. Sequence verified plasmids were transformed intoE. coliStellar strain (Takara) and mobilized intoR. rubrumby triparental conjugation with helper strainE. coliJM109/pRK2013. Transconjugants were selected on 16% PYE agar plates with 2 μg/ml tetracycline and 50 μg/ml streptomycin under aerobic growth at 30° C. Isolates were then tested for their ability to grow anaerobically with sulfate, MT-EtOH, or DMS as sole sulfur source.R. rubrum Δ0772:3/Δ0793:6 transconjugants with plasmids that complemented for growth on MT-EtOH and DMS were also quantified for restoration of ethylene and methane production by GC as described below.

Whole-cell VOSC utilization and gas production assays: Cells were initially grown aerobically in 150 ml serum bottles containing sulfur-free Ormerod's malate minif al medium supplemented with 50 μM ammonium sulfate (methylthio-alkane reductase inducing conditions) to mid log phase (O.D.660nmof 0.7-0.8). Cultures were washed anaerobically three times by centrifugation and resuspension in sulfur-free Ormerod's malate minimal medium. Cells were resuspended to a final O.D.660nmof ˜2.0 (higher cell densities suppressed methylthio-alkane reductase activity), dispensed in 20 ml aliquots in 60 ml serum vials, fed with 1 mM of DMS, EMS, or MT-EtOH, sealed, and incubated at 30° C. under 2000 lux incandescent illumination for 12 h. Produced methane, ethane, and ethylene gas was quantified by GC as described below.

Whole-cell nitrogenase and methylthio-alkane reductase specific rate assays:R. rubrumwild type and NFL gene deletion (Δ0772:3/Δ0793:6) strains were grown anaerobically under argon headspace to late log phase (O.D.660nm0.9-1.1) in Ormerod's malate minimal medium with 15 mM ammonium chloride or sodium glutamate as sole N-source and 50 μM or 1 mM sodium sulfate as sole S-source, For whole-cell nitrogenase assays (46), 2 ml of culture was transferred via syringe to an anaerobic 7.5 ml serum vial flushed with argon. Assays were initiated by the addition of 0.06 atm acetylene and allowed to proceed for 10 min under 2000 lux illumination at 30° C. Assays were quenched with 100% (w/v) trichloroacetic acid to 10% final and ethylene was quantified by GC as described below. Similarly, for whole-cell methylthio-alkane reductase assay, 4 ml of culture were transferred via syringe to an anaerobic 8 ml serum vial flushed with argon. Assays were initiated by the addition of EMS to 1 mM final concentration and allowed to proceed for 30 min under 2000 lux illumination at 30° C. Assays were quenched with TCA and ethane was quantified by GC.

GC analysis of hydrocarbons: Quantification of methane, ethane, and ethylene was performed using a Shimazdu GC-14A with Restek Rt-Alumina BOND/Na2SO4column. Gaseous culture headspace after feeding or growth experiments was injected (250-500 μl) at 180° C. and separated isothermally at 30° C. Eluted compounds were detected by flame ionization detector at 180° C. and identified based on retention time of methane, ethane, and ethylene standard (Praxair). The total amount of each hydrocarbon present was calculated from the peak area as compared to standard concentration curves of the corresponding reference standard.

Targeted metabolomics:R. rubrumwild type and Rru_A0793-Ru_A0796 deletion strains were grown anaerobically to an O.D.660nmof 0.8 (mid log phase) in Ormerod's malate minimal medium supplemented with 50 μM ammonium sulfate to induce ethylene production. Cultures were washed anaerobically three times by centrifugation and resuspension in sulfur-free Ormerod's malate minimal medium. Cells were resuspended to a final O.D.660nmof ˜2.0 (higher concentrations repressed methylthio-alkane reductase activity), supplemented with 100 μM 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent for trapping free thiols), and sealed as 1 ml aliquots in 1.5 ml anaerobic serum vials. Cells were then fed with 10 μM MT-EtOH and 1 μM (2-[methyl-C14]thio)ethanol and incubated under 2000 lux incandescent light at 30° C. Metabolism was stopped by flash freezing in liquid nitrogen; cells were pelleted, media supernatant reserved, and the cell pellet was extracted with 80% acetonitrile+0.04N ammonium hydroxide with vortexing for 5 min followed by 20 min incubation at −20° C. Acetonitrile was removed by vacuum concentration, and the extracted metabolites were combined with the reserved media supernatant. Metabolites were separated by reverse phase HPLC and identified by inline scintillation detector based on retention time compared to reference standards as previously described for N=2 biological replicates (10).

Free-energy calculations: Standard free energies of formation and reaction were determined using electronic structure calculations with continuum solvent models. Specifically, density functional theory with the B3YLP (47 , 48) exchange correlation functional was used with the 6-311++G(2d, 2p) basis set. The geometries were optimized and harmonic frequencies determined in a continuum model solvent using the COSMO self-consistent reaction field method (49). All calculations were performed with the NWChem computational chemistry package (50) using the EMSL Arrows interface (5.1). H2was used as the electron donor in each redox reaction since the actual electron donor is not known. The relative difference in the reaction free energies will not change if, for example, ferredoxin or any other redox pair were used as the electron donor, since the electrochemical potential of the actual electron donor would be measured relative to the standard hydrogen electrode.

Phylogenetics: TheR. rubrumMarH, MarD, and MarK proteins were separately queried against the NCBI reference genome database using the translated nucleotide blast (tblastn) algorithm and filtered for protein subjects with e-value<e-50. Each identified MarH, MarD, and MarK candidate was correlated with its reference genome and only genomes were retained that contained all three homologues on the same contig and with MarD and MarK being adjacent. These candidates, along with recently discovered Group VI representatives from metagenome assembled genomes (28) were then appended to a reference nitrogenase (Groups I, II, III) and NFL sequence (Groups IV and V) database (9) with additional sequences identified from genomes in the JGI IMG/M database. Amino acid sequences were aligned using MAFFT (52) (v7.394) (—auto). Alignments were trimmed using TrimAl (53) (v1.4.rev22) (—gappyont). Maximum likelihood trees were constructed using IQ-TREE (54) (v1.6.8) (−alrt 1000-bb 1000) using best-fit models (NifH: LG+R10; NifD: LG+R6) identified by ModelFinder (55) as implemented in IQ-TREE with ultrafast bootstrap (UFBoot) (56).

Pairwise alignment of NifB, NifH, NifD, and NifK superfamily sequences for conserved active site residue analysis (FIG.10-FIG.13) was performed using Clustal Omega (EMBL-EBI) (57) and visualized with Jalview (58). Gene synteny (FIG.18) was visualized using R package (R Foundation, Vienna, Austria) ‘gggenes’ (59) for an ˜28 kbp neighborhood centered on the NifD homologs identified in selected genomes representing the Nif and NFL clades.

To identify organisms with native ethylene capacity (DHAP Shunt plus marBHDK genes,FIG.17), organisms with a putative MarHDK complex, as indicated by the phylogenetic tree analysis (FIG.4andFIG.16), were then analyzed for the presence of DHAP shunt homologues by querying each genome (tblastn) with theR. rubrumandE. coliDHAP Shunt genes (10, 25), MtnK, MtnP, MtnA, and Ald2, with a cutoff of e-value <−20. For organism phylogenetic analysis (FIG.17), 113 genomic sequences includingR. rubrum, R. palustris, B. viridis,and additional random organisms with MarHDK genes were downloaded from NCBI (Genome or Assembly databases). This set of genomes was aligned to a set of reference bacteria using GTDB-TK (de_novo_wf) (60). The non-redundant subset of organisms as shown inFIG.17together with Chloroflexota sequences as the outgroup from the reference database were extracted from the alignment and a maximum likelihood tree was built using IQ-TREE (54) (−alrt 1000-bb 1000) using the best-fit model LG+F+R6 identified by ModelFinder (55) as implemented in IQ-TREE with ultrafast bootstrap (UFBoot) (56).

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