Synthesis of 1-butanol from carbon dioxide by engineered hydrogen bacteria

Abstract

Bacterial strains can be engineered which are capable of producing 1-butanol using carbon dioxide (CO2) and hydrogen gas (142), wherein the strain naturally utilizes Calvin-Benson-Bassham (CBB) cycle, and further wherein the strain has been engineered so that expression of products from the CBB pathway are increased compared to a non-engineered version of the strain. These strains can be used in methods to produce 1-butanol. CbbR mutants can also be tested for their ability to produce 1-butanol.

Description

Attorney Docket No.103362-020WO1 SYNTHESIS OF 1-BUTANOL FROM CARBON DIOXIDE BY ENGINEERED HYDROGEN BACTERIA CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No.63 / 678,755, filed August 2, 2024, incorporated herein by reference in its entirety. GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under DE-FG02-08ER15976 and DE-AR0000095 awarded by the Department of Energy. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING

[0003] The sequence listing submitted on August 1, 2025, as an .XML file entitled 103362-020WO1_ST26.xml” created on July 30, 2025, and having a file size of 86,995 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND

[0004] 1-butanol is an essential platform chemical for both the manufacturing and transportation industries. It is used in solvents, as well as in manufacturing of textile polymers and paints, and can be used as a drop-in fuel replacement for conventional gasoline. Initial industrial biological production of 1-butanol, or “biobutanol”, was developed during World War I, utilizing Clostridium sp. Acetone-Butanol-Ethanol sugar fermentation pathways. Biobutanol production declined in the 1950’s due to advances in chemical synthesis from petrochemicals. However, increasing concerns about fossil fuel usage have renewed interest in sustainable and renewable biobutanol. Metabolic engineering of microbes has enabled the potential upcycling of industrial waste streams, such as food waste and glycerol from biodiesel manufacturing, into biobutanol to mitigate anthropogenic CO2release into the atmosphere. For example, C. pasteurianum, K. pneumoniae, and E. coli have been engineered to heterotrophically utilize glycerol (5-15 g / L butanol). Clostridia sp. and C. necator have been engineered to utilize sugars other than glucose for 1-butanol production (20 g / L, and 0.2 g / L, respectively). For autotrophic carbon fixation via the Wood-Ljungdahl pathway, C. ljungdahlii and C. autoethanogenum have been engineered to convert CO-rich syngas into 1-butanol (0.2-2 g / L butanol), and for autotrophic carbon fixation viaAttorney Docket No.103362-020WO1 the Calvin-Benson-Bassham cycle, R. palustris (3 mg / L) and cyanobacteria ( 0.014 - 4 g / L) have been engineered to produce1-butanol from sunlight and carbon dioxide.

[0005] However, biological carbon capture and synthesis of 1-butanol from abundant industrial CO2-rich waste streams like flue gas have yet to be realized. Next to water, cement is the most consumed resource in the world (4.4 billion metric tons annually) to enable modern civilization. As a result, CO2-containing flue gas from the thermal reformation of limestone for cement production contributes to ~6% of the global annual CO2emissions. Unrefined flue gases contain compounds such as SO2, NOx, H2S, CN and CO, which are inhibitory to the growth of many organisms. To date only photosynthetic microalgae have been extensively studied for growth on flue gas and subsequent biofuel production from algal biomass. Furthermore, specific to 1-butanol production, high concentrations of oxygen often present in flue gas are suspected to inhibit the activity of one or more 1-butanol pathway enzymes.

[0006] Thus, there exists a need for production of 1-butanol from engineered strains of microorganisms. This is at least partially satisfied by the present disclosure. SUMMARY

[0007] Disclosed herein are engineered bacterial strains capable of producing 1-butanol using carbon dioxide (CO2) and hydrogen gas (H2), wherein the strain naturally utilizes Calvin-Benson- Bassham (CBB) cycle, and further wherein the strain has been engineered so that expression of products from the CBB pathway are increased compared to a non-engineered version of the strain. Also disclosed are methods of using these strains to produce 1-butanol, and methods of engineering bacterial strains within the CbbR.

[0008] Disclosed herein are engineered bacterial strains capable of producing 1-butanol using carbon dioxide (CO2) and hydrogen gas (H2), wherein the strain naturally utilizes Calvin-Benson- Bassham (CBB) cycle, and further wherein the strain has been engineered so that expression of products from the CBB pathway are increased compared to a non-engineered version of the strain. Also disclosed herein is a method of producing 1-butanol from an engineered bacteria using carbon dioxide (CO2) and hydrogen gas (H2), wherein the strain naturally utilizes Calvin-Benson- Bassham (CBB) cycle, and further wherein the strain has been engineered so that expression of products from the CBB pathway are increased compared to a non-engineered version of the strain. Also disclosed herein is a method of selecting and determining a mutation within CbbR which can increase 1-butanol production in an engineered bacterial strain, the method comprising: providing a library of mutations within CbbR into bacterial strains engineered to produce 1-butanol, determining which mutations provide an increase in 1-butanol synthesis, and selecting said strain.Attorney Docket No.103362-020WO1

[0009] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

[0011] Figure 1A-D shows engineered butanol production pathways in C. necator. A) Engineered butanol production pathways (this work) using non-native enzyme from the clostridium ABE pathway (Hbd, Crt, AhdE2) and the trans-2-enoyl-CoA reductase from T. denticola (Ter) (black arrows) versus those using native PHB synthesis enzymes (PhaB, PhaJ, Bcd) and E. coli alcohol / aldehyde dehydrogenases (EutE / YqhD) (gray arrows) in previous works. B-D) Organization of engineered butanol-production operons. Cbb, Calvin-Benson-Bassham; H2ase, hydrogenase.

[0012] Figure 2A-D shows engineering autotrophic butanol production in C. necator. A) Nile Red stain for detection of PHB (black pigment). B) RT-PCR of inserted butanol production genes. C) Production of butanol during autotrophic and heterotrophic growth on various carbon substrates. Error bars are standard deviation of n=3 independent experiments. D) Growth and autotrophic butanol production by engineered C. necator strains using defined CO2 / H2 gas mixture. Error bars are standard deviation for n=3 independent experiments. For D, dashed lines correspond to Optical Density at 600nm; solid lines correspond to 1-butanol.

[0013] Figure 3A-C shows different alcohol / aldehyde dehydrogenase homologs control butanol to ethanol ratios. A) Screen of alcohol / aldehyde dehydrogenases in an E. coli butanol- production strain RKE12 for preference of butanol versus ethanol synthesis. B and C) Transfer of eutE and yqhD aldehyde / alcohol dehydrogenases from E. coli screens that increase butanol to ethanol ratios into C. necator coordinately increase butanol to ethanol ratios in C. necator under both heterotrophic (B) and autotrophic (C) conditions, and increase overall yields under (B) heterotrophic conditions. In all graphs error bars are standard deviation for n=3 independent experiments.

[0014] Figure 4A-D shows modified CBB pathway expression through CbbR amino acid variants increases autotrophic butanol production. A) C. necator screen of CbbR* variants for alterations in CBB pathway expression as measured by beta-galactosidase activity and overall carbon fixation as measured by RubisCO activity. B) Incorporation of select CbbR* variants intoAttorney Docket No.103362-020WO1 indicated butanol production strains increases overall RubisCO activity. C) Incorporation of CbbR* D144N variant to form butanol production strains OB311:BuOH(19) and BuOH(33) increases butanol yields versus parent strains with wild type CbbR, strains OB311:BuOH(7), BuOH(13) and BuOH(32). D) Incorporation of cbbR* D144N to form strains OB311:BuOH(19), BuOH(28) and BuOH(29) increases overall butanol production from petroleum upgrader gas compared to parent strains OB311:BuOH(13), BuOH(26) and BuOH(27). In all graphs, error bars are the standard deviation of n=3 independent experiments.

[0015] Figure 5 shows heterotrophic growth on gluconate and corresponding alcohol and organic production in engineered C. necator strains. Strains OB311:BuOH(7,14,15) pertain to organic acid production knockouts. Strains OB311:BuOH(20, 25) compare alternative aldehyde / alcohol dehydrogenases.

[0016] Figure 6 shows heterotrophic growth on gluconate and corresponding alcohol and organic production in C. necator strains H16, OB311, H16:BuOH(1), and OB311:BuOH(7) Error bars are the standard deviation of n=4 independent experiments.

[0017] Figure 7A-B shows autotrophic growth on 5% CO2, 10.5% O2, 45% H2 and corresponding alcohol production in engineered C. necator strains. A) Strains OB311:BuOH(27, 29, 31). B) Strains OB311:BuOH(26, 28, 30). Error bars are the standard deviation of n=3 independent experiments.

[0018] Figure 8 shows reproducibility of autotrophic growth on 5% CO2, 10.5% O2, 45% H2 and corresponding alcohol production by engineered C. necator. Error bars are the standard deviation of n=3 independent experiments. DETAILED DESCRIPTION

[0019] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. 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. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. DEFINITIONS

[0020] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:Attorney Docket No.103362-020WO1

[0021] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0022] 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. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0023] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0024] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.Attorney Docket No.103362-020WO1

[0025] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub- ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0026] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. GENERAL DESCRIPTION

[0027] Carbon dioxide present in flue gas from cement and petroleum refining industries represents a significant percentage of the annual anthropogenic greenhouse gas emissions into the environment. Direct biological removal of CO2from these waste steams for carbon capture and upcycling to renewable biofuels and bioproducts has remained challenging due to the presence of oxygen and toxic compounds in the flue gas, which limits the number of industrially amenable organisms that can be employed. To address this problem, the aerobic chemolithoautotrophic bacterium Cupriavidus necator strain H16 (formerly Ralstonia eutropha) was first engineered for 1-butanol synthesis from CO2and H2by inactivating polyhydroxybutyrate synthesis and introducing a modified Clostridium acetobutylicum 1-butanol pathway at the phaCAB locus.Attorney Docket No.103362-020WO1 Subsequently, an industrial flue gas adapted strain, C. necator OB311 (developed by Oakbio Inc. (DBA Novonutrients), Sunnyvale, California) was engineered for 1-butanol synthesis and further optimized the 1-butanol pathway and Calvin-Benson-Bassham (CBB) cycle for 1-butanol production through CbbR transcription factor mutagenesis, phaCAB promoter replacement, and alcohol / aldehyde dehydrogenase enzyme selection. Autotrophic 1-butanol production was achieved with yields up to 300 mg / L from defined gas mixtures similar to those achieved for phototrophic cyanobacteria, and up to 30 mg / L from untreated cement kiln and oil upgrader gas. These results establish an engineered pathway for autotrophic biobutanol production in C. necator, a useful host organism for industrial biofuel production, and demonstrate direct carbon capture to synthesize a key industrial chemical and transportation fuel from industrial waste gas without any manipulation beyond addition of H2or O2to meet growth requirements.

[0028] Based on these findings, disclosed herein is an engineered bacterial strain capable of producing 1-butanol using carbon dioxide (CO2) and hydrogen gas (H2), wherein the strain naturally utilizes Calvin-Benson-Bassham (CBB) cycle, and further wherein the strain has been engineered so that expression of products from the CBB pathway are increased compared to a non-engineered version of the strain. Example 1 provides a variety of ways to optimize this engineered bacterial strain to maximize production of 1-butanol.

[0029] The Calvin–Benson–Bassham (CBB) cycle, which utilizes the CO2fixation enzyme ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO), is a key biological pathway for converting atmospheric CO2to organic matter. The mutations disclosed herein can be within CbbR (CBB regulator).

[0030] Any strain of bacteria can be used which naturally comprises the CBB cycle. Examples include, but are not limited to, photoautotrophic bacteria such as Cupriavidus necator, Rhodospirillum rubrum, Cereibacter spheroides, Rhodobacter Capsulatus, and Rhodospeudomonas palustris. A specific example includes the C. necator strain OB311.

[0031] The engineered bacterial strain can have at least one mutation in CbbR. Mutations of CbbR are described in Dangel et al. Microbiology (Reading).2015 Sep;161(9):1816-1829, which generally described CbbR mutations, but not those which can specifically increase 1-butanol production to a level greater than 30 mg / L (such as 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg / L or more). The mutation can be a single point mutation, multiple point mutations, or can be a splice, an insertion, or a deletion within CbbR. Specifically, described herein are mutations in C. necator strain OB311 (wherein the native CbbR is represented by SEQ ID NO: 1), wherein the mutationsAttorney Docket No.103362-020WO1 occur in at least one of H249Y, L79F, and D144N (any of SEQ ID NOS: 2-8). The reference strain, C. necator H16, can be found in NCBI database as ASM479872v1; GenBank Assembly GCA_004798725.1; RefSeq Assembly GCF_004798725.1. Mutations in these genes are described in more detail below.

[0032] As mentioned above, the engineered bacterial strain can be engineered in a number of ways which can increase 1-butanol synthesis. Such methods can be found in Example 1.

[0033] Also disclosed herein are methods of producing 1-butanol from bacteria, the method comprises using the engineered bacterial strains disclosed herein.

[0034] Further disclosed are methods of selecting and determining a mutation within CbbR which can increase 1-butanol production in an engineered bacterial strain, the method comprising: providing a library of mutations within CbbR into bacterial strains engineered to produce 1- butanol; determining which mutations provide an increase in 1-butanol synthesis; and selecting the strain.

[0035] C. necator wild-type strain H16 (formerly Ralstonia / Hydrogenomonas / Wautersia eutropha and Alcaligenes eutrophus) and the Oakbio flue-gas adapted strain (OB311, Sunnyvale, CA) can be used as chassis for engineering autotrophic synthesis of biobutanol from CO2-rich industrial waste streams. The organism is a fast-growing facultative aerobe and chemolithoautotroph. It can assimilate CO2into precursory metabolites through the Calvin- Benson-Bassham (CBB) cycle, acquire electrons for NAD(P)H synthesis and energy metabolism through hydrogenases, and synthesize ATP through aerobic oxidative phosphorylation or anaerobic nitrate respiration (Fig.1A). C. necator is also capable of utilizing organic acids, some sugars and oils, and can synthesize up to 90% by cell mass of poly(3-hydroxybutyrate) (PHB) for carbon storage. Unlike microalgae and cyanobacteria, C. necator does not require light for growth, and can be cultivated to high cell densities (>100 g / L). Most notably, C. necator is tolerant of and can detoxify a large number of heavy metals, chlorinated aromatics, sulfur dioxide (SO2), and NOx species. Therefore, it is useful for growth and biofuel production directly from CO2-rich industrial gas waste streams.

[0036] As can be seen in Example 1, type-strain C. necator H16 was initially employed for facile genetic engineering to establish autotrophic 1-butanol production from purified CO2and H2. However, due to poor growth and 1-butanol production by engineered C. necator H16 strains on industrial waste gases, the flue gas-adapted strain, OB311, was subsequently employed as the host to engineer autotrophic 1-butanol synthesis from cement kiln and petroleum upgrader gas streams as feedstocks. Through metabolic and genetic regulation engineering of the 1-butanol and CBB pathways, biobutanol production was enabled at average yields of 250 mg / L culture from purifiedAttorney Docket No.103362-020WO1 gas and 30 mg / L from both cement kiln and petroleum processing gas. This represents the first microbial production of biobutanol directly from unrefined flue gas. In addition, biobutanol yields up to 300 mg / L culture from CO2 / H2by engineered C. necator are comparable to previously engineered C. necator systems for 1-butanol production from sugars.

[0037] In certain embodiments, the engineered bacterial strains described herein comprise mutations within a CbbR regulator gene sequence, such as that provided in SEQ ID NO: 1. SEQ ID NO: 1 corresponds to the wild-type Cupriavidus necator OB311 CbbR coding sequence, which encodes a LysR-type transcriptional regulator responsible for controlling expression of key genes within the Calvin-Benson-Bassham (CBB) cycle, including the cbbL, cbbS, and cbbP genes encoding RuBisCO and phosphoribulokinase. Modifications to this sequence can include single nucleotide substitutions, insertions, deletions, or a combination thereof, particularly in regions corresponding to known or predicted DNA-binding and regulatory domains.

[0038] In some embodiments, the CbbR regulator is modified to include single point mutations, such as H249Y, L79F, and D144N, each of which individually or in combination can enhance CbbR-mediated transcriptional activation of the CBB operon. These mutations may reside within the helix-turn-helix domain or effector-binding domain of the protein, and may alter DNA-binding affinity, effector responsiveness, or allosteric regulation. Other amino acid substitutions may be introduced at positions 50–300 of the protein, particularly in regions conserved among homologs of Cupriavidus, Ralstonia, Hydrogenophaga, and Burkholderia species.

[0039] In certain embodiments, the nucleotide sequence encoding CbbR, or the polypeptide itself, exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. Sequence identity can be calculated over the full length of the gene or over functionally relevant domains (e.g., N-terminal DNA-binding domain or C-terminal regulatory domain). Identity is assessed using algorithms such as ClustalW or BLAST, with default parameters. In some cases, functional homologs of CbbR from closely related autotrophic bacteria may be substituted for SEQ ID NO: 1, provided they retain the ability to activate CBB genes and are compatible with C. necator expression machinery. Suitable homologs may include CbbR sequences from Ralstonia eutropha, Cupriavidus taiwanensis, or Alcaligenes species, which share greater than 85% amino acid identity to SEQ ID NO: 1.

[0040] Variants of the CbbR gene also include codon-optimized versions for expression in different bacterial hosts, such as E. coli, Clostridium, or Pseudomonas, depending on the chassis organism used for synthetic biology applications. In some embodiments, the CbbR sequence is engineered with synonymous mutations that maintain amino acid sequence while improving mRNA stability, translation efficiency, or compatibility with cloning methods. Additionally,Attorney Docket No.103362-020WO1 epitope tags (e.g., FLAG, His-tag, HA-tag) or regulatory fusions (e.g., GFP, degradation tags, riboswitch-controlled 5' UTRs) may be added to facilitate detection or tunable expression.

[0041] The invention also encompasses combinatorial libraries of CbbR variants, including saturation mutagenesis libraries where each amino acid position is randomized (e.g., using NNK codons), scanning alanine mutagenesis libraries to map functional residues, and error-prone PCR libraries to introduce random mutations across the entire sequence. These libraries may be screened in high-throughput assays to identify beneficial variants that enhance 1-butanol production when introduced into autotrophic strains.

[0042] In some cases, chimeric regulators may be constructed by replacing one or more domains of SEQ ID NO: 1 with corresponding regions from other LysR-type regulators, creating synthetic regulators with altered binding specificity or activation profiles. For example, the N- terminal DNA-binding domain may be retained from SEQ ID NO: 1, while the effector-binding domain is derived from a different LysR family member with desirable allosteric properties.

[0043] Unless otherwise specified, the amino acid sequence corresponding to SEQ ID NO: 1 is defined using standard IUPAC single-letter codes and is understood to include conservative substitutions, where one amino acid is replaced with another having similar physicochemical properties (e.g., valine to isoleucine, aspartate to glutamate, lysine to arginine). Such substitutions may preserve secondary structure, charge, or hydrophobicity and are considered within the scope of functional variants.

[0044] In all embodiments, the described CbbR variants are intended to result in enhanced expression of Calvin cycle enzymes relative to wild-type strains. Enhancement can be measured at the transcriptional level (e.g., via qPCR or RNA-seq), translational level (e.g., via proteomics or reporter constructs), or functional level (e.g., increased RuBisCO activity, CO₂ fixation rate, or downstream 1-butanol production). This increased flux through the CBB pathway supports more efficient conversion of CO₂ and H₂ into biomass and target products under autotrophic growth conditions.

[0045] In certain embodiments, the CbbR regulator may be engineered using non-canonical amino acids (ncAAs) incorporated into the protein sequence to alter structure-function relationships in ways not achievable with the standard 20 amino acids. These non-canonical residues can include amino acids with expanded side chains, photo-reactive groups, keto or azido moieties, fluorinated analogs, or metal-coordinating functionalities. Incorporation of such residues may be achieved via orthogonal tRNA / synthetase systems or chemical mutagenesis, and can enable fine-tuning of protein stability, DNA-binding affinity, or allosteric response to metabolic signals. For example, incorporation of a photo-crosslinkable ncAA at or near the DNA-bindingAttorney Docket No.103362-020WO1 domain of CbbR may allow for light-controlled modulation of gene expression. Similarly, inclusion of redox-sensitive residues can be used to engineer CbbR variants responsive to intracellular NADH / NADPH ratios, directly linking redox state to Calvin cycle regulation and biofuel production efficiency.

[0046] Additionally, CbbR variants may be generated through the use of non-canonical or chemically modified nucleotides in the mutagenesis process. These include base analogs such as inosine, 5-methylcytosine, pseudouridine, 2-aminopurine, and others that alter hydrogen bonding, base-pairing specificity, or transcriptional fidelity. When introduced during oligonucleotide- directed mutagenesis or PCR amplification, such analogs can increase sequence diversity, promote error-prone replication, or enhance the structural diversity of encoded proteins. In some cases, non-standard codons may be introduced using expanded genetic codes, enabling programmed insertion of ncAAs at specific positions in the CbbR gene. These strategies allow for the generation of hypervariable CbbR libraries with enhanced functional diversity, enabling discovery of variants with superior performance in CO₂ fixation or synthetic pathway activation. EXAMPLES Example 1: Autotrophic Synthesis of 1-butanol from Industrial Waste Carbon Dioxide by Engineered Cupriavidus necator. Materials and Methods Bacterial strains, culture conditions and plasmids

[0047] All C. necator strains used are listed in Table 1. E. coli strain JM109 was used for propagation of plasmids, strain S17-1 (Biomedal) was used for conjugative plasmid transfer to C. necator, and strain RKE12 , a derivative of BW25113, was used for screening functional alcohol dehydrogenases. Routine growth of Escherichia coli was performed aerobically in liquid or on solid LB medium at 37°C. Routine growth of C. necator strain H16 (type strain) and OB311 (flue gas adapted strain of H16; Oakbio, Sunnyvale, California) was performed aerobically in liquid or on solid Ormerod’s minimal medium containing 30 mM DL-malate or 20 mM succinate at 30°C. Antibiotics were used at the following concentrations (μg per ml): for E. coli, ampicillin, 125; tetracycline, 12.5; for C. necator, kanamycin, 25; tetracycline, 3-25.

[0048] 1-butanol production cultures were grown under chemoheterotrophic and chemoautotrophic conditions in Ormerod’s medium at 30°C. For chemoheterotrophic butanol production, 250 ml Erlenmeyer flasks containing 30 ml of medium with 110 mM gluconate wereAttorney Docket No.103362-020WO1 inoculated from several colonies, topped with a rubber stopper vented by an 18-gauge filtered needle, and then shaken at 110 rpm. For small-scale chemoautotrophic butanol production under defined gas atmosphere, a mixture of 0.2 μm filtered 5% CO2, 45% H2, 50% air was used. Tubes with 20 ml Ormerod’s medium were inoculated from several colonies, stoppered, sparged with an18-gauge needle inserted into the media, and vented by a second 18-gauge needle. Bubbling rate was approximately 250 bubbles per minute to ensure proper aeration. Growth on Cement Kiln and Petroleum Upgrader Gas

[0049] Cells were cultured autotrophically in 1 L scale pilot vessels with continuous bubbling and agitation in a temperature-controlled incubator at 30 °C. At regular intervals, small culture samples (~1.5 ml) were aseptically removed for optical density and 1-butanol measurements. The optical density of each culture was recorded in duplicate by an ICN Titertek microplate reader by measuring the absorbance of 200 ul samples at 620 nm. For growth and 1-butanol production on cement kiln gas, kiln flue gas (LehighSouthwest, Heidelberg Cement Group, Cupertino, CA) was directly mixed 50% with H2and introduced to the culture without any further modification. Similarly for petroleum upgrader gas, the raw gas stream of hydrogen, CO2 and light hydrocarbons from a heavy oil refinery and upgrader (Scotford, Alberta, Canada) was collected for use before it entered the inlet of a Hydrogen Manufacturing Unit pressure swing absorbance stage for purified hydrogen production. The collected, pressurized petroleum upgrader gas was then mixed 50% with air to supply the necessary oxygen and introduced to the culture without any further modification. Genetic Modifications

[0050] DNA oligonucleotide primers and plasmids used are listed in Table 2 and 3. PCR amplification for cloning was performed using Phusion®DNA polymerase (Thermo Scientific), and PCR amplification of genomic DNA isolated from modified C. necator strains to verify presence and absence of target genes was perform using PrimeSTAR®GXL DNA polymerase (Clontech) following manufacturer protocols. Restriction endonucleases and T4 DNA ligase were from New England Biolabs. Site-directed mutagenesis was performed using the QuikChange Lightning site-directed kit (Agilent). Replacement of phaC1 and insertion of hbd, crt, and ter

[0051] In all engineered strains, the phaC1 locus was deleted, and in the case of butanol production strains [BuOH(##)] was replaced with 3-hydroxybutyryl-CoA dehydrogenase (hbd)Attorney Docket No.103362-020WO1 and 3-hydroxybutyryl-CoA dehydratase (crt) from C. acetobutylicum, and trans-enoyl-CoA reductase (ter) from T. denticola using homologous recombination (Fig. 1 B-D). The 829 bp region upstream from phaC1 and the 501 bp region downstream region from phaC1 were amplified and cloned into pUC19 using EcoRI / SacI and SalI / HindIII, respectively, to construct pUC19:upC1_dnC1. The hbd, crt, and ter genes were first cloned into pBBR1MCS-3 using KpnI / XhoI for hbd, XhoI / SmaI for crt, and SmaI / SpeI for ter. The hbd-crt-ter fragment was then amplified and cloned into pUC19:upC1_dnC1 using SacI and SalI to construct pUC19:upC1_h.c.t._dnC1, wherein hbd-crt-ter was flanked by the phaC1 upstream and downstream regions.

[0052] For deletion of phaC1, the upstream and downstream fragment from pUC19:upC1_dnC was subcloned into the pJQ200mp18Tet suicide vector using XbaI yielding pJQTet:ΔC. Similarly, for deletion of phaC1 and simultaneous insertion of hbd, crt, and ter, genes and phaC1 flanking regions were subcloned from pUC19:upC1_h.c.t._dnC1 to pJQ200mp18Tet with XbaI yielding pJQTet:ΔC:h.c.t. In addition, codon optimized versions of hbd, crt, and ter based on C. necator codon usage frequencies (denoted as hbd*, crt*, and ter*) were synthesized, assembled into pUC19:upC1_dnC1 to construct pUC19:upC1_h*.c*.t*._dnC1 and subcloned into pJQ200mp18Tet to from pJQTet:ΔC:h*.c*.t*. Plasmids were mobilized into C. necator via biparental mating and conjugative transfer using E. coli S17-1 Transconjugants were selected by kanamycin and tetracycline resistance, and homologous recombinants were selected by loss of sensitivity to sucrose and PCR amplification of the C. necator genome region of interest to verify gene replacement. Replacement of phaB1 with adhE2 alcohol dehydrogenase

[0053] Using a similar strategy, the phaB1 gene was inactivated and replaced with an alcohol dehydrogenase. The 618 bp region upstream of phaB1 and the 562 bp region downstream of phaB1 were amplified and cloned into pUC19 using EcoRI / SacI and SalI / HindIII, respectively, to construct pUC19:upB1_dnB1. AdhE2 was first cloned into pBBR1MCS-3 using SpeI and XbaI, then amplified and cloned into pUC19:upB1_dnB1 using SacI and SalI to construct pUC19:upB1_adhE2_dnB1. AdhE2 and flanking regions were subcloned into pJQ200mp18Tet using XbaI yielding pJQTet:ΔB:adhE2. Mobilization into C. necator and selection of recombinants were performed as described above.Attorney Docket No.103362-020WO1 Screening and insertion of alternate alcohol / aldehyde dehydrogenase genes at the phaB1 locus

[0054] Screening of alternative alcohol / aldehyde dehydrogenase enzymes was initially performed in E. coli RKE12 (BW25113ΔadhE, ΔldhA, ΔfrdABCD, Δpta), and deletions were constructed using methods previously described. AdhE2 in p91:BuOH(1) was replaced with alternative aldehyde and alcohol dehydrogenases and tested in E. coli according to previous methods. Top candidates were selected for testing in C. necator.

[0055] Alternative enzymes to adhE2 in C. necator were placed at the phaB1 locus. For these strains, alternative aldehyde dehydrogenases were amplified and cloned into pUC19:upB1_dnB1 with SacI / BamHI, and alternative alcohol dehydrogenases were amplified and cloned into pUC19:upB1_dnB1 using BamHI / SalI, resulting in different versions of pUC19:upB1_ald_alc_dnB1. Further steps to place these genes at the phaB1 locus were carried out as described for adhE2, and respective suicide plasmids were pJQTet:ΔB:02201,02210 and pJQTet:ΔB:02201,yqhD. Insertion of an additional copy of adhE2 at the cbb locus

[0056] To insert adhE2 at the cbb locus on chromosome 2 in butanol production strains, the 1010 nt upstream region for recombination was amplified, which included most of cbbR and all of the cbbR / cbbL promoter region, and the 864 nt downstream region for recombination was amplified, which included the cbbL native ribosome binding site (RBS) and part of cbbL, and cloned into pUC19 to construct pUC19:upcbbL_dnPcbbL. Then adhE2 was amplified and clone into pUC19:upcbbL_dnPcbbL using SacI and SphI, resulting in pUC19:upcbbL_ adhE2_dnPcbbL. The adhE2 gene with flanking regions was subcloned into pJQ200mp18Tet via XbaI, yieldingfor integration into C. necator. This placed adhE2 in front of cbbL (RubisCO large subunit) with both genes under control of the cbb promoter. Additional gene deletions

[0057] Phosphotransacetylase genes, pta1 and pta2; acetylkinase gene, ackA; propionate CoA- transferase gene, pct; and transhydrogenase genes, pntAa1, ptnAb1, pntB1, pntAb3, and ptnB3 were deleted by homologous recombination similar to methods used for deleting phaC1. Genomic regions upstream and downstream of target genes were amplified by PCR using primers listed in Table 2 and cloned into pUC19 using the indicated restriction enzymes. The combined upstream and downstream fragments were then subcloned into the XbaI site of pJQ200mp18Tet to construct the final suicide vector for target gene deletion as listed in Table 4.Attorney Docket No.103362-020WO1 Replacement of the phaC1 promoter with the cbb promoter

[0058] The phaC1 promoter was replaced with the cbb promoter by homologous recombination. The 660 nt upstream region of the phaC1 promoter and the 730 nt region downstream region (hbd gene) were each amplified and cloned into pUC19 to construct plasmid pUC19:upPphaC1_dnPphaC1. For the cbb promoter, the 207 nt region between cbbL and cbbR plus 40 nt of cbbR were amplified from chromosome 2 of the C. necator genome and cloned into pUC19:upPphaC1_dnPphaC1 using SalI to construct pUC19:upPphaC1_Pcbb_dnPphaC1. This fragment containing the phaC1 promoter upstream and downstream regions flanking the cbb promoter was subcloned using XbaI into pJQ200mp18Tet to construct pJQTet:ΔPphaC1:Pcbb for promoter replacement in C. necator. Replacement of native cbbR with modified cbbR in C. necator

[0059] CbbR single point mutation and multiple point mutation variants (CbbR*) for enhancing CBB pathway expression were selected based on a prior screen of point mutations that resulted in constitutively active CbbR* variants. Single and multiple point mutation CbbR* variants (Fig.4A) were constructed by site directed mutagenesis and screened in C. necator strain 76-cbbLacZ (Table 1) for their ability to alter lacZ and RubisCO expression levels as done previously. RubisCO activity assays were performed as previously described by Satagopan et al, 2009, and LacZ beta-galactosidase activity assays were performed as previously described by Dangel & Tabita, 2015.

[0060] To replace the native cbbR with cbbR* variants on the chromosome in C. necator butanol production strains, a 3.5 kb fragment containing cbbR, the cbbR / cbbL promoter regions, cbbL and most of cbbS was first amplified from C. necator genomic DNA by PCR using PfuTurbo DNA polymerase (Agilent Technologies) and primers RLSXba3 and DD227. The fragment was cloned into plasmid pCR-BluntII-TOPO using the Invitrogen Zero Blunt TOPO PCR cloning kit to make TOPO-4G and mutations were incorporated by site directed mutagenesis. The 3.5 kb fragment containing each CbbR* mutation was digested with XbaI and cloned into the pJQsm2MCS suicide vector, resulting in pJQsm2-4G-360 (with D144N) and pJQsm2-4G-362 (with A167V).

[0061] For generation of CbbR-3* (H249Y / L79F / D144N), site directed mutagenesis was performed on pJQsm2-4G-360 to add the CbbR* H249Y mutation, resulting in pJQsm2-4G- 360 / 534. This plasmid was further modified through site-directed mutagenesis to add the CbbR* L79F mutation, resulting in pJQsm2-4G-360 / 534 / 540.Attorney Docket No.103362-020WO1

[0062] For construction of CbbR* mutants in strains with an altered cbb operon (e.g. strains 26 and 27 with adhE2 between Pcbb and cbbL), the cbbR gene and part of its promoter were amplified through PCR from existing CbbR* strains [Strain OB311:BuOH(19) with CbbR* D144N and Strain OB311:BuOH(24) with CbbR-3*] using primers DD560 and DD561. The resulting PCR products were digested with XbaI and cloned into pJQ200mp18Tet, resulting in plasmids pJQtet560 / 561 / D144N and pJQtet560 / 561 / 3*. The modifications to strain OB311:BuOH(26) resulted in strains OB311:BuOH(28) and OB311:BuOH(30), and the modifications to strain OB311:BuOH(27) resulted in strains OB311:BuOH(29) and OB311:BuOH(31), respectively. Polyhydroxybutyrate staining

[0063] PHB present in whole cells was visualized by treating culture plates with 10 μg / ml Nile Red stain in PBS for 15 minutes, followed by bright field imaging to observe strains that darken in the presence of Nile Red due to the presence of PHB. Analysis of compounds by GC

[0064] Alcohols and organic acids were quantified with a Shimadzu GC-2014 gas chromatograph (GC) equipped with a flame ionization detector, manual injection port, and Restek Stabilwax®-DA capillary column (30 m, 0.32 mm i.d., 0.25 μm df). 0.5 μl of culture supernatant was injected with a 1:5 split ratio, and compounds were separated by a thermal gradient of 50 °C to 125° C at 10 °C / min followed by a gradient of 125 °C to 220 °C at 20 °C / min and finally held at 220° C for 8 min with helium as carrier gas at 20 cm / sec and a purge flow of 3 mL / min. Standard curves were obtained for quantification of ethanol and butanol based on peak area of standards of defined concentration. Results Engineering autotrophic 1-butanol production in C. necator H16

[0065] Enzymatic 1-butanol synthesis pathways involve the coupling of 2 molecules of acetyl- CoA, followed by dehydration, reduction, and deacylation steps to yield CoA and 1-butanol (Fig. 1A). Previous work in C. necator employed native enzymes from the NADPH-dependent PHB pathway (PhaA, PhaB1, PhaJ) along with non-native NADPH-dependent aldehyde / alcohol dehydrogenases (EutE, YqhD) for biobutanol synthesis (Fig.1A, gray arrows). However, given the requirement of the CBB cycle for NADPH to sustain autotrophic carbon assimilation, an alternate NADH-dependent route via the Clostridium acetobutylicum acetone-butanol-ethanolAttorney Docket No.103362-020WO1 (A.B.E.) fermentation pathway enzymes, Hbd, Crt, and AdhE2, was devised (Fig. 1A, black arrows), to prevent competition between the CBB and 1-butanol synthesis pathways for reductant. In addition, the A.B.E. route synthesizes non-native (S)-3-hydroxybutyryl-CoA as an intermediate, which is poised to avoid potential funneling of native (R)-3-hydroxybutyryl-CoA by competing pathways for 1-butanol production. To demonstrate, initially PHB synthesis was inactivated via phaC1 deletion (Fig.2A) to accumulate (R)-3-hydroxybutyryl-CoA via the native NADPH-dependent route. Then when the C. acetobutylicum adhE2 gene was expressed, encoding a bifunctional butyryl-CoA aldehyde / alcohol dehydrogenase, from a plasmid (pRPS- MCS3:adhE2) to synthesize 1-butanol (Fig.1A), there was 10-fold less 1-butanol produced under autotrophic growth conditions from CO2and H2versus heterotrophic growth with gluconate (strain H16 ΔphaC1; Table 1). This indicated that NADPH or 1-butanol precursory metabolites were limited during autotrophic growth via the native PHB pathway. Therefore, to enhance autotrophic biobutanol synthesis using the A.B.E. pathway, phaC1 was replaced on the chromosome with the alternate butyryl-CoA synthesis genes (hbd, crt, ter), such that transcription was controlled by the phaC1 promoter. Hbd translation utilized the native PhaC1 ribosome binding site (RBS), and Crt and Ter translation each utilized the C. necator NrdD RBS. Coordinately, phaB1 was replaced with adhE2 such that translation utilized the native PhaB1 RBS (strain H16:BuOH(1); Fig. 1C, Table 1). Pathway expression was verified by RT-PCR (Fig. 2B), and the strain produced 1- butanol at similar levels both heterotrophically with fructose or gluconate (25-50 mg / L; Fig.2C, Table 1) and autotrophically with CO2and H2. This established the ability of C. necator to synthesize biobutanol autotrophically from CO2and H2at modest levels that were only ~ 4-fold less than yields previously observed when the native PHB pathway genes and fructose as carbon source were used (200 mg / L).

[0066] To investigate the balance of electron carriers for biobutanol synthesis and the CBB cycle, deletion of the C. necator transhydrogenases, which convert NADH to NADPH was targeted. During autotrophic growth with CO2and H2and heterotrophic growth with gluconate, reducing equivalents in the form of NADH are generated via hydrogenases and the Entner- Doudoroff pathway, respectively (Fig.1A). NADH and NADPH are then interconverted through one of three transhydrogenase complexes. Notably, the third transhydrogenase complex, Pnt3, shows the highest increase in abundance under autotrophic conditions. Inactivation of the third transhydrogenase complex through deletion of components PntAb3 and PntB3, followed by deletion of the first transhydrogenase complex through deletion of components PntAa1, PntAb1, and PntB1 increased heterotrophic 1-butanol production from gluconate up to 2-fold, but did not affect autotrophic yields from CO2and H2(Strains H16:BuOH(5,8); Table 1). This indicates thatAttorney Docket No.103362-020WO1 while under heterotrophic growth NADH may be limiting for biobutanol production via the NADH-dependent synthetic pathway (hbd, crt, ter, Fig.1A), it is not the primary factor limiting yields from CO2and H2.Engineering C. necator OB311 flue gas-adapted strain for increased biobutanol yields

[0067] Before pursuing further strain optimization of the H16 lineage for biobutanol synthesis, it was anticipated that deployment of this strain for direct conversion of industrial waste gas streams to product would be problematic due to inhibitory SO2, NOxand other compounds present. Indeed, C. necator H16 grew poorly under chemolithoautotrophic conditions using 50 % H2and 50 % flue gas directly from the Lehigh Southwest Cement Kiln in 1 L pilot reactors, as compared to growth on defined gases (Td_flue-gas= 3.8 ± 0.4 d, n=2 vs. Td-CO2 / H2= 0.17 ± 0.03 d, n=4; Fig.1D and Fig. 1E). Together, these results demonstrated that the C. necator H16 lineage was not an ideal chassis for biobutanol production from industrial waste gas.

[0068] To overcome this challenge, the Oakbio cement kiln flue gas-adapted strain, C. necator OB311 was leveraged. This strain exhibits a 2.5-fold enhancement both in growth rate and total biomass yield compared to H16 grown on flue gas (OB311 Td_flue-gas= 1.4 ± 0.3 vs. H16 Td_flue-gas= 3.8 ± 0.4, n=3; Fig. 1D). Therefore the same biobutanol engineering paradigm in strain OB311 as with H16 to insert hbd, crt, ter, and adhE2 was performed (OB311:BuOH(7); Fig.1B, Table 1). This OB311 engineered strain exhibited 1.6-fold faster growth kinetics compared H16 engineered strains during autotrophic growth with defined CO2 / H2mixture [OB311:BuOH(7) Td_CO2 / H2= 0.17 ± 0.03 vs. H16 Td_ CO2 / H2= 0.27 ± 0.04, n=3, P=0.0257, 2-tailed T-test], and produced 2.5-fold more 1-butanol (Fig. 1E). Coordinately, when strain OB311:BuOH(7) was grown in 1 L pilot reactors with cement kiln flue gas, titers of 30 mg / L were observed, ~10-fold more than for engineered H16 strains (Fig.1F). Thus, use of a flue gas-adapted strain of C. necator for growth on inhibitory industrial CO2-rich waste gas followed by metabolic engineering enabled biobutanol synthesis from cement flue gas at levels identical to those obtained by engineering H16 wild type strain to produce biobutanol from a defined gas mixture (H16:BuOH(1) on CO2 / H2= 26 ± 4 mg / L, n=3 vs. OB311:BuOH(7) on flue gas = 30 mg / L, n=1; P=0.10, 2-tailed T-test; Table 1). Selective production of 1-butanol via specific aldehyde and alcohol dehydrogenases

[0069] In addition to 1-butanol, engineered strains also accumulated ethanol, acetate, and butyrate when grown heterotrophically on gluconate. Production of these compounds wasAttorney Docket No.103362-020WO1 negligible in the parent H16 and OB311 strains under the same conditions (Fig. 3B and Figs 5 and 6). Similarly, ethanol was also produced during autotrophic growth (Fig. 3C and Fig. 8). Under oxygen limiting and fermentative conditions, wild type C. necator has been previously observed to produce these compounds, presumably via phosphate acetyltransferases (Pta), acetyl kinase (Ack), and propionate CoA-transferase (Pct). Deletion of the acetyl kinase and CoA- transferase genes in biobutanol strain OB311:BuOH(7) did not have a significant impact on the production of unwanted alcohols and acids during heterotrophic growth with gluconate, nor during autotrophic growth [strains OB311:BuOH(13,14,15), Table 1, Fig. 5-6]. This indicates that production of these compounds may arise due to compensating CoA-transferases that may be upregulated in the absence of specific CoA-transferases, or due to the promiscuity of the butanol pathway dehydrogenases.

[0070] Previously, two strategies have been employed to deacetylate and reduce butyryl-CoA to 1-butanol; the bifunctional aldehyde / alcohol dehydrogenase AdhE2, or a separate CoA- acylating butanol dehydrogenase (EutE) and broad-substrate range alcohol dehydrogenase from E. coli (YqhD) (Fig.1A). A previously developed E. coli butanol production strain was employed to efficiently screen several alcohol / aldehyde dehydrogenase candidates and quantify the yields and ratio of butanol to ethanol under aerobic growth with glucose. Both the bifunctional AdhE2 from C. acetobutylicum and the putative butyryl-CoA dehydrogenase EutE from Rhodobacter capsulatus (RCAP_rcc02201) in combination with the broad substrate range native E. coli alcohol dehydrogenase YqhD produced similar levels of 1-butanol (~3 g / L), but strikingly, 4-fold less ethanol was produced when using EutE and YqhD versus AdhE2 (Fig. 3A). In addition, expression of the R. capsulatus iron-containing alcohol dehydrogenase homolog associated with EutE (AdhFe, RCAP_rcc02210) did not alter 1-butanol yields, but increased ethanol by 1.5-fold (n=3, P=0.01, 2-tailed T-test), indicating butyraldehyde does not serve as an efficient substrate. Together, this revealed that in vivo the combination of EutE and YqhD dehydrogenases was more specific for butyryl-CoA versus AdhE2. Therefore, adhE2 on the chromosome of the engineered flue-gas tolerant strain OB311:BuOH was replaced(7). Consistent with the E. coli screen, the resulting C. necator strain [OB311:BuOH(20) Fig. 1C, Table 1] produced butanol:ethanol at a ratio of 2:1 versus 1:1 in the parent strain OB311:BuOH(7) and reached butanol titers up to 300 mg / L from gluconate (Fig. 3B), which is 50% more than previous benchmarks for C. necator using fructose. Acetate and butyrate production was unaffected, indicating that these arise from alternate, albeit unknown, competing pathways. Similarly, under autotrophic conditions, the EutE / YqhD strain OB311:BuOH(20) produced only butanol, in contrast to the AdhE2-containing parent strain OB311:BuOH(7) (Fig.3C). However, overall butanol yields were 3-fold less in theAttorney Docket No.103362-020WO1 EutE / YqhD versus AdhE2-containing strain. One possibility is that compared to AdhE2, EutE is more oxygen sensitive as seen for other aldehyde dehydrogenases, and subtle differences in culture oxygenation between autotrophic and heterotrophic growth could render EutE less active under autotrophic conditions. Increased biobutanol yields through altering CBB pathway activity

[0071] In attempt to increase autotrophic biobutanol pathway activity and yields, multiple gene copies of AdhE2 and EutE / YqhD were expressed, which resulted in no further improvement (Table 1). This, along with the lack of effect on autotrophic biobutanol production upon deletion of the transhydrogenases (vida supra; Table 1) indicated that during autotrophic growth there were other key limiting factors. Carbon assimilation through the CBB pathway to synthesize glyceraldehyde-3-phosphate and ultimately acetyl-CoA for biobutanol was focused on. Previous work has shown that cbb operon gene expression in numerous organisms is under control of the lysR-type transcriptional regulator CbbR, co-regulatory proteins (e.g. RegA), and effector molecules (e.g. PEP, ATP, NADPH, RuBP). An increase in CBB pathway activity through increased gene expression, particularly of the key rate-limiting enzyme RubisCO, is poised to increase CO2assimilation and product yields. Through prior mutagenesis studies of the C. necator cbbR gene, amino acid substitutions in the CbbR (SEQ ID NO: 1) DNA-binding linker region (L79F (SEQ ID NO: 2) and D84N), dimerization domain (D144N (SEQ ID NO: 3) and A167V), and effector molecule regulatory domain (H249Y (SEQ ID NO: 4)) that increased cbb operon expression and thus cellular RubisCO activity were identified. Of the multiple substitutions found in each region, those listed above conferred the largest increases in CBB pathway activity.

[0072] Extending this work, it was hypothesized that combinations of specific substitutions from each CbbR region (linker, dimerization, regulatory) would function cumulatively to further increase CBB pathway expression and activity. Using the previously developed C. necator selection strain (strain 76), in which the native cbbR gene is replaced by a modified cbbR gene of interest and lacZ is inserted under control of the cbb promoter (Pcbb, Fig.1D), the capability of individual substitutions and combinations thereof to increase cbb operon expression as monitored through LacZ activity, and carbon assimilation as measured through cellular RubisCO activity was quantified (Fig.4A). Indeed, individual point mutations increased CBB pathway activity by up to 4-fold, and specific combinations up to 7-fold. Next, these modified cbbR genes were transferred to biobutanol production strains by direct chromosomal replacement of the native cbbR gene (Fig. 1D). The single CbbR point mutations D144N (SEQ ID NO: 3) and A167V each increased RubisCO activity 4-fold using defined gas mixtures, consistent with the initial screeningAttorney Docket No.103362-020WO1 results (Fig. 4B). Coordinately, the D144N (SEQ ID NO: 3) point mutation increased butanol yields up to 3-fold on defined gas mixtures (OB311:BuOH(19); Fig.4C and Table 1), reaching titers of 188 ± 39 mg / L. When the original butanol pathway promoter, PPhaC1, was also replaced with the cbb promoter such that both the cbb operon and butanol pathway operon were under control of the D144N (SEQ ID NO: 3) constitutive CbbR variant (Fig.1D), biobutanol titers did not significantly increase (245 ± 31 mg / L, P=0.1, 2-tailed T-test), but ethanol production was significantly reduced over 2-fold (OB311:BuOH(33); Fig. 4C and Table 1). These biobutanol yields from autotrophic growth on CO2and H2are on par with C. necator strains optimized for heterotrophic growth with gluconate (Fig 3B) and fructose, and engineered photosynthetic cyanobacteria. This furthermore indicates that the butanol pathway enzymes and gene expression are prime targets for future optimization. The high performing CbbR triple substitution variants from the reporter strain screens (Fig. 4A) were unstable in the biobutanol production strains. Butanol production consistently decreased nearly 5-fold (Table 1), and RubisCO activities, after an initial increase, reverted to wild type levels post several passages of strains containing the modified cbbR gene. This is likely due to the metabolic burden of both operating the butanol pathway and CBB pathway at high levels of expression.

[0073] Employing top-performing single CbbR* variant butanol production strains, their stability in multiple batch experiments in pilot trials with petroleum upgrader gas was tested. Strain OB311:BuOH(33), which possessed the CbbR* D144N (SEQ ID NO: 3) modified regulator and both the CBB cycle and hbt, crt, ter, 1-butanol pathway genes under control of the cbb promoter (Pcbb), produced an average of 189±28, 245±31, and 282±18 mg / L biobutanol using defined gas mixtures amongst three different batch trials, demonstrating the stability and reproducibility of the strain for product yields (Fig.4C and Fig.8). Subsequently, 1 L pilot studies with cement flue gas supplemented with H2and petroleum upgrader gas supplemented with O2were performed. Consistent with initial tests on defined gas mixtures, RubisCO activity increased 8-fold compared to the parent strain with the native CbbR (Fig.4C). Discussion

[0074] Direct microbial utilization of CO2from industrial waste streams for bioproduct synthesis poses an efficient means for greenhouse gas mitigation and renewable materials production. It circumvents costly CO2purification from other inhibitory compounds present in flue gas and streamlines implementation of microbial carbon capture technology directly at the industrial waste source. While multiple bacteria have emerged as promising chassis forAttorney Docket No.103362-020WO1 engineering high-yielding biofuel production systems, particularly 1-butanol and isobutanol as drop-in gasoline replacements, the limitations imposed by inhibitory compounds present in unrefined CO2feedstock have remained a major challenge.

[0075] In this study, initial engineering of type strain C. necator H16 for 1-butanol production resulted in yields of 25 mg / L from CO2 / H2and 3 mg / L from cement kiln flue gas. Previously, first generation engineered cyanobacteria produced 2-14 mg / L 1-butanol, similar to C. necator. The initial 10-fold lower 1-butanol yields from flue gas versus CO2 / H2was clearly due, in part, to the slow growth rate and low biomass yield of C. necator H16 on flue gas (Fig.2D). Indeed, use of the Oakbio flue gas-adapted C. necator OB311 strain for growth on cement kiln flue gas resulted in a 10-fold increase of 1-butanol yield from flue gas to 30 mg / L, and a 2.5-fold increase of 1-butanol yield from CO2 / H2to 65 mg / L. This increase is likely due to OB311 strain tolerance to NOx, SO2and other compounds in flue gas and redirection of carbon flow toward acetyl-CoA, given that the 1-butanol yields are higher for the OB311:BuOH(7) versus the H16:BuOH(1) strain for both flue gas and pure CO2 / H2feedstocks (Table 1 and Fig. 2). Coordinately, this increase does not appear to be due to any increase in carbon assimilation rates, as the original type-strain C. necator H16 and OB311 strain show the same growth rate and total biomass yields on CO2 / H2(Fig.2D) and exhibit the same RubisCO activities (Fig.4C).

[0076] It was previously established that photosynthetic 1-butanol production in cyanobacteria from CO2employed oxygen tolerant alcohol / aldehyde dehydrogenases that increased biobutanol titers by 50-fold to 400 mg / L. Modular design and tuned expression of the butanol pathway genes resulted in further increases to 4.3 g / L. In the present study, a comparison screening of the canonical bifunctional aldehyde / alcohol dehydrogenase (AdhE2) from the A.B.E. pathway versus the putative long-chain alcohol and aldehyde dehydrogenases from E. coli and R. capsulatus revealed that the putative long chain aldehyde dehydrogenase EutE from R. capsulatus (RCAP_rcc02201), in conjunction with the alcohol dehydrogenase YqhD from E. coli, substantially increased the butanol:ethanol ratio from 1:1 to 2:1 during heterotrophic growth on gluconate, and nearly eliminated ethanol production during autotrophic growth with CO2 / H2(Fig. 3B-C). However, these enzymes appear to still suffer from oxygen sensitivity, as indicated by the dramatic 4-fold decrease in 1-butanol yields during autotrophic growth with CO2 / H2where culture oxygenation is higher as a result of sparging the culture with gas versus heterotrophic growth..Therefore, as with cyanobacteria, future isolation of suitable long chain alcohol and aldehyde dehydrogenases that are oxygen tolerant can increase 1-butanol titers in C. necator.

[0077] Lastly, modular engineering of the expression of the 1-butanol synthesis pathway genes and the CBB pathway for carbon fixation revealed that, in C. necator, both carbon flux into theAttorney Docket No.103362-020WO1 cell and through the 1-butanol pathway are key limiting factors for 1-butanol yields. Increased expression of the CBB pathway for CO2fixation through a constitutive CbbR regulator (D144N, SEQ ID NO: 3) alone increased 1-butanol yield 3-fold to 188 mg / L. Subsequent increase in 1- butanol pathway gene expression through use of the cbb promoter and constitutive CbbR regulator (D144N, SEQ ID NO: 3) further increased 1-butanol production 1.5-fold to 245 mg / L. These yields stably range from 200-300 mg / L in multiple batch trials. Further increases in pathway expression through stronger constitutive regulators (e.g. CbbR*-H249Y / L79F / D144N, SEQ ID NO: 8) appears to present a metabolic burden that decreases 1-butanol production and can even render the pathway unstable. This can be due to the energetic load of synthesizing increased amounts of protein, or due to an imbalance in redox carriers, such as NAD(P)H, that need to be in the reduced form to drive both carbon fixation and 1-butanol synthesis (Fig.1).

[0078] In total, these engineering efforts were effective in generating 1-butanol synthesis from CO2 / H2by C. necator, resulting in a 3000-fold increase in biobutanol titers from the initial strains (0.07 mg / L, H16∆phaC1::pBBRMSC3-AdhE2, Table 1) to strains engineered for increased expression of 1-butanol and CBB pathways (245 mg / L, OB311:BuOH(33), Table 1). Furthermore, it demonstrates the advantages of Oakbio’s flue-gas adapted strains of C. necator for engineering autotrophic biobutanol production directly from various industrial CO2waste streams. Interestingly, after initial engineering of the Oakbio C. necator OB311 for biobutanol production from flue gas and petroleum upgrader gas [OB311:BuOH(7)], further efforts to upregulate pathway activity resulted in no more than a 1.5-fold increase in 1-butanol titer on flue gas versus up to a 5-fold increases on pure CO2 / H2feedstocks. This suggests that the overall cell metabolism, and thus biobutanol production, is likely still partially inhibited by NOx, SO2and other compounds present in the CO2-rich industrial waste stream. Regardless, these experiments present the first successful biological process for direct production of 1-butanol from abundant cement and petroleum refinery industrial waste streams.Attorney Docket No.103362-020WO1 TABLES Table 1. Strains used in this study and corresponding autotrophic butanol produced from defined gas mixtures (in mg / L). Error bars are the standard deviation for n=3 independent experiments.Attorney Docket No.103362-020WO1Attorney Docket No.103362-020WO1TABLE 2. Primers used in this study.Attorney Docket No.103362-020WO1Attorney Docket No.103362-020WO1Attorney Docket No.103362-020WO1Attorney Docket No.103362-020WO1TABLE 3. Plasmids used in this study.Attorney Docket No.103362-020WO1Attorney Docket No.103362-020WO1SEQUENCES SEQ ID NO: 1 CbbR MSSFLRALTLRQLQIFVTVARHASFVRAAEELHLTQPAVSMQVKQLESVVGMALFERV KGQLTLTEPGDRLLHHASRILGEVKDAEEGLQAVKDVEQGSITIGLISTSKYFAPKLLAG FTALHPGVDLRIAEGNRETLLRLLQDNAIDLALMGRPPRELDAVSEPIAAHPHVLVASPR HPLHDAKGFDLQELRHETFLLREPGSGTRTVAEYMFRDHLFTPAKVITLGSNETIKQAV MAGMGISLLSLHTLGLELRTGEIGLLDVAGTPIERIWHVAHMSSKRLSPASESCRAYLLE HTAEFLGREYGGLMPGRRVA SEQ ID NO: 2 CbbR L79F MSSFLRALTLRQLQIFVTVARHASFVRAAEELHLTQPAVSMQVKQLESVVGMALFERV KGQLTLTEPGDRLLHHASRIFGEVKDAEEGLQAVKDVEQGSITIGLISTSKYFAPKLLAG FTALHPGVDLRIAEGNRETLLRLLQDNAIDLALMGRPPRELDAVSEPIAAHPHVLVASPR HPLHDAKGFDLQELRHETFLLREPGSGTRTVAEYMFRDHLFTPAKVITLGSNETIKQAV MAGMGISLLSLHTLGLELRTGEIGLLDVAGTPIERIWHVAHMSSKRLSPASESCRAYLLE HTAEFLGREYGGLMPGRRVA SEQ ID NO: 3 CbbR D144N MSSFLRALTLRQLQIFVTVARHASFVRAAEELHLTQPAVSMQVKQLESVVGMALFERV KGQLTLTEPGDRLLHHASRILGEVKDAEEGLQAVKDVEQGSITIGLISTSKYFAPKLLAG FTALHPGVDLRIAEGNRETLLRLLQNNAIDLALMGRPPRELDAVSEPIAAHPHVLVASPR HPLHDAKGFDLQELRHETFLLREPGSGTRTVAEYMFRDHLFTPAKVITLGSNETIKQAV MAGMGISLLSLHTLGLELRTGEIGLLDVAGTPIERIWHVAHMSSKRLSPASESCRAYLLE HTAEFLGREYGGLMPGRRVA SEQ ID NO: 4 CbbR H249Y MSSFLRALTLRQLQIFVTVARHASFVRAAEELHLTQPAVSMQVKQLESVVGMALFERV KGQLTLTEPGDRLLHHASRILGEVKDAEEGLQAVKDVEQGSITIGLISTSKYFAPKLLAG FTALHPGVDLRIAEGNRETLLRLLQDNAIDLALMGRPPRELDAVSEPIAAHPHVLVASPR HPLHDAKGFDLQELRHETFLLREPGSGTRTVAEYMFRDHLFTPAKVITLGSNETIKQAVAttorney Docket No.103362-020WO1 MAGMGISLLSLYTLGLELRTGEIGLLDVAGTPIERIWHVAHMSSKRLSPASESCRAYLLE HTAEFLGREYGGLMPGRRVA SEQ ID NO: 5 CbbR L79F D144N MSSFLRALTLRQLQIFVTVARHASFVRAAEELHLTQPAVSMQVKQLESVVGMALFERV KGQLTLTEPGDRLLHHASRIFGEVKDAEEGLQAVKDVEQGSITIGLISTSKYFAPKLLAG FTALHPGVDLRIAEGNRETLLRLLQNNAIDLALMGRPPRELDAVSEPIAAHPHVLVASPR HPLHDAKGFDLQELRHETFLLREPGSGTRTVAEYMFRDHLFTPAKVITLGSNETIKQAV MAGMGISLLSLHTLGLELRTGEIGLLDVAGTPIERIWHVAHMSSKRLSPASESCRAYLLE HTAEFLGREYGGLMPGRRVA SEQ ID NO: 6 CbbR L79F H249Y MSSFLRALTLRQLQIFVTVARHASFVRAAEELHLTQPAVSMQVKQLESVVGMALFERV KGQLTLTEPGDRLLHHASRIFGEVKDAEEGLQAVKDVEQGSITIGLISTSKYFAPKLLAG FTALHPGVDLRIAEGNRETLLRLLQDNAIDLALMGRPPRELDAVSEPIAAHPHVLVASPR HPLHDAKGFDLQELRHETFLLREPGSGTRTVAEYMFRDHLFTPAKVITLGSNETIKQAV MAGMGISLLSLYTLGLELRTGEIGLLDVAGTPIERIWHVAHMSSKRLSPASESCRAYLLE HTAEFLGREYGGLMPGRRVA SEQ ID NO: 7 CbbR D144N H249Y MSSFLRALTLRQLQIFVTVARHASFVRAAEELHLTQPAVSMQVKQLESVVGMALFERV KGQLTLTEPGDRLLHHASRILGEVKDAEEGLQAVKDVEQGSITIGLISTSKYFAPKLLAG FTALHPGVDLRIAEGNRETLLRLLQNNAIDLALMGRPPRELDAVSEPIAAHPHVLVASPR HPLHDAKGFDLQELRHETFLLREPGSGTRTVAEYMFRDHLFTPAKVITLGSNETIKQAV MAGMGISLLSLYTLGLELRTGEIGLLDVAGTPIERIWHVAHMSSKRLSPASESCRAYLLE HTAEFLGREYGGLMPGRRVA SEQ ID NO: 8 CbbR L79F D144N H249Y MSSFLRALTLRQLQIFVTVARHASFVRAAEELHLTQPAVSMQVKQLESVVGMALFERV KGQLTLTEPGDRLLHHASRIFGEVKDAEEGLQAVKDVEQGSITIGLISTSKYFAPKLLAG FTALHPGVDLRIAEGNRETLLRLLQNNAIDLALMGRPPRELDAVSEPIAAHPHVLVASPR HPLHDAKGFDLQELRHETFLLREPGSGTRTVAEYMFRDHLFTPAKVITLGSNETIKQAV MAGMGISLLSLYTLGLELRTGEIGLLDVAGTPIERIWHVAHMSSKRLSPASESCRAYLLE HTAEFLGREYGGLMPGRRVA SEQ ID NO: 9 EcoRIXbaIUP01F CT GAATTC TCTAGA CTTGCCGACATCTATGCGCT SEQ ID NO: 10 EcoRIXbaIUP01F CT GAATTC TCTAGA CTTGCCGACATCTATGCGCT SEQ ID NO: 11 UpSacI03R CT GAGCTC GATTTGATTGTCTCTCTGCCGTCA SEQ ID NO: 12 phaAprmtrSaII01F CT GTCGAC CGCTTGCATGAGTGCCGAttorney Docket No.103362-020WO1 SEQ ID NO: 13 5phaAXbaIHindIII02R CT AAGCTT TCTAGA CAGCTTGGCATCGCCCAT SEQ ID NO: 14 hbdKpnI01F CT GGTACC AAGGAAGGACTGATCATGAAAAAGGTATGTGTTATAGG SEQ ID NO: 15 hbdXhoI03R CT CTCGAG TTATTTTGAATAATCGTAGAAACC SEQ ID NO: 16 crtXhoI01F CT CTCGAG AAGGAAGGACTGATC ATGGAACTAAACAATGTCATC SEQ ID NO: 17 crtSmaI02R CT CCCGGG CTATCTATTTTTGAAGCCTTC SEQ ID NO: 18 terSmaI01F CT CCCGGG AAGGAAGGACTGATC ATGATTGTAAAACCAATGGT SEQ ID NO: 19 terSpeI02R CT A CT ACTAGT TTAAATCCTGTCGAACCTTT SEQ ID NO: 20 hbdSacI01F CT GAGCTC ATGAAAAAGGTATGTGTTATAGG SEQ ID NO: 21 SalIter04R CT GTCGAC TTAAATCCTGTCGAACCTTT SEQ ID NO: 22 EcoRIXbaI3phaA01F CT GAATTC TCTAGA CTTCAAGACCGACGAGTTCGTG SEQ ID NO: 23 phaBprmtSacI01R CT GAGCTC GTCCACTCCTTGATTGGCTTCG SEQ ID NO: 24 SalIDown01F CT GTCGAC CCTGCCGGCCTGGTTCAAC SEQ ID NO: 25 DownXbaIHindIII02R CT AAGCTT TCTAGA TTCTGAATCCATGACCAGCTGCTTG SEQ ID NO: 26 adhE2SpeI01F CT ACTAGT AAGGAAGGACTGATC ATGAAAGTTACAAATCAAAAAGAA SEQ ID NO: 27 adhE2XbaI02R CT TCTAGA TTAAAATGATTTTATATAGATATCCTTAAGAttorney Docket No.103362-020WO1 SEQ ID NO: 28 adhE2SacI01F CT GAGCTC ATGAAAGTTACAAATCAAAAAGAA SEQ ID NO: 29 adhE2SalI05R CT GTCGAC TTAAAATGATTTTATATAGATATCCTTAAG SEQ ID NO: 30 SacIXbaIpta1 F GAT GAGCTC TCTAGA CGATGCGATGACCGAATACAC SEQ ID NO: 31 SalIpta1 R GAT GTCGAC CTTCTCATGCTTGGCGTTCAC SEQ ID NO: 32 SalIackA F GAT GTCGAC GAGGAACTGATGATTGCCAGG SEQ ID NO: 33 HindIIIXbaIpta1 R GAT AAGCTT TCTAGA GCATCAAATCGAAGCTCTTAAGC SEQ ID NO: 34 UPpta2F CGATTT GAGCTC TCTAGA GCAGGAGCGCACGCCCACCG SEQ ID NO: 35 UPpta2R CGATTT GTCGAC GGCGCGGTCGATGATGCGGAGG SEQ ID NO: 36 DNpta2F CGATTT GTCGAC CTCTCGCGCGGCTGCAGCGCG SEQ ID NO: 37 DNpta2R CGATTT AAGCTT TCTAGA GCCGCCCCAGCAACGTGGCG SEQ ID NO: 38 UPpct_F CGATTT GAATTC TCTAGA GCTCACCAGCACACAGTAGCCG SEQ ID NO: 39 UPpct_R CGATTT GGATCC GATCACCTTCATTGCTGTCTCCG SEQ ID NO: 40 DNpct_F CGATTT GGATCC GATCACCTTCATTGCTGTCTCCG SEQ ID NO: 41 DNpct_R CGATTT AAGCTT TCTAGA CCATCATGTCCTTGCCTATCAGC SEQ ID NO: 42 UPpntAb3B3_01F CAT GAGCTC TCTAGA CGTGCTGGACTTCCTCAAGCAttorney Docket No.103362-020WO1 SEQ ID NO: 43 UPpntAb3B3_02R CAT GGATCC CTCCATCGACTTCTCCTCCGTT SEQ ID NO: 44 DNpntAb3B3_03F CAT GGATCC GCCAAGAACGACCCCCAGTC SEQ ID NO: 45 DNpntAb3B3_04R CAT AAGCTT TCTAGA ATTGCAGCGCTTCACGCGG SEQ ID NO: 46 UPpntAB1_Re01F CAT GAATTC TCTAGA CAACTCGGTGCAGAGGTTCAC SEQ ID NO: 47 UPpntAB1_Re02R CAT GGATCC CTCGCCTCGCAGTGTTTCC SEQ ID NO: 48 DNpntAB1_Re03F CAT GGATCC GCCAAGAAGGTGGTCGAAGATATG SEQ ID NO: 49 DNpntAB1_Re04R CAT AAGCTT TCTAGA CAGCGTCTCCGATGCATACTC SEQ ID NO: 50 HindIIIXbaI_C1up_F CGATTT AAGCTT TCTAGA ATTGCCTGCCGGTGCTGCTATG SEQ ID NO: 51 SalI_C1up_R CGATTT GTCGAC ATGTACATCGTGGCCATTGGCTG SEQ ID NO: 52 SacI_hbd_F CGATTT GAGCTC ATGAAAAAGGTATGTGTTATAG SEQ ID NO: 53 EcoRIXbaI_hbd_R CGATTT GAATTC TCTAGA CATCCATTATAGCAAGACATATATCAAGAC SEQ ID NO: 54 SalI_Pcbb_F CT GTCGAC TTCATCCTTCTCGCCTACTGGCGAAGGGTAAGGGCG SEQ ID NO: 55 Pcbb_RBS_SacI_R CGATTT GAGCTC GCTTGTCTCCTTGCGTGGTTGAG SEQ ID NO: 56 EcoRI_XbaI_cbbR_F CGATTT GAATTC TCTAGA GCTGGACATATGCGCAACATGC SEQ ID NO: 57 pcbb_RBS_SacI_R CGATTT GAGCTC GCTTGTCTCCTTGCGTGGTTGAGCAttorney Docket No.103362-020WO1 SEQ ID NO: 58 SphI_RBS_RbcL_F CGATTT GCATGC AAGGAGACAAGCATGAACGCACC SEQ ID NO: 59 RbcL_XbaI_HindIII_R CGATTT AAGCTT TCTAGA GTTGCTCATGGACTGGATACAGG SEQ ID NO: 60 SacI_adhE2_F CGATTT GAGCTC ATGAAAGTTACAAATCAAAAAGAACTAAAACAAAAGC SEQ ID NO: 61 adhE2_SphI_R CGATTT GCATGC TTAAAATGATTTTATATAGATATCCTTAAGTTCAC SEQ ID NO: 62 RLSXba3 CAGCCGG TCTAGA TCAGTAGCGGCTGCCCTCGGGGC SEQ ID NO: 63 DD227 GCATTT TCTAGA AAGCTTAACCAGACGGCAACGCTGACCG SEQ ID NO: 64 DD560 GCATTT TCTAGA GTATAAGCAATCCCTTAAGTGCGGACC SEQ ID NO: 65 DD561 GCATTT TCTAGA CTTGGGCGTTACCCGTTCGAAATCGGC REFERENCES 1. da Silva Trindade, W.R. and R.G. dos Santos, Review on the characteristics of butanol, its production and use as fuel in internal combustion engines. Renewable and Sustainable Energy Reviews, 2017.69: p.642-651. 2. Moon, H.G., et al., One hundred years of clostridial butanol fermentation. FEMS microbiology letters, 2016.363(3). 3. Shen, C.R., et al., Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Applied and environmental microbiology, 2011.77(9): p.2905-2915. 4. Lan, E.I. and J.C. Liao, Microbial synthesis of n-butanol, isobutanol, and other higher alcohols from diverse resources. Bioresource technology, 2013.135: p.339-349. 5. Wang, M., L. Fan, and T. Tan, 1-Butanol production from glycerol by engineered Klebsiella pneumoniae. Rsc Advances, 2014.4(101): p.57791-57798. 6. Laguna, R., et al., Development of a plasmid addicted system that is independent of co- inducers, antibiotics and specific carbon source additions for bioproduct (1-butanol) synthesis in Escherichia coli. Metabolic Engineering Communications, 2015.2: p.6-12.Attorney Docket No.103362-020WO1 7. Black, W.B., et al., Rearrangement of coenzyme A-acylated carbon chain enables synthesis of isobutanol via a novel pathway in Ralstonia eutropha. ACS Synthetic Biology, 2018.7(3): p.794-800. 8. Dürre, P., Butanol formation from gaseous substrates. FEMS microbiology letters, 2016. 363(6): p. fnw040. 9. Karim, A.S., et al., In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design. Nature Chemical Biology, 2020.16(8): p.912-919. 10. Bai, W., et al., n-Butanol production by Rhodopseudomonas palustris TIE-1. Communications biology, 2021.4(1): p.1-16 %@ 2399-3642. 11. Lan, E.I. and J.C. Liao, ATP drives direct photosynthetic production of 1-butanol in cyanobacteria. Proceedings of the National Academy of Sciences, 2012.109(16): p.6018-6023. 12. Lan, E.I. and J.C. Liao, Metabolic engineering of cyanobacteria for 1-butanol production from carbon dioxide. Metabolic engineering, 2011.13(4): p.353-363. 13. Lan, E.I., S.Y. Ro, and J.C. Liao, Oxygen-tolerant coenzyme A-acylating aldehyde dehydrogenase facilitates efficient photosynthetic n-butanol biosynthesis in cyanobacteria. Energy & Environmental Science, 2013.6(9): p.2672-2681. 14. Liu, X., et al., Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO 2 in cyanobacteria. Energy & Environmental Science, 2019.12(9): p.2765-2777. 15. Ellis, L.D., et al., Toward electrochemical synthesis of cement—An electrolyzer-based process for decarbonating CaCO3 while producing useful gas streams. Proceedings of the National Academy of Sciences, 2020.117(23): p.12584-12591. 16. Van Oss, H.G. and A.C. Padovani, Cement manufacture and the environment part II: environmental challenges and opportunities. Journal of Industrial ecology, 2003.7(1): p.93- 126. 17. Lara-Gil, J.A., C. Senés-Guerrero, and A. Pacheco, Cement flue gas as a potential source of nutrients during CO2 mitigation by microalgae. Algal research, 2016.17: p.285-292. 18. Nagappan, S., et al., Enhancement of biofuel production by microalgae using cement flue gas as substrate. Environmental Science and Pollution Research, 2020.27(15): p.17571-17586. 19. Cheah, W.Y., et al., Biosequestration of atmospheric CO2 and flue gas-containing CO2 by microalgae. Bioresource technology, 2015.184: p.190-201. 20. Huang, G., et al., Current techniques of growing algae using flue gas from exhaust gas industry: a review. Applied biochemistry and biotechnology, 2016.178(6): p.1220-1238. 21. Yadav, G., S.K. Dash, and R. Sen, A biorefinery for valorization of industrial waste- water and flue gas by microalgae for waste mitigation, carbon-dioxide sequestration and algal biomass production. Science of the total environment, 2019.688: p.129-135.Attorney Docket No.103362-020WO1 22. Zeiler, K.G., et al., The use of microalgae for assimilation and utilization of carbon dioxide from fossil fuel-fired power plant flue gas. Energy Conversion and Management, 1995. 36(6-9): p.707-712. 23. Noguchi, S., et al., Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production. Metabolomics, 2016.12(2): p.1- 10. 24. Kircher, M. and T. Schwarz, CO2 und CO-Nachhaltige Kohlenstoffquellen für die Kreislaufwirtschaft.2020: Springer. 25. Pohlmann, A., et al., Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature biotechnology, 2006.24(10): p.1257-1262. 26. Huschner, F., et al., Development of a feeding strategy for high cell and PHA density fed- batch fermentation of Ralstonia eutropha H16 from organic acids and their salts. Process Biochemistry, 2015.50(2): p.165-172. 27. Diels, L. and M.A.X. Mergeay, DNA probe-mediated detection of resistant bacteria from soils highly polluted by heavy metals. Applied and Environmental Microbiology, 1990.56(5): p. 1485-1491. 28. Vollmer, M.D., K. Stadler-Fritzsche, and M. Schlömann, Conversion of 2- chloromaleylacetate in Alcaligenes eutrophus JMP134. Archives of microbiology, 1993.159(2): p.182-188. 29. Lin, J., et al., Continuous desulfurization and bacterial community structure of an integrated bioreactor developed to treat SO2 from a gas stream. Journal of Environmental Sciences, 2015.37: p.130-138. 30. Pohlmann, A., et al., A novel NO‐responding regulator controls the reduction of nitric oxide in Ralstonia eutropha. Molecular microbiology, 2000.38(3): p.626-638. 31. Yanisch-Perron, C., J. Vieira, and J. Messing, Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene, 1985.33(1): p. 103-119. 32. Ormerod, J.G., K.S. Ormerod, and H. Gest, Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Archives of biochemistry and biophysics, 1961.94(3): p.449-463. 33. Boynton, Z.L., G.N. Bennet, and F.B. Rudolph, Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824. Journal of bacteriology, 1996.178(11): p.3015-3024. 34. Tucci, S. and W. Martin, A novel prokaryotic trans-2-enoyl-CoA reductase from the spirochete Treponema denticola. FEBS letters, 2007.581(8): p.1561-1566 %@ 0014-5793.Attorney Docket No.103362-020WO1 35. Dangel, A.W. and F.R. Tabita, Amino acid substitutions in the transcriptional regulator CbbR lead to constitutively active CbbR proteins that elevate expression of the cbb CO2 fixation operons in Ralstonia eutropha (Cupriavidus necator) and identify regions of CbbR necessary for gene activation. Microbiology, 2015.161(9): p.1816-1829. 36. Satagopan, S., et al., A Rubisco mutant that confers growth under a normally “inhibitory” oxygen concentration. Biochemistry, 2009.48(38): p.9076-9083. 37. Fontaine, L., et al., Molecular characterization and transcriptional analysis of adhE2, the gene encoding the NADH-dependent aldehyde / alcohol dehydrogenase responsible for butanol production in alcohologenic cultures of Clostridium acetobutylicum ATCC 824. Journal of bacteriology, 2002.184(3): p.821-830. 38. Siedow, A., et al., A megaplasmid-borne anaerobic ribonucleotide reductase in Alcaligenes eutrophus H16. Journal of bacteriology, 1999.181(16): p.4919-4928. 39. Kohlmann, Y., et al., Analyses of soluble and membrane proteomes of Ralstonia eutropha H16 reveal major changes in the protein complement in adaptation to lithoautotrophy. Journal of proteome research, 2011.10(6): p.2767-2776. 40. Sznajder, A., D. Pfeiffer, and D. Jendrossek, Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16. Applied and environmental microbiology, 2015.81(5): p.1847-1858. 41. Lindenkamp, N., M. Schürmann, and A. Steinbüchel, A propionate CoA-transferase of Ralstonia eutropha H16 with broad substrate specificity catalyzing the CoA thioester formation of various carboxylic acids. Applied microbiology and biotechnology, 2013.97(17): p.7699- 7709. 42. Volodina, E., et al., Characterization of propionate CoA-transferase from Ralstonia eutropha H16. Applied microbiology and biotechnology, 2014.98(8): p.3579-3589. 43. Pantazopoulous, P.E. and M.T. Madigan, Primary alcohols and di-alcohols as growth substrates for the purple nonsulfur bacterium Rhodobacter capsulatus. Canadian journal of microbiology, 2000.46(12): p.1166-1170. 44. Dangel, A.W. and F.R. Tabita, CbbR, the master regulator for microbial carbon dioxide fixation. Journal of bacteriology, 2015.197(22): p.3488-3498. 45. Gibson, J.L. and F.R. Tabita, Nucleotide sequence and functional analysis of CbbR, a positive regulator of the Calvin cycle operons of Rhodobacter sphaeroides. Journal of bacteriology, 1993.175(18): p.5778-5784. 46. Gibson, J.L. and F.R. Tabita, The molecular regulation of the reductive pentose phosphate pathway in Proteobacteria and Cyanobacteria. Archives of microbiology, 1996. 166(3): p.141-150. 47. Paoli, G.C., et al., Expression of the cbbLcbbS and cbbM genes and distinct organization of the cbb Calvin cycle structural genes of Rhodobacter capsulatus. Archives of microbiology, 1995.164(6): p.396-405.Attorney Docket No.103362-020WO1 48. Romagnoli, S. and F.R. Tabita, A novel three-protein two-component system provides a regulatory twist on an established circuit to modulate expression of the cbbI region of Rhodopseudomonas palustris CGA010. Journal of bacteriology, 2006.188(8): p.2780-2791. 49. Chia, S.R., et al., CO2 mitigation and phycoremediation of industrial flue gas and wastewater via microalgae-bacteria consortium: Possibilities and challenges. Chemical Engineering Journal, 2021.425: p.131436. 50. Gleizer, S., et al., Engineering microbes to produce fuel, commodities, and food from CO2. Cell Reports Physical Science, 2020.1(10): p.100223. 51. Chakravarty, J. and C.J. Brigham, Solvent production by engineered Ralstonia eutropha: channeling carbon to biofuel. Applied microbiology and biotechnology, 2018.102(12): p.5021- 5031. 52. Tichi, M.A. and F.R. Tabita, Maintenance and control of redox poise in Rhodobacter capsulate strains deficient in the Calvin-Benson-Bassham pathway. Archives of microbiology, 2000.174(5): p.322-333 %@ 1432-072X. 53. Tichi, M.A. and F.R. Tabita, Interactive control of Rhodobacter capsulatus redox- balancing systems during phototrophic metabolism. Journal of bacteriology, 2001.183(21): p. 6344-6354 %@ 1098-5530. 54. Wang, X., D.L. Falcone, and F.R. Tabita, Reductive pentose phosphate-independent CO2 fixation in Rhodobacter sphaeroides and evidence that ribulose bisphosphate carboxylase / oxygenase activity serves to maintain the redox balance of the cell. Journal of bacteriology, 1993.175(11): p.3372-3379 %@ 0021-9193. 55. Gordon, G.C. and J.B. McKinlay, Calvin cycle mutants of photoheterotrophic purple nonsulfur bacteria fail to grow due to an electron imbalance rather than toxic metabolite accumulation. Journal of bacteriology, 2014.196(6): p.1231-1237 %@ 0021-9193. 56. McKinlay, J.B. and C.S. Harwood, Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria. Proceedings of the National Academy of Sciences, 2010. 107(26): p.11669-11675 %@ 0027-8424. 57. Kovach, M.E., Elzer, P.H., Hill, D.S., Robertson, G.T., Farris, M.A., Roop II, R.M. and Peterson, K.M., 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene, 166(1), pp.175-176. 58. Quandt, J. and Hynes, M.F., 1993. Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene, 127(1), pp.15-21.

Claims

Attorney Docket No.103362-020WO1 CLAIMS What is claimed is:

1. An engineered bacterial strain capable of producing 1-butanol using carbon dioxide (CO2) and hydrogen gas (H2), wherein the strain naturally utilizes Calvin-Benson- Bassham (CBB) cycle, and further wherein the strain has been engineered so that expression of products from the CBB pathway are increased compared to a non- engineered version of the strain.

2. The engineered bacterial strain of claim 1, wherein the mutation(s) are within CbbR (CBB regulator).

3. The engineered bacterial strain of claim 1 or 2, wherein the strain is Cupriavidus necator.

4. The engineered bacterial strain of claim 3, wherein the C. necator strain is OB311, and further wherein the mutations are within CbbR, wherein the CbbR comprises SEQ ID NO:

1.

5. The engineered bacterial strain of any one of claims 1-4, wherein said mutation comprises one or more single point mutations.

6. The engineered bacterial strain of claim 4, wherein the mutations within SEQ ID NO: 1 comprise at least one of H249Y, L79F, and D144N.

7. The engineered bacterial strain of any one of claims 1-6, wherein the strain is further engineered by inactivating native polyhydroxybutyrate synthesis.

8. The engineered bacterial strain of any one of claims 1-7, wherein the strain is further engineered by introducing a 1-butanol pathway.

9. The engineered bacterial strain of claim 8, wherein the 1-butanol pathway is a modified version of Clostridium acetobutylicum 1-butanol pathway.

10. The engineered bacterial strain of any one of claims 1-9, wherein a native phaCAB promoter is replaced.

11. The engineered bacterial strain of any one of claims 8-10, wherein the 1-butanol pathway is introduced at phaCAB locus.

12. The engineered bacterial strain of claim 11, wherein phaC1 is replaced with alternate butyryl-CoA synthesis genes.

13. The engineered bacterial strain of claim 12, wherein the alternate butyryl-CoA synthesis genes comprise hbd, crt, and ter.

14. The engineered bacterial strain of claim 12 or 13, wherein phaB1 is replaced with adhE2.

15. The engineered bacterial strain of any one of claims 1-14, wherein expression of at least one native transhydrogenase is reduced or eliminated in the bacterial strain.Attorney Docket No.103362-020WO1 16. The engineered bacterial strain of claim 15, wherein expression at least one of PntAb3, PntB3, PntAa1, PntAb1, and PntB1is reduced or eliminated.

17. The engineered bacterial strain of claim 1 in which carbon dioxide (CO2) and hydrogen gas (H2), are provided by industrial waste gas.

18. A method of producing 1-butanol from an engineered bacteria using carbon dioxide (CO2) and hydrogen gas (H2), wherein the strain naturally utilizes Calvin-Benson- Bassham (CBB) cycle, and further wherein the strain has been engineered so that expression of products from the CBB pathway are increased compared to a non- engineered version of the strain.

19. The method of claim 18, wherein autotrophic 1-butanol production is achieved at a rate of at least 30 mg / L.

20. The method of claim 18, wherein autotrophic 1-butanol production is achieved at a rate of at least 100 mg / L.

21. The method of claim 18, wherein autotrophic 1-butanol production is achieved at a rate of at least 200 mg / L.

22. The method of claim 18, wherein autotrophic 1-butanol production is achieved at a rate of at least 300 mg / L.

23. The method of any one of claims 18-22, wherein the method is carried out in presence of industrial waste gas.

24. A method of selecting and determining a mutation within CbbR which can increase 1- butanol production in an engineered bacterial strain, the method comprising: a. Providing a library of mutations within CbbR into bacterial strains engineered to produce 1-butanol; b. Determining which mutations provide an increase in 1-butanol synthesis; and c. Selecting the strain of step b).

25. The method of claim 24, wherein the CbbR sequence provided to the library of mutations consists of any one of SEQ ID NO: 1-7.

Images