Consolidated ch4 and co2 conversion by methanotrophic bacteria

Abstract

Methanotrophic bacteria (methanotrophs) are well known for their unique capacity to utilize CH4 as a carbon and energy source, acting as the primary biological sink of this greenhouse gas in nature. These organisms have also garnered interests for biotechnology applications targeting conversion of natural gas or anaerobic digestion-derived biogas to valuable products, including single cell protein, liquid fuels derived from methanotroph fatty acids, and valuable chemical precursors. There are a subset of methanotrophs that use both CH4 and CO2 as carbon sources. However, improvements in carbon assimilation, flux, and conversion efficiency to target molecules is needed to advance this potentially transformative biotechnology. Disclosed herein are engineered methanotroph strains with increased co-expression of varying combinations of native CO2-associated enzymes, including RubisCO, inorganic carbon transporters, and five carbonic anhydrase isoforms previously shown to be essential for methanotroph cultivation.

Description

CONSOLIDATED CH4 AND CO2 CONVERSION BY METHANOTROPHIC BACTERIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 511 ,732, filed July 3, 2023, which is hereby incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a sequence listing filed in ST.26 format entitled “921402-2020 Sequence Listing” created on June 18, 2024, having 82,903 bytes. The content of the sequence listing is incorporated herein in its entirety.BACKGROUND OF THE INVENTION

[0003] Methanotrophic bacteria (methanotrophs) are well known for their unique capacity to utilize CH4as a carbon and energy source, acting as the primary biological sink of this greenhouse gas in nature. These organisms have also garnered interests for biotechnology applications targeting conversion of natural gas or anaerobic digestion-derived biogas to valuable products, including single cell protein, liquid fuels derived from methanotroph fatty acids, and valuable chemical precursors. There are a subset of methanotrophs that use both CH4and CO2as carbon sources. However, improvements in carbon assimilation, flux, and conversion efficiency to target molecules is needed to advance this potentially transformative biotechnology.SUMMARY OF THE INVENTION

[0004] Disclosed herein are engineered methanotroph strains with increased coexpression of varying combinations of native CO2-associated enzymes, including RubisCO, inorganic carbon transporters, and five carbonic anhydrase isoforms previously shown to be essential for methanotroph cultivation (Figure 3).

[0005] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF FIGURES

[0006] Figures 1 A and 1 B show M. capsulatus CA knock-out strains show growth defects. Figure 1A shows DNA agarose gel confirming replacement of wild-type (WT) locus with antibiotic resistance cassette. Figure 1 B shows growth of WT and CA knock-out strains after 10 days cultivation on NMS solid medium.

[0007] Figure 2A and 2B show M. capsulatus CA overexpression strains do not alter CO2evolution / consumption. Figure 2A shows quantitative real-time PCR evaluating CA transcription in CA-overexpressing strains. Figure 1 B shows the ratio of CO2evolved compared to CH4 consumed in serum vial cultures of overexpressing strains.

[0008] Figure 3 shows genes to be overexpressed in M. capsulatus to improve CO2assimilation. Genes encoding the ribulose bisphosphate carboxylase / oxygenase (RubisCO) enzyme (cbbLS) and its associated chaperone (cbbQ) and activase (cbbX) proteins will be coordinately expressed with genes encoding an inorganic anionic transporter (sulP) and one each or combinations of five carbonic anhydrases (caa1-5). A total of 10 engineered strains will evaluated for improved CO2assimilation capacity compared to wild-type. Strain biomass production and gas consumption capacity will be quantified in serum vials supplied with 20% CH4in air.

[0009] Figures 4A to 4E show M. capsulatus expresses five carbonic anhydrase (CA) isoforms. Figure 4A is a phylogenetic tree of the five annotated CAs encoded by the M. capsulatus Bath genome. Locus tags are indicated in parentheses. Figure 4B shows CA activity measured in whole-cell lysates in the presence (+) or absence (-) of the CA inhibitor acetazolamide (AZM). Relative transcription of the CA isoforms determined by qRT-PCR during M. capsulatus cultivation in (Fig. 4C) serum vials with 20% CH4 in air or (Fig. 4D) a gas reactor supplied with a continuous mixture of 20% CH4 / 2% CO2in air. Figure 4E shows relative CA transcription a a continuous gas reactor 3h hours after CO2 depletion from the gas mixture. Data in Figs. 4B-4E represent the mean ± SEM from at least two independent experiments (n=3-6). ** p < 0.01.

[0010] Figures 5A to 5E show M. capsulatus is hypersusceptible to carbonic anhydrase inhibition. Figure 5A shows bacterial growth in liquid NMS medium with the acetazolamide (AZM) CA inhibitor compared to an untreated control. Figure 5B shows survival of M. capsulatus after exposure to 100pM AZM. Figure 5C shows bacterial growth of AZM-adapted cells in the presence of AZM compared to naive and untreated controls. Collective (Fig. 5D) or individual (Fig. 5E) CA transcription in ten AZM-adapted transformants compared to wild-type controls was evaluated using qRT-PCR. The data represent the mean ± SEM from at least two independent experiments (n=3-10). *** p < 0.001.

[0011] Figures 6A to 6C show CA-deficient M. capsulatus exhibit defective growth. Figure 5A shows confirmation of individual CA knock-out strains via PCR. CA genes were replaced with antibiotic resistance cassettes via marker-exchange mutagenesis and knock-out (A) strains were confirmed via PCR compared to wild-type (WT). Figure 5B shows comparisonof colony formation one week after incubation of a dilution plate of equivalent WT and CA knock-out strains (CA1-5). Figure 5C shows bacterial growth of wild-type and ACA2, ACA3, and ACA5 knock-out strains ectopically expressing a respective CA from a replicative plasmid. We were unable to complement the ACA1 and ACA4 strains. Data in C represent the mean ± SEM from at two independent experiments (n=4).

[0012] Figures 7A to 7C show CA overexpression boosts M. capsulatus growth. Figure 7A shows CA isoforms were overexpressed from the pCAHOI broad-host-range expression vector. Individual CA transcription in overexpression strains was compared to wild-type (WT) via qRT-PCR. Figure 7B shows bacterial growth of wild-type (WT) and CA overexpressing strains in serum vials supplied with 20% CH4 in air. Figure 7C shows the ratio of CO2evolved to CF consumed by WT and CA overexpression strains to reach mid logarithmic growth (CD600 ~ 0.5) determined by gas chromatography. The data represent the mean ± SD (Fig. 7A) or SEM (Figs. 7B,7C) from at least two independent experiments (n=2-6). * p < 0.05. ** p < 0.01.

[0013] Figures 8A to 8C show CA overexpression improves Methylococcus carbon conversion efficiency. Figure 8A shows high-resolution cell growth quantification and Figure 8B shows biomass yield calculated using the CO2evolved / CH4consumed ratio of wild-type (WT) and strains overexpressing CA2 (pCA2) or CA3 (pCA3) alone or in combination (pCA2-3). Figure 8C shows mixed cultures consisting of WT M. capsulatus and either pCA2, pCA3, or pCA2-3 were cultivated for 48h to evaluate bacterial fitness. The ratio of engineered strain (pCAx) to WT after 48h was compared to the ratio of the inoculum by replica dilution plating on NMS solid medium with (pCAx only) or without kanamycin (WT + pCAx).DETAILED DESCRIPTION

[0014] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0015] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0016] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0017] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0018] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0019] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0020] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. 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, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0021] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describingparticular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.Definitions

[0022] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0023] As used herein, the term “recombinant” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that has at least one genetic alternation or has been modified by the introduction of an exogenous nucleic acid, or refers to a cell that has been altered such that the expression of an endogenous nucleic acid molecule or gene can be controlled, where such alterations or modifications are introduced by genetic engineering. Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins or enzymes, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, or other functional disruption of the cell's genetic material. Such modifications include, for example, coding regions and functional fragments thereof for heterologous or homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Genetic modifications to nucleic acid molecules encoding enzymes, or functional fragments thereof, can confer a biochemical reaction capability or a metabolic pathway capability to the recombinant cell that is altered from its naturally occurring state.

[0024] As used herein, the term “host bacterium” or “host” refers to a microorganism (e.g., methanotrophic bacterium) that has not yet been genetically modified as described herein.

[0025] As used herein, the term “methanotrophic bacterium” refers to a methylotrophic bacterium that has the ability to oxidize methane as its sole carbon and energy source.

[0026] As used herein, the term “Ci substrate” or “Ci compound” refers to any organic compound that lacks a carbon to carbon bond. Ci substrates include methane, methanol, formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide, methylated amines (e.g., methylamine, dimethylamine, trimethylamine, etc.), methylated thiols, methyl halogens (e.g., bromomethane, chloromethane, iodomethane, dichloromethane, etc.), and cyanide.

[0027] As used herein, “exogenous” means that the referenced molecule (e.g., nucleic acid) or referenced activity (e.g., enzyme activity or membrane transport) is introduced into a host methanotrophic bacterium by genetic engineering. The molecule can be introduced, for example, by introduction of a nucleic acid into the host genetic material such as by integration into a host chromosome or by introduction of a nucleic acid as non-chromosomal geneticmaterial, such as on a plasmid. When the term is used in reference to expression of an encoding nucleic acid, it refers to introduction of the encoding nucleic acid in an expressible form into the host methanotrophic bacterium. When used in reference to an enzymatic or protein activity, the term refers to an activity that is introduced into the host reference bacterium. Therefore, the term “endogenous” or “native” refers to a referenced molecule or activity that is present in the host bacterium. The term “chimeric” when used in reference to a nucleic acid refers to any nucleic acid that is not endogenous, comprising sequences that are not found together in nature. For example, a chimeric nucleic acid may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences that are derived from the same source, but arranged in a manner different than that found in nature. The term “heterologous” refers to a molecule or activity that is derived from a source other than the referenced species or strain whereas “homologous” refers to a molecule or activity derived from the host bacterium. Accordingly, a methanotrophic bacterium comprising an exogenous nucleic acid as provided in the present disclosure can utilize either a heterologous or homologous nucleic acid or both.

[0028] It is understood that when more than one exogenous nucleic acid is included in a bacterium that the more than one exogenous nucleic acid refers to the referenced encoding nucleic acid or protein activity, as discussed above. It is also understood that such more than one exogenous nucleic acid can be introduced into the host bacterium on separate nucleic acid molecules, on a polycistronic nucleic acid molecule, on a single nucleic acid molecule encoding a fusion protein, or a combination thereof, and still be considered as more than one exogenous nucleic acid. Similarly, it is understood that more than two exogenous nucleic acid molecules can be introduced into a host bacterium in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or enzymatic activities refers to the number of encoding nucleic acids or the number of protein activities, not the number of separate nucleic acid molecules introduced into the host bacterium.

[0029] As used herein, “nucleic acid”, also known as polynucleotide, refers to a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acids include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), both of which may be single or double stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semisynthetic DNA.

[0030] As used herein, “transformation” refers to the transfer of a nucleic acid (e.g., exogenous nucleic acid) into the genome of a host bacterium, resulting in genetically stable inheritance. Host bacteria containing the transformed nucleic acids are referred to as “recombinant” or “non-naturally occurring” or “genetically engineered” or “transformed” or “transgenic” bacteria.Methanotrophic Bacteria

[0031] In certain embodiments, methanotrophic bacteria are genetically engineered to express exogenous CO2-associated enzymes, including RubisCO, inorganic carbon transporters, and carbonic anhydrase isoforms. Methanotrophic bacteria have the ability to oxidize methane as a carbon and energy source. Methanotrophic bacteria are classified into three groups based on their carbon assimilation pathways and internal membrane structure: type I (gamma proteobacteria), type II (alpha proteobacteria, and type X (gamma proteobacteria). Type I methanotrophs use the ribulose monophosphate (RuMP) pathway for carbon assimilation whereas type II methanotrophs use the serine pathway. Type X methanotrophs use the RuMP pathway but also express low levels of enzymes of the serine pathway. Methanotrophic bacteria are grouped into several genera: Methylomonas, Methylobacter, Methylococcus, Methylocystis, Methylosinus, Methylomicrobium, Methanomonas, and Methylocella. Methanotrophic bacteria include obligate methanotrophs, which can only utilize Ci substrates for carbon and energy sources, and facultative methanotrophs, which naturally have the ability to utilize some multi-carbon substrates as a sole carbon and energy source. Facultative methanotrophs include some species of Methylocella, Methylocystis, and Methylocapsa (e.g Methylocella Silvestris, Methylocella palustris, Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystis bryophila, and Methylocapsa aurea KYG), and Methylobacterium organophilum (ATCC 27,886). Exemplary obligate methanotrophic bacteria include: Methylococcus capsulatus Bath strain, Methylomonas 16a (ATCC PTA 2402), Methylosinus trichosporium OB3b (NRRL B-11 ,196), Methylosinus sporium (NRRL B-11 , 197), Methylocystis parvus (NRRL B-11 , 198), Methylomonas methanica (NRRL B-11 ,199), Methylomonas albus (NRRL B-11 ,200), Methylobacter capsulatus (NRRL fil l , 201), Methylomonas flagellata sp AJ-3670 (FERM P-2400), Methylacidiphilum infernorum and Methylomicrobium alcaliphilum 20Z.

[0032] In some embodiments, the disclosed enzymes can be expressed in any methanotrophic strain for coordinated CH4and CO2assimilation. However, in methanotrophs that do not natively express these enzymes, the phosphoribulokinase may also need to be expressed.

[0033] A selected methanotrophic host bacteria may also undergo strain adaptation under selective conditions to identify variants with improved properties for production. Improved properties may include increased growth rate, yield of desired products, and tolerance of likely process contaminants.

[0034] Each of the methanotrophic bacteria of this disclosure may be grown as an isolated pure culture, with a heterologous organism(s) that may aid with growth, or one or more different strains of methanotrophic bacteria may be combined to generate a mixed culture.Recombinant Methanotrophic Bacteria

[0035] Provided in the present disclosure are recombinant methanotrophic bacteria that may be produced by introducing (e.g., by transformation) into the host bacteria at least one expressible exogenous nucleic acid encoding an exogenous RubisCO, an exogenous inorganic carbon transporter, and one or more an exogenous carbonic anhydrases. Alternatively, if a selected methanotrophic host bacterium exhibits endogenous expression of one of more RubisCO, inorganic carbon transporter, or carbonic anhydrases genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient component(s) to achieve the desired capability. Additionally, a host methanotrophic bacterium may possess other genetic modifications conferring it with other desirable properties, unrelated to CO2utilization. For example, a host methanotrophic bacterium may possess genetic modifications conferring high growth, tolerance of contaminants or particular culture conditions, ability to metabolize additional carbon substrates, or ability to synthesize desirable products or intermediates.

[0036] Polypeptide sequences and encoding nucleic acids for proteins, protein domains, and fragments thereof described herein may also include natural and recombinantly engineered variants. A nucleic acid variant refers to a nucleic acid that may contain one or more substitutions, additions, deletions, insertions, or may be or comprise fragment(s) of a reference nucleic acid. A variant nucleic acid may have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference nucleic acid, as long as the variant nucleic acid can still perform its requisite function or biological activity in the CO2utilization pathway. A variant polypeptide may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a reference protein, as long as the variant polypeptide can still perform its requisite function or biological activity (e.g., membrane transport).

[0037] These variants may have improved function and biological activity (e.g., higher enzymatic activity or improved specificity for substrate) than the parent (or wildtype) protein. Due to redundancy in the genetic code, nucleic acid variants may or may not affect amino acid sequence. A nucleic acid variant may also encode an amino acid sequence comprising one or more conservative substitutions compared to a reference amino acid sequence. A conservative substitution may occur naturally in the polypeptide (e.g., naturally occurring genetic variants) or may be introduced when the polypeptide is recombinantly produced. A conservative substitution is where one amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art would expect that the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, and / or the amphipathic nature of the residues, and is known in the art. Amino acid substitutions, deletions, and additions may be introduced into a polypeptide using well-known and routinely practiced mutagenesis methods (see, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY 2001). Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired. Deletion or truncation variants of proteins may also be constructed by using convenient restriction endonuclease sites adjacent to the desired deletion. Alternatively, random mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis may be used to prepare polypeptide variants (see, e.g., Sambrook et al., supra). Variant nucleic acids may be naturally occurring or genetically engineered.

[0038] Nucleic acids encoding CO2utilization pathway components may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, restriction enzyme sites, multiple cloning sites, other coding segments, and the like.

[0039] Differences between a wild type (or parent or reference) nucleic acid or polypeptide and the variant thereof, may be determined by methods routinely practiced in the art to determine identity, which are designed to give the greatest match between the sequences tested. Methods to determine sequence identity can be applied from publicly available computer programs. Computer program methods to determine identity between two sequences include, for example, BLASTP, BLASTN (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990), and FASTA (Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988). The BLASTfamily of programs is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.

[0040] Assays for determining whether a polypeptide variant folds into a conformation comparable to the non-variant polypeptide or fragment include, for example, the ability of the protein to react with mono- or polyclonal antibodies that are specific for native or unfolded epitopes, the retention of ligand-binding functions, the retention of enzymatic activity (if applicable), and the sensitivity or resistance of the mutant protein to digestion with proteases (see Sambrook et al., supra). Polypeptides, variants and fragments thereof, can be prepared without altering a biological activity of the resulting protein molecule (i.e., without altering one or more functional activities in a statistically significant or biologically significant manner). For example, such substitutions are generally made by interchanging an amino acid with another amino acid that is included within the same group, such as the group of polar residues, charged residues, hydrophobic residues, and / or small residues, and the like. The effect of any amino acid substitution may be determined empirically merely by testing the resulting modified protein for the ability to function in a biological assay, or to bind to a cognate ligand or target molecule.Codon Optimization

[0041] Expression of recombinant proteins is often difficult outside their original host. For example, variation in codon usage bias has been observed across different species of bacteria (Sharp et al., 2005, Nucl. Acids. Res. 33:1141-1153). Over-expression of recombinant proteins even within their native host may also be difficult. In certain embodiments, at least one nucleic acid encoding a CO2utilization pathway component that is to be introduced into host methanotrophic bacteria according to any of the embodiments described herein is codon optimized to enhance protein expression in the methanotrophic bacteria. Codon optimization refers to alteration of codons in genes or coding regions of nucleic acids for transformation of a methanotrophic bacterium to reflect the typical codon usage of the host bacteria species without altering the polypeptide for which the DNA encodes. Codon optimization methods for optimum gene expression in heterologous hosts have been previously described (see, e.g., Welch et al., 2009, PLoS One 4:e7002; Gustafsson et al., 2004, Trends Biotechnol. 22:346-353; Wu et al., 2007, Nucl. Acids Res. 35:D76-79; Villalobos et al., 2006, BMC Bioinformatics 7:285; U.S. Patent Publication 2011 / 0111413; U.S. Patent Publication 2008 / 0292918; disclosure of which are incorporated herein by reference, in their entirety). One, two, three, or more nucleic acids encoding CO2utilization pathway components may be codon optimized. For example, wherein a recombinant methanotrophic bacterium comprises two glycerol utilization pathway components, one nucleic acid molecule (e.g., glycerol kinase) may be codon optimized, while the secondnucleic acid molecule (e.g., glycerol-3-phosphate dehydrogenase) is not codon optimized, or vice versa. Alternatively, all nucleic acids encoding CO2utilization pathway components may be codon optimized.Transformation Methods

[0042] Any of the recombinant methanotrophic bacteria described herein may be transformed to comprise at least one exogenous nucleic acid to provide the host bacterium with a new or enhanced activity (e.g., enzymatic activity) or may be genetically modified to remove or substantially reduce an endogenous gene function using a variety of methods known in the art.

[0043] Transformation refers to the transfer of a nucleic acid (e.g., exogenous nucleic acid) into the genome of a host bacterium, resulting in genetically stable inheritance. Host bacteria containing the transformed nucleic acid molecules are referred to as “non-naturally occurring” or “recombinant” or “transformed” or “transgenic” bacteria.

[0044] Expression systems and expression vectors useful for the expression of heterologous nucleic acids in methanotrophic bacteria are known.

[0045] Electroporation of Ci metabolizing bacteria has been previously described in Toyama et al., 1998, FEMS Microbiol. Lett. 166:1-7; Kim and Wood, 1997, Appl. Microbiol. Biotechnol. 48:105-108; Yoshida et al., 2001 , Biotechnol. Lett. 23:787-791 , and US2008 / 0026005.

[0046] Bacterial conjugation, which refers to a particular type of transformation involving direct contact of donor and recipient cells, is more frequently used for the transfer of nucleic acids into C1 metabolizing bacteria. Bacterial conjugation involves mixing “donor” and “recipient” cells together in close contact with each other. Conjugation occurs by formation of cytoplasmic connections between donor and recipient bacteria, with unidirectional transfer of newly synthesized donor nucleic acid molecules into the recipient cells. A recipient in a conjugation reaction is any cell that can accept nucleic acids through horizontal transfer from a donor bacterium. A donor in a conjugation reaction is a bacterium that contains a conjugative plasmid, conjugative transposon, or mobilized plasmid. The physical transfer of the donor plasmid can occur through a self-transmissible plasmid or with the assistance of a “helper” plasmid. Conjugations involving Ci metabolizing bacteria have been previously described in Stolyar et al., 1995, Mikrobiologiya 64:686-691 ; Motoyama et al., 1994, Appl. Micro. Biotech. 42:67-72; Lloyd et al., 1999, Archives of Microbiology 171 :364-370; and Odom et al., PCT Publication WO 02 / 18617; Ali et al., 2006, Microbiol. 152:2931-2942.

[0047] Expression of heterologous nucleic acids in Ci metabolizing bacteria is known in the art (see, e.g., U.S. Pat. No. 6,818,424, US2003 / 0003528). Mu transposon basedtransformation of methylotrophic bacteria has been described (Akhverdyan et al., 2011 , Appl. Microbiol. Biotechnol. 91 :857-871). A mini-Tn7 transposon system for single and multicopy expression of heterologous genes without insertional inactivation of host genes in Methylobacterium has been described (US2008 / 0026005).

[0048] Various methods for inactivating, knocking-out, or deleting endogenous gene function in C1 metabolizing bacteria may be used. Allelic exchange using suicide vectors to construct deletion / insertional mutants in slow growing C1 metabolizing bacteria have also been described in Toyama and Lidstrom, 1998, Microbiol. 144:183-191 ; Stolyar et al., 1999, Microbiol. 145:1235-1244; Ali et al., 2006, Microbiology 152:2931-2942; Van Dien et al., 2003, Microbiol. 149:601-609.

[0049] Suitable homologous or heterologous promoters for high expression of exogenous nucleic acids may be utilized. For example, U.S. Pat. No. 7,098,005 describes the use of promoters that are highly expressed in the presence of methane or methanol for heterologous gene expression in C1 metabolizing bacteria. Additional promoters that may be used include deoxy-xylulose phosphate synthase methanol dehydrogenase operon promoter (Springer et al., 1998, FEMS Microbiol. Lett. 160:119-124); the promoter for PHA synthesis (Foellner et al. 1993, Appl. Microbiol. Biotechnol. 40:284-291); or promoters identified from native plasmid in methylotrophs (EP296484). Non-native promoters include the lac operon Plac promoter (Toyama et al., 1997, Microbiology 143:595-602) or a hybrid promoter such as Ptrc (Brosius et al., 1984, Gene 27:161-172). In certain embodiments, promoters or codon optimization are used for high constitutive expression of exogenous nucleic acids encoding glycerol utilization pathway enzymes in host methanotrophic bacteria. Regulated expression of an exogenous nucleic acid in the host methanotrophic bacterium may also be utilized. In particular, regulated expression of exogenous nucleic acids encoding glycerol utilization enzymes may be desirable to optimize growth rate of the non-naturally occurring methanotrophic bacteria.

[0050] Methods of screening are disclosed in Brock, supra. Selection methods for identifying allelic exchange mutants are known in the art (see, e.g., U.S. Patent Publication No. 2006 / 0057726, Stolyar et al., 1999, Microbiol. 145:1235-1244; and Ali et al., 2006, Microbiology 152:2931-2942.Culture Methods

[0051] A variety of culture methodologies may be used for the recombinant methanotrophic bacteria described herein. For example, methanotrophic bacteria may be grown by batch culture and continuous culture methodologies. In certain embodiments, the culturesare grown in a controlled culture unit, such as a fermentor, bioreactor, hollow fiber membrane bioreactor, or the like.

[0052] A classical batch culturing method is a closed system where the composition of the media is set at the beginning of the culture and not subject to external alterations during the culture process. Thus, at the beginning of the culturing process, the media is inoculated with the desired methanotrophic bacteria and growth or metabolic activity is permitted to occur without adding anything to the system. Typically, however, a “batch” culture is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Within batch cultures, cells moderate through a static lag phase to a high growth logarithmic phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in logarithmic growth phase are often responsible for the bulk production of end product or intermediate in some systems. Stationary or post-exponential phase production can be obtained in other systems.

[0053] The Fed-Batch system is a variation on the standard batch system. Fed-Batch culture processes comprise a typical batch system with the modification that the substrate is added in increments as the culture progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed- Batch systems is difficult and is therefore estimated on the basis of the changes of measureable factors, such as pH, dissolved oxygen, and the partial pressure of waste gases. Batch and Fed- Batch culturing methods are common and known in the art (see, e.g., Thomas D. Brock, Biotechnology: A Textbook of Industrial Microbiology, 2ndEd. (1989) Sinauer Associates, Inc., Sunderland, Mass.; Deshpande, 1992, Appl. Biochem. Biotechnol. 36:227, herein incorporated by reference in its entirety).

[0054] Continuous cultures are “open” systems where a defined culture media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in logarithmic phase growth. Alternatively, continuous culture may be practiced with immobilized cells where carbon and nutrients are continuously added and valuable products, by-products, and waste products are continuously removed from the cell mass. Cell immobilization may be performed using a wide range of solid supports composed of natural and / or synthetic materials.

[0055] Continuous or semi-continuous culture allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limited nutrient, such as the carbon source or nitrogen level, at a fixed rate and allow all other parameters to modulate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to media being drawn off must be balanced against the cell growth rate in the culture. Methods of modulating nutrients and growth factors for continuous culture processes, as well as techniques for maximizing the rate of product formation, are well known in the art, and a variety of methods are detailed by Brock, supra.

[0056] Culture media must contain suitable carbon substrates for the methanotrophic bacteria. A culture media may comprise CO2as the sole carbon source for the non-naturally occurring methanotrophic bacteria as described herein. Alternatively, a culture media may comprise two or more carbon substrates (mixed carbon substrates). For example, the culture media may comprise a mixture of CO2and methane.Gene sequences

[0057] Disclosed herein is a methanotroph engineered to express an exogenous RubisCO enzyme and it associated chaperone and activase proteins. In some embodiments, the RubisCO enzyme is encoded by a gene from a Methanotrophic bacteria. For example, the RubisCO enzyme can be encoded by a gene from a Methylococcus, Methylocaldum, Methylocapsa, Methylocella, Methyloferula, or Methylacidiphilum bacteria.

[0058] In some embodiments, the RubisCO enzyme is encoded by the MCA_RS13440- RS13450 genes from a Methanotrophic bacteria. Therefore, in some embodiments, the RubisCO enzyme is encoded by the nucleic acid sequence: ATGGCTGTCAAAACATACAACGCGGGCGTCAAGGAATACCGCGAAACCTACTGGGACCCG AACTACACTCCCGCCGACACCGATCTGCTGGCGGTCTTCAAGATCACCCCTCAGCCGGGT GTGCCGCGCGAAGAAGCCGCCGCCGCCGTGGCCGCGGAATCGTCGACCGGCACCTGGA CCACCGTCTGGACCGACCTGCTGACCGACCTCGACTATTACAAGGGCCGCGCCTACCGGA TCGAGGACGTGCCCGGCCAGGACGAACAGTTCTACGCCTTCATCGCCTATCCCATCGACC TGTTCGAGGAGGGCTCCGTCGTCAACGTGTTCACCTCGCTGGTGGGCAACGTGTTCGGCT TCAAGGCCGTGCGCGGCCTGCGCCTGGAAGACGTGCGCTTCCCCCTCGCCTACGTGATG ACCTGCGGCGGCCCGCCCCACGGCATCCAGGTGGAGCGCGACATCATGAACAAGTACGG CCGTCCGTTGCTCGGCTGCACCATCAAGCCCAAGCTGGGCCTGTCGGCCAAGAACTACG GCCGCGCCGTGTACGAGTGCCTCAGAGGCGGCCTGGACTTCACCAAGGACGACGAGAACGTCAACAGCCAGCCGTTCATGCGCTGGCGCCAGCGCTTCGACTTCGTGATGGAGGCGATCGAGAAGGCCGAGGCCGAAACCGGCGAGCGCAAGGGCCACTACCTCAACGTGACCGCCCCGACTCCGGAAGAGATGTACAAACGTGCCGAGTACGCCAAGGAAATCGGCGCGCCCATCATCATGCACGACTTCATCACCGGGGGCTTCTGCGCCAACACGGGTCTCGCCAACTGGTGCCGCAACAACGGCATGCTGCTGCACATCCACCGCGCCATGCACGCCGTGATGGACCGCAATCCCAACCACGGCATCCACTTCCGCGTCTTCACCAAGATGCTGCGCCTTTCTGGCGGCGACCATCTGCACACCGGCACCGTGGTCGGCAAGCTGGAAGGCGACCGCCAGGCCACCCTGGGGTGGATCGACCTGCTGCGCGAACGCAGCATCAAGGAAGACCGCAGCCGCGGCATCTTCTTCGACCAGGAATGGGGCGCCATGCCCGGCGTGTTCGCGGTCGCCTCCGGCGGCATCCACGTCTGGCACATGCCGGCACTGCTTTCGATCTTCGGCGACGACGCGGTGTTCCAGTTCGGTGGCGGCACCCTGGGCCATCCTTGGGGCAACGCGGCCGGCGCCGCTGCCAACCGCGTGGCGCTGGAAGCCTGCGTGGAGGCCCGCAACGAAGGCCGCCAGCTCGAGAAGGAAGGCAAGGAAATCCTGACCGAAGCCGCCAAGAGCAGCCCGGAACTCAAGGCCGCAATGGAGACCTGGAAGGAAATCAAATTCGAGTTCGACACCGTCGACAAGCTCGACGTGGCCCACCGGTAAGCCTCAAGAGGATTCGGGAAATACCATGAGCGATATGCAAGACTACAAATCCAGCCTGTCCGACAGCGGCTCGCGCAAGTTCGAGACTTTCTCCTACCTGCCGCCGATGAACCCGGAGAAGATCCGCCGGCAGGTGGAATACATCGTCAGCCGCGGCTGGAATCCGGCCATCGAACATACCGAGCCGGAAAACGCCTTCGACCACTACTGGTACATGTGGAAACTGCCGATGTTCGGCGAGACCGACGTCGATGCGATCCTGGCCGAGGCCGAGGCCTGCCACAAGGCGCACCCCAACAACCACGTCCGACTGGTCGGCTACGACAACTTCAGGCAGACCCAGGGCGCCGCCATGGTGATCTATCGGGGGCCGTCGGTCTGACTCTGCGGTAACCCCGGGAACGCCTGGAAGCCCCTGTCCGCCCGGCGGATCGGGGCTTCTTCATGGACAACATCCAGAAGCATTCAGTCCGCACCTTCGTTGCGAGGAGGTTCTTCGATGTCCAGCACAGAGCAATACCTGATTCAAGAACGACCCTATTACCGCGCCACCGCCGACGAAGTCGAGCTGTTCGAGGCCGCCTATGCCGCGCGCATGCCGGTGATGCTCAAGGGCCCGACCGGCTGCGGCAAGACCCGGTTCGTGGAATACATGGCCTGGAAGCTGAAGCGCCCGCTGATCACGGTGGCCTGCAACGAAGACATGACGGCCTCGGACCTGGTCGGCCGCTTCCTGCTCGACGCCAGCGGCACCCGCTGGCAGGACGGCCCCCTCACCCTGGCCGCCCGCATCGGCGCCATCTGCTATCTCGACGAGGTCGTGGAGGCCCGCCAGGACACCACCGTGGTCATCCACCCCCTGACCGATCACCGCCGCACCCTGCCGCTGGACAAGAAAGGCGAGCTGGTGCATGCCCACCCGGATTTCCAGTTGGTGATTTCCTACAATCCCGGCTACCAGAACCTGCTGAAGGACCTCAAGCAATCGACCAAGCAGCGCTTCGGCGCGCTGGATTTCGGCTATCCCGAGACGGCTGTCGAGGTCGATGTGGTGTCGCACGAGACCGGCATCGATCCCAAGATCGCCGAGAAGCTGGTGCAGATCGCCCACCGCGCCCGGAACCTCAAGGGCCACGGCCTGGACGAAGGCATCTCGACCCGCTTGCTGGTCTATGCCGGCCATCTCATCGCCAAGGGTATCGAGCCCAGAGCGGCCTGCATGATGACGCTCGTCCGCCCCCTCACCGACGACC CGGATATGCGCGATACCCTCGACGCCGCCGTAGCGACGTTTTTCTGA (SEQ ID NO:1).

[0059] In some embodiments, the chaperone is cbbQ and is encoded by the MCA_ RS13250 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the RubisCO enzyme is encoded by the nucleic acid sequence: ATGTCCAGCACAGAGCAATACCTGATTCAAGAACGACCCTATTACCGCGCCACCGCCGAC GAAGTCGAGCTGTTCGAGGCCGCCTATGCCGCGCGCATGCCGGTGATGCTCAAGGGCCC GACCGGCTGCGGCAAGACCCGGTTCGTGGAATACATGGCCTGGAAGCTGAAGCGCCCGC TGATCACGGTGGCCTGCAACGAAGACATGACGGCCTCGGACCTGGTCGGCCGCTTCCTG CTCGACGCCAGCGGCACCCGCTGGCAGGACGGCCCCCTCACCCTGGCCGCCCGCATCG GCGCCATCTGCTATCTCGACGAGGTCGTGGAGGCCCGCCAGGACACCACCGTGGTCATC CACCCCCTGACCGATCACCGCCGCACCCTGCCGCTGGACAAGAAAGGCGAGCTGGTGCA TGCCCACCCGGATTTCCAGTTGGTGATTTCCTACAATCCCGGCTACCAGAACCTGCTGAAG GACCTCAAGCAATCGACCAAGCAGCGCTTCGGCGCGCTGGATTTCGGCTATCCCGAGACG GCTGTCGAGGTCGATGTGGTGTCGCACGAGACCGGCATCGATCCCAAGATCGCCGAGAA GCTGGTGCAGATCGCCCACCGCGCCCGGAACCTCAAGGGCCACGGCCTGGACGAAGGCA TCTCGACCCGCTTGCTGGTCTATGCCGGCCATCTCATCGCCAAGGGTATCGAGCCCAGAG CGGCCTGCATGATGACGCTCGTCCGCCCCCTCACCGACGACCCGGATATGCGCGATACC CTCGACGCCGCCGTAGCGACGTTTTTCTGA (SEQ ID NO: 10).

[0060] In some embodiments, the chaperone is cbbX and is encoded by the MCA_ RS13480 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the RubisCO enzyme is encoded by the nucleic acid sequence: TTGAGCGACTACCAAGAACATCTCGAACGACTCGATCCTCGCGTCAAGGACACGCTGGAG GCGAGCTTCCACGAAGCCGCACGGGTCATGTCGCCGGGCGCCCTCAAGAACTACATCGA GGGCGCGCGGGCCTTGTCGGAACTCGGGCGCGGCGCAGAGCTGGTGCTGGCCTATGTC CAGCAGATGCCGCTGGTGTGCAAGGAGGTCGGCGACGAGGTGATCGCGGACGTCGTCAG CGGCGTGATGAAACTGGCGTCCATGGTCAGCGGGGCGGTGATCGAGCGGCTGTTCGACA GCCTTCCCACCGCGGCCCGGCGGCTGGGCGACGGCGAACTTTTGCGGCAGTACCTCGGC CTGATCCACCAGCTTTCCGCCAAGGCACCGCGGGGCCTGCGACCGATGCTGGACCACCT CGACCCGCTGTTCGACAAACTGACCCTGGGGGGACTGCGGCGCTGGGCATTGTGGGGCG CTCAGGCCCATGCCCGCGACTTCGAGACCCTGGCAAAGTACTTCGCCCTGGAGACTGCCG ACAGCCTGGCGGTGCTGCAGCAGGAGCGGCGCGGCACCCTGTTCGTCGACACCCAGCGC AAGCTCAACTTCTACCTGCGCGCGCTGTGGGGCCGGGATTTCTACCTGCGCCCCACCTCT GGCGATTTCGAGACGCGCGAAGGCTACAAACCGTACATCGAACACGGGGTGATCTTCGTGCCCGATGCGTACGACGACTTCGGCCCGGTATCGGGCAAGGAACTCTACCGGGCAACCGC CGCCCATGCCGCCGCCCACCTGGCTTACACCATCCGCCCGCTGTCCGCGGAGCAACTGA CGCCGGTGCAGATGTTCCTGATCGGTCTGTTCGAGGACGCCCGCGTCGAGGCGCTGGCG ATCCGCGAATTCCCCGGGCTCAAACAGCTCTGGGCGCCTTTGTTCGAAGCCGCCCTCGGC AGCGACCGCAACGCCGACCCGATGGTACCCTGGCTGGAGCGGCTGGCTCACGCTCTGCT CGATGAACGGATAGGAGCCGACGATCCGATCGTGGCCGAGCTGCGCGAATCCTTCCATG CCGCGCTGGCCAAGGATCCGGCCGACAACCGGCTGAGCTGGGACCTGGGCGTCACCCTC TCCAACCGCATCAAGGACCGGTGGGGCCTGCCCTCGCTGCGCGTCCTGGAAAGCTTCGG CGTGCCGTACCGGGACGACAACCGCTTCCTCTGGACCTTCGGCGAGACCGAATGGGCCG AGGCCGACCACATCGCCGCCAGCCAGCGCCAGGTGCGCAGGAAGGTATCCCTGATGGAG ATGATCGACGAGATCGATTGCGAACTGGCCGGGGACGACGCCCAGGAAATCTGGACCCT GGAAACGCCGTTCTATCTCGATCAGGAAGGCTGCACCATCAACGAACTGGAAGGCAAGGA ACCGGTCAGCGACCCTTACCACTATCCGGAATGGGACTACCAGGTCCAGTTGCACCGCCC CAACTGGGCCACCGTGCTGGAGCGGCGCCAGGCCAAGGACGACCCGGCTCTCGTCGAAC GGGTCCTGCTCGAATACAAGCCCGTCGCCAGCCGCCTCAAGCACATCATCGACGCCCTGC AGCCGCAGGGTGTGATCCGCCAACGGCGGCGGGAAGACGGCGACGAGATCGACCTGAA CGCCGCCATCCGGGCCATGATCGACATCCGCATGGGCGAGATGCCCGACCCCCGCATCA ACATCCGCACGATCCGCAAGACCCGCGACCTCGCGGTACTGCTGCTGCTCGACTTGTCGG AATCCACCAACGAAACCTTGCCCGGCAGCGATCGGCCAATCATTCAGCTGGCACGCGAGG CCTGCACCCTCCTGGCCTGGGCCATCGACGGCATCGGCGATCCCTTCGCCATCCACGGC TTCGCCTCCGACGGCCGTCACGACGTGCAGTACTACCGCTTCAAGGATTTCGACCAGCCT TTCGACGACCAGGCCAAGTCCCGTATCGCCGGGATGAAGGGCGGGCTGTCCACCCGGAT GGGCGCCGCCATGCGGCATGCCGCGCACCATCTGCGACATCGGCCCCAGCAGAGGAAAT TGCTGCTCCTGGTCAGCGACGGCGAACCGGCCGACATCGACGAGCGCGACCCGCAGTAT CTCCGCCTCGACACCAAGAAGGCAGTCGAGGAACTGGCCTCCTCGGGCATCATGACCTAC TGCCTGACGCTGGACCCGGATGCCGACGCTTACGTCTCCCGGATTTTCGGCCCCAACCGC TATACGGTCGTGGACCACGTCCAGCGCCTGCCCGAACGGCTGCCCATGCTGTTCGCCGGCCTGACGGCCTGA (SEQ ID NO: 11).

[0061] Disclosed herein is a methanotroph engineered to express an exogenous Carbonic anhydrase 1 enzyme. In some embodiments, the Carbonic anhydrase 1 enzyme is encoded by the MCA_04475 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the Carbonic anhydrase 1 enzyme is encoded by the nucleic acid sequence: ATGTCACAGATTCTCAGCGAAGTACTGGCAGCCAACCAGGGCTATGCGTCCACTTTCGACA AGGGCGGTCTGCCCATGCCGCCCGGCAGGCGTTTCGCTATATTGACCTGCATGGACGCGCGCCTGGACCCGGCCAAGTATGCAGGTCTTAGCGAAGGCGATGCCCATGTCATCCGTAAC GCCGGCGGGCGTGCCAGCGACGATGCCATCCGTTCGCTGGTCATTTCCTACAAACTGCTG GGCACCCGCGAATGGTTCGTGATCCACCATACCGACTGCGGCATGGAAACCTTCACCAAC GAGATCATGAACGACCTCCTCGCCAGCAGTCTGAAGACCGCAACGATCGACGCGAACGGC TGGCACGACTGCTGTGAAGGTCCCGGCTGCACCGAGGGCAAGTACATCAATTGGCTGACG ATCGCGAACCAGCAGCAAAGCGTGCTGGAGGACGTGACACGCATCCGCCGTCATCCCCT GGTTCCCGCCGACATCCCGATCTACGGTTACATTTACGACGTCCGCACCGGCCGGCTGGT CGAAGTGCCAGAGGCCACGGCCGCGGGCAGAGCCGCTTAG (SEQ ID NO:2).

[0062] Disclosed herein is a methanotroph engineered to express an exogenous Carbonic anhydrase 2 enzyme. In some embodiments, the Carbonic anhydrase 2 enzyme is encoded by the MCA_07100 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the Carbonic anhydrase 2 enzyme is encoded by the nucleic acid sequence: ATGGTACGGCGAACCATTTGGGCCGCACTGGCCCTCATCTGTATCCTGAGTTTTTCGCGC CCGGCCGGAGCCGCGGGATGCCCAAGCGAACCCGACTGGTCCTACAGCGGCGCCAACG GCCCCAGCAACTGGAACACCCTGTTTCCCATTCAGTGCGGGACAGGTTCGCGGCAGTCTC CATTCGATATCGTCACCGCCAACGTCACCAAGGCCGACCTGCCCAAGCTTAGCTTTCACTA CAATGTCGCCGACGTGTTGTATCTCGACCACGACGATCAGGTCGCCTTCAACCACGGCAA CGGCAACTTCATAAAGATCGGGAAGGATCGCTTCGACCTTATCAACATCCACGAGCATACC CCGAGCGAATACACTCTAAACGGCCAACAGTTTCCGATGGAGATTCATCTCGTCCATCAGA ACAAGCAGACCGGGCAGCTCGCGGTGATTGGCGTCTTCGTCGTCACGGGCAAGGAAAAC AAGGGCGTCAAGGAGGCTCCGAGCTTCAACGACCCCTCGACGGTCGCTCTTAAGCTGACC GGGTTGATTCCCAAAAAGCGCAATTATGTGCGCTTCAACGGTTCCCTTACCACCCCAGGCG AAAACATCGCTCTGCCCAGATGCGCTGAAGGTGTGCTGTGGACGGAGATGCTGACGCCGA TCGCGATGTCCTCCGACCAGATCGCCGCTTTCCAGGACTCGGCCCAGGTCTGCTGGGGC ACCAGTATCACGAATCGTCCCCTCCAGCCCTCCAACAACAGGTTCCTGCTCTCGCCCAGG AACGTCGATCCGTGA (SEQ ID NO:3).

[0063] Disclosed herein is a methanotroph engineered to express an exogenous Carbonic anhydrase 3 enzyme. In some embodiments, the Carbonic anhydrase 3 enzyme is encoded by the MCA_08185 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the Carbonic anhydrase 3 enzyme is encoded by the nucleic acid sequence: GTGCAAGAGTCTGACAGCAAAGGCAAGGATGAATGCGAGGTCGAAGGCATCTGCTGCGAT CTCGAACGTTTCATTTCCGGCGTCAAAGGATTCAAGGAACGTTTCTACCGGCGTTACCCGG AACACATGCAGCAGCTCGTCGAGCGGGGGCAGCGGCCGGTCGCGCTCATGATCGCCTGC AGCGATTCGCGGGTCGATCCGGCTTTGCTCATGAACGTGGCGCCGGGCGATCTGTTCGTGGTGCGCAACGTCGCCAATCTCGTTCCGCCTTACCATGGCTCGTCCGCCCCCGATGGTGTA GGCGCAGCGCTGGAATATGCCGTCCGTCATCTCCATGTGCCCCATGTCATCGTCCTCGGT CACGCCCAGTGCGGTGGTATCAAGGCCTTGCTCGACATGGCGACGGGAGCCGAGTTCGA GAGCGACTTCATCGGCGAGTGGGTCTCGATGGCCATGGAAGCCTGCTGGCAGTATGTGC CGGATGCCAGCGGGGAGGGACTCAGACGGGTCTCACTGGAGCGGCTGAAAGACTACTCC TATCTGGTCGAGCGTGCGGCCATTCAAGGTTCGCTGGGGAATCTGCTGACCTATCCCTGG GTCCGGGAGGCCGTGGAGAAAGAGCGGCTTTTCCTGCAGGGCTGGTGGTTCGATCTGGA GTCTGGAGACCTGTGGGTCACCAGCGGCGACAACTCGCAGTTCTTGCCTGTGACCGATTG A (SEQ ID NO:4).

[0064] Disclosed herein is a methanotroph engineered to express an exogenous Carbonic anhydrase 4 enzyme. In some embodiments, the Carbonic anhydrase 4 enzyme is encoded by the MCA_13695 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the Carbonic anhydrase 4 enzyme is encoded by the nucleic acid sequence: ATGGCGATTCAGACCTACAATGGTATCCACCCCACACTGGGCCATAACGTATTCGTGGCG GAAAGCGCCTTCGTGGCGGGCGACGTGAGCCTGGGCGACGATGTTTCGATCTGGCCCTG CGTGGTCGCCCGCGGCGACGTCCACCGCATCGAAATCGGCCCCGCCACCAACATCCAGG ACGGCAGCGTGCTGCACGTCACCCAGCCCAGCGCCTTCAACGAAGCCGGATTCCCCTTGA TCGTCGGCGCCGGCGTCACCGTCGGCCACCGCGCCGTGCTGCACGGCTGCACCATCGG CGACCTGTGCCTGATCGGCATCGGCGCCATCGTCATGGACGGCGCCGTCATCGAGGACC GCGTCATGGTCGGTGCCGGTACCCTGGTCCCGCCGGGCAAGCGCCTGGAAAGCGGCTAC CTCTACGTCGGATCGCCCGTCAGACAGGCGCGGCCACTGAAGGACTCCGAACTCGAGTTC CTCGTCCATTCCCGCGACGGTTACGTCAGGCTAAAGGAGCGGTATTTGCGCGACGGTGTT ATGTTGTGA (SEQ ID NO:5).

[0065] Disclosed herein is a methanotroph engineered to express an exogenous Carbonic anhydrase 5 enzyme. In some embodiments, the Carbonic anhydrase 5 enzyme is encoded by the MCA_15330 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the Carbonic anhydrase 5 enzyme is encoded by the nucleic acid sequence: ATGGTACGGCGAACCATTTGGGCCGCACTGGCCCTCATCTGTATCCTGAGTTTTTCGCGC CCGGCCGGAGCCGCGGGATGCCCAAGCGAACCCGACTGGTCCTACAGCGGCGCCAACG GCCCCAGCAACTGGAACACCCTGTTTCCCATTCAGTGCGGGACAGGTTCGCGGCAGTCTC CATTCGATATCGTCACCGCCAACGTCACCAAGGCCGACCTGCCCAAGCTTAGCTTTCACTA CAATGTCGCCGACGTGTTGTATCTCGACCACGACGATCAGGTCGCCTTCAACCACGGCAA CGGCAACTTCATAAAGATCGGGAAGGATCGCTTCGACCTTATCAACATCCACGAGCATACC CCGAGCGAATACACTCTAAACGGCCAACAGTTTCCGATGGAGATTCATCTCGTCCATCAGAACAAGCAGACCGGGCAGCTCGCGGTGATTGGCGTCTTCGTCGTCACGGGCAAGGAAAAC AAGGGCGTCAAGGAGGCTCCGAGCTTCAACGACCCCTCGACGGTCGCTCTTAAGCTGACC GGGTTGATTCCCAAAAAGCGCAATTATGTGCGCTTCAACGGTTCCCTTACCACCCCAGGCG AAAACATCGCTCTGCCCAGATGCGCTGAAGGTGTGCTGTGGACGGAGATGCTGACGCCGA TCGCGATGTCCTCCGACCAGATCGCCGCTTTCCAGGACTCGGCCCAGGTCTGCTGGGGC ACCAGTATCACGAATCGTCCCCTCCAGCCCTCCAACAACAGGTTCCTGCTCTCGCCCAGG AACGTCGATCCGTGA (SEQ ID NO:6).

[0066] Disclosed herein is a methanotroph engineered to express an exogenous SulP inorganic anion transporter. In some embodiments, the SulP inorganic anion transporter is encoded by the MCA_07105 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the SulP inorganic anion transporter is encoded by the nucleic acid sequence: ATGGTCAAAAAGCATCTCGCGTACTATCTCGCACATCTCACCCAGGACATCCCCGCCGGC ATCGTGGTGTTCCTGGTGGCGCTCCCTCTCTGTCTGGGTATTGCGCTGGCGTCCGGCGCG CCGCTGCTTTCAGGCGTGGTTGCCGGCATCATCGGCGGACTGGTTGTCGCTTGGGCCAG CGGTTCGCAGTTGAGTGTGTCGGGCCCGGCCGCGGGCCTGACGGTCATCGTGCTGCAGG GTATCGAAAAGCTCGGCGGCTTCGAGCACTTTCTGCTCGCCCTGATCCTGGCGGGGCTGA TGCAGCTCGCTCTGGGCTTCCTCAAGGCGGGAACCATCGGCGCCTATTTCCCGTCGTCCG TCATCAAGGGCATGCTTTCGGCGATCGGCCTGATCTTGATTGCCAAACAGCTCCCCCACG CCGTCGGCTATGGCCGGGACATCCTGGGCGAGGAAACCTATCTGCCTCAGGACACGGAG GGCACTTTCTCGGAACTGATGCACGCCATGGACTCGATTTCCCCGGGCGCGACCGTCGTG AGCGCCATTGCCATCGTCATCATGGTGCTGTGGGAGTCCCGGCTTGTCCGTGCCGTCCCG TTGCTGGCGTGGATTCCGGGGCCGCTGGCGGCGATTGCCTGGGCGGTGGCGTTCAATCT CTCGATGGCGGGATCGTCTTGGGAGATCGCGCCGGAGCACATGGTCCAGCTTCCAGACAT CCGGGGAGTCGGTGATCTGGCCGGACGGCTCGTCTTTCCGGATTTCAGCCGGATCATGG ATCCGGCGGTGTACAGGGTGGCGTTCACGATTGCCGTCATTGCCAGCCTCGAAACCCTGC TCAGCTTGGAAGCAGTCGACAAGCTCGACCCCCTGAAGCGGGTGGCGCCGACCAACCGG GAGCTGAAGGCACAGGGTATCGGCAACCTGTTGGCCGGCTTGCTGGGCGGTCTACCGCT GACGGCGGTGATTGTGCGCTCGTCGGCCAACATCAATGCCGGCGGGCGTACCAAGGTGG CCTGCTTCATCCACGGCCTTCTACTGCTGGTAAGCGTGAGCTTCCTCGCCCGCTACCTCAA CCACATCCCTCTGGCCGTGCTGGCCGCGATCCTGCTGATGACGGGCTATAAACTGGTCAA GCCGGCCTTGGTGGCAGAGATGTACCACAAGGGCGTGAGCCAGTTTATTCCGTTCGCCGT CACGGTCATGGCGATACTGGCCACCGACCTGCTCATAGGGATCGCCGTCGGCATCGCCT GTGGCCTATACTACGTCGTCCGAGCGAATTTCCATGCCGCCATTTCTCTCACCCGCCATGG GAACCACTATCTGCTGCGACTGCGCAAAGACGTCTCGTTCCTGAACAAGGCACTGTTGCGCGAGCAACTGGACCAGGTCGAACCCGACAGCGAGCTGATCATCGACGGCACCTACGCGG AATTCGTGGATCAGGACATCCTCGAAACCATCGAGAACTTCGTCGAAGCCGCGCGCGACG ACCGTATCGTGGTTTATCTGAAAAATTTCAAAGCCGCGTACGCCGGCAGCACCGCCAAGAA CCGGCAAGACAACACCGATACCACCCCTTTGTACAGGATTTCCGGCTGA (SEQ ID NO:7).

[0067] In some embodiments, the SulP inorganic anion transporter is encoded by the MCA_12125 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the SulP inorganic anion transporter is encoded by the nucleic acid sequence:ATGAACACATCGACGATCGCCGATAGCATGCCGCTGGGCGGTATCGCCGGCTTGCAAGCC CACTGGAGAAACGACCTGGTCTCCGGCTTTCTGGTCTTCCTGATCGCTCTGCCGCTGTGC CTCGGCATCGCCATGGCTTCCGGCTTTCCCCCCATGGCCGGGATCATTACCGCGGTGGTG GGCGGTCTGCTGGTATCCCGTATCAACGGTTCCCACGTGACCATCAACGGCCCCGCAGCC GGGCTCATCGTGGTGATCCTCGCCGCGACGCAGACGCTGGGCCAGGGGGATGCGATGCA GGGCTATCGCTGTACCCTGGCTGCCATCGTGGTGGCTTCGGTGCTACAGATCGCGCTGG GCCTGTTCAAAGCGGGACGCCTCAACGCCTATTTCCCCGCCTCGGTGGTCCACGGCATGC TCGCCGCCATCGGCATCATCATCATGGCCAAGCAGATCCACGTCATGGTTGGCGTCAAAC CGGAAGCCGCCAGCGACGATCTCCTGGCGACGATCGCCGAGATCCCCCACAGCCTGCTC AATCCCAACCCGGAAATCGCGCTGATCGGCTCTGTGGGTCTTTTGATCCTCATCGCCTGGT CGCTGATACGGAGCCGCCATCTGAAGATGGTGCCGGCGCCGCTGCTGGTCGTGATCGCC GGGATGGCCCTCGGCAAGTATTTCGACCTGGATCATGAACATATTTATATGTTCCATCCCG ACAACCCCATTTTGCCGCACCATGAATATACCGTCGGCCCCAAGTTCCTGGTGACCATTCC GGAAGATTTCACTGCAGGCTTCTATTTCCCGGACTTTTCCAAAGTCTGGACGGCGGAGTTC TGGGGAGCCGTGATCAGCATCTGTCTGGTGGGCAGCCTGGAAACACTGCTCAGCGCGGT GGCGGTAGACAAGCTCGATCCCTATAAACGCTATTCCGACCTGAATCGTGATCTCACGGC CGTCGGGGTCGGGAATCTGCTTTGCGGCATGATCGGGGGACTGCCAATGATCGCGGAAA TCGTCCGCAGTTCGGCCAACATCACCAATGAAGCCAAGACCGGCTGGGCCAATTTCTTCC ACGGCGCTTTCCTGCTGCTGTTCGTCGCGCTGTTTCCGCACGTGATCCACGAGATTCCTCT GGCTTCGCTGGCGGCGCTGCTGGTTTACACCGGCTTCCGGCTGGCATCACCCCGGGAAT TCGCCAAAACCATGGACATCGGCTGGGAGCAGTTGACACTGTTCGTGGTGACCATCTTCG GGGTGCTTGCCACGGATCTCCTGGTCGGCGTCGGCATCGGCATCTTCGCCAAACTGGTCA TCCACATGCTGCGGGGCGTTCACCCCAAGCATCTGCTCAAGATTTCCTACCGCGTCGAAG AGCATCCGGCCGGCACCTTCGTGATCCATGTCGACGGCTCTGCGATCTTCTCCAATTTCAT CGCGCTCAAGAGCGAAGTCGCGGATCTGCCCGTGGGGAAAACGGTCATCTTCGACCTGTC GAAGGCCGATTTCATCGACCACACCGTCATGGAGTTCATCGACCATTTCCGGCACGACTAT GTGCAGCGACACGGCCGCTGCGAGATAAGGGGCCTCGAGTCCCACGTGCCGTATTCGAATCATCCGCTGGCCGCGAGAAAGCGCAAACCGCAGGCCAAGGCCGCCGACAAAGTGAAAC TCGGCGGGCAAACCGGCTGA (SEQ ID NO:8).

[0068] In some embodiments, the SulP inorganic anion transporter is encoded by the MCA_ RS07105 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the SulP inorganic anion transporter is encoded by the nucleic acid sequence: ATGGTCAAAAAGCATCTCGCGTACTATCTCGCACATCTCACCCAGGACATCCCCGCCGGC ATCGTGGTGTTCCTGGTGGCGCTCCCTCTCTGTCTGGGTATTGCGCTGGCGTCCGGCGCG CCGCTGCTTTCAGGCGTGGTTGCCGGCATCATCGGCGGACTGGTTGTCGCTTGGGCCAG CGGTTCGCAGTTGAGTGTGTCGGGCCCGGCCGCGGGCCTGACGGTCATCGTGCTGCAGG GTATCGAAAAGCTCGGCGGCTTCGAGCACTTTCTGCTCGCCCTGATCCTGGCGGGGCTGA TGCAGCTCGCTCTGGGCTTCCTCAAGGCGGGAACCATCGGCGCCTATTTCCCGTCGTCCG TCATCAAGGGCATGCTTTCGGCGATCGGCCTGATCTTGATTGCCAAACAGCTCCCCCACG CCGTCGGCTATGGCCGGGACATCCTGGGCGAGGAAACCTATCTGCCTCAGGACACGGAG GGCACTTTCTCGGAACTGATGCACGCCATGGACTCGATTTCCCCGGGCGCGACCGTCGTG AGCGCCATTGCCATCGTCATCATGGTGCTGTGGGAGTCCCGGCTTGTCCGTGCCGTCCCG TTGCTGGCGTGGATTCCGGGGCCGCTGGCGGCGATTGCCTGGGCGGTGGCGTTCAATCT CTCGATGGCGGGATCGTCTTGGGAGATCGCGCCGGAGCACATGGTCCAGCTTCCAGACAT CCGGGGAGTCGGTGATCTGGCCGGACGGCTCGTCTTTCCGGATTTCAGCCGGATCATGG ATCCGGCGGTGTACAGGGTGGCGTTCACGATTGCCGTCATTGCCAGCCTCGAAACCCTGC TCAGCTTGGAAGCAGTCGACAAGCTCGACCCCCTGAAGCGGGTGGCGCCGACCAACCGG GAGCTGAAGGCACAGGGTATCGGCAACCTGTTGGCCGGCTTGCTGGGCGGTCTACCGCT GACGGCGGTGATTGTGCGCTCGTCGGCCAACATCAATGCCGGCGGGCGTACCAAGGTGG CCTGCTTCATCCACGGCCTTCTACTGCTGGTAAGCGTGAGCTTCCTCGCCCGCTACCTCAA CCACATCCCTCTGGCCGTGCTGGCCGCGATCCTGCTGATGACGGGCTATAAACTGGTCAA GCCGGCCTTGGTGGCAGAGATGTACCACAAGGGCGTGAGCCAGTTTATTCCGTTCGCCGT CACGGTCATGGCGATACTGGCCACCGACCTGCTCATAGGGATCGCCGTCGGCATCGCCT GTGGCCTATACTACGTCGTCCGAGCGAATTTCCATGCCGCCATTTCTCTCACCCGCCATGG GAACCACTATCTGCTGCGACTGCGCAAAGACGTCTCGTTCCTGAACAAGGCACTGTTGCG CGAGCAACTGGACCAGGTCGAACCCGACAGCGAGCTGATCATCGACGGCACCTACGCGG AATTCGTGGATCAGGACATCCTCGAAACCATCGAGAACTTCGTCGAAGCCGCGCGCGACG ACCGTATCGTGGTTTATCTGAAAAATTTCAAAGCCGCGTACGCCGGCAGCACCGCCAAGAA CCGGCAAGACAACACCGATACCACCCCTTTGTACAGGATTTCCGGCTGA (SEQ ID NO:9).

[0069] In some embodiments, the disclosed enzymes can be expressed in any methanotrophic strain for coordinated CH4and CO2assimilation. However, in methanotrophsthat do not natively express these enzymes, the phosphoribulokinase may also need to be expressed. In some embodiments, the phosphoribulokinase is encoded by the MCA_ RS14965 gene from a Methanotrophic bacteria. Therefore, in some embodiments, the SulP inorganic anion transporter is encoded by the nucleic acid sequence:ATGTCCAAGAAACACCCCATCATCGCCATCACCGGTTCGTCCGGCGCGGGCACGACCACC GTAAAATGCGCCTTCGAGCATATTTTCTTCAGGCTCGGACTCAAACCACTGGTCATCGAGG GCGACAGCTTTCATCGCTATGACCGTGTGGAAATGCGTGCGCAGATCGACAAGGCCAGAC GTGAAGGACGTCATTTCAGCCATTTTTCGATCGAGGCCAACATCCTGGACGAGCTCGAAAA CGTTTTTCGGCACTACGGGGAAACGGGAACGGCTCGCCGCCGTTTTTATGTCCACAATGA AGCCGAGAGTCAGCGGCTGGGGGGTTACAAACCGGGAACCTTCACCCCATGGGAAGAAG TACCTGCAGGGACGGATCTGCTGTTTTACGAAGGTCTGCACGGAGGCGTGATCACCGACC GCATCAATGTCAGAAAATACGTCGATCTTCTCGTCGGCGTGGTTCCCATCGTCAATTTGGA ATGGATTCAAAAAATCCATCGCGATACCGCCGAACGCGGCTATAAGCCCGAGGACGTTAC GGAAACGATACTGCGCCGCATGGATGACTACGTCAAGGTCATCACCCCCCAATTTTCACAG ACCGACATCAATTTCCAGCGTGTCCCGACGGTCGATACTTCCAATCCGTTCATTGCCCGGG ATATTCCCACCCCGGATGAGAGTTTCGTCATCATCCGCTTCAAAGAGCCTGGCAAATTCAA CGTGGATTTCCCTTATCTCCTGGCCATGCTGCAAAACTCCTTCATGTCACGACATAATTCGA TCGTGATTCCGGGAGGCAAGATGGGACTCGCAATGGAGATCATTTTCCGGCCGATACTCG AACGCATGATGGCGGATCGTGAGAAAGGCTGA (SEQ ID NO: 12).Embodiments

[0070] Embodiment 1. A recombinant methanotrophic bacterium, comprising an exogenous polynucleotide comprising a gene encoding a ribulose-1 ,5-bisphosphate carboxylase / oxygenase (RubisCO) enzyme and an associated chaperone protein and an associated activase protein; an exogenous polynucleotide comprising a gene encoding a carbonic anhydrase, and an exogenous polynucleotide comprising a gene encoding a an inorganic carbon transporter, wherein each of the gene encoding the RubisCO enzyme, the gene encoding the carbonic anhydrase, and the gene encoding a the inorganic carbon transporter are operably linked to an expression control sequence.

[0071] Embodiment 2. The bacterium of embodiment 1 , wherein the RubisCO enzyme is cbbLs.

[0072] Embodiment 3. The bacterium of embodiment 2, wherein the RubisCO enzyme is encoded by the nucleic acid sequence SEQ ID NO:1.

[0073] Embodiment 4. The bacterium of any one of embodiments 1 to 3, wherein the chaperone protein is cbbQ.

[0074] Embodiment 5. The bacterium of embodiment 4, wherein the chaperone protein is encoded by the nucleic acid sequence SEQ ID NO:10.

[0075] Embodiment 6. The bacterium of any one of embodimentw 1 to 5, wherein the activase protein is cbbX.

[0076] Embodiment 7. The bacterium of embodiment 6, wherein the chaperone protein is encoded by the nucleic acid sequence SEQ ID NO:11 .

[0077] Embodiment 8. The bacterium of any one of embodiments 1 to 7, wherein carbonic anhydrase comprises carbonic anhydrase 1 (caa1), carbonic anhydrase 2 (caa2), carbonic anhydrase 3 (caa3), carbonic anhydrase 4 (caa4), carbonic anhydrase 5 (caa5), or any combination thereof.

[0078] Embodiment 9. The bacterium of embodiment 8, wherein the carbonic anhydrase is encoded by the nucleic acid sequence SEQ ID NO:2, 3, 4, 5, or 6.

[0079] Embodiment 10. The bacterium of any one of embodiments 1 to 9, wherein the inorganic carbon transporter is a SulP transporter.

[0080] Embodiment 11. The bacterium of embodiment 10, wherein the inorganic carbon transporter is encoded by the nucleic acid sequence SEQ ID NO:7 or 8.

[0081] Embodiment 12. The bacterium of any one of embodiments 1 to 11 , wherein the methanotrophic bacterium is a gammaproteobacterial or alphaproteobacterial methanotroph.

[0082] Embodiment 13. The bacterium of any one of embodiments 1 to 11 , wherein the methanotrophic bacterium is a Verrucomicrobia / Candidate phylum NC10 methanotroph.

[0083] Embodiment 14. The bacterium of any one of embodiments 1 to 11 , wherein the methanotrophic bacterium is selected from Methylococcus capsulatus, Methylomonas sp. 16A, Methylosinus trichosporium, Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus, Methylomonas flagellata, Methylacidiphilum infernorum, Methylomicrobium alcaliphilum Methylocella Silvestris, Methylocella palustris, Methylocella tundrae, Methylocystis daltona, Methylocystis bryophila, or Methylocapsa aurea.

[0084] Embodiment 15. The bacterium of any one of embodiments 1 to 11 , wherein the methanotrophic bacterium is further engineered to express a heterologous phosphoribulokinase.

[0085] Embodiment 16. The bacterium of embodiment 15, wherein the phosphoribulokinase is encoded by the nucleic acid sequence SEQ ID NO: 12.

[0086] Embodiment 17. The bacterium of any one of embodiments 1 to 16, wherein the gene encoding the RubisCO enzyme, the gene encoding the carbonic anhydrase, and the gene encoding a the inorganic carbon transporter are in the same expression vector.

[0087] Embodiment 18. The bacterium of embodiment 17, wherein the gene encoding the RubisCO enzyme, the gene encoding the carbonic anhydrase, and the gene encoding a the inorganic carbon transporter are operably linked to the same expression control sequence.

[0088] Embodiment 19. The bacterium of any one of embodiments 1 to 18, wherein each of the gene encoding the RubisCO enzyme, the gene encoding the carbonic anhydrase, and the gene encoding a the inorganic carbon transporter are integrated into the methanotrophic bacterium genome.

[0089] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1:

[0090] Methanotrophic bacteria (methanotrophs) can use methane (CH4) as both a carbon and energy source. These unique microbes are ubiquitous in terrestrial and aquatic ecosystems where they play a vital role in the biogeochemical cycling of CH4, a potent greenhouse gas (GHG). Due to their unique metabolism, methanotrophs are used industrially for the conversion of CH4-rich gas to valuable products, including single cell protein and polyhydroxyalkanoates (Nguyen, A., et al. Trends Biotechnol. 2021 39:381-396). Bacterial CH4assimilation is mediated by cytoplasmic or membrane-associated CH4monooxygenases (MMO), which use molecular oxygen to oxidize CH4to methanol (CH3OH). CH3OH is further oxidized to formaldehyde (CH2O) in the periplasm by calcium- or lanthanide-dependent CH3OH dehydrogenases. CH2O can enter central metabolism by 1) directly condensing with a C5 sugar in the ribulose monophosphate (RuMP) cycle, 2) conversion to methylene tetrahydrofolate (H4F) via methylene tetrahydromethanopterin- and H4F-C1 transfer pathways followed by condensation with glycine in the serine cycle, or 3) complete oxidation to carbon dioxide (CO2), which can be assimilated via autotrophic pathways. Methanotrophs are broadly categorized based on their route of CH2O metabolism, wherein gammaproteobacterial methanotrophs use the RuMP pathway, alphaproteobacterial methanotrophs use the serine cycle, and Verrucomicrobia / Candidate phylum NC10 methanotrophs use the ribulose-1 ,5-bisphophate carboxylase / oxygenase (RubisCO) and the Calvin cycle to reassimilate CH4-derived CO2. Notably, all aerobic methanotrophs completely oxidize a percentage (Gammaproteobacteria and Alphaprotebacteria) or all (Verrucomicrobia / Candidate phyla NC10) of assimilated CH4to CO2.

[0091] Methylococcus capsulatus str. Bath is a model methanotroph that has served to elucidate several fundamental aspects of methanotrophy, including structural elucidation of the particulate and soluble forms of MMO and characterization of the generalizable “copper switch” physiological response to copper starvation. M. capsulatus belongs to a phylogenetical distinct lineage of methanotrophs within the Methylococcales family of gammaproteobacterial methanotrophs (Type lb) that includes the Methylocaldum, Methylococcus, Methylogaea, Methylomagnum, and Methyloterricola genera (Orata, F. D., et al. Front. Microbiol. 2018 9:3162). These methanotrophs encode RubisCO, but, in contrast to Verricomicrobia, M. capsulatus derives its biomass from CH4-derived formaldehyde via the RuMP cycle and CO2via RubisCO (Henard, C. A., et al. Appl. Environ. Microbiol. 2021 87:e008812; Baxter, N. J., et al. Arch. Microbiol. 2002 177:279-289; Stanley, S. H., et al. Microbiol. 1982 128:2927-2935). Previous studies show that the M. capsulatus RubisCO is expressed and active, and that both the enzyme and CO2are essential for bacterial cultivation (Henard, C. A., et al. Appl. Environ. Microbiol. 2021 87:e008812).

[0092] Most RubisCO enzymes exhibit poor efficiency with low catalytic rate constants and substrate specificity (Erb, T. J., et al. Curr. Opin. Biotechnol. 2018 49:100-107). Thus, many autotrophic bacteria have developed inorganic carbon concentrating mechanisms (CCM) to improve CO2assimilation. On of the most characterized CCMs are carboxysomes in photoautotrophic cyanobacteria that is a proteinaceious “organelle” that houses RubisCO, a bicarbonate transporter, and carbonic anhydrases (CA). Carboxysomes have not been visualized in M. capsulatus electron micrographs and the genome does not contain genes that encode the carboxysome structural shell proteins. In microbes lacking carboxysomes, CAs can independently mediate a CCM by sequestering CO2. CAs are zinc metalloenzymes found in all domains of life that interconvert CO2and H2O to a H+and bicarbonate (HCO3). Bacteria typically encode one or more of the a-, [3- y-classes of carbonic anhydrase. In addition to CO2sequestration, CAs also play a role in regulating intracellular pH, energy production, and signaling (Supuran, C. T., et al. Metabolites 2017 7). CA activity can also enhance CO2assimilation by conversion to HCO3_, which does not readily diffuse from the cell. In turn, intracellular HCO3_can be converted back to CO2and increase RubisCO substrate availability. As such, CAs are being evaluated as a means to sequester atmospheric CO2and mitigate greenhouse gases (Effendi, S. S. W., et al. Process Biochemistry 2019 87:55-65). M. capsulatus encodes several CA isoforms, but their involvement in M. capsulatus CO2metabolism is unknown. In this study, the expression of the five distinct CAs encoded by M. capsulatus were evaluated and, using a genetics approach, their roles assessed in M.capsulatus CO2metabolism. M. capsulatus expresses all five CA isoforms during cultivation with CH4in serum vials. We observed that M. capsulatus growth is significantly inhibited by the CA inhibitor acetazolamide, underscoring the importance of CAs for M. capsulatus growth. Individual CA knock-out strains exhibited marked growth retardation, showing that the CAs play important, non-redundant roles in M. capsulatus metabolism. M. capsulatus strains overexpression CA isoforms did not show increased CO2consumption or biomass accumulation.Example 2:Materials and Methods

[0093] Bacterial Cultivation. Bacterial strains used in this study are shown in Table 1. M. capsulatus Bath cultures were routinely maintained with nitrate mineral salts (NMS) solid medium in stainless steel gas chambers supplied with 20% CH4in the gas phase as previously described (Tapscott et al. 2019). Strains were grown in 150 mL vials containing 10 mL of liquid NMS medium or 250 mL vials containing 20 mL of liquid NMS medium. After inoculation with plate-derived biomass or diluted seed culture, vials were crimped with gray butyl stoppers to create gas-tight seals. CH4was added to the headspace to reach a final CH4of 20% in air (vol / vol), and cultures were incubated at 37°C at 200 rpm orbital shaking. Continuous gas cultivation was performed using a custom mid-throughput gas fermentation reactor fitted with 150-mL, 38 mm Kimax cultivation tubes supplied with mixed gas via rotameters and stainless steel sparge stones. 100 mL cultures were inoculated with plate-derived biomass to an OD6oo of 0.1 and supplied with 20% CH4 / 2% CO2in air (vol / vol) at a flow rate of 1 volume gas / volume culture / min (vvm) premixed with gas-specific mass flow controllers. Culture samples (~100 pL) were periodically extracted from vials with a syringe to measure bacterial growth via dilution plating or spectrophotometrically at OD6oo using a Nanodrop spectrophotometer. High resolution bacterial growth was measured every 20 seconds by Cell Growth Quantifier (CGQ) optical sensors (Scientific Bioprocessing) to determine growth rates. The backscatter arbitrary units measured by the CGQ sensors were transformed to OD6oo using linear regression of measured initial and final OD6oo measurements.

[0094] Carbonic anhydrase knockout and overexpression strain construction. Primers used for cloning and genetic manipulations are shown in Table 2. All PCRs were performed with Q5 High-Fidelity 2X Master Mix following the manufacturer’s recommendations (New England Biolabs) and PCR annealing temperatures were determined using the NEB Tm Calculator tool. Plasmids were assembled using NEBuilder HiFi DNA Assembly Master Mix following the manufacturer’s vectoninsert molar ratio recommendations, but total reaction volumes were minimized and typically between 4-10 pL, depending on the amount of assembled fragments (New England Biolabs). M. capsulatus Bath carbonic anhydrase (MCA0910, MCA1080, MCA1422, MCA1665, and MCA2797) knock-out strains were created by amplifying 1000 bp DNA regions upstream and downstream flanking the genes of interest from genomic DNA and an Flp recombinase recognition target (FRT)-flanked gentamicin or kanamycin resistance cassette from pPS856 or pKD13, respectively. The 1 kb upstream-antibiotic resistance- 1 kb downstream was assembled with pK18mobpheS linearized via PCR using primers OCAH576 / 577 and transformed into chemically competent E. coli DH10b. Sequence-confirmed plasmids were then transferred into S17-1 E. coli via chemical transformation to mediate biparental conjugation with M. capsulatus as previously described (Nath S, et al. Methods Mol Biol 2022 2489:421-434). M. capsulatus transformants were selected on NMS containing 50 pg / mL kanamycin or 20 pg / mL gentamicin and 10 mM 4-chloro-phenylalanine to select for double-crossover events. Antibiotic- and 4-chloro-phenyalanine-resistant transformant colonies were screened by PCR to confirm the replacement of a CA locus with the antibiotic resistance cassette. The pK18mobpheS plasmid was generated following methods found in Ishikawa et al. (Ishikawa M, et al. Appl Environ Microbiol 2018 84). Here, a M. capsulatus mutated pheS gene (minimally codon-optimized to enable synthesis) encoding the a-subunit of phenylalanyl-tRNA synthetase containing two missense mutations (A306G and T252A) was synthesized as a gblock by Integrated DNA Technologies and assembled with pK18mobsacB linearized with primers oCAH572 / oCAH573 to replace sacB with the mutated pheS gene.

[0095] Overexpression plasmids and strains were created by amplifying each CA open reading frame from M. capsulatus Bath gDNA using the corresponding primers (oCAH169- OCAH178) and assembling with pCAHOI linearized via PCR with primers oCAH3 / oCAH4 to create pCA1-pCA5, placing the genes under transcriptional control of the anhydrotetracycline (aTc)-inducible Ptefpromoter. To generate the pCA2 / 3 plasmid for co-expression of CA2 and CA3, the CA3 ORF with its native ribosome binding site was amplified from gDNA with primers oCAH1431 / oCAH170 and assembled with pCA2 linearized with primers oCAH1424 / oCAH4, generating a CA2-CA3 operon. Overexpression constructs were then transferred to M. capsulatus Bath via biparental mating. Antibiotic-resistant M. capsulatus transformants were screened via colony PCR using plasmid-specific primers oCAH5 / oCAH6 and the resultant PCR amplicon sequence was verified via Sanger sequencing.

[0096] Phylogenetic analysis. CA sequences were Percent identity of each CA isoform was gathered using NCBI blastN across known type I and type II methanotroph families with the resulting heatmap generated in Graphpad Prism 9.

[0097] CA activity assay. Wildtype M. capsulatus Bath cultivated in 10 mL liquid NMS to OD6oo ~ 1 .0 were pelleted at 4080 x g for 10 minutes and the supernatant was removed viaaspiration. Cells were lysed using the B-PER bacterial protein extraction reagent (ThermoFisher) and total protein was quantified on a Nanodrop spectrophotometer using the Pierce 660nmProtein Assay Reagent (Thermo-Fisher) compared to known BSA standards. Lysates were normalized to 10 mg / mL protein in 10 mM Tris-HCI and 100 pg of lysate was used to determine CA activity following the Abeam Carbonic Anhydrase Colorimetric Activity Assay Kit protocol (Abeam). Briefly, cell lysate and kit reagents were added to microtiter plates and allowed to incubate for 10 minutes at room temperature covered / protected from light and then production of nitrophenol was monitored at A405nm every 30 seconds for 10 minutes using a BioTek Synergy Mx microplate reader. Nitrophenol was quantified via regression analysis compared to known nitrophenol standards which correlates to CA activity based on activity curves previously generated by the manufacturer. Acetazolamide (AZM) was added to a final concentration of 100 pM to inhibit CA activity in reactions where indicated.

[0098] AZM treatment. Wild-type or AZM-adapted M. capsulatus Bath were grown in liquid medium to OD6oo ~1 and diluted to OD6oo = 0.1 in fresh medium with or without AZM (Sigma-Aldrich). Cell death and / or growth was monitored via dilution plating and spectrophotometrically at OD6oo using a Nanoddrop spectrophotometer. % killing was calculated by comparing the colony forming units after treatment to colony forming units at T = 0.

[0099] CA transcription analysis via quantitative reverse-transcriptase PCR. CA transcription was determined in wild-type M. capsulatus cultivated in serum vials or a continuous gas bioreactor. Serum vial seed cultures were inoculated to OD6oo = 0.1 in liquid NMS from plate biomass and cultivated 24 h. Seed cultures were diluted in fresh medium to OD6oo = 0.01 (serum vials) or OD6oo = 0.1 (bioreactor). Serum vial cultivations were grown until mid-log phase (OD6oo = 0.5) for RNA extraction. To evaluate CA transcription in response to CO2 starvation, bioreactor cultures were cultivated with continuous 20% CH4 / 2% CO2in air for 3 h (CO2-replete) after which the CO2was removed from the gas composition (CO2-deplete) and cells were cultivated for an additional 3 h prior to RNA extraction. Cell cultures were mixed 5:1 with ice-cold 5% phenol / 95% ethanol and incubated for 20 minutes on ice prior to RNA isolation using the High Pure RNA Isolation Kit with optional on-column DNase treatment (Roche). Total RNA (1 pg) was used to generate cDNA using the Superscript Vilo IV reverse transcriptase kit following the manufacturer’s protocol (Thermo). Primers used for qPCR are listed in Table 2. Reactions were prepared using Powertrack SYBR Green master mix (Life Technologies) using recommended cycling conditions and amplification detection with a QuantStudio 3 qPCR system. Expression of each CA determined by cycle threshold (Ct) was normalized to the housekeeping gene rpoDand relative expression between targets or conditions was calculated using the AACt method (Panagiotou et al., 2009).

[0100] Gas Chromatography and biomass yield calculations. Wild-type M. capsulatus and overexpression strains (pCA1-pCA5) were grown overnight in liquid medium (OD6oo ~ 0.1) with 50 pg / mL kanamycin and 0.2 pg / mL aTc for CA induction, where applicable. Seed cultures were sub-cultured to OD6oo = 0.1 in fresh medium without antibiotics (to avoid any growth inhibition) and 0.2 pg / mL aTc for CA induction in serum vials, which were stoppered, sealed, and 20% CH4added to the headspace via syringe. The CH4and CO2composition in the headspace was determined 10 min after gas addition and every 24 h during methanotroph cultivation using an SRI 8610c gas chromatograph combined with an SRI S.S HayeSep D 6’ x 1 / 8” column, SRI on-column injector, and TCD and FID detectors (SRI Instruments). Runs were 3 minutes in length, isocratic at 50°C. Gases were quantified as % composition by comparison to known standards. % gas was converted to weight using the specific gravities of CH4(0.622 mg / mL) or CO2(1 .47 mg / mL) at 37°C. CH4uptake and CO2evolution rates per dry cell weight (DCW) were determined by converting OD6oo to DCW using a formula empirically determined by comparing OD6oo measurements to colony forming units during serum vial cultivation (OD6oo = 1 is equivalent to 0.254 ± 0.0249 g DCW / L).

[0101] Competitive Growth Index. Wild-type M. capsulatus and antibiotic-resistant CA overexpression strains were equally mixed / subcultured to OD6oo ~ 0.01 in NMS liquid medium and cultured for 48 h to late logarithmic phase. Serial dilutions of the cultures were replica plated on NMS and NMS containing 50 pg / mL kanamycin before and after cultivation, and colony forming units (CFUs) were enumerated after 1 week of incubation. Competitive indices were calculated by determining the ratio of CA-overexpression strain to wild-type cells after cultivation (output ratio) divided by the inoculum ratio (input ratio) using the following equation: output ratio [NMS Kn CFUs- (NMS CFUs - NMS Kn CFUs)] I input ratio [NMS Kn CFUs - (NMS CFUs - NMS Kn CFUs)].Results

[0102] M. capsulatus Bath encodes five carbonic anhydrase isoforms. Five CA-encoding genes are annotated in the M. capsulatus Bath genome: MCA0910 (CA1), MCA1080 (CA2), MCA1442 (CA3), MCA1665 (CA4), and MCA2797 (CA5). The genes encode a-(CA2), y-(CA5), and p-CAs (CA1 , clade D; CA3, clade A, CA4, clade B) with limited amino acid sequence identity (Figure 4A). Phylogenetic analyses indicate that [3-CA1 , [3-CA3, and y-CA5 are highly conserved in methanotrophic bacteria, and the CA phylogenetic trees are congruent with 16s- and pmoA-based methanotroph classifications. In contrast, a-CA2 and [3-CA4 isoform orthologsshare limited sequence identity (<30%) with other methanotroph CAs, while orthologs in non- methanotrophic bacteria with higher sequence identity (>50%) were identified, suggesting these isoforms may have been acquired via horizontal gene transfer. The phylogenetic analysis of the M. capsulatus a-CA2 shows that sequences with significant identity and similarity are abundant in caprophilic and autotrophic bacteria. Further, a-CA2, but not the other isoforms, contains a predicted Sec-specific signal sequence identified by SignalP (Teufel F, et al. Nat Biotechnol 2022 40:1023-1025), suggesting that this CA is secreted to the periplasm. CA activity can be detected in M. capsulatus whole-cell lysates (0.74 ± 0.05 mll / mg protein), which could be partially inhibited by the carbonic anhydrase inhibitor acetazolamide (Figure 4B).

[0103] Quantitative real-time PCR established that all CA isoforms were transcribed during bacterial cultivation in a serum vial with CH4as the only supplied carbon source and in a bioreactor with continuous CH4and CO2supply (Figures 4C and 4D). [3-CA4 showed the highest relative expression in serum vials, which was 9.3-fold higher than the lowest expressed y-CA5. Relative expression of [3-CA1 was 3.6-fold higher (p < 0.05) in a bioreactor culture compared to a serum vial, but expression of the other CA genes was comparable between these two growth conditions (Figure 5C). The difference in expression between the highest (P- CA1) and lowest (y-CA5) expressed CA genes was 32.7-fold in bioreactor-grown cells (Figure 4D). To evaluate whether M. capsulatus Bath regulates CA expression in response to CO2availability, we compared CA transcription in bioreactor-cultivated cells after removal of CO2from the gas stream (CO2-deplete) to those cultured with continuous 20% CH4and 2% CO2in air (CO2-replete) (Figure 4E). In support of the role of the CAs in CO2metabolism, transcription of all isoform genes was significantly increased within 3h after CO2starvation (CA1 2.6-fold; CA2 3.4-fold; CA3 3.8-fold; CA4 2.8-fold, and CA5 2.0-fold).

[0104] The carbonic anhydrase inhibitor acetazolamide inhibits M. capsulatus growth. The importance of CA activity for bacterial growth was initially tested by treating cells with increasing concentrations of AZM. AZM showed dose-dependent M. capsulatus growth inhibition with 100 pM AZM-treated cultures exhibiting a 3-day lag phase followed by growth with similar kinetics to untreated cells (open circles, Figure 5A). Dilution plating of AZM-treated cells supported that the CA inhibitor is bactericidal, killing 98% of the treated cells within 72 h after exposure (Figure 5B). However, a population of cells survived and began to expand after 96 h (Figure 5B), which was not due to AZM degradation given that AZM maintains its growth inhibitory effect after abiotic incubation in NMS medium at 37°C for up to 10 days (data not shown). We did not rule out the possibility that AZM was biologically consumed or degraded under our experimental conditions. However, the hyper resistance of AZM-treated cells to asubsequent exposure to AZM concentrations that completely inhibited growth of naive controls indicated that M. capsulatus adapts to overcome AZM-mediated toxicity (Figure 5C). To test whether the CAs may be related to the AZM adaptation, we quantified CA transcription in ten independent colonies from the AZM-adapted population and found that each strain showed increased overall CA expression ranging from 6.8- to 25.8-fold compared to collective wild-type CA expression levels (Figure 5D), linking AZM resistance to increased CA expression. However, there was variation between which CA isoforms were induced, and to what extent, in the adapted strains preventing a direct correlation of AZM resistance to a particular CA isoform (Figure 5E).

[0105] Carbonic anhydrase mutants exhibit defective growth. To assess the role of the individual CAs, five CA-deficient strains were constructed lacking each individual CA isoform via marker-exchange mutagenesis (ACA1 ::KnR, ACA2::GmR, ACA3::GmR, ACA4::KnR, ACA5::GmR). Antibiotic-resistant transformants were isolated on solid NMS medium and individual CA knockout strains were confirmed via PCR (Figure 6A). These CA knock-out transformants appeared after 4-8 weeks incubation compared to isogenic wild-type M. capsulatus Bath that form visible colonies within 1-2 weeks, underscoring that each CA isoform has a critical, non-redundant role in M. capsulatus metabolism and physiology. The extreme growth defects of the CA knock-out strains precluded reasonable growth comparison in liquid NMS medium; thus, we spotted dilution series of plate-derived wild-type and CA KO M. capsulatus strains on solid medium (Figure 6B). After 10 days of incubation, wild-type colonies were readily visible, but the CA KO strains had no observable growth, even in the undiluted spots (Figure 6B). A plasmid with tetracycline promoter (Ptet) / aTc-inducible expression of a CA was introduced into its respective CA KO background strain to confirm that the observed growth defects were attributed to removal of the CA and not unintended genetic defects. Growth defects were complemented by ectopic expression of CA2, CA3, and CA5 in strains pCA2 ACA2::GmR, pCA3 ACA3::GmR, and pCA5 ACA5::GmR, respectively (Figure 6C). However, we were unable to recover growth of ACA1 ::KnRand ACA4::KnRstrains, potentially due to dysregulated expression of CA1 or CA4 from the pCAHOI Ptefpromoter or off-target effects resulting from removal of the CA gene. An 86 bp intergenic region exists between CA1 and the downstream gene and CA4 appears to be the first gene in an operon with a downstream gene (the CA4 stop codon and downstream MCA1664 start codon are separated by 11 bp). However, the putative ribosomal binding site at the 3’ end of the CA4 open reading frame was maintained in the ACA4::KnR strain to ensure translation of the downstream gene transcript, but it’s possible that removal of the CA1 or CA4 sequences could disrupt regulatory elements that control expression of the downstream genesresulting in the observed growth defects. Collectively, the AZM toxicity towards M. capsulatus and the significant growth defects of the CA knock-out strains underscore that CA activity is critical for optimal M. capsulatus growth.

[0106] Carbonic anhydrase overexpression improves M. capsulatus growth and increases carbon conversion efficiency. The Ptet-inducible CA expression plasmids developed for complementation of the KO strains were also transferred to wild-type M. capsulatus to analyze the effect of each isoform on M. capsulatus growth and C1 gas consumption / evolution. Overexpression of each isoform was confirmed via qRTPCR, with each isoform being induced ~ 10-fold over baseline when transcribed from the Ptet promoter (Figure 7A). Growth analyses of the CA overexpression strains in serum vials with CPU supplied as the only carbon source showed that increased expression of CA2 (pCA2) or CA3 (pCA3) isoforms enhanced growth compared to wild-type cultures with basal level expression (Figure 7B). The growth enhancement of pCA2 and pCA3 was isolated to the early lag phase of growth since all CA overexpression strains had similar specific growth rates as wild-type M. capsulatus, ranging from 0.094-0.112 h-1during logarithmic growth. Also, the CA overexpression strains entered stationary phase at similar CD as wild-type M. capsulatus and accumulated comparable biomass over the duration of cultivation (Figure 7B). Since the positive effect of CA overexpression on growth was attributed to lag phase, we evaluated gas consumption / evolution dynamics within the first 48 h of cultivation, which is the time required for cells to reach mid- logarithmic growth / metabolic steady state under our growth conditions (Table 3). M. capsulatus and the pCAx strains consumed between 106 to 133 mmol CH4 / g DW with no statistical difference between the overexpression strains and wild-type cultures (Table 3). Wild-type M. capsulatus evolved 76 ± 8 mmol / g DW CO2 within 48 h, which is consistent with prior gas consumption / evolution measurements reported by this methanotroph (Leak DJ, et al. Appl Microbiol Biotechnol 1986 23:470-476). However, CA overexpression strain cultures showed an 8-21 mmol CO2 / g DW-1decrease in gas evolution, with pCA2 and pCA3 evolving 55 ± 7 and 56 ± 3 mmol CO2 / g DW-1, respectively (Table 3). As such, pCA2 and pCA3 evolved less CO2per CH4consumed (0.41 ± 0.05 and 0.45 ± 0.04 mol / mol, respectively) compared to wild-type (0.73 ± 0.10 mol / mol) (Figure 7C and Table 3). We did not measure significant differences in biomass yield from CH4between any of the pCAx overexpression strains and wild-type M. capsulatus, indicating that the amount of formaldehyde condensation with ribulose monophosphate as part of the RuMP pathway was unaltered by CA overexpression (Table 3). However, overexpression of CA1 , CA2, CA3, and CA5 variants significantly improved carbon conversion to biomass efficiency (CCE) when the CO2evolved / CH4consumed ratio was considered as the overallcarbon assimilated to biomass (Table 3). The pCA2 and pCA3 strains showed the best overall CCE (pCA2, 3.25 ± 0.57 mg DW / mol CO2 / mol CH4; pCA3, 3.09 ± 0.34 mg DW / mol CO2 / mol CH4) which correlated to 2.2- and 2.1-fold improvement in biomass yield compared to wild-type (1.47 ± 0.25 mg DW / mol CO2 / mol CH4), respectively (Table 3), consistent with the boosted growth observed in these strains (Figure 7A). Collectively, these results show that wild-type M. capsulatus and CA overexpression strains consumed comparable amounts of CH4and had similar biomass yield directly from CH4, but CA2 and CA3 overexpression decreased CO2accumulation in the serum vial headspace (Table 3). The decrease in CO2was not due to a decrease in complete CH4oxidation to CO2with associated higher formaldehyde flux to biomass, but rather is attributed to increased CO2re-uptake, which improved biomass yield of the CA overexpression strains (Figure 5).

[0107] The effect of CA2 and CA3 co-overexpression on M. capsulatus growth and gas consumption / evolution dynamics was next evaluated. High-resolution cell growth quantification supported that overexpression of both CA2 and CA3 improved growth compared to overexpression of either variant individually, with the pCA2-3 strain exhibiting a 14-hour decreased lag phase and reaching logarithmic growth earlier than wild-type (Figure 8A). Like the individual CA overexpression strains, pCA2-3 did not accumulate more biomass / reach higher OD than wild-type. The pCA2-3 strain’s improved productivity was attributed to a 2.6-fold improvement in CCE (3.82 ± 0.34 mg DW / mol CO2 / mol CH4) over wild-type (Figure 7B and Table 3), which was also significantly higher than the CCE improvements observed via overexpression of CA2 or CA3 individually. To further validate these observed CCE improvements, we performed a competition assay to compare fitness of the pCA2, pCA3, orpCA2-3 engineered strains to wild-type in a mixed culture with the hypothesis that the overexpression strains would outperform wild-type M. capsulatus due to increased CO2uptake. Indeed, pCA2, pCA3, and pCA2-3 outperformed wild-type 1.49-, 2.25-, and 2.51-fold, respectively (Figure 8C), which are fitness enhancements consistent with the growth enhancements and CCE improvements measured in axenic cultures (Figure 8B). These data highlight that CA overexpression is a viable strategy to improve CCE in a RubisCO-encoding methanotrophic biocatalyst. Further, the pCA2-3 strain represents an engineered biocatalyst with enhanced biomass productivity that could improve the techno-economics of M. capsulatus- based single cell protein production.Discussion

[0108] Methanotrophic bacteria play vital roles in biogeochemical cycling across the globe and mitigate climate change by direct capture of the greenhouse gas CH4. M. capsulatus and other Type lb Gammaproteobacterial methanotrophs encode RubisCO which mediates CO2assimilation essential for their growth, thereby limiting the overall CO2evolution from biological CH4oxidation. This study provides several lines of evidence that CAs have vital roles in the M. capsulatus inorganic metabolism needed for growth of this obligate methanotroph. These results show that increasing methanotroph CA levels improves biomass productivity while paradoxically decreasing CO2evolution, improving overall CCE to biomass. Thus, the biocatalysts with enhanced CCE and the insights developed here can be leveraged for improving CH4bioconversion processes and climate change mitigation.

[0109] M. capsulatus expresses five CA isoforms with phylogenetic relationships that provide insight into their possible function(s) in general methanotroph metabolism and / or the specialized dual CH4and CO2metabolism occurring in distinct methanotroph clades that express RubisCO. The M. capsulatus [3-CA1 , [3-CA3, [3-CA4, and y-CA5 isoforms have orthologs in Gamma- and Alphaproteobacterial methanotrophs with significant (>30%) sequence identify. The presence of these orthologs in the Gammaproteobacterial methanotroph Methylotuvimicrobium alcaliphilum 20Z that does not require CO2for growth (Henard CA, et al. Appl Environ Microbiol 2021 87:e0088121) suggests that these CAs may perform more generalized functions or have evolved unique functions in M. capsulatus. In contrast, M. capsulatus a-CA2 orthologs are not common in methanotrophs but are found in an array of chemolithoautotrophic bacteria, highlighting a strong relationship of this CA isoform with autotrophic inorganic carbon metabolism. Indeed, a-CA2 overexpression increased M. capsulatus CO2assimilation (Figures 7 and 8). The a-CA2 isoform shares 36% identity with an a-CA in the chemolithoautotrophic sulfur oxidizing bacteria Ectothiorhodospira. Notably, the M.capsulatus RubisCO large subunit’s closest non-methanotroph ortholog is found in Ectothiorhodospira mobilis (92% sequence identity) (Tourova TP, et al. Microbiology 2005 74:321-328), so perhaps several interspecies genetic exchanges have occurred between Methylococcus and this purple sulfur bacteria, playing a part in development of the Methylococcus dual C1 metabolism.

[0110] The five CA genes are appreciably expressed, and our data indicate that CA transcription can be induced in response to a decrease in CO2availability (Figure 4). Further, bacterial treatment with the CA inhibitor, AZM, elicited an adaptive response resulting in an AZM-resistant phenotype that, at least partially, is attributed to CA overexpression (Figure 5). The AZM-resistant phenotype is maintained in the absence of AZM selective pressure, pointing to genetic mutation(s) as a mechanism underlying the phenotype. AZM and other sulfonamide CA inhibitors are currently being considered as antibacterial therapeutics for treatment of antibiotic-resistant bacterial infections (Nocentini A, et al. Antibiotics (Basel) 2023 12). These data show bacteria can develop resistance to AZM; thus, the M. capsulatus AZM adaptation may provide insight into pathogenic bacterial resistance strategies. Whole-genome sequencing of the AZM-resistant strains may identify mutations leading to dysregulation of CA expression or other adaptive mechanisms. For applied methanotroph biotechnologies, the identified mutations / genes would represent putative metabolic engineering targets to further improve CO2uptake and conversion.

[0111] It was hypothesized that the M. capsulatus cytoplasmic CAs (e.g. [3-CA1 , [3-CA3, P-CA4, and y-CA5) would complement the loss of other isoforms, so we were surprised to find that each CA KO strain had a substantial growth defect compared to wild-type (Figure 8). These data highlight that each CA plays a unique, critical role in M. capsulatus metabolism and physiology, but the underlying mechanisms remain undetermined. CO2and HCO3are required for several biochemical reactions; thus, some M. capsulatus CAs may play distinct roles in maintaining the intracellular CO2 / HCO3_balance for biosynthetic processes or other physiological functions rather than capturing external CO2. Each CA isoform could have unique binding partners or localization in Methylococcus, which could also explain a lack of redundancy. The M. capsulatus a-CA2 is the only secreted CA, and based on the physiological role of a-CAs in other bacteria, likely converts CO2to HCO3in the periplasmic space (Gai CS, et al. AMB Express 2014 4:2; Dobrinski KP, et al. Appl Environ Microbiol 2010 76:3561-3567). The M. capsulatus gene encoding [3-CA3 is in an operon with a gene annotated to encode a SulP family inorganic anion transporter, and members of this protein family have been demonstrated to transport HCO3in cyanobacteria (Felce J, et al. J Mol Microbiol Biotechnol2004 8:169-176; Price GD, et al. Proc Natl Acad Sci USA 2004 101 :18228-18233; Shelden MO, et al. Mol Membr Biol 2010 27:12-22). Additionally, the [3-CA4 gene is in an operon with a downstream gene (MCA1664) annotated as a xanthine / uracil permease, which is thought to be the ancestral protein family of the SulP bicarbonate transporters (Nayak N, et al. Res Sq. 2023). The M. capsulatus [3-CA3 and [3-CA4 genomic context implies that they may coordinate with a SulP transporter to either import or export HCO3‘ across the periplasmic membrane. Notably, we observed an additive growth enhancement with improved CO2assimilation when the a-CA2 and P-CA3 were co-overexpressed (Figure 7 and 8). These data lead us to a model wherein the a- CA2 converts CO2to bicarbonate in the periplasm, which is imported across the periplasmic membrane by the SulP family transporter and converted to CO2in the cytoplasm by the [3-CA3, increasing RubisCO substrate availability (Figure 9). This speculative carbon concentration mechanism needs to be experimentally tested but is a possible explanation for the phenotypes observed here.

[0112] Technoeconomic analyses have identified COE and productivity as primary cost drivers in CH4bioconversion processes using methanotrophic bacteria (Fei Q, et al. Biochem Eng J 2020 107500). A two-fold improvement in COE in a natural gas to lactate conversion bioprocess can decrease the lactate minimal selling price ($ / kg) by 34%, which is primarily due to an overall decrease in capital expenditures (Fei Q, et al. Biochem Eng J 2020 107500). The engineered strain developed here (pCA2-3) has a 2.5-fold increase in COE (Figure 8). As such, using this strain to convert CP -rich natural gas or anaerobic digestion-derived biogas to valuable products (e.g. lactate) or microbial biomass / single cell protein would immediately reduce production costs. Further strain improvements to enhance CO2assimilation / CCE, increase product titer and productivity, and bioprocess optimization could lead to dramatic cost reductions to facilitate the commercialization of CH4bioconversion processes.

[0113] Methanotroph-dependent conversion of CH4represents an approach to capture GHGs and mitigate climate change. These data advance understanding of the dual CH4and CO2metabolism occurring in the methanotroph, Methylococcus capsulatus, a methanotroph currently used industrially to produce single cell protein. These and future insights into the M. capsulatus metabolism and physiology will guide strain improvement strategies required to realize the effective use of these biocatalysts in biotechnology applications.

[0114] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0115] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. A recombinant methanotrophic bacterium, comprising an exogenous polynucleotide comprising a gene encoding a ribulose-1 ,5-bisphosphate carboxylase / oxygenase (RubisCO) enzyme and an associated chaperone protein and an associated activase protein; an exogenous polynucleotide comprising a gene encoding a carbonic anhydrase, and an exogenous polynucleotide comprising a gene encoding a an inorganic carbon transporter, wherein each of the gene encoding the RubisCO enzyme, the gene encoding the carbonic anhydrase, and the gene encoding a the inorganic carbon transporter are operably linked to an expression control sequence.

2. The bacterium of claim 1 , wherein the RubisCO enzyme is cbbLs.

3. The bacterium of claim 2, wherein the RubisCO enzyme is encoded by the nucleic acid sequence SEQ ID NO:1.

4. The bacterium of claim 1 , wherein the chaperone protein is cbbQ.

5. The bacterium of claim 4, wherein the chaperone protein is encoded by the nucleic acid sequence SEQ ID NO:10.

6. The bacterium of claim 1 , wherein the activase protein is cbbX.

7. The bacterium of claim 6, wherein the chaperone protein is encoded by the nucleic acid sequence SEQ ID NO:11.

8. The bacterium of claim 1 , wherein carbonic anhydrase comprises carbonic anhydrase 1 (caa1), carbonic anhydrase 2 (caa2), carbonic anhydrase 3 (caa3), carbonic anhydrase 4 (caa4), carbonic anhydrase 5 (caa5), or any combination thereof.

9. The bacterium of claim 8, wherein the carbonic anhydrase is encoded by the nucleic acid sequence SEQ ID NO:2, 3, 4, 5, or 6.

10. The bacterium of claim 1 , wherein the inorganic carbon transporter is a SulP transporter.11 . The bacterium of claim 10, wherein the inorganic carbon transporter is encoded by the nucleic acid sequence SEQ ID NO:7 or 8.

12. The bacterium of claim 1 , wherein the methanotrophic bacterium is a gammaproteobacterial or alphaproteobacterial methanotroph.

13. The bacterium of claim 1 , wherein the methanotrophic bacterium is a Verrucomicrobia / Candidate phylum NC10 methanotroph.

14. The bacterium of claim 1 , wherein the methanotrophic bacterium is selected from Methylococcus capsulatus, Methylomonas sp. 16A, Methylosinus trichosporium, Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus, Methylomonas flagellata, Methylacidiphilum infernorum, Methylomicrobiumalcaliphilum Methylocella Silvestris, Methylocella palustris, Methylocella tundrae, Methylocystis daltona, Methylocystis bryophila, or Methylocapsa aurea.

15. The bacterium of claim 1 , wherein the methanotrophic bacterium is further engineered to express a heterologous phosphoribulokinase.

16. The bacterium of claim 15, wherein the phosphoribulokinase is encoded by the nucleic acid sequence SEQ ID NO: 12.

17. The bacterium of claim 1 , wherein the gene encoding the RubisCO enzyme, the gene encoding the carbonic anhydrase, and the gene encoding a the inorganic carbon transporter are in the same expression vector.

18. The bacterium of claim 17, wherein the gene encoding the RubisCO enzyme, the gene encoding the carbonic anhydrase, and the gene encoding a the inorganic carbon transporter are operably linked to the same expression control sequence.

19. The bacterium of claim 1 , wherein each of the gene encoding the RubisCO enzyme, the gene encoding the carbonic anhydrase, and the gene encoding a the inorganic carbon transporter are integrated into the methanotrophic bacterium genome.

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