MICROBIAL APPROACH FOR THE PRODUCTION OF LONG CHAIN COMPOUNDS

This disclosure describes recombinant Megasphaera microbes designed to include increased consumption of acetate, increased carbon flux to butyryl-CoA and/or hexanoyl-CoA, increased production of butyrate and/or hexanoate, or a combination thereof, than a comparable control. This disclosure also describes methods that generally include growing such recombinant microbes under conditions effective for the recombinant microbes to consume greater amounts of acetate, produce increased amounts of butyryl-CoA and/or hexanoyl-CoA, produce increased amounts of butyrate and/or hexanoate, or a combination thereof.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an XML file entitled “0235000295US01.xml” having a size of 20 kilobytes and created on 9 Aug. 2022. The information contained in the Sequence Listing is incorporated by reference herein.

BACKGROUND

Greenhouse gas emissions and the enormous carbon footprint of transportation have contributed to the impending climate crisis of the planet. The advancement of the biofuel industry to create carbon-neutral processes such as drop-in and replacement fuels for petroleum-based gasoline is critical in resolving the transportation industry's contribution to the climate crisis (Nelson 2017, Lamsen 2012). The current model for drop-in fuels is largely based on ethanol produced industrially from starches in corn feedstocks. Though this ethanol produced from starch has a positive energy balance and already contributes to lowered emissions when added to gasoline, farming practices, including land use, is a concern for corn-based bioethanol production and have the potential to be greatly lowered (afdc.energy.gov/fuels/ethanol_fuel_basics.html) (Tracy 2011). The development of next-generation biofuels such as longer chain-length alcohols like butanol and hexanol offers the opportunity for increasing the efficiency of the biofuels produced and reducing carbon-based fuel emissions (Choi, Lamsen 2021). Longer-chain alcohols and fatty acid production has been seen in a variety of organisms, including but not limited to many Clostridia (Choi, Tracy, Weimer). Heterologous chain-elongation pathways have been expressed inE. coli, but organisms with native flux condensing acetyl-coA groups are more robust in their ability to form the end-products, and, ultimately, engineering these pathways inE. coliwas not seen to produce these alcohols in industrially relevant fluxes (Dekishima 2011, Clomburg 2012, Kataoaka 2017, Dollomonaco 2011, Kim 2015).

SUMMARY OF THE APPLICATION

Megasphaera elsdeniiis a ruminal mesophilic obligate anaerobe that is a member of the Negativicutes class of bacteria that natively possesses high-flux energy metabolism from a variety of carbon sources to fatty acid pre-cursors of alcohols with longer chain-length than acetate, including butyric acid, hexanoic acid, and even octanoic acid (Nelson 2017, Prabhu 2012). These native metabolic abilities makeM. elsdeniia uniquely promising organism for metabolic engineering and optimization of drop-in biofuel production of the longer-chain alcohol biofuels of the future. Additionally, this work takes full advantage of this novel organism that has already been used in fermentation studies (Nelson 2017, Prabhu 2012), but has not been genetically tractable to genetic engineering until recently. With advanced sequencing technologies, transcriptomics, and cross-phylogeny implementation of other rapid genetic tools now available, the abilities ofM. elsdeniican be exploited to serve as a platform for the production on longer chain alcohols. These tools include the development of a strategy for the use of a counter-selectable marker in this work for targeted enzyme deletions to increase flux towards targeted products, utilizing the gene upp (uracil phosphoribosyltransferase), which allows for the use of counter-selection against an integrated vector using 5-fluorouracil, and the gene pyrF (orotidine-5′-phosphate decarboxylase), which allows for the use of counter-selection against an integrated vector using 5-fluoroorotic acid (5-FOA).

Relatively little is known aboutMegasphaera elsdeniimetabolism and the biochemical activities of its chain elongation and organic acid production enzymes. Metabolic engineering efforts in this organism will be greatly bolstered by a better understanding of the roles of these enzymes in metabolism and its flux, and, as a result of the ambiguous nature of genome annotation, the functions of its putative organic acid production enzymes are unknown, including its various acyl-CoA transferases. As described herein, genetic links between putative chain elongation enzymes and organic acid fermentation products inM. elsdeniiare identified using newly developed genetic tools to construct a chromosomal marker replacement of a putative propionyl-CoA transferase gene, locus tag: MELS_0742, and others. The resulting phenotype of this mutant strain is then observed to elucidate the resulting fermentation profile and growth phenotypes of this single mutant.

Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below.

As used herein, the terms “genetically engineered” microbe and “recombinant” microbe are used interchangeably and refer to a microbe that has been altered by human intervention. For example, a “recombinant” microbe refers to a microbe that has been genetically manipulated. In one or more embodiments, a recombinant microbe is one that includes alteration of endogenous nucleotides. For example, a microbe is a genetically modified microbe by virtue of introduction of an alteration of endogenous nucleotides. For instance, an endogenous coding region could be deleted or mutagenized. Another example of a recombinant microbe is one into which has been introduced an exogenous polynucleotide and expresses a protein from the exogenous polynucleotide. Yet another example of a genetically modified microbe is one having an altered regulatory sequence, such as a promoter, to result in increased or decreased expression of an operably linked endogenous coding region.

As used herein, an “exogenous polynucleotide” refers to a polynucleotide that is not normally or naturally found in a microbe. An exogenous polynucleotide includes a coding region that is not normally found in a microbe, and a coding region that is normally found in a microbe but is operably linked to a regulatory region to which it is not normally linked. An “endogenous polynucleotide” is also referred to as a “native polynucleotide.”

As used herein, the term “protein” refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term “protein” also includes molecules which contain more than one protein joined by a disulfide bond, or complexes of proteins that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, enzyme, and polypeptide are all included within the definition of protein and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the protein is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.

As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded RNA and DNA. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide may be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. A polynucleotide may include nucleotide sequences having different functions, including, for instance, coding regions, and non-coding regions such as regulatory regions.

As used herein, the terms “coding region,” “coding sequence,” and “CDS,” are used interchangeably and refer to a nucleotide sequence that encodes a protein and, when placed under the control of appropriate regulatory sequences expresses the encoded protein. The boundaries of a coding region are generally determined by a translation start codon at its 5′ end and a translation stop codon at its 3′ end. A “regulatory sequence” is a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators. The term “operably linked” refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner. A regulatory sequence is “operably linked” to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.

Genetic loci of aMegasphaera, such as anM. elsdenii, are referred to herein using one of two locus tag identifiers. One locus tag identifier has the prefix “MELS_” followed by the number of the CDS in theMegasphaera elsdeniistrain DSM 20460 draft genome, GenBank accession HE576794.1. The second locus tag identifier has the prefix “MELS_RS” followed by the number of the CDS in theMegasphaera elsdeniistrain DSM 20460 complete sequence, GenBank accession NC_015873.1.

As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements. The use of “and/or” in some instances does not imply that the use of “or” in other instances may not mean “and/or.”

As used herein, “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” or the like are used in their open ended inclusive sense, and generally mean “include, but not limited to,” “includes, but not limited to,” or “including, but not limited to.”

It is understood that wherever embodiments are described herein with the language “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” and the like, otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided. The term “consisting of” means including, and limited to, whatever follows the phrase “consisting of.” That is, “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of” indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

Conditions that are “suitable” for an event to occur, such as replication of a microbe or positive selection of a marker, or “suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event.

In the description herein particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.

DETAILED DESCRIPTION

The present disclosure provides microbes, compositions, and methods useful for increasing microbial production of longer chain organic acids. The disclosure includes metabolically engineering a microbial host. The microbial host is engineered to increase carbon flux to butyryl-CoA and/or hexanoyl-CoA, to produce increased amounts of butyrate and/or hexanoate, or a combination thereof. The microbial host can be further metabolically engineered to convert butyryl-CoA to butyraldehyde and butanol, hexanoyl-CoA to hexanaldehyde and hexanol, or a combination thereof. The microbial host can also be further metabolically engineered to produce other molecules derived from the chain elongation pathway. Recombinant microbes can be referred to herein as “metabolically engineered” microbes when the genetic engineering is directed to disruption or alteration of a metabolic pathway so as to cause a change in the microbe's metabolism compared to a comparable control.

Recombinant Microbes

A microbial host that is used to engineer a recombinant microbe of the present disclosure is a member of the genusMegasphaera. Examples of members of the genusMegasphaeraincludeM. hominis, M. cerevisiae, M. elsdenii, M. micronuciformis, M. paucivorans, andM. sueciensis. Megasphaeraspp., are readily available. In one embodiment, theMegasphaeraisM. elsdenii, and in one embodiment theM. elsdeniimicrobial host that is used to engineer a recombinant microbe is ATCC 25940. Methods for metabolically engineering aMegasphaerasp. are described herein.

A recombinantMegasphaeramicrobe of the present disclosure includes one or more mutations designed to increase carbon flux towards the targeted products butyrate (the straight-chain alkyl carboxylic acid with the chemical formula CH3(CH2)2CO2H, also referred to as butyric acid) and/or hexanoate (the straight-chain alkyl carboxylic acid with the chemical formula CH3(CH2)4CO2H, also referred to as hexanoic acid). The increased carbon flux towards the targeted products butyrate and/or hexanoate can be referred to as increased carbon flux to butyryl-CoA and/or hexanoyl-CoA, an intermediate step in the pathway to butyrate and/or hexanoate.

In one embodiment, a recombinantMegasphaeramicrobe, such asM. elsdenii, includes a mutation, such as a deletion, of a coding region encoding a CoA-transferase. Examples of CoA-transferases include those encoded by the coding regions MELS_0742, MELS_0464, and MELS_0034 ofM. elsdeniiATCC 25940. MELS_0742 and MELS_0464 encode a propionate CoA transferase, and MELS_0034 encodes an acetate CoA/Acetoacetate CoA-transferase alpha subunit. In another embodiment, a recombinantMegasphaerasp., such asM. elsdenii, includes a mutation, such as a deletion, of a coding region encoding a member of the glyoxalase family, for instance MELS_0743. In another embodiment, a recombinantMegasphaerasp., such asM. elsdenii, includes a mutation, such as a deletion, of a coding region encoding a lyase such as lactoyl-CoA dehydratase subunit alpha, for instance MELS_0745. When aMegasphaerasp. other thanM. elsdeniiATCC 25940 is used, closely related CoA-transferases, glyoxalases, and lyases can be identified using routine methods such as homology searches or RNAseq and proteomic analyses, and mutations of one or more coding regions can be engineered into the microbe using the methods described herein.

A recombinantMegasphaeramicrobe can include reduced expression of a propionyl-CoA transferase. In one or more embodiments, when the microbe isM. elsdeniiATCC 25940, the mutation is in the coding region MELS_0742. For instance,M. elsdeniiATCC 25940 was found to include four coding regions encoding putative propionate-CoA transferases, MELS_0742, MELS_0464, MELS_1631, and MELS_1130, but analysis of expression suggested that MELS_0742 was most highly expressed. Subsequent deletion of MELS_0742 resulted in a recombinantM. elsdeniiwith undetectable propionate-CoA transferase activity as inferred from fermentation profile data (Example 1).

A recombinantMegasphaeramicrobe having a mutation in one or more coding regions described herein can have increased carbon flux to butyryl-CoA, hexanoyl-CoA, or the combination thereof. In one or more embodiments, a recombinantMegasphaeradescribed herein can produce increased amounts of butyrate, hexanoate, or the combination thereof, compared to a comparable control microbe. In one or more embodiments, a recombinantMegasphaeradescribed herein can be further engineered to convert butyryl-CoA to butanol, hexanoyl-CoA to hexanol, or the combination thereof, and likely increased production of other molecules of longer chain length. In one or more embodiments, a recombinantMegasphaeradescribed herein can consume increased amounts of acetate compared to a comparable control microbe. As used herein, a comparable control microbe is a microbe that is genetically identical to the recombinantMegasphaeraexcept for the mutation or mutations being evaluated. The increased production of butyrate and/or hexanoate can be observed when the recombinantMegasphaerais grown on lactate (also referred to as lactic acid). In some embodiments, increased production of butyrate and/or hexanoate is not observed when the recombinantMegasphaerais grown on glucose. The increase of each of butyrate and/or hexanoate can be at least 2-fold, at least 4-fold, at least 6-fold, at least 8-fold, at least 10-fold, at least 100-fold, at least 1000-fold, or more compared to a comparable control microbe. In one or more embodiments, the increase of each of butyrate and/or hexanoate is no greater than 10,000-fold compared to a comparable control microbe. In some embodiments there is not a theoretical maximum fold-increase. For instance, where a microbe is engineered to produce a new compound that is not produced at detectable levels by the comparable control, the fold-increase can be extremely high even if the absolute amount of the new compound did not increase substantially. The increased consumption of acetate can be observed when the recombinantMegasphaerais grown on lactate (also referred to as lactic acid). In some embodiments, increased consumption of acetate is not observed when the recombinantMegasphaerais grown on glucose. The increase of acetate consumption can be at least 2-fold, at least 4-fold, at least 6-fold, at least 8-fold, at least 10-fold, at least 100-fold, at least 1000-fold, or more compared to a comparable control microbe.

A recombinantMegasphaeramicrobe having reduced propionate-CoA transferase activity produces decreased amounts of valerate. The presence of decreased amounts of valerate was surprising, as the expected result was a much greater decrease in valerate or no detectable valerate. The enzyme deleted, MELS_0742, is thought to be responsible also for the lactate conversion to lactoyl-CoA. The inventors predicted interrupting this reaction would interrupt the entire acrylate cycle, which provides the propionyl-CoA that leads to valerate production.

A model was constructed using the Department of Energy (DOE) KBase and represents glucose fermentation to butyrate and lactate fermentation to butyrate and propionate (FIG.1). A map adaptation was constructed from the metabolic reconstruction of central carbon metabolism (FIG.2). The map adaptation represents steps to target in the engineering ofMegasphaera, such asM. elsdenii, to alter its native metabolism to genetically funnel carbon towards longer chain-length targets such as butyryl-CoA and/or hexanoyl-CoA, enabling increased production of butyrate and hexanoate. Using the constructed map and the genomic DNA sequence forM. elsdenii, several coding regions annotated as putative acyl-CoA transferase genes were identified. The inventors predict that mutations in one or more of these coding regions will increase flux withinMegasphaerasp. towards longer chain-length targets such as butyrate and hexanoate. Examples of coding regions that can be targeted for mutation, in addition to those discussed herein, include MELS_0034, MELS_0437, MELS_0464, MELS_0430, MELS_0033, MELS_0341, MELS_1130, and MELS_1631. The mutation can be any mutation that reduces expression of the protein encoded by a coding region, or a mutation that reduces the activity of the protein, including a point mutation an insertion, or a deletion. A recombinantMegasphaerahaving a mutation in one or more of a coding region described herein can produce, or is expected to produce, increased amounts of butyrate, hexanoate, or the combination thereof, compared to a comparable control microbe. Accordingly, the present disclosure includes a recombinantMegasphaera, such asM. elsdenii, that includes a mutation in one or more of the coding regions described herein, in any combination. In one embodiment, a recombinantMegasphaeraincludes a mutation in a coding region encoding a propionyl-CoA transferase, such as a mutation in the coding region MELS_0742.

In one embodiment, a recombinantMegasphaeramicrobe, such asM. elsdenii, includes one or more mutations in coding regions responsible for the acrylate cycle pathway. The inventors used the map adaptation and the genomic DNA sequence forM. elsdeniito identify a cluster of several coding regions annotated as responsible for the acrylate cycle pathway. The inventors predict that mutations in these coding regions will increase flux withinMegasphaerasp. towards longer chain-length targets such as butyryl-CoA and/or hexanoyl-CoA, leading to increased production of butyrate and hexanoate. The cluster of locus tags that will be targeted for mutation include MELS_0742-0747. The mutation can be any mutation that reduces expression of the protein encoded by a coding region, or a mutation that reduces the activity of the protein, including a point mutation, an insertion or a deletion. A recombinantMegasphaerahaving a mutation in one or more of these coding regions involved in the acrylate cycle pathway can produce increased amounts of butyrate, hexanoate, or the combination thereof, compared to a comparable control microbe. Accordingly, the present disclosure includes a recombinantMegasphaera, such asM. elsdenii, that includes a mutation in one or more of the putative acrylate cycle pathway genes.

In one embodiment, a recombinantMegasphaeramicrobe includes a mutation, such as a deletion, of a coding region encoding uracil phosphoribosyltransferase (Upp). An example of one upp coding region is MELS_2191. Uracil phosphoribosyltransferase converts the uracil analogue, 5-fluorouracil (5-FU), to the toxic product fluorodeoxyuridylate which kills growing microbes that are synthesizing uracil. As described herein, mutants of upp are, therefore resistant to 5-FU, providing 5-FU resistance as a counter selectable marker (Guss and Riley, US Published Patent Application No. 2021/0024965).

In one embodiment, a recombinantMegasphaeramicrobe includes a mutation, such as a deletion, of a coding region encoding orotidine-5′-phosphate decarboxylase (PyrF). An example of one pyrF coding region is MELS_RS04415. Deletion and complementation of orotidine-5′-phosphate decarboxylase (pyrF) inMegasphaera, such asM. elsdeniiallows for counter-selection of transformants growing in media containing uracil. In the presence of 5-Fluoroorotic acid (5-FOA), the pyrE gene product orotate phosphoribosyltransferase adds a phosphate group via phosphoribosyl pyrophosphate to 5-FOA becoming 5′-fluoroorotidine monophosphate and the substrate for the pyrF gene. pyrF cleaves the carboxylic group from 5′-fluoroorotidine monophosphate creating 5′-fluorouridine monophosphate (5′-FUMP), a toxic analog of uridine monophosphate and a precursor to both the RNA nucleotide uracil and the DNA nucleotide thymine. The addition of fluorine at the 5′ carbon becomes a lethal inhibitor to thymidylate synthetase, preventing the methylation and conversion of 5′-fluorouracil to thymidine. These toxic nucleotide analogs prevent the translation of RNA, the replication of DNA, and ultimately cause cell death. Nonconservative pyrF mutants are uracil auxotrophs and resistant to 5-FOA. The inventors have established 5-FOA minimal inhibitory concentrations (MICs) in Reinforced clostridial medium (RCM), a rich complex media, at 1 mg/mL and 2.5 mg/mL in liquid and solid media, respectively. 5-FOA allows for counter-selection with sufficient uracil present in the growth medium.

In one embodiment a recombinantMegasphaeramicrobe includes an exogenous coding region encoding a bifunctional aldehyde-alcohol dehydrogenase. An example of a coding region encoding aldehyde-alcohol dehydrogenase is adhE2. An adhE2 coding region can be obtained from a member of the genusClostridium, such asC. acetobutylicum. Examples of adhE2 coding regions and AdhE2 proteins, and engineeringM. elsdeniito include adhE2, are described in Guss and Riley (US Published Patent Application No. 2021/0024965). A bifunctional aldehyde-alcohol dehydrogenase can catalyze the production of butanol from butyryl-CoA.

In one embodiment a recombinantMegasphaeramicrobe includes an exogenous coding region encoding an acyl-CoA reductase. An example of a coding region encoding acyl-CoA reductase is acr. An acr coding region can be obtained from a member of the genusClostridium, such asC. acetobutylicum, C. carboxidivorans, andC. saccharoperbutylacetonicum. An acyl-CoA reductase can catalyze the conversion of hexanoyl-CoA to hexanaldehyde.

In one embodiment a recombinantMegasphaeramicrobe includes an exogenous coding region encoding an alcohol dehydrogenase. An example of a coding region encoding alcohol dehydrogenase is adh. An adh coding region can be obtained from a member of the genusClostridium, such asC. acetobutylicum, C. carboxidivorans, andC. saccharoperbutylacetonicum. An alcohol dehydrogenase can catalyze the conversion of hexanaldehyde to hexanol.

Methods of Making a RecombinantMegasphaeraMicrobe

Megasphaerasp. typically include robust restriction systems that cleave a DNA polynucleotide introduced into aMegasphaerasp. This has prevented the establishment of methods for genetic analysis ofMegasphaerasp. Provided herein are methods for genetically engineeringMegasphaera, includingM. elsdenii, to construct the recombinantMegasphaeradescribed herein.

Microbial strains have been created that methylate DNA for introduction intoMegasphaeracells and the successful identification of transformants. The methylated DNA obtained from the methylating microbial strains can be introduced intoMegasphaeracells and is protected from theMegasphaerarestriction systems. One example of a methylating microbial strain includes the coding regions MELS_0050-0051 and MELS_1615-1616. In one embodiment, a methylating microbial strain expressing these coding regions is anE. colisuch as TOP10 (ThermoFisher) further genetically modified to be dcm−. The TOP10 strain can be further modified to include a poly-attB cassette, for instance integrated at the Hong Kong phage attachment locus, and MELS_0050-0051 integrated at the R4 site of the poly-attB and MELS_1615-1616 integrated at the lambda attB locus. The proteins encoded by MELS_0050-0051 and MELS_1615-1616 are useful in preparing DNA for transformation intoM. elsdeniiATCC 25940 and potentially otherMegasphaeraspp. Methylating microbial strains are described in Guss and Riley (US Patent Application No. 2021/0024965).

Also provided herein is a recombinantMegasphaerathat has been engineered to introduce a mutation to result in decrease expression of one or more of the coding regions MELS_0050, MELS_0051, MELS_1615, or MELS_1616. In one embodiment, a recombinantMegasphaeraincludes a mutation of both MELS_0050 and MELS_0051, both MELS_1615 and MELS_1616, or a mutation of MELS_0050, MELS_0051, MELS_1615, and MELS_1616. The mutation can be any mutation that reduces expression of the protein encoded by the coding region, or a mutation that reduces the activity of the protein, including a point mutation, an insertion, or a deletion. A recombinantMegasphaeraincluding a mutation in one or more of these coding regions has a reduced or inactive restriction system, and DNA can be transformed into such a recombinantMegasphaerawith the expectation that transformants will be identified.

Accordingly, producing a recombinantMegasphaera, for instanceM. elsdenii, includes transforming with DNA that has been obtained from a methylating microbial strain. DNA used to transformMegasphaerasp. is typically in the form of a vector. A vector is a replicating polynucleotide, such as a plasmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, or artificial chromosome vectors. Typically, a vector is capable of replication in a microbial host, for instanceE. coli. In some embodiments a vector is capable of replication in aMegasphaerasp. In some embodiments the vector is a plasmid. Origins of replication that are useful inMegasphaerainclude, but are not limited to, those present in the plasmids pIM13 (Projan et al., 1987), pBC1 (De Rossi et al., 1992), and pVJL1 (Liu et al., 2012).

DNA used to transform aMegasphaerasp. optionally includes one or more marker sequences, which typically encode a molecule that inactivates or otherwise detects or is detected by a compound in the growth medium. For example, the inclusion of a marker sequence can render the transformed microbe resistant to an antibiotic, or it can confer compound-specific metabolism on the transformed microbe. Examples of marker sequence that confer resistance and can be used inMegasphaerasp. include those encoding resistance to chloramphenicol and to erythromycin.

In one embodiment, theMegasphaerasp. to be transformed includes reduced expression, such as undetectable expression, of an upp coding region. In one embodiment, the upp coding region is MELS_2191. Use of a recipientMegasphaerasp. with an upp mutation is helpful in engineering targeted mutations, such as deletions, of specific coding regions, or targeted insertions of DNA, such as a coding region (Guss and Riley, US Published Patent Application No. 2021/0024965). The polynucleotide used to transform aMegasphaerasp. includes an upp coding region, e.g., MELS_2191, which encodes uracil phosphoribosyltransferase and converts 5-fluorouracil (5-FU), to the toxic product fluorodeoxyuridylate, which kills growing microbes. For instance, transformation of aMegasphaerasp. with a plasmid containing sequences of DNA sharing homology to sequences flanking a coding region targeted for deletion can result in the integration of the plasmid into the microbe's chromosome to produce a specific genetic lesion. If theMegasphaerasp. includes a deletion of upp and the vector includes an upp coding region, incubating the transformants in 5-FU can counter-selection against the integrated vector (see Example 1). Accordingly, the present disclosure also includes methods for using aMegasphaerahaving an upp mutation, including methods for constructing aMegasphaerahaving a targeted mutation. The targeted mutation can be the production of a deletion of the target coding region. The production of a deletion greatly reduces the likelihood of reversion.

A method for using an upp-Megasphaerato construct aMegasphaerahaving a targeted mutation includes transforming theMegasphaerawith a plasmid that includes an upp coding region that will complement the upp mutation. The plasmid is replication incompetent in the recipientMegasphaera. The plasmid also includes a mutagenic cassette. A mutagenic cassette is designed to target a specific location, typically a coding region, where a mutation is to be introduced.

In one embodiment, theMegasphaerasp. to be transformed includes reduced expression, such as undetectable expression, of a pyrF coding region. In one embodiment, the pyrF coding region is MELS_RS04415. Use of a recipientMegasphaerasp. with a pyrF mutation is helpful in engineering targeted mutations, such as deletions, of specific coding regions, or targeted insertions of DNA, such as a coding region (see, for instance, Lipscomb et al., 2016, Applied and Environmental Microbiology, 82(14):4421-4428). The polynucleotide used to transform aMegasphaerasp. includes a pyrF coding region, e.g., MELS_RS04415, which encodes orotidine-5′-phosphate decarboxylase and converts 5-Fluoroorotic acid (5-FOA) to a toxic product that kills the cells. Deleting pyrF results in a strain that is a uracil auxotroph resistant to 5-FOA, allowing prototrophic selection and counter-selection of the wild-type pyrF. For instance, transformation of aMegasphaerasp. with a plasmid containing sequences of DNA sharing homology to sequences flanking a coding region targeted for deletion can result in the integration of the plasmid into the microbe's chromosome to produce a specific genetic lesion. If theMegasphaerasp. includes a deletion of pyrF and the vector includes a pyrF coding region, incubating the transformants in 5-FOA can counter-selection against the integrated vector. Accordingly, the present disclosure also includes methods for using aMegasphaerahaving a pyrF mutation, including methods for constructing aMegasphaerahaving a targeted mutation. The targeted mutation can be the production of a deletion of the target coding region. The production of a deletion greatly reduces the likelihood of reversion.

A method for using a pyrF-Megasphaerato construct aMegasphaerahaving a targeted mutation includes transforming theMegasphaerawith a plasmid that includes a pyrF coding region that will complement the pyrF mutation. The plasmid is replication incompetent in the recipientMegasphaera. The plasmid also includes a mutagenic cassette. A mutagenic cassette is designed to target a specific location, typically a coding region, where a mutation is to be introduced.

The mutagenic cassette includes two distinct DNA sequences that will permit homologous recombination between the plasmid and the microbe's chromosome. The two distinct DNA sequences typically flank the targeted coding region in the microbe's chromosome. On the plasmid, the two distinct DNA sequences flank the lesion that is to be introduced into the chromosome. The lesion, in combination with the two distinct DNA sequences, can be one that will result in deletion of the coding region. The mutagenic cassette also includes a marker, such as a coding region encoding an antibiotic marker, present within the lesion. Optionally, the mutagenic cassette also includes two attachment sites that flank the lesion. In one embodiment, the structure of a mutagenic cassette is first homologous DNA sequence-first attachment site-lesion/marker-second attachment site-second homologous DNA sequence. The attachment sites are sequence that can be identified by a recombinase, such as an integrase and, in the presence of a suitable recombinase, the region between the attachment sites—the marker—can be removed from the chromosome.

Following transformation, recipients of the plasmid are selected using the marker present with the lesion. The result is transformants that are likely to include the plasmid integrated in the chromosome by virtue of a crossover event between one of the two DNA sequences and the appropriate sequences on the microbe's chromosome. After selection for successful transformants, subsequent exposure of the transformants to 5-FU or 5-FOA selects for a second crossover event that eliminates the upp coding region or the pyrF coding region and the plasmid sequences, resulting in the insertion of the lesion, such as a deletion, of the targeted coding region. The coding region encoding the marker can then be removed by use of a recombinase.

AMegasphaerasp. can also be engineered to include an exogenous polynucleotide, such as, but not limited to, a bifunctional aldehyde-alcohol dehydrogenase such as one encoded by adhE2, an acyl-CoA reductase such as one encoded by acr, or an alcohol dehydrogenase such as one encoded by adh. Methods for use of upp and use ofpyrF to engineer insertions into microbe are described in Guss and Riley (US Published Patent Application No. 2021/0024965) and (Lipscomb et al., 2016, Applied and Environmental Microbiology, 82(14):4421-4428). A coding region present on a polynucleotide that is to be introduced into aMegasphaerasp. can be codon optimized for expression in theMegasphaerasp.

Methods for transformation ofMegasphaeraspp. include electroporation. Examples of producing electrocompetentMegasphaeraspp. are described in Example 1 and in Guss and Riley (US Patent Application No. 2021/0024965).

Methods of Using RecombinantMegasphaera

The present disclosure also includes methods of using a recombinantMegasphaera. In one embodiment, a method includes using a recombinantMegasphaerato produce butyrate and/or hexanoate. The method includes incubating a recombinantMegasphaeraunder conditions suitable for fermentation. Typically, the medium used includes lactate as the primary carbon source, and in one embodiment, the sole carbon source.

In one embodiment, the method can include further processing of the butyryl-CoA and/or hexanoyl-CoA product. For instance, if the recombinantMegasphaerainclude an exogenous coding region encoding a bifunctional aldehyde-alcohol dehydrogenase, such as adhE2, butyryl-CoA is converted to butanol (Guss and Riley, US Patent Application No. 2021/0024965). Alternatively, if the recombinantMegasphaerainclude an exogenous coding region encoding an acyl-CoA reductase, such as acr, hexanoyl-CoA is converted to hexanaldehyde, and if the recombinantMegasphaeraalso includes an exogenous coding region encoding an alcohol dehydrogenase, such as adh, hexanaldehyde is converted to hexanol.

Exemplary Aspects

The invention is defined in the claims. However, below there is provided a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

Aspect 1. A recombinantMegasphaeramicrobe genetically modified to (i) consume a greater amount of acetate, (ii) produce a greater amount of butyrate, hexanoate, or a combination thereof, than a comparable control microbe, or (iii) increase carbon flux to butyryl-CoA and/or hexanoyl-CoA than a comparable control microbe, or a combination thereof, wherein the recombinantMegasphaeramicrobe comprises a mutation of a CoA-transferase coding region, a glyoxalase coding region, or a lyase coding region.

Aspect 3. The recombinantMegasphaeramicrobe of Aspect 1 or 2, wherein theM. elsdeniiis a modified ATCC 25940.

Aspect 4. The recombinantMegasphaeramicrobe of any one of Aspects 1-3, wherein the mutation of the CoA-transferase coding region comprises a deletion of the CoA transferase coding region.

Aspect 5. The recombinantMegasphaeramicrobe of any one of Aspects 1-4, wherein the CoA-transferase coding region encodes a propionyl-CoA transferase.

Aspect 6. The recombinantMegasphaeramicrobe of any one of Aspects 1-5, wherein propionate production by the recombinantMegasphaeramicrobe is undetectable.

Aspect 7. The recombinantMegasphaeramicrobe of any one of Aspects 1-6, wherein the propionyl-CoA transferase coding region is MELS_0742, MELS_0464, MELS_1631, or MELS_1130, or a combination thereof.

Aspect 8. The recombinantMegasphaeramicrobe of any one of Aspects 1-7, wherein the recombinantMegasphaeramicrobe comprises a mutation of at least 1, at least 2 propionyl-CoA transferase coding regions selected from MELS_0742, MELS_0464, and MELS_0034, or mutation of all 3 propionyl-CoA transferase coding regions.

Aspect 9. The recombinantMegasphaeramicrobe of any one of Aspects 1-8, wherein the mutation of the glyoxalase coding region comprises a deletion of the glyoxalase coding region.

Aspect 10. The recombinantMegasphaeramicrobe of any one of Aspects 1-9, wherein the glyoxalase coding region is MELS_0743.

Aspect 11. The recombinantMegasphaeramicrobe of any one of Aspects 1-10, wherein the mutation of the lyase coding region comprises a deletion of the lyase coding region.

Aspect 12. The recombinantMegasphaeramicrobe of any one of Aspects 1-11, wherein the lyase coding region is MELS_0745.

Aspect 13. The recombinantMegasphaeramicrobe of any one of Aspects 1-12, wherein the increase of butyrate or hexanoate is at least 2-fold greater than the comparable control microbe.

Aspect 14. The recombinantMegasphaeramicrobe of any one of Aspects 1-13, wherein the increase of acetate consumption is at least 2-fold greater than the comparable control microbe.

Aspect 15. A recombinantMegasphaeramicrobe comprising a mutation of a pyrF coding region.

Aspect 16. The recombinantMegasphaeramicrobe of any one of Aspects 1-15, wherein the mutation is a deletion of at least a portion of the pyrF coding region.

Aspect 17. The recombinantMegasphaeramicrobe of any one of Aspects 1-16, wherein theMegasphaeramicrobe isM. elsdenii.

Aspect 18. The recombinantMegasphaeramicrobe of any one of Aspects 1-17, wherein theM. elsdeniiis a modified ATCC 25940.

Aspect 19. The recombinantMegasphaeramicrobe of any one of Aspects 1-18, wherein the pyrF coding region is MELS_RS04415.

Aspect 20. A method for increasing carbon flux to acetoacetyl-CoA, comprising: incubating a recombinantMegasphaeramicrobe with lactate as a carbon source under conditions suitable for replication, wherein the carbon flux to acetoacetyl-CoA in the recombinantMegasphaerais at a level greater than a comparable control, and, wherein the recombinantMegasphaeramicrobe comprises a mutation of a CoA-transferase coding region, a glyoxalase coding region, or a lyase coding region a mutation of a CoA-transferase.

Aspect 21. A method for producing butyrate, hexanoate, or combination thereof, comprising: incubating a recombinantMegasphaeramicrobe with lactate as a carbon source under conditions suitable for replication, wherein the recombinantMegasphaeraproduces butyrate, hexanoate, or combination thereof at a level greater than a comparable control, wherein the recombinantMegasphaeramicrobe comprises a mutation of a CoA-transferase coding region, a glyoxalase coding region, or a lyase coding region a mutation of a CoA-transferase.

Aspect 22. A method for genetically engineering aMegasphaera, comprising: providing the recombinantMegasphaeraof any one of Aspects 1-19, transforming the recombinantMegasphaerawith a plasmid comprising a pyrF coding region and a mutagenic cassette, wherein the mutagenic cassette of the plasmid comprises a marker flanked by DNA sequences, wherein the DNA sequences are selected to result in homologous recombination between the plasmid and two regions of DNA present in the recombinantMegasphaerathat flank a coding region targeted for mutation; and incubating the transformed recombinantMegasphaeraunder conditions suitable for positive selection of the transformed recombinantMegasphaeraand counter selection of the transformed recombinantMegasphaerato select for those that have lost the pyrF coding region, wherein the transformed recombinantMegasphaeraidentified by the positive and counter selection comprise a mutation of the targeted coding region.

Aspect 23. The method of Aspect 22, wherein the mutagenic cassette of the plasmid further comprises attachment sites flanking the marker, wherein the attachment sites are between the marker and the DNA sequences, and wherein the attachment sites are identified by a recombinase that can promote recombination between the two attachment sites and deletion of the marker located between the attachment sites.

Aspect 24. The method of Aspect 22 or 23, wherein the mutation of the targeted coding region comprises a deletion of the targeted coding region.

EXAMPLES

Methods for Engineering Deletions inMegasphaera: Deletion of upp Gene

A coding sequence (CDS) of anM. elsdeniiis referred to herein using one of two locus tag identifiers. One locus tag identifier has the prefix “MELLS_” followed by the number of the CDS in theMegasphaera elsdeniistrain DSM 20460 draft genome, GenBank accession HE576794.1. The second locus tag identifier has the prefix “MELS_RS” followed by the number of the CDS in theMegasphaera elsdeniistrain DSM 20460 complete sequence, GenBank accession NC_015873.1.

Deletion of MELS_2191, a uracil phosphoribosyltransferase (upp), in theM. elsdeniichromosome. To construct a deletion of MELS_2191 in theM. elsdeniiATCC 25940 genome (Hatmaker, 2019), plasmid pLAR151 was constructed by Gibson assembly (Table 1) via an intermediate plasmid, pLAR147. The pBC1 origin of replication was amplified from the pBC1 plasmid (DeRossi, 1992) cloned in place of pIM13 in the shuttle plasmid pMTL82151 (Heap, 2009), resulting in plasmid pLAR147. Then, 490 bp each that are DNA regions upstream and downstream of the upp gene (MELS_2191) were amplified and cloned into the MCS of pLAR147, resulting in the plasmid pLAR151. Additionally, a point mutation was inadvertently obtained in the pBC1 origin of replication, potentially rendering it non-functional. All PCR amplifications were performed using Phusion Master Mix (Thermo Fisher). The plasmid pLAR151 was transformed intoE. colistrain AG4157 (Table 1) which expresses two methyltransferases and their corresponding specificity subunits (MELS_0051-0052, MELS_1615-1616) fromM. elsdeniicloned into theE. colichromosome. Electrocompetent cells ofM. elsdeniiATCC 25940 were prepared (Guss 2021), and methylated plasmid DNA was subsequently isolated and used to transformM. elsdeniiATCC 25940 according to the transformation procedure of Guss 2021. Transformants were selected on RCM (HIMEDIA) agar plates with 5 μg/mL thiamphenicol (TM) incubated for 72 hours. Colonies were picked into RCM (BD Difco) with 5 μg/mL TM and incubated overnight. The liquid cultures were passaged into RCM (HIMEDIA) liquid cultures and subsequently plated in RCM (BD Difco) with 20 μg/mL 5-fluorouracil. The plates were incubated overnight, and colonies were streaked on RCM plates. Single colonies were picked into RCM (HIMEDIA) and PCR screened for the chromosomal deletion of upp (FIGS.3A and3B).

Results

Deletion of theM. elsdeniiuracil phosphoribosyltransferase gene allows counter-selection of the wild type allele using 5-fluorouracil. Uracil phosphoribosyltransferase (upp) converts the uracil analogue, 5-fluorouracil (5-FU), to a toxic product, fluorodeoxyuridylate (HdUMP, Singh 2015). To test whetherM. elsdeniiwas sensitive to 5-FU, cells were grown in liquid medium with 5-200 μg of 5-FU and found to be sensitive to 5 μg/mL 5-FU. Growth of the wild type strain on 5-FU selecting resistance also resulted in spontaneous mutations in this gene, indicating that it is responsible for conversion of 5-FU to FdUMP. A deletion of the uracil phosphoribosyltransferase (upp, MELS_2191) in theM. elsdeniichromosome resulted in a strain, AG5855, that is resistant to 50 μg/ml 5-FU. This chromosomal deletion allowed for the counter-selection of plasmids containing a copy of the wild type allele. This is the first counter-selection strategy developed inM. elsdenii.

Methods for Engineering Deletions inMegasphaera: Deletion of a Putative Propionyl-CoA Transferase Gene

Deletion of a putative propionyl CoA transferase (MELS_0742) from theM. elsdeniichromosome. Plasmid pLAR179 was constructed via Gibson assembly. The plasmid's backbone was amplified from plasmid pMTL85141 (Heap, 2009) to linearize the plasmid, excluding the cat gene. MELS_2191, the uracil phosphoribosyltransferase, was amplified from theM. elsdeniichromosome and was cloned in place of the cat gene for the purposes of counter-selection. Additionally, a cassette containing ˜800 bp upstream and downstream of MELS_0742 flanking a cat gene driven by the promoter region of MELS_0747 (PMELS_0747) and PhiC31 attB/P sites, was synthesized by Twist Bioscience. Subsequently, the cassette was amplified and cloned into the final construct downstream of the upp gene. The plasmid was transformed intoE. colistrain AG4157 (Table 1), and isolated. Electrocompetent cells ofM. elsdeniiATCC 25940 were prepared (Guss 2021), and methylated plasmid pLAR179 was subsequently isolated and used to transform AG5855 (M. elsdeniiATCC25940 Δupp). Colonies were selected on RCM agar plates containing 5 μg/mL thiamphenicol and incubated at 37° C. for 72 hours. Colonies were picked into liquid medium containing RCM medium (Difco) with 5 μg/mL TM, incubated overnight, and then plated on RCM (Difco) with 5 μg/mL TM and 50 μg/mL 5-fluorouracil for counter-selection of the plasmid. Plates were incubated for 48 hours at 37° C. and single colonies were picked into RCM (Difco) liquid medium with 5 μg/mL TM and screened for the marker-replacement (FIGS.4A and4B). Three single colony purifications were needed to generate a pure culture of the deletion from the initial merodiploid.

Results

Deletion of a putative propionyl-CoA reductase gene results in loss of propionate production and decreased valerate production.

There are nine annotated propionate-CoA transferases, (ptc) inM. elsdenii. We performed RNAseq and proteomic analysis on wild typeM. elsdenii, and it revealed that expression of MELS_0742 was highest during growth on lactic acid, and MELS_1130 was not detectable. To investigate the role of the most highly expressed of these, MELS_0742, a deletion of this gene was constructed in the AG5855 (Δupp) background strain, generating strain JWME04. The plasmid designed to generate this deletion contained a cat gene with upstream and downstream homology to MELS_0742 with ΦC31 attachment sites flanking the cat gene. The cat gene allowed selection of marker replacement events and the ΦC31 attachment sites allows subsequent removal of the cat gene for subsequent engineering. Growth of AG5855 (Δupp) and JWME04 (Δupp Δpct::phiC31 attB-cat-ΦC31 attB) was compared to the wild type during growth on both glucose and lactate (FIG.5). While both strains grew better on glucose, growth of the deletion strains was indistinguishable from wildtype suggesting that these deletions had no effect on growth on either glucose or lactate.

Evaluation of Fatty Acid Production and Carbon Substrate Usage

To investigate the effect MELS_0742 on organic acid production, High Performance Liquid Chromatography (HPLC) analysis was performed on cells grown in either glucose or lactate. As shown inFIGS.6A and6B, there was a complete loss of propionate production and a significant reduction in valerate production in the strain lacking MELS_0742. M. elsdeniiATCC 25940 wild type,M. elsdeniiATCC 25940 Δupp (strain AG5855), andM. elsdeniiATCC 25940 MELS_0742::ΦC31-cat-ΦC31 (strain JWME04) were grown in 5 mL RCM (HiMedia)+5 μg/mL thiamphenicol, if necessary, overnight. 50 μL of each strain was added to Balch tubes containing 10 mL of modified RCM with lactate and, separately, with glucose. Each strain was cultured in duplicate for 72 hours at 37° C. Samples were taken at 24-hour intervals, optical densities measurements taken, and fermentation products were quantified using HPLC. Lactate, glucose, acetic acid, butyric acid, valeric acid, propionic acid, (and octanoic acid) were quantified on Agilent 1260 infinity series HPLC with the Aminex-HPX-87H column (Bio-Rad). The mobile phase was 5 mM sulfuric acid. The column was heated at 65° C., the flow rate was 0.6 mL/min, and the chromatograph was visualized using an RI detector.

Seed cultures ofM. elsdeniiATCC 25940 were grown, inoculated and grown to stationary phase in 500 mL RCM. Competent cells were prepared at room temperature, and the washes were performed with a 250 mM sucrose, 10% glucose solution. Electrocompetent cells (20 μL) were electroporated with 1 μg of DNA. A 1 mM cuvette was used and electroporated with a square wave at 1200 v and 1.5 ms using a Bio-Rad GenePulser. After electroporation, cells were recovered in 1 mL RCM (BD Difco) and incubated for 3 hours. Cells were then plated in molten RCM+1.5% agar, and, once the agar solidified, they were incubated for 2-3 days at 37° C. in sealed boxes in an anaerobic chamber.

Deletion of pyrF inMegasphaera elsdeniiResults in Resistance to 5-FOA, Allowing Selection and Counter Selection of Genetic Markers and Facilitates Strain Construction

The native ability to condense acetyl-CoA groups to efficiently generate C4 to C8 compounds makesMegasphaera elsdeniia compelling platform for the production of fuels and chemicals from lactate and plant carbohydrates. Our overall objective is to developM. elsdeniias a platform for the conversion of lignocellulosic biomass sugars and organic acids into longer chain alcohols such as hexanol as well as other valuable chemicals. While progress has been made in developing basic genetic tools in this strain methods for DNA transformation rely on in vivo methylation of DNA in a strain ofE. colithat contains two methyltransferases fromM. elsdenii. A deletion of pyrF that allows counter selection of plasmids containing the wild type allele is constructed as shown inFIG.7.

Defined Medium forMegasphaera

A defined medium for any strain ofMegasphaerawas developed. A defined medium allows for mass balance analysis and manipulation of carbon and nitrogen sources to study and manipulate increased production of organic acids. This defined medium is also useful for the selection of uracil prototrophy, making pyrF both a selectable and a counter-selectable marker. The defined medium is produced by combining the following:

CITATIONS

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.