SYNTHETIC PATHWAY FOR BIOLOGICAL CARBON DIOXIDE SEQUESTRATION

Abstract

This invention relates to methods for increasing carbon fixation and/or increasing biomass production in a plant, comprising: introducing into a plant, plant part, and/or plant cell heterologous polynucleotides encoding (1) a succinyl CoA synthetase, (2) a 2-oxoglutarate:ferredoxin oxidoreductase, (3) a 2-oxoglutarate carboxylase, (4) an oxalosuccinate reductase, or (5) an isocitrate lyase, or (6) a succinyl CoA synthetase and a 2-oxoglutarate:ferredoxin oxidoreductase, (7) a 2-oxoglutarate carboxylase and an oxalosuccinate reductase polypeptide, and/or (8) a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and an isocitrate lyase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein said heterologous polynucleotides are from a bacterial and/or an archaeal species. Additionally, transformed plants, plant parts, and/or plant cells are provided as well as products produced from the transformed plants, plant parts, and/or plant cells.

Description

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 5051-887_ST25.txt, 266,968 bytes in size, generated on Jul. 18, 2016 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures.

FIELD OF THE INVENTION

The present invention relates to methods for increasing carbon fixation and biomass production in plants.

BACKGROUND

All life depends on photosynthetic carbon fixation in which CO₂ is converted to organic compounds in the presence of water and light. However, this is an inefficient process, particularly in C₃ plants, because of a competing process called photorespiration. Photorespiration results in the release of about a quarter of the carbon that is fixed by photosynthesis. The inefficiency of C₃ photosynthesis is largely due to the enzyme ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) that catalyzes two competing reactions, carboxylation and oxygenation. Carboxylation leads to net fixed carbon dioxide and oxygenation utilizes oxygen and results in a net loss of carbon. The relative concentrations of carbon dioxide and oxygen and the temperature as well as water availability determine which reaction occurs or dominates. Thus, C₃ plants do not grow efficiently in hot and/or dry areas because, as the temperature increases, Rubisco incorporates more oxygen. Some plants, such as C₄ and CAM (Crassulacean acid metabolism) plants, have developed mechanisms that reduce the effect of photorespiration by more efficiently delivering carbon dioxide to Rubisco, thereby outcompeting the oxygenase activity.

SUMMARY OF THE INVENTION

This invention is directed to methods for improving the efficiency of CO₂ fixation and increasing biomass production in plants.

Thus, in one aspect, the present invention provides a method for increasing carbon fixation and/or increasing biomass production in a plant, comprising: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide to produce a stably transformed plant, plant part, and/or plant, wherein the heterologous polynucleotide is from a bacterial or an archaeal species.

In another aspect, the present invention provides a method for increasing carbon fixation and/or increasing biomass production in a plant, comprising: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide to produce a stably transformed plant, plant part, and/or plant, wherein the heterologous polynucleotide is from a bacterial or an archaeal species.

In still another aspect, a method for increasing carbon fixation and/or increasing biomass production in a plant is provided, comprising: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein said heterologous polynucleotide is from a bacterial or an archaeal species.

In a further aspect, a method for increasing carbon fixation and/or increasing biomass production in a plant, comprising: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein said heterologous polynucleotide is from a bacterial or an archaeal species.

In another aspect, the invention provides a method for increasing carbon fixation and/or increasing biomass production in a plant, comprising: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding an isocitrate lyase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein said heterologous polynucleotide is from a bacterial or an archaeal species.

In an additional aspect of the invention, a method for increasing carbon fixation and/or increasing biomass production in a plant is provided, comprising: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein said heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and said heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide are from a bacterial or an archaeal species.

In a further aspect, a method for increasing carbon fixation and/or increasing biomass production in a plant is provided, comprising: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein said heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and said heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide are from a bacterial and/or an archaeal species. In a further aspect, the method optionally additionally comprises introducing into the plant, plant part, and/or plant cell a heterologous polynucleotide encoding an isocitrate lyase polypeptide, wherein said heterologous polynucleotide encoding isocitrate lyase is from a bacterial or archaeal species.

In a further aspect, the present invention provides stably transformed plants, plant parts and/or plant cells, seeds from said stably transformed plants, and crops comprising said stably transformed plants.

In additional aspects, the present invention provides products produced from the transformed plants, plant parts and/or plant cells, seeds and/or crops of this invention.

The foregoing and other objects and aspects of the present invention are explained in detail in the drawings and specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic for the condensed reverse tricarboxylic acid (crTCA) cycle.

FIG. 2 shows a schematic view of 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) enzyme assay.

FIG. 3 shows a schematic view of reductive carboxylation catalyzed by 2-oxoglutarate carboxylase/isocitrate dehydrogenase (OGC/ICDH) (adapted from Aoshima et al. Mol. Microbiol. 62:748-759 (2006)).

FIG. 4 shows construct 1 comprising the elements for cloning, selection and expression of chloroplast targeted isocitrate dehydrogenase/oxalosuccinate reductase (ICDH/OSR), isocitrate lyase (ICL) and the chloroplast targeted two subunits of OGC.

FIG. 5 shows construct 2 comprising the elements for cloning, selection and expression of chloroplast targeted two subunits each of succinyl CoA synthetase (SCS) and 2-oxoglutarate:ferredoxin oxidoreductase (KOR).

FIG. 6 shows the amount of glyoxylate detected by LC-MS over time for the full crTCA cycle reaction conducted under anaerobic conditions (diamonds, unlabeled glyoxylate; squares, 1-¹³C-glyoxylate; and triangles, 2-¹³C-glyoxylate). The results were corrected based on the natural abundance of the ¹³C glyoxylate.

FIG. 7 shows the amount of succinate detected by LC-MS over time for the full crTCA cycle reaction conducted under anaerobic conditions (diamonds, unlabeled succinate; squares, 1-¹³C-succinate; and triangles, 2-¹³C-succinate). The results were corrected based on the natural abundance of the ¹³C succinate.

FIG. 8 shows the amount of 2-oxoglutarate detected by LC-MS over time for the full crTCA cycle reaction conducted under anaerobic conditions (diamonds, unlabeled 2-oxoglutarate; squares, 1-¹³C-2-oxoglutarate; and triangles, 2-¹³C-2-oxoglutarate). The results were corrected based on the natural abundance of the ¹³C 2-oxoglutarate.

FIG. 9 shows plant heights at 30 DAP. C1×C2 F1 plants are significantly taller than wild type and C2 T3 but not significantly taller than C1 T3. Values labeled with the same letter are not significantly different according to Fisher's LSD test (p=0.05). C2 plants were transformed with Construct 2 and C1 plants were transformed with Construct 1.

FIG. 10 shows C. sativa plants at 36 days after planting (DAP). Panel A: Wild-type, Panel B; C1×C2 F1 plants, Panel C; C1 T3 plants, and Panel D: C2 T3 plants. Both parental lines, C1 and C2 and the F1 of their cross exhibit a significant increase in plant height over wild type plants.

FIG. 11 shows C. sativa plants at 48 DAP. Panel A: C1 T3 plants; Panel B: C2 T3 plants; Panel C: C1×C2 F1 plants; and Panel D: Wild-type.

FIG. 12 shows expression of transgenes in the respective transgenic Camelina lines (C1-1, C1-2, C1-3, C2-1, C2-5, C2-8) as compared to wild type (WT1, WT2).

FIG. 13 provides a schematic of potential partial pathways function in shifting plant metabolism in the partial crTCA expressing lines.

FIG. 14 shows metabolites in both the synthetic pathway and the Krebs cycle (left panel) and metabolites exclusive to the Krebs cycle (right panel).

FIG. 15 shows steady-state levels of the Benson Calvin cycle (left panel) and sugar levels (right panel) in WT and transgenic lines.

FIG. 16 shows serine and glycine levels for WT and transgenic lines.

FIG. 17 shows pyruvate levels for WT and transgenic lines.

FIG. 18 provides a schematic showing the role of PEP carboxylase, PEP carboxy kinase, malic enzyme and pyruvate carboxylase (upper panel) and the pyruvate and oxaloacetate levels (left panel) and pyruvate and alanine levels in in WT and transgenic lines.

FIG. 19 shows the effect of the synthetic cycle on redox balance.

FIG. 20 shows the growth phenotype and height of Camelina wild type (wt) plants and C1 and C2 parent plants. While there is variation in the height of the parent plants, the wt plants are fairly uniform. We chose the tallest wt plant (WT3) as a visual comparison in the images with the integrated crosses (FIG. 21).

FIGS. 21A-21C shows the growth phenotype of full cycle for three integrated lines: C1 (FIG. 21A), C2 (FIG. 21B), C3 (FIG. 21C). For comparison, the tallest wt plant, WT3, was included in the images.

FIGS. 22A-22C shows photosynthetic CO₂ fixation rates (FIG. 22A-22B) and dark respiration (FIG. 22C) in the integrated crosses (X13, X36, X50) compared to wt or parent lines when measured under ambient CO₂ (400 ppm) (FIG. 22A) or under elevated CO₂ (1600 ppm) (FIG. 22B).

FIG. 23 shows transcript abundance of C1 transgenes in the C1 parent lines and the integrated crosses.

FIG. 24 shows transcript abundance of C2 transgenes in the C2 parent lines and the integrated crosses.

FIG. 25 shows transcript abundance of C3 transgenes in the integrated crosses.

FIGS. 26A-26C shows respective protein levels (western blots) for each transgene; immunoblot developed with colorimetric staining (FIG. 26A) and immunoblot developed with ECL (FIG. 26B, FIG. 27C).

FIGS. 27A-27B shows seed yield (g seed/plant) (FIG. 27A) and seed weight (FIG. 27B) of wt, parent and integrated, crossed lines.

DETAILED DESCRIPTION

The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

Unless otherwise defined, 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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art.

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

The term “about,” as used herein when referring to a measurable value such as a dosage or time period and the like, means variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

As used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y. As used herein, phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”

The terms “comprise,” “comprises” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

“Complement” as used herein can mean 100% complementarity or identity with the comparator nucleotide sequence or it can mean less than 100% complementarity (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like, complementarity).

The terms “complementary” or “complementarity,” as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence “5′-A-G-T-3” binds to the complementary sequence “3′-A-C-T-5′.” Complementarity between two single-stranded molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

As used herein, the terms “express,” “expresses,” “expressed” or “expression,” and the like, with respect to a nucleotide sequence (e.g., RNA or DNA) indicates that the nucleotide sequence is transcribed and, optionally, translated. Thus, a nucleotide sequence may express a polypeptide of interest or a functional untranslated RNA. A “functional” RNA includes any untranslated RNA that has a biological function in a cell, e.g., regulation of gene expression. Such functional RNAs include but are not limited to RNAi (e.g., siRNA, shRNA), miRNA, antisense RNA, ribozymes, RNA aptamers, and the like.

As used herein, the terms “fragment” when used in reference to a polynucleotide will be understood to mean a nucleic acid molecule or polynucleotide of reduced length relative to a reference nucleic acid molecule or polynucleotide and comprising, consisting essentially of and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 30%, 35%, 40%, 45%, 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%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.

As used herein, a “functional” polypeptide or “functional fragment” is one that substantially retains at least one biological activity normally associated with that polypeptide. In particular embodiments, the “functional” polypeptide or “functional fragment” substantially retains all of the activities possessed by the unmodified peptide. By “substantially retains” biological activity, it is meant that the polypeptide retains at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide). A “non-functional” polypeptide is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%).

As used herein, the term “gene” refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, and the like. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and 5′ and 3′ untranslated regions). A gene may be “isolated” by which is meant a nucleic acid molecule that is substantially or essentially free from components normally found in association with the nucleic acid molecule in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid molecule.

“Genome” as used herein can refer to the nuclear genome, the chloroplast genome, the mitochondrial genome and/or a plasmid genome.

A “heterologous” or a “recombinant” nucleotide sequence is a nucleotide sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence.

Different nucleic acids or proteins having homology are referred to herein as “homologues.” The term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species. “Homology” refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins. Thus, the compositions and methods of the invention further comprise homologues to the nucleotide sequences and polypeptide sequences of this invention. “Orthologous,” as used herein, refers to homologous nucleotide sequences and/or amino acid sequences in different species that arose from a common ancestral gene during speciation. A homologue of a nucleotide sequence of this invention has a substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to said nucleotide sequence of the invention.

As used herein, hybridization, hybridize, hybridizing, and grammatical variations thereof, refer to the binding of two complementary nucleotide sequences or substantially complementary sequences in which some mismatched base pairs are present. The conditions for hybridization are well known in the art and vary based on the length of the nucleotide sequences and the degree of complementarity between the nucleotide sequences. In some embodiments, the conditions of hybridization can be high stringency, or they can be medium stringency or low stringency depending on the amount of complementarity and the length of the sequences to be hybridized. The conditions that constitute low, medium and high stringency for purposes of hybridization between nucleotide sequences are well known in the art (See, e.g., Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; M. R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Two nucleotide sequences can also be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. A non-limiting example of “stringent” hybridization conditions include conditions represented by a wash stringency of 50% formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C. “Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York (1993). In some representative embodiments, two nucleotide sequences considered to be substantially identical hybridize to each other under highly stringent conditions. Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH.

An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. As used herein, the term “percent sequence identity” or “percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). In some embodiments, “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence.

As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

Optimal alignment of sequences for aligning a comparison window is well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, Mass.). The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of this invention “percent identity” may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

The percent of sequence identity can be determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software Package™ (Version 10; Genetics Computer Group, Inc., Madison, Wis.). “Gap” utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, J Mol. Biol. 48:443-453, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. “BestFit” performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482-489, 1981, Smith et al., Nucleic Acids Res. 11:2205-2220, 1983).

Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo et al. (Applied Math 48:1073(1988)). More particularly, preferred computer programs for determining sequence identity include but are not limited to the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (e.g., NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., e.g., NCBI, NLM, NIH; (Altschul et al., J Mol. Biol. 215:403-410 (1990)); version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and for polynucleotide sequence BLASTN can be used to determine sequence identity.

The terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement” (and grammatical variations thereof), as used herein, describe an elevation in, for example, carbon fixation and/or biomass production, and/or an elevation in CO₂ uptake in a plant, plant part or plant cell, or in a photosynthetic bacterium. This increase can be observed by comparing the increase in the plant, plant part or plant cell, or photosynthetic bacterium transformed with, for example, a heterologous polynucleotide encoding a bacterial or archaeal succinyl CoA synthetase, 2-oxoglutarate:ferredoxin oxidoreductase, 2-oxoglutarate carboxylase, oxalosuccinate reductase or isocitrate lyase as compared to an appropriate control (e.g., the same organism (e.g., the same species of plant, plant part or plant cell, or photosynthetic bacterium) lacking (i.e., not transformed with) said heterologous polynucleotide(s)). Thus, as used herein, the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement” (and grammatical variations thereof), and similar terms indicate an elevation of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more, or any range therein, as compared to a control (e.g., a plant, plant part and/or plant cell, or photosynthetic bacterium that does not comprise said heterologous polynucleotide).

“Increased biomass production” as used herein refers to a transformed plant or plant part having a greater dry weight over the entire plant or any organ of the plant (leaf, stem, roots, seeds, seed pods, flowers, etc), increased plant height, leaf number, and/or seed number or increased root volume compared to the native or wild type (e.g., a plant, plant part that is not transformed with the heterologous polynucleotides of the invention (e.g., heterologous polynucleotides encoding a succinyl CoA synthetase polypeptide, a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and/or an isocitrate lyase polypeptide). “Increased biomass production” can also refer to a greater dry weight of cells (e.g., tissue culture, cell suspension (e.g., algal culture, photosynthetic bacterial culture) and the like) as compared to cells not transformed with the heterologous polynucleotides of the invention.

“Increased carbon fixation” as used herein refers to a greater conversion of CO₂ to organic carbon compounds in a transgenic plant (e.g., a plant, plant part that is not transformed with the heterologous polynucleotides of the invention (e.g., heterologous polynucleotides encoding a succinyl CoA synthetase polypeptide, a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide, and/or an isocitrate lyase polypeptide) when compared to the native or wild type (e.g., not transformed with said heterologous polynucleotides). “Increased carbon fixation” can be measured by analyzing CO₂ fixation rates using a Licor System or radiolabeled ¹⁴CO₂ or by quantifying dry biomass. Increased carbon fixation can also occur for transformed cells (e.g., tissue culture, cell suspension (e.g., algal culture, photosynthetic bacterial culture), and the like) as compared to cells not transformed with the heterologous polynucleotides of the invention.

In some embodiments, the recombinant nucleic acids molecules, nucleotide sequences and polypeptides of the invention are “isolated.” An “isolated” nucleic acid molecule, an “isolated” nucleotide sequence or an “isolated” polypeptide is a nucleic acid molecule, nucleotide sequence or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule, nucleotide sequence or polypeptide may exist in a purified form that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide. In representative embodiments, the isolated nucleic acid molecule, the isolated nucleotide sequence and/or the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more pure.

In other embodiments, an isolated nucleic acid molecule, polynucleotide or polypeptide may exist in a non-native environment such as, for example, a recombinant host cell. Thus, for example, with respect to nucleotide sequences, the term “isolated” means that it is separated from the chromosome and/or cell in which it naturally occurs. A polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs in and is then inserted into a genetic context, a chromosome and/or a cell in which it does not naturally occur (e.g., a different host cell, different regulatory sequences, and/or different position in the genome than as found in nature). Accordingly, the polynucleotides and their encoded polypeptides are “isolated” in that, by the hand of man, they exist apart from their native environment and therefore are not products of nature, however, in some embodiments, they can be introduced into and exist in a recombinant host cell.

As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence” and “polynucleotide” refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2′-hydroxy in the ribose sugar group of the RNA can also be made.

As used herein, the term “nucleotide sequence” refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5′ to 3′ end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded or double stranded. The terms “nucleotide sequence” “nucleic acid,” “nucleic acid molecule,” “oligonucleotide” and “polynucleotide” are also used interchangeably herein to refer to a heteropolymer of nucleotides. Nucleic acid sequences provided herein are presented herein in the 5′ to 3′ direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR §§ 1.821-1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25.

By “operably linked” or “operably associated,” it is meant that the indicated elements are functionally related to each other, and are also generally physically related. Thus, the term “operably linked” or “operably associated” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Therefore, a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For instance, a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence. Those skilled in the art will appreciate that the control sequences (e.g., promoter) need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered “operably linked” to the nucleotide sequence.

Any plant (or groupings of plants, for example, into a genus or higher order classification) can be employed in practicing this invention including an angiosperm, a gymnosperm, a monocot, a dicot, a C3, C4, CAM plant, a microalgae, and/or a macroalgae. The methods of the present invention may additionally be practiced with photosynthetic bacteria.

The term “plant part,” as used herein, includes but is not limited to reproductive tissues (e.g., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower bud, ovules, seeds, embryos, nuts, kernels, ears, cobs and husks); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristem, axillary bud, cotyledon, hypocotyls, and leaves); vascular tissues (e.g., phloem and xylem); specialized cells such as epidermal cells, parenchyma cells, chollenchyma cells, schlerenchyma cells, stomata, guard cells, cuticle, mesophyll cells; callus tissue; and cuttings. The term “plant part” also includes plant cells, including plant cells that are intact in plants and/or parts of plants, plant protoplasts, plant tissues, plant organs, plant cell tissue cultures, plant calli, plant clumps, and the like. As used herein, “shoot” refers to the above ground parts including the leaves and stems. As used herein, the term “tissue culture” encompasses cultures of tissue, cells, protoplasts and callus.

As used herein, “plant cell” refers to a structural and physiological unit of the plant, which typically comprise a cell wall but also includes protoplasts. A plant cell of the present invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue (including callus) or a plant organ. In some embodiments, a plant cell can be an algal cell.

In some embodiments of this invention, a plant, plant part or plant cell can be from a genus including, but not limited to, the genus of Camelina, Sorghum, Gossypium, Brassica, Allium, Armoracia, Poa, Agrostis, Lolium, Festuca, Calamogrostis, Deschampsia, Spinacia, Beta, Pisum, Chenopodium, Helianthus, Pastinaca, Daucus, Petroselium, Populus, Prunus, Castanea, Eucalyptus, Acer, Quercus, Salix, Juglans, Picea, Pinus, Abies, Lemna, Wolffia, Spirodela, Oryza or Gossypium.

In other embodiments, a plant, plant part or plant cell can be from a species including, but not limited to, the species of Camelina alyssum (Mill.) Thell., Camelina microcarpa Andrz. ex DC., Camelina rumelica Velen., Camelina sativa (L.) Crantz, Sorghum bicolor (e.g., Sorghum bicolor L. Moench), Gossypium hirsutum, Brassica oleracea, Brassica rapa, Brassica napus, Raphanus sativus, Armoracia rusticana, Allium sative, Allium cepa, Populus grandidentata, Populus tremula, Populus tremuloides, Prunus serotina, Prunus pensylvanica, Castanea dentate, Populus balsamifer, Populus deltoids, Acer Saccharum, Acer nigrum, Acer negundo, Acer rubrum, Acer saccharinum, Acer pseudoplatanus or Oryza sativa. In additional embodiments, the plant, plant part or plant cell can be, but is not limited to, a plant of, or a plant part, or plant cell from wheat, barley, oats, turfgrass (bluegrass, bentgrass, ryegrass, fescue), feather reed grass, tufted hair grass, spinach, beets, chard, quinoa, sugar beets, lettuce, sunflower (Helianthus annuus), peas (Pisum sativum), parsnips (Pastinaca sativa), carrots (Daucus carota), parsley (Petroselinum crispum), duckweed, pine, spruce, fir, eucalyptus, oak, walnut, or willow. In particular embodiments, the plant, plant part and/or plant cell can be from Camelina sativa.

In further embodiments, a plant and/or plant cell can be an alga or alga cell from a class including, but not limited to, the class of Bacillariophyceae (diatoms), Haptophyceae, Phaeophyceae (brown algae), Rhodophyceae (red algae) or Glaucophyceae (red algae). In still other embodiments, a plant and/or plant cell can be an algae or algae cell from a genus including, but not limited to, the genus of Achnanthidium, Actinella, Nitzschia, Nupela, Geissleria, Gomphonema, Planothidium, Halamphora, Psammothidium, Navicula, Eunotia, Stauroneis, Chlamydomonas, Dunaliella, Nannochloris, Nannochloropsis, Scenedesmus, Chlorella, Cyclotella, Amphora, Thalassiosira, Phaeodactylum, Chrysochromulina, Prymnesium, Thalassiosira, Phaeodactylum, Glaucocystis, Cyanophora, Galdieria, or Porphyridium. Additional nonlimiting examples of genera and species of diatoms useful with this invention are provided by the US Geological Survey/Institute of Arctic and Alpine Research at westerndiatoms.colorado.edu/species.

In some embodiments, photosynthetic bacteria useful with this invention includes, but is not limited to, cyanobacteria, Synechococcus spp., Synechocystis spp., Prochlorococcus spp., purple bacteria including but not limited to Rhodospirillaceae or nitrogen-fixing cyanobacteria including but not limited to Anabena spp.

As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” “suppress,” and “decrease” (and grammatical variations thereof) mean a decrease of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or any range therein, as compared to a control.

As used herein, the term “substantially identical” means that two nucleotide sequences have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Thus, for example, a homolog of a polynucleotide of the invention can have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, for example, a polynucleotide encoding a succinyl CoA synthetase polypeptide, a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, a 2-oxoglutarate carboxylase polypeptide, a oxalosuccinate reductase polypeptide and/or a isocitrate lyase polypeptide.

As used herein, the terms “transformed” and “transgenic” refer to any plant, plant part, and/or plant cell that contains all or part of at least one recombinant (e.g., heterologous) polynucleotide. In some embodiments, all or part of the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations. For the purposes of the invention, the term “recombinant polynucleotide” refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering. Examples include any cloned polynucleotide, or polynucleotides, that are linked or joined to heterologous sequences. The term “recombinant” does not refer to alterations of polynucleotides that result from naturally occurring events, such as spontaneous mutations, or from non-spontaneous mutagenesis followed by selective breeding.

The term “transgene” as used herein, refers to any nucleotide sequence used in the transformation of an organism. Thus, a transgene can be a coding sequence, a non-coding sequence, a cDNA, a gene or fragment or portion thereof, a genomic sequence, a regulatory element and the like. A “transgenic” organism, such as a transgenic plant, transgenic yeast, or transgenic bacterium, is an organism into which a transgene has been delivered or introduced and the transgene can be expressed in the transgenic organism to produce a product, the presence of which can impart an effect and/or a phenotype in the organism.

The term “transformation” as used herein refers to the introduction of a heterologous polynucleotide into a cell. Transformation of a plant, plant part, plant cell, yeast cell and/or bacterial cell may be stable or transient.

“Transient transformation” in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell.

By “stably introducing” or “stably introduced” in the context of a polynucleotide introduced into a cell it is intended that the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.

“Stable transformation” or “stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. “Genome” as used herein also includes the nuclear and the plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast genome. Stable transformation as used herein can also refer to a transgene that is maintained extrachromosomally, for example, as a minichromosome. The phrase “a stably transformed plant, plant part, and/or plant cell expressing said one or more polynucleotide sequences” and similar phrases used herein, means that the stably transformed plant, plant part, and/or plant cell comprises the one or more polynucleotide sequences and that said one or more polynucleotide sequences are functional in said stably transformed plant, plant part, and/or plant cell.

Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a plant). Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a plant or other organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and/or hybridization protocols that are well known in the art.

Accordingly, the present invention is directed to compositions and methods for increasing carbon fixation and biomass production in a plant, plant cell and/or plant part, or in a photosynthetic bacterium by introducing in the plant, plant cell and/or plant part, or into the photosynthetic bacterium heterologous polynucleotides that encode polypeptides for the biological carbon sequestration/crTCA cycle enzymes as described herein.

Thus, in some embodiments, the present invention provides a method for increasing carbon fixation and/or increasing biomass production in a plant, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide to produce a stably transformed plant, plant part, and/or plant cell expressing said heterologous polynucleotide, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed plant, plant part, and/or plant cell as compared to a control (e.g., a plant not comprising said heterologous polynucleotide), wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In another embodiment, a method for increasing carbon fixation and/or increasing biomass production in a plant is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide to produce a stably transformed plant, plant part, and/or plant cell expressing said heterologous polynucleotide, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed plant, plant part, and/or plant cell as compared to a control, wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In additional embodiments, a method for increasing carbon fixation and/or increasing biomass production in a plant is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide to produce a stably transformed plant, plant part, and/or plant cell expressing said heterologous polynucleotide, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed plant, plant part, and/or plant cell as compared to a control, wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In some embodiments, a method for increasing carbon fixation and/or increasing biomass production in a plant is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding an isocitrate dehydrogenase/oxalosuccinate reductase polypeptide (both isocitrate dehydrogenase (ICDH) and oxalosuccinate reductase (OSR) are appropriate names for the enzyme catalyzing step four of the crTCA cycle) to produce a stably transformed plant, plant part, and/or plant cell expressing said heterologous polynucleotide, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed plant, plant part, and/or plant cell as compared to a control, wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In further embodiments, a method for increasing carbon fixation and/or increasing biomass production in a plant is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding an isocitrate lyase polypeptide to produce a stably transformed plant, plant part, and/or plant cell expressing said heterologous polynucleotide, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed plant as compared to a control, wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In still further embodiments, a method for increasing carbon fixation and/or increasing biomass production in a plant is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide to produce a stably transformed plant, plant part, and/or plant cell expressing said heterologous polynucleotides, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed plant, plant part, and/or plant cell as compared to a control, wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide are from a bacterial and/or an archaeal species.

In some embodiments, a method for increasing carbon fixation and/or increasing biomass production in a plant is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide to produce a stably transformed plant, plant part, and/or plant cell expressing said heterologous polynucleotides, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed plant, plant part, and/or plant cell as compared to a control, wherein the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide are from a bacterial and/or an archaeal species.

In further embodiments, a method for increasing carbon fixation and/or increasing biomass production in a plant is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and a heterologous polynucleotide encoding an isocitrate lyase polypeptide to produce a stably transformed plant, plant part, and/or plant cell expressing said heterologous polynucleotides, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed plant, plant part, and/or plant cell as compared to a control, wherein the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and the heterologous polynucleotide encoding an isocitrate lyase polypeptide are from a bacterial and/or an archaeal species.

Thus, in some embodiments, the present invention provides a method for increasing carbon fixation and/or increasing biomass production in a photosynthetic bacterium, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide to produce a stably transformed photosynthetic bacterium expressing said heterologous polynucleotide, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed photosynthetic bacterium as compared to a control (e.g., a photosynthetic bacterium not comprising said heterologous polynucleotide), wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In another embodiment, a method for increasing carbon fixation and/or increasing biomass production in a photosynthetic bacterium is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide to produce a stably transformed photosynthetic bacterium expressing said heterologous polynucleotide, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed photosynthetic bacterium as compared to a control, wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In additional embodiments, a method for increasing carbon fixation and/or increasing biomass production in a photosynthetic bacterium is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide to produce a stably transformed photosynthetic bacterium expressing said heterologous polynucleotide, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed photosynthetic bacterium as compared to a control, wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In some embodiments, a method for increasing carbon fixation and/or increasing biomass production in a photosynthetic bacterium is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding an isocitrate dehydrogenase/oxalosuccinate reductase polypeptide (both isocitrate dehydrogenase (ICDH) and oxalosuccinate reductase (OSR) are appropriate names for the enzyme catalyzing step four of the crTCA cycle) to produce a stably transformed photosynthetic bacterium expressing said heterologous polynucleotide, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed photosynthetic bacterium as compared to a control, wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In further embodiments, a method for increasing carbon fixation and/or increasing biomass production in a photosynthetic bacterium is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding an isocitrate lyase polypeptide to produce a stably transformed photosynthetic bacterium expressing said heterologous polynucleotide, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed photosynthetic bacterium as compared to a control, wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In still further embodiments, a method for increasing carbon fixation and/or increasing biomass production in a photosynthetic bacterium is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide to produce a stably transformed photosynthetic bacterium expressing said heterologous polynucleotides, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed photosynthetic bacterium as compared to a control, wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide are from a bacterial and/or an archaeal species.

In some embodiments, a method for increasing carbon fixation and/or increasing biomass production in a photosynthetic bacterium is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide to produce a stably transformed photosynthetic bacterium expressing said heterologous polynucleotides, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed photosynthetic bacterium as compared to a control, wherein the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide are from a bacterial and/or an archaeal species.

In further embodiments, a method for increasing carbon fixation and/or increasing biomass production in a photosynthetic bacterium is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and a heterologous polynucleotide encoding an isocitrate lyase polypeptide to produce a stably transformed photosynthetic bacterium expressing said heterologous polynucleotides, thereby increasing carbon fixation and/or increasing biomass production in said stably transformed photosynthetic bacterium as compared to a control, wherein the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and the heterologous polynucleotide encoding an isocitrate lyase polypeptide are from a bacterial and/or an archaeal species.

In some aspects, the methods of the invention further comprise, consist essentially of, or consist of regenerating a stably transformed plant or plant part from the stably transformed plant cell, wherein expression of one or more of the heterologous polynucleotides in said regenerated and stably transformed plant or plant part results in the stably transformed plant and/or plant part having increased carbon fixation and/or increased biomass production as compared to a control (e.g., a plant or plant part not transformed with and stably expressing said heterologous polynucleotides).

Thus, in some embodiments, a method for producing a plant having increased carbon fixation and/or increased biomass production is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide or an isocitrate lyase polypeptide, thereby producing a stably transformed plant, plant part, and/or plant cell having increased carbon fixation and/or increased biomass production as compared to a control (e.g., a plant not comprising said heterologous polynucleotide), wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In other embodiments, a method for producing a plant having increased carbon fixation and/or increased biomass production is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, thereby producing a stably transformed plant, plant part, and/or plant cell having increased carbon fixation and/or increased biomass production as compared to a control, wherein said heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and said heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide are from a bacterial and/or an archaeal species.

In further embodiments, a method for producing a plant having increased carbon fixation and/or increased biomass production is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide, thereby producing a stably transformed plant, plant part, and/or plant cell having increased carbon fixation and/or increased biomass production as compared to a control, wherein said heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and said heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide are from a bacterial and/or an archaeal species.

In additional embodiments, a method for producing a plant having increased carbon fixation and/or increased biomass production is provided, comprising, consisting essentially of, or consisting of: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and a heterologous polynucleotide encoding an isocitrate lyase polypeptide, thereby producing a stably transformed plant, plant part, and/or plant cell having increased carbon fixation and/or increased biomass production as compared to a control, wherein said heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, said heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and said heterologous polynucleotide encoding an isocitrate lyase polypeptide are from a bacterial and/or an archaeal species.

In additional embodiments, the preset invention provides methods for producing stably transformed photosynthetic bacteria having increased carbon fixation and/or increased biomass production

Thus, in some embodiments, a method for producing a photosynthetic bacterium having is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide or an isocitrate lyase polypeptide, thereby producing a stably transformed photosynthetic bacterium having increased carbon fixation and/or increased biomass production as compared to a control (e.g., a photosynthetic bacterium not comprising said heterologous polynucleotide), wherein the heterologous polynucleotide is from a bacterial and/or an archaeal species.

In other embodiments, a method for producing photosynthetic bacterium having increased carbon fixation and/or increased biomass production is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, thereby producing a stably transformed photosynthetic bacterium having increased carbon fixation and/or increased biomass production as compared to a control, wherein said heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and said heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide are from a bacterial and/or an archaeal species.

In further embodiments, a method for producing a photosynthetic bacterium having increased carbon fixation and/or increased biomass production is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide, thereby producing a a stably transformed photosynthetic bacterium having increased carbon fixation and/or increased biomass production as compared to a control, wherein said heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and said heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide are from a bacterial and/or an archaeal species.

In additional embodiments, a method for producing a photosynthetic bacterium having increased carbon fixation and/or increased biomass production is provided, comprising, consisting essentially of, or consisting of: introducing into a photosynthetic bacterium a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and a heterologous polynucleotide encoding an isocitrate lyase polypeptide, thereby producing a stably transformed photosynthetic bacterium having increased carbon fixation and/or increased biomass production as compared to a control, wherein said heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, said heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and said heterologous polynucleotide encoding an isocitrate lyase polypeptide are from a bacterial and/or an archaeal species.

The polypeptides succinyl CoA synthetase, 2-oxoglutarate:ferredoxin oxidoreductase, 2-oxoglutarate carboxylase, oxalosuccinate reductase, and isocitrate lyase (i.e., the biological carbon sequestration/crTCA cycle polypeptides of the invention), and the polynucleotides that encode said polypeptides are known in the art and are produced by many different organisms. Thus, in some embodiments, a biological carbon sequestration polypeptide (crTCA cycle polypeptide) useful with this invention can be any archaeal or bacterial polypeptide useful for biological carbon sequestration and having the enzyme activity of succinyl CoA synthetase, 2-oxoglutarate:ferredoxin oxidoreductase, 2-oxoglutarate carboxylase, oxalosuccinate reductase, or isocitrate lyase. Selection of a particular polypeptide for use with this invention is based on a number of factors including, for example, the number of subunits in the enzyme (e.g., selecting those with the fewest number of subunits) and the kinetic properties of the individual polypeptides (e.g., a polypeptide with a high kcat value). Examples of organisms from which these polypeptides and polynucleotides can be derived include, but are not limited to, Escherichia coli (e.g., E. coli MG1655), Azotobacter vinelandii (e.g., A. vinelandii DJ), Bradyrhizobium sp. (e.g., Bradyrhizobium sp. BTAi1), Azospirillum sp (e.g., Azospirillum sp. B510), Paenibacillus sp. (e.g. Paenibacillus sp. JDR-2), Halobacterium sp. (e.g., Halobacterium sp NRC-1), Hydrogenobacter thermophilus (e.g., H. thermophilus TK-6), Bacillus sp (e.g., Bacillus sp M3-13), Paenibacillus larvae subsp. larvae (e.g., Paenibacillus larvae subsp. larvae B-3650), Haladaptus paucihalophilus (e.g., H. paucihalophilus DX253), Magnetococcus sp. (e.g., Magnetococcus sp. MC-1), Candidatus Nitrospira defluvii (e.g., Candidatus Nitrospira defluvii NIDE1204), Thiocystis violascens (e.g., T. violascens DSM198), Mariprofundus ferroxydans (e.g., M. ferroxydans PV-1), Pseudomonas stutzeri (e.g., P. stutzeri ATCC14405), Acinetobacter baumannii (e.g. A. baumannii ABT07, A. baumannii ACICU), Chlorobium limicola (e.g. C. limicola DSM 245), Kosmotoga olearia (e.g. K. olearia TBF 19.5.1), Marine gamma proteobacterium (e.g. Marine gamma proteobacterium HTCC2080), Corynebacterium glutamicum (e.g. C. glutamicum ATCC 13032), Gordonia alkanivorans (e.g. G. alkanivorans NBRC 16433), Nocardia farcinica (e.g. N. farcinica IFM 10152), Rhodococcus pyridinivorans (e.g. R. pyridinivorans AK37), Rhodococcus jostii (e.g. R. jostii RHA1) and Clostridium ljungdahlii.

Thus, in some embodiments, a polypeptide and/or polynucleotide encoding a polypeptide having the enzyme activity of succinyl CoA synthetase can be from Escherichia coli, Azotobacter vinelandii, Bradyrhizobium sp., Azospirillum sp., or any combination thereof. In some embodiments, the polypeptide having the enzyme activity of succinyl CoA synthetase can be a two subunit enzyme. In other embodiments, a polypeptide and/or polynucleotide encoding a polypeptide having the enzyme activity of 2-oxoglutarate:ferredoxin oxidoreductase can be from Paenibacillus sp., Halobacterium sp., Hydrogenobacter thermophilus, Bacillus sp, Paenibacillus larvae subsp. larvae, Haladaptus paucihalophilus, Magnetococcus sp., or any combination thereof. In further embodiments, the polypeptide having the enzyme activity of 2-oxoglutarate:ferredoxin oxidoreductase can be a two subunit enzyme. In still other embodiments, a polypeptide and/or polynucleotide encoding a polypeptide having the enzyme activity of 2-oxoglutarate carboxylase can be from Candidatus Nitrospira defluvii, Hydrogenobacter thermophilus, Thiocystis violascens, Mariprofundus ferroxydans, Pseudomonas stutzeri, or any combination thereof. In some embodiments, the polypeptide having the enzyme activity of 2-oxoglutarate carboxylase can be a two subunit enzyme. In additional embodiments, a polypeptide and/or polynucleotide encoding a polypeptide having the enzyme activity of oxalosuccinate reductase can be from Acinetobacter baumannii, Chlorobium limicola, Kosmotoga olearia, Marine gamma proteobacterium, or any combination thereof. In further embodiments, a polypeptide and/or polynucleotide encoding a polypeptide having the enzyme activity of isocitrate lyase can be from Corynebacterium glutamicum, Gordonia alkanivorans, Nocardia farcinica, Rhodococcus pyridinivorans, Rhodococcus jostii, or any combination thereof.

More particularly, in some embodiments, a polynucleotide encoding a succinyl CoA synthetase polypeptide useful with this invention includes, but is not limited to, a nucleotide sequence from E. coli strain K-12 substr. MG1655 (e.g., NCBI Accession Nos. NC_000913.2 (772,237 . . . 763,403), NC_000913.2 (763,403 . . . 764,272); see, e.g., SEQ ID NO:3); from Azotobacter vinelandii DJ (e.g., NCBI Accession Nos. NC_012560.1 (3,074,152 . . . 3,075,321), NC_012560.1 (3,073,268 . . . 3,074,155); see, e.g., SEQ ID NO:6); from Bradyrhizobium sp. BTAi1 (e.g., NCBI Accession Nos. NC_009485.1 (393,292 . . . 394,488), NC_009485.1 (394,545 . . . 395,429); see, e.g., SEQ ID NO:9); and/or from Azospirillum sp. B510 (e.g., NCBI Accession Nos. NC_013854.1 (2,941,010 . . . 2,942,206), NC_013854.1 (2,942,208 . . . 2,943,083); see, e.g., SEQ ID NO:12). In other embodiments, a succinyl CoA synthetase polypeptide can have an amino acid sequence that includes but is not limited to an amino acid sequence from E. coli strain K-12 substr. MG1655 (e.g., NCBI Accession Nos. NP_415256.1 and NP_415257.1); see, e.g., SEQ ID NO:1 and SEQ ID NO:2); from Azotobacter vinelandii DJ (e.g., NCBI Accession Nos. YP_002800115.1 and YP_002800114.1); see, e.g., SEQ ID NO:4 and SEQ ID NO:5); from Bradyrhizobium sp. BTAi1 (e.g., NCBI Accession Nos. YP_001236586.1 and YP_001236587.1); see, e.g., SEQ ID NO:7 and SEQ ID NO:8); and/or from Azospirillum sp. B510 (e.g., NCBI Accession Nos. YP_003449758.1 and YP_003449759.1); see, e.g., SEQ ID NO:10 and SEQ ID NO:11. In some embodiments, a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide can be from E. coli strain K-12 substr. MG1655. In some particular embodiments, a heterologous polynucleotide encoding a succinyl CoA synthetase from E. coli strain K-12 substr. MG1655 comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:3.

In other embodiments, a polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide useful with this invention includes, but is not limited to, a nucleotide sequence from Halobacterium sp. NRC-1 (e.g., NCBI Accession Nos. NC_002607.1 (856,660 . . . 858,582), NC_002607.1 (855,719 . . . 856,657); see, e.g., SEQ ID NO:15); from Hydrogenobacter thermophilus TK-6 (e.g., NCBI Accession Nos. NC_013799.1 (997,525 . . . 999,348), NC_013799.1 (996,624 . . . 997,511); see, e.g., SEQ ID NO:18); from Bacillus sp. M3-13 (e.g., NCBI Accession Nos. NZ_ACPC01000013.1 (932 . . . 2,668), NZ_ACPC01000013.1 (65 . . . 931); see, e.g., SEQ ID NO:21); from Paenibacillus larvae subsp. larvae B-3650 (e.g., NCBI Accession Nos. NZ_ADZY02000226.1 (7,939 . . . 9,687), NZ_ADZY02000226.1 (7,085 . . . 7,951); see, e.g., SEQ ID NO:24); from Haladaptatus paucihalophilus DX253 (e.g., NCBI Accession Nos. NZ_AEMG01000009.1 (157,678 . . . 159,432), NZ_AEMG01000009.1 (156,818 . . . 157,681); see, e.g., SEQ ID NO:27); and/or from Magnetococcus sp. MC-1 (e.g., NCBI Accession Nos. NC_008576.1 (2,161,258 . . . 2,162,979), NC_008576.1 (2,162,976 . . . 2,163,854); see, e.g., SEQ ID NO:30). In other embodiments, a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide can have an amino acid sequence that includes, but is not limited to, an amino acid sequence from Halobacterium sp. NRC-1 (e.g., NCBI Accession Nos. AAG19514.1, AAG19513.1, NP_280034.1 and NP_280033.1); see, e.g., SEQ ID NO:13 and SEQ ID NO:14); from Hydrogenobacter thermophilus TK-6 (e.g., NCBI Accession Nos. YP_003432752.1 and YP_003432751.1); see, e.g., SEQ ID NO:16 and SEQ ID NO:17); from Bacillus sp. M3-13 (e.g., NCBI Accession Nos. ZP_07708142.1 and ZP_07708141.1); see, e.g., SEQ ID NO:19 and SEQ ID NO:20); from Paenibacillus larvae subsp. larvae B-3650 (e.g., NCBI Accession Nos. ZP_09070120.1 and ZP_09070119.1); see, e.g., SEQ ID NO:22 and SEQ ID NO:23); from Haladaptatus paucihalophilus DX253 (e.g., NCBI Accession Nos. ZP_08044530.1 and ZP_08044529.1); see, e.g., SEQ ID NO:25 and SEQ ID NO:26); and/or from Magnetococcus sp. MC-1 (e.g., NCBI Accession Nos. YP_865663.1 and YP_865664.1); see, e.g., SEQ ID NO:28 and SEQ ID NO:29). In some embodiments, a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide can be from Paenibacillus sp. subsp. larvae B-3650. In particular embodiments, a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide from Paenibacillus sp. subsp. larvae B-3650 comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:24.

In further embodiments, a polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide useful with this invention includes, but is not limited to, a nucleotide sequence from Hydrogenobacter thermophilus TK-6 (e.g., NCBI Accession Nos. NC_013799.1 (1,271,487 . . . 1,273,445), NC_013799.1 (1,273,469 . . . 1,274,887); see, e.g., SEQ ID NO:33); from Candidatus Nitrospira defluvii (e.g., NCBI Accession Nos. NC_014355.1 (1,174,721 . . . 1,176,652), NC_014355.1 (1,176,781 . . . 1,178,199); see, e.g., SEQ ID NO:36); from Hydrogenobacter thermophilus TK-6 (e.g., NCBI Accession Nos. NC_013799.1 (1,271,487 . . . 1,273,445), NC_013799.1 (1,273,469 . . . 1,274,887); see, e.g., SEQ ID NO:39); from Thiocystis violascens DSM198 (e.g., NCBI Accession Nos. NZ_AGFC01000013.1 (61,879 . . . 63,297) and (63,889 . . . 65,718); see, e.g., SEQ ID NO:42); from Mariprofundus ferrooxydans PV-1 (e.g., NCBI Accession Nos. NZ_AATS01000007.1 (81,967 . . . 83,385) and (83,475 . . . 85,328); see, e.g., SEQ ID NO:45); and/or from Pseudomonas stutzeri ATCC14405 (AGSL01000085.1 (52,350 . . . 53,765) and (50,522 . . . 52,339); see, e.g., SEQ ID NO:48). In further embodiments, a 2-oxoglutarate carboxylase polypeptide can have an amino acid sequence that includes, but is not limited to, an amino acid sequence from Hydrogenobacter thermophilus TK-6 (e.g., NCBI Accession Nos. YP_003433044.1 and YP_003433045.1); see, e.g., SEQ ID NO:31 and SEQ ID NO:32); from Candidatus Nitrospira defluvii (e.g., NCBI Accession Nos. YP_003796887.1 and YP_003796888.1); see, e.g., SEQ ID NO:34 and SEQ ID NO:35); from Hydrogenobacter thermophilus TK-6 (e.g., NCBI Accession Nos. YP_003433044.1 and YP_003433045.1); see, e.g., SEQ ID NO:37 and SEQ ID NO:38); from Thiocystis violascens DSM198 (e.g., NCBI Accession Nos. ZP_08925050.1 and ZP_08925052.1); see, e.g., SEQ ID NO:40 and SEQ ID NO:41 and/or SEQ ID NO:43 and SEQ ID NO:44); from Mariprofundus ferrooxydans PV-1 (e.g., NCBI Accession Nos. ZP_01452577.1 and ZP_01452578.1); see, e.g., SEQ ID NO:46 and SEQ ID NO:47); and/or from Pseudomonas stutzeri ATCC14405 (e.g., NCBI Accession Nos. EHY78621.1 and EHY78620.1); see, e.g., SEQ ID NO:49 and SEQ ID NO:50). In some embodiments, a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide can be a 2-oxoglutarate carboxylase from Candidatus Nitrospira defluvii. In some particular embodiments, a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide from Candidatus Nitrospira defluvii comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:36. In other embodiments, a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide can be a 2-oxoglutarate carboxylase polypeptide from Hydrogenobacter thermophilus TK-6. In some particular embodiments, a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide from Hydrogenobacter thermophilus TK-6 comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:33, SEQ ID NO:39 and/or SEQ ID NO:42.

In still further embodiments, a polynucleotide encoding a oxalosuccinate reductase polypeptide useful with this invention includes, but is not limited to, a polynucleotide from Chlorobium limicola DSM 245 (e.g., NCBI Accession Nos. AB076021.1); see, e.g., SEQ ID NO:53); from Kosmotoga olearia TBF 19.5.1 (e.g., NCBI Accession Nos. NC_012785.1 (1,303,493 . . . 1,304,695); see, e.g., SEQ ID NO:55); from Acinetobacter baumannii ACICU (e.g., NCBI Accession Nos. NC_010611.1 (2,855,563 . . . 2,856,819); see, e.g., SEQ ID NO:57); from Marine gamma proteobacterium HTCC2080 (e.g., NCBI Accession Nos. NZ_AAVV01000002.1 (123,681 . . . 124,934); see, e.g., SEQ ID NO:59); and/or from Nitrosococcus halophilus Nc4 (e.g., NCBI Accession Nos. NC_013960.1 (2,610,547 . . . 2,611,815); see, e.g., SEQ ID NO:61). In other embodiments, an oxalosuccinate reductase polypeptide can have an amino acid sequence that includes, but is not limited to, an amino acid sequence from Chlorobium limicola DSM 245 (e.g., NCBI Accession Nos. BAC00856.1); see, e.g., SEQ ID NO:52); from Kosmotoga olearia TBF 19.5.1 (e.g., NCBI Accession Nos. YP_002940928.1); see, e.g., SEQ ID NO:54); from Acinetobacter baumannii ACICU (e.g., NCBI Accession Nos. YP_001847346.1); see, e.g., SEQ ID NO:56); from Marine gamma proteobacterium HTCC2080 (e.g., NCBI Accession Nos. ZP_01625318.1); see, e.g., SEQ ID NO:58); and/or from Nitrosococcus halophilus Nc4 (e.g., NCBI Accession Nos. YP_003528006.1); see, e.g., SEQ ID NO:60). In some embodiments, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide can be from Acinetobacter baumannii. In some particular embodiments, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide from Acinetobacter baumannii comprises, consists essentially of, or consists of nucleotide sequence of SEQ ID NO:57. In other embodiments, a heterologous polynucleotide encoding an oxalosuccinate reductase can be from Chlorobium limicola. In some particular embodiments, a heterologous polynucleotide encoding an oxalosuccinate reductase from Chlorobium limicola comprises, consists essentially of, or consists of nucleotide sequence of SEQ ID NO:53. In further embodiments, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide can be from Kosmotoga olearia TBF 19.5.1. In some particular embodiments, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide from Kosmotoga olearia TBF 19.5.1 comprises, consists essentially of, or consists of nucleotide sequence of SEQ ID NO:55. In still further embodiments, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide can be from Nitrosococcus halophilus Nc4. In some particular embodiments, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide from Nitrosococcus halophilus Nc4 comprises, consists essentially of, or consists of nucleotide sequence of SEQ ID NO:60.

In additional embodiments, a heterologous polynucleotide encoding an isocitrate lyase polypeptide useful with this invention includes, but is not limited to, a polynucleotide from Corynebacterium glutamicum ATCC 13032 (e.g., NCBI Accession Nos. NC_003450.3 (2,470,741 . . . 2,472,039); see, e.g., SEQ ID NO:63); from Gordonia alkanivorans NBRC 16433 (e.g., NCBI Accession Nos. NZ_BACI01000050.1 (37,665 . . . 38,960); see, e.g., SEQ ID NO:65); Nocardia farcinica IFM 10152 (e.g., NCBI Accession Nos. NC_006361.1 (5,525,226 . . . 5,526,515); see, e.g., SEQ ID NO:67); that from Rhodococcus pyridinivorans AK37 (e.g., NCBI Accession Nos. NZ_AHBW01000053.1 (20,169 . . . 21,458); see, e.g., SEQ ID NO:69); and/or from Rhodococcus jostii RHA1 (e.g., NCBI Accession Nos. NC_008268.1 (2,230,309 . . . 2,231,598); see, e.g., SEQ ID NO:71). In other embodiments, an isocitrate lyase polypeptide can have an amino acid sequence that includes, but is not limited to, an amino acid sequence from Corynebacterium glutamicum ATCC 13032 (e.g., NCBI Accession Nos. NP_601531.1); see, e.g., SEQ ID NO:62); from Gordonia alkanivorans NBRC 16433 (e.g., NCBI Accession Nos. ZP_08765259.1); see, e.g., SEQ ID NO:64); Nocardia farcinica IFM 10152 (e.g., NCBI Accession Nos. YP_121446.1); see, e.g., SEQ ID NO:66); that from Rhodococcus pyridinivorans AK37 (e.g., NCBI Accession Nos. ZP_09310682.1); see, e.g., SEQ ID NO:68); and that from Rhodococcus jostii RHA1 (e.g., NCBI Accession Nos. YP_702087.1); see, e.g., SEQ ID NO:70). In some embodiments, a heterologous polynucleotide encoding an isocitrate lyase polypeptide can be from Corynebacterium glutamicum. In some particular embodiments, a heterologous polynucleotide encoding an isocitrate lyase polypeptide from Corynebacterium glutamicum comprises, consists essentially of, or consists of nucleotide sequence of SEQ ID NO:63. In further embodiments, a heterologous polynucleotide encoding an isocitrate lyase polypeptide can be from Rhodococcus pyridinivorans AK37. In some particular embodiments, a heterologous polynucleotide encoding an isocitrate lyase polypeptide from Rhodococcus pyridinivorans AK37 comprises, consists essentially of, or consists of nucleotide sequence of SEQ ID NO:68.

In further embodiments, polypeptides and the polynucleotides encoding said polypeptides can be modified for use with this invention. For example, a native or wild type intergenic spacer sequence in a selected polynucleotide can be substituted with another known spacer or a synthetic spacer sequence. Thus, for example, the intergenic spacer sequence in the 2-oxoglutarate carboxylase polynucleotide sequence from Candidatus Nitrospira defluvii and/or Thiocystis violascens DSM198 can be substituted with the 26 base pair spacer from the 2-oxoglutarate carboxylase Hydrogenobacter thermophilus polynucleotide sequence (see, e.g., the spacer sequence in SEQ ID NO:33) resulting in a 2-oxoglutarate carboxylase polypeptide having the nucleotide sequence of SEQ ID NO: 36 or SEQ ID NO:45, respectively.

Other polypeptide modifications useful with this invention include amino acid substitutions (and the corresponding base pair changes in the respective polynucleotide encoding said polypeptide). Thus, in some embodiments, a polypeptide and/or polynucleotide sequence of the invention can be a conservatively modified variant. As used herein, “conservatively modified variant” refers to polypeptide and polynucleotide sequences containing individual substitutions, deletions or additions that alter, add or delete a single amino acid or nucleotide or a small percentage of amino acids or nucleotides in the sequence, where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

As used herein, a conservatively modified variant of a polypeptide is biologically active and therefore possesses the desired activity of the reference polypeptide (e.g., succinyl CoA synthetase, 2-oxoglutarate:ferredoxin oxidoreductase, 2-oxoglutarate carboxylase, oxalosuccinate reductase, isocitrate lyase) as described herein. The variant can result from, for example, a genetic polymorphism or human manipulation. A biologically active variant of the reference polypeptide can have at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity (e.g., about 30% to about 99% or more sequence identity and any range therein) to the amino acid sequence for the reference polypeptide as determined by sequence alignment programs and parameters described elsewhere herein. An active variant can differ from the reference polypeptide sequence by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

Naturally occurring variants may exist within a population. Such variants can be identified by using well-known molecular biology techniques, such as the polymerase chain reaction (PCR), and hybridization as described below. Synthetically derived nucleotide sequences, for example, sequences generated by site-directed mutagenesis or PCR-mediated mutagenesis which still encode a polypeptide of the invention, are also included as variants. One or more nucleotide or amino acid substitutions, additions, or deletions can be introduced into a nucleotide or amino acid sequence disclosed herein, such that the substitutions, additions, or deletions are introduced into the encoded protein. The additions (insertions) or deletions (truncations) may be made at the N-terminal or C-terminal end of the native protein, or at one or more sites in the native protein. Similarly, a substitution of one or more nucleotides or amino acids may be made at one or more sites in the native protein.

For example, conservative amino acid substitutions may be made at one or more predicted, preferably nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an “essential” amino acid is required for biological activity. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue with a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity.

In some embodiments, amino acid changes can be made to alter the catalytic activity of an enzyme. For example, amino acid substitutions can be made to a thermoactive enzyme that has little activity at room temperature (e.g., about 20° C. to about 50° C.) so as to increase activity at these temperatures. A comparison can be made between the thermoactive enzyme and a mesophilic homologue having activity at the desired temperatures. This can provide discrete differences in amino acids that can then be the focus of amino acid substitutions.

Thus, in some embodiments, amino acid sequence variants of a reference polypeptide can be prepared by mutating the nucleotide sequence encoding the enzyme. The resulting mutants can be expressed recombinantly in plants, and screened for those that retain biological activity by assaying for the enzyme activity (e.g., succinyl CoA synthetase activity, 2-oxoglutarate:ferredoxin oxidoreductase activity, 2-oxoglutarate carboxylase activity, oxalosuccinate reductase activity, isocitrate lyase activity) using standard assay techniques as described herein. Methods for mutagenesis and nucleotide sequence alterations are known in the art. See, e.g., Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; and Techniques in Molecular Biology (Walker & Gaastra eds., MacMillan Publishing Co. 1983) and the references cited therein; as well as U.S. Pat. No. 4,873,192. Clearly, the mutations made in the DNA encoding the variant must not disrupt the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington, D.C.).

In a representative embodiment, the large subunit from the 2-oxoglutarate carboxylase polypeptide (cfiA) from Hydrogenobacter thermophilus TK-6 can be modified at residue 203 to be alanine (A) instead of methionine (M), at residue 205 to be valine (V) instead of phenylalanine (F), at residue 234 to be methionine (M) instead of threonine (T), at residue 236 to be isoleucine (I) instead of threonine (T), at residue 240 to be leucine (L) instead of methionine (M), at residue 274 to be arginine (R) instead of glutamic acid (E) and/or at residue 288 to be glutamine (Q) instead of aspartic acid (D) as shown, for example, in the amino acid sequences of SEQ ID NO:38 and SEQ ID NO:41 and the corresponding codon changes as shown, for example, in the nucleotide sequences of SEQ ID NO:39 or SEQ ID NO:42. Such changes result in a thermophilic 2-oxoglutarate carboxylase that can function at lower temperatures than the native H. themophilus TK-6 2-oxoglutarate carboxylase. The amino acids targeted for substitution were identified by comparing the H. themophilus TK-6 2-oxoglutarate carboxylase with its nearest mesophilic homolog from Candidatus Nitrospira defluvii.

The deletions, insertions and substitutions in the polypeptides described herein are not expected to produce radical changes in the characteristics of the polypeptide (e.g., the temperature at which the polypeptide is active). However, when it is difficult to predict the exact effect of the substitution, deletion or insertion in advance of doing so, one of skill in the art will appreciate that the effect can be evaluated by routine screening assays for the particular polypeptide activities of interest (e.g., succinyl CoA synthetase, 2-oxoglutarate:ferredoxin oxidoreductase, 2-oxoglutarate carboxylase, oxalosuccinate reductase, isocitrate lyase) as described herein.

In some embodiments, the compositions of the invention can comprise functional fragments of the polypeptide. As used herein, “functional fragment” means a portion of the reference polypeptide that retains the polypeptide activity of, for example, succinyl CoA synthetase, 2-oxoglutarate:ferredoxin oxidoreductase, 2-oxoglutarate carboxylase, oxalosuccinate reductase, or isocitrate lyase. A fragment also means a portion of a nucleic acid molecule encoding the reference polypeptide. An active fragment of the polypeptide can be prepared, for example, by isolating a portion of a polypeptide-encoding nucleic acid molecule that expresses an encoded fragment of the polypeptide (e.g., by recombinant expression in vitro), and assessing the activity of the fragment. Nucleic acid molecules encoding such fragments can be at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2000 contiguous nucleotides, or up to the number of nucleotides present in a full-length polypeptide-encoding nucleic acid molecule. As such, polypeptide fragments can be at least about 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 contiguous amino acid residues, or up to the total number of amino acid residues present in the full-length polypeptide.

Methods for assaying the activities of the crTCA cycle enzymes of the invention (e.g., succinyl CoA synthetase, 2-oxoglutarate:ferredoxin oxidoreductase, 2-oxoglutarate carboxylase, oxalosuccinate reductase and isocitrate lyase) are known in the art. Exemplary activity assays for these enzymes are set forth below.

Succinyl CoA Synthetase.

The succinyl CoA synthetase assay is a spectrophotometric method that measures the increase of absorbance at 232 nm in response to thioester formation. The standard reaction solution consists of 10 mM sodium succinate, 10 mM MgCl₂, 0.1 mM CoA, 0.1 mM DTT, 0.4 mM nucleotide (ATP or GTP) and 0.1 M KCl in 50 mM Tris-HCl (pH 7.4). The reaction is started with the addition of purified succinyl CoA synthetase or crude extract containing SCS. The reaction is monitored in a spectrophotometer set at 232 nm at 25° C. (See, e.g., Bailey et al. A dimeric form of Escherichia coli succinyl-CoA synthetase produced by site-directed mutagenesis. J. Mol. Biol. 285:1655-1666 (1999); Bridger et al. Succinyl coenzyme A synthetase from Escherichia coli. Methods Enzymol. 13:70-75 (1969)).

For the LC/MS method of detection of succinyl CoA produced (LC-ESI-IT), the enzyme reactions are stopped by the addition of 30 μL of 15% (wt/vol) trifluoroacetic acid. A Nucleosil RP C18 (5 μm, 100-Å pores; Knauer GmbH, Berlin, Germany) reverse-phased column serves to separate the CoA esters at 30° C. A 50 mM concentration of ammonium acetate (pH 5.0) adjusted with acetic acid (eluent A) and 100% (vol/vol) methanol (eluent B) serves as eluents. Elution occurs at a flow rate of 0.3 ml/min. Ramping is performed as follows: equilibration with 90% eluent A for 2 min before injection and 90 to 45% eluent A for 20 min, followed by holding for 2 min, and then a return to 90% eluent A within 5 min after injection. Detection of CoA esters occurs at 259 nm with a photodiode array detector. The instrument is tuned by direct infusion of a solution of 0.4 mM CoA at a flow rate of 10 μL/min into the ion source of the mass spectrometer to optimize the ESI-MS system for maximum generation of protonated molecular ions (parents) of CoA derivatives. The following tuning parameters are retained for optimum detection of CoA esters: capillary temperature, 300° C.; sheet gas flow, 12 liters/h; auxiliary gas flow, 6 liters/h; and sweep gas flow, 1 liter/h. The mass range is set to m/z 50 to 1,000 Da when running in the scan mode. The collision energy in the MS mode is set to 30 V. See, e.g., Schurmann et al. Novel Reaction of Succinyl Coenzyme A (Succinyl-CoA) Synthetase: Activation of 3-Sulfinopropionate to 3-Sulfinopropionyl-CoA in Advenella mimigardefordensis Strain DPN7T during Degradation of 3,3-Dithiodipropionic Acid. J Bacteriol. 193(12):3078 (2011).

2-Oxoglutarate:ferredoxin oxidoreductase. The assay for the forward reaction for 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) is a coupled spectrophotometric assay based in the changes of NADH levels, which are measured at 340 nm. As shown in FIG. 2, the OGOR enzyme reaction is coupled with GDH catalyzed conversion of 2-oxoglutarate to glutamate, consuming NADH to NAD+. The pyruvate oxoreductase (POR) reaction reproduces reduced form of ferredoxin (Yamamoto et al. Carboxylation reaction catalyzed by 2-oxoglutarate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus. Extremophiles. 14:79-85 (2010)). In some aspects, the transgenic plants of this invention additionally comprise heterologous/recombinant ferredoxin from, for example, Hydrogenobacter thermophilus TK-6 and/or from Clostridium ljungdahlii, to assist OGOR in catalyzing the conversion of succinyl-CoA to 2-oxoglutarate as described herein. In other embodiments, the plant's endogenous levels of ferredoxin are sufficient to assist OGOR in catalyzing the conversion of succinyl-CoA to 2-oxoglutarate and thus, introduction of a heterologous ferredoxin is unnecessary.

For the reverse reaction for OGOR, enzymatic activity of recombinant OGOR in the cell-free extract is determined by 2-oxoglutarate dependent reduction of methyl viologen at 578 nm. The standard assay mixture contains 10 mM MOPS (pH 6.8), 1 mM MgCl₂, 1 mM DTT, 20 mM NaHCO3, 5 mM NH₄C1, 0.25 mM CoA, 0.26 mM NADH, 100 mM pyruvate, 1 mM succinyl-CoA, and proteins (OGOR, POR, ferredoxin, and GDH). The gas phase in the quartz cell is replaced with argon. The reaction is initiated by addition of succinyl-CoA. The change in A340 (representing a decrease in the consumption of NADH) is measured using a spectrophotometer. The measurement is taken 30 s following succinyl-CoA addition. The reaction mixtures contain 50 mM Tris/HCl, pH 7.5.5 mM sodium 2-oxoglutarate, 1 mM MgCl₂, 2.5 mM DTT, 0.1 mM CoA, 50 uM TPP, and 1 mM methyl viologen in a final volume of 2 ml. The reduction of methyl viologen is monitored at 578 nm. (See, e.g., Yun et al. Biochem. Biophys. Res. Comm. 282: 589-594 (2001); Wahl et al. J Biol Chem. 262: 10489-10496 (1987).

For the GC/MS method for the measurement of targeted metabolites including succinate, 2-oxoglutarate, glyoxylate, and citrate (GC-EI), the enzyme reactions are stopped by the addition of 30 μL of 15% (wt/vol) trifluoroacetic acid. GC/GC/MS experiments are performed using a LECO Pegasus III time-of-flight mass spectrometer with the 4D upgrade (LECO Corp., St. Joseph, Mich., USA). Column 1 is a 20m Rtx-5 capillary column with an internal diameter of 250 μm and a film thickness of 0.5 μm and column 2 was a 2m Rtx-200 (Restek, Bellefonte, Pa., USA) with a 180 μm internal diameter and 0.2 μm film thickness. The two columns are joined by a cryogenic modulator with a modulation period of 1.5 s with a hot pulse time of 0.40 s. Ultra high purity helium is used as the carrier gas at constant flow mode of 1 mL/min. 1 μL of a given sample is injected in triplicate in split-less mode via an Agilent 7683 autosampler. The inlet temperature is set at 280° C. The temperature program used for column 1 begins at 60° C. with a hold time of 0.25 min, then increased at 8° C./min to 280° C. with a hold time at 280° C. for 10 min. Column 2 is held in a separate oven which is initially set at 70° C. and followed the same temperature program as column 1. The ion source temperature is set to 250° C. Mass spectra are collected from m/z 40 to 600 at 100 spectra/s with a 5 min solvent delay (Yang et al. Journal of Chromatography A, 1216:3280-3289 (2009)).

2-Oxoglutarate Carboxylase.

The assay for 2-oxoglutarate carboxylase is a spectrophotometric assay in which the reductive carboxylation of 2-oxoglutarate to isocitrate is monitored indirectly at 340 nm (measuring NADH oxidation). See FIG. 3. Note that this assay is actually measuring the combined reactions of crTCA Cycle Reaction #3 and #4 (OGC and oxalosuccinate reductase). The reaction mixture for this assay (total volume of 250 μl) is composed of 100 mM Bicine-KOH (prepared from 1 M stock solution of pH 8.5, adjusted at room temperature), 50 mM NaHCO₃, 10 mM 2-oxoglutarate, 10 mM Mg-ATP, 0.25 mM NADH, 3.6 mg of ICDH (from H. thermophilus, recombinant) and OGC. The reaction is started by the addition of NADH and OGC. NADH oxidation is monitored at 340 nm (e=6.3 mM-1cm-1) for 1 min. One unit of activity is defined as 1 mmol of NADH oxidized per min (Aoshima et al. Mol. Microbiol. 62:748-759 (2006)). The GC/MS method for OGC is the same as that set forth for crTCA cycle reaction #2 above.

Oxalosuccinate Reductase.

The assay provided herein for crTCA cycle reaction #3 (see, e.g., (Aoshima et al. Mol. Microbiol. 62:748-759 (2006)). For the LC/MS method for the detection of isocitrate produced (LC-ESI), chromatographic separation is carried out using a 250×4.6 mm (5 μm) Allure Organic Acids column (Restek Corp., Bellefonte, Pa.) fitted with a 10×4.6 mm (5 μm) guard column at 30° C. Mobile phase is water/methanol (85:15) containing 0.5% formic acid, delivered at 0.7 mL/min. The column effluent is split in a ratio of 1:1 before the ionization source. The injection volume is 10 μL. Two multiple reaction monitoring (MRM) transitions in the negative ion mode are used. The dwell time, interchannel delay, and interscan delay are 0.1, 0.02, and 0.1 s, respectively. Other operating parameters are as follows: capillary voltage, 3 kV; source and desolvation temperature, 120 and 350° C.; desolvation and cone gas flow rates, 900 and 50 L/h, respectively; cone voltage, 20 V; collision energy, 20 eV. (See, e.g., Ehling et al. J. Agric. Food Chem. 59:2229-2234(2011)).

Isocitrate lyase.

This is a continuous spectrophotometric rate determination in which isocitrate lyase (ICL) converts isocitrate to succinate and glyoxylate. The glyoxylate is chemically converted to glyoxylate phenylhydrazone in the presence of phenylhydrazine. The glyoxylate phenylhydrazone is measured at 324 nm. The reaction mixture contains 30 mM imidazole (pH 6.8), 5 mM MgCl₂, 1 mM EDTA, 4 mM phenylhydrazine and 10 mM isocitrate. The reaction was performed at room temperature. After adding ICL, the reaction was continuously monitored at 324 nm (See, e.g., Chell et al. Biochemical Journal 173:165-177 (1978)).

These assays can be performed on protein extracts from plants, plant parts (e.g., leaf, stem, seed, and the like) and plant cells (e.g., cell cultures comprising tissue culture, a suspension of plant cells such as algal cells, protoplasts and the like) or protein extracts from cells and/or cell cultures of photosynthetic bacteria.

In some embodiments, a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, a 2-oxoglutarate carboxylase, oxalosuccinate reductase polypeptide and/or a isocitrate lyase polypeptide (e.g., the polynucleotides encoding the biological carbon sequestration/crTCA cycle enzymes) as well as any other heterologous polynucleotide encoding a polypeptide or functional nucleic acid of interest can be comprised within an expression cassette. As used herein, “expression cassette” means a recombinant nucleic acid molecule comprising at least one polynucleotide sequence of interest (e.g., a heterologous polynucleotide encoding a biological carbon sequestration polypeptide, and the like), wherein said recombinant nucleic acid molecule is operably associated with at least one control sequence (e.g., a promoter). Thus, some embodiments of the invention provide expression cassettes designed to express a recombinant nucleic acid molecule/heterologous polynucleotide encoding polypeptides having the enzyme activity of succinyl CoA synthetase, 2-oxoglutarate:ferredoxin oxidoreductase, 2-oxoglutarate carboxylase, oxalosuccinate reductase, and/or isocitrate lyase.

An expression cassette comprising a recombinant nucleic acid molecule may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.

In some embodiments, heterologous polynucleotides encoding a succinyl CoA synthetase polypeptide, a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and/or an isocitrate lyase polypeptide can be comprised in a single expression cassette. In some embodiments, the single expression cassette can further comprise a heterologous polynucleotide encoding any other polypeptide or functional nucleic acid of interest. The expression cassette can be operably linked to a promoter that drives expression of all of the polynucleotides comprised in the expression cassette and/or the expression cassette can comprise one or more promoters operably linked to one or more of the heterologous polynucleotides for driving the expression of said heterologous polynucleotides individually or in combination. In other embodiments, the heterologous polynucleotides encoding a succinyl CoA synthetase polypeptide, a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and/or an isocitrate lyase polypeptide (and/or any other polypeptide or functional nucleic acid of interest) can be comprised in more than one expression cassette.

When the heterologous polynucleotides are comprised within more than one expression cassette, said heterologous polynucleotides encoding the crTCA cycle polypeptides of this invention can be introduced into plants singly or more than one at a time using co-transformation methods as known in the art. In addition to transformation technology, traditional breeding methods as known in the art can be used to assist in introducing into a single plant each of the polynucleotides encoding the crTCA cycle enzymes alone or in combination as described herein and/or any additional polynucleotides of interest to produce a plant, plant part, and/or plant cell comprising and expressing each of the desired heterologous polynucleotides of interest.

Any promoter useful for initiation of transcription in a cell of a plant can be used in the expression cassettes of the present invention. A “promoter,” as used herein, is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i.e., a coding sequence) that is operably associated with the promoter. The coding sequence may encode a polypeptide and/or a functional RNA. Typically, a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. In general, promoters are found 5′, or upstream, relative to the start of the coding region of the corresponding coding sequence. The promoter region may comprise other elements that act as regulators of gene expression. These include a TATA box consensus sequence, and often a CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50:349). In plants, the CAAT box may be substituted by the AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).

Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, i.e., “chimeric genes” or “chimeric polynucleotides.” A promoter can be identified in and isolated from the organism to be transformed and then inserted into the nucleic acid construct to be used in transformation of the organism.

The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the host cell to be transformed. Thus, for example, expression of the heterologous polynucleotide encoding the polypeptides of the crTCA cycle as described herein can be in any plant, plant part, (e.g., in leaves, in stalks or stems, in ears, in inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, seeds and/or seedlings, and the like), or plant cells (including algae cells). For example, in the case of a multicellular organism such as a plant where expression in a specific tissue or organ is desired, a tissue-specific or tissue preferred promoter can be used (e.g., a root specific/preferred promoter). In contrast, where expression in response to a stimulus is desired a promoter inducible by stimuli or chemicals can be used. Where continuous expression at a relatively constant level is desired throughout the cells or tissues of an organism a constitutive promoter can be chosen.

Thus, promoters useful with the invention include, but are not limited to, those that drive expression of a nucleotide sequence constitutively, those that drive expression when induced, and those that drive expression in a tissue- or developmentally-specific manner. These various types of promoters are known in the art. Promoters can be identified in and isolated from the plant to be transformed and then inserted into the expression cassette to be used in transformation of the plant.

Non-limiting examples of a promoter include the promoter of the RubisCo small subunit gene 1 (PrbcS1), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdcal) (See, Walker et al. Plant Cell Rep. 23:727-735 (2005); Li et al. Gene 403:132-142 (2007); Li et al. Mol Biol. Rep. 37:1143-1154 (2010)). PrbcS1 and Pactin are constitutive promoters and Pnr and Pdcal are inducible promoters. Pnr is induced by nitrate and repressed by ammonium (Li et al. Gene 403:132-142 (2007)) and Pdcal is induced by salt (Li et al. Mol Biol. Rep. 37:1143-1154 (2010)).

Examples of constitutive promoters useful for plants include, but are not limited to, cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well as U.S. Pat. No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA 84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:6624-6629), sucrose synthase promoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148), and the ubiquitin promoter. The constitutive promoter derived from ubiquitin accumulates in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al., 1991. Plant Science 79: 87-94), maize (Christensen et al., 1989. Plant Molec. Biol. 12: 619-632), and arabidopsis (Norris et al. 1993. Plant Molec. Biol. 21:895-906). The maize ubiquitin promoter (UbiP) has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926. The ubiquitin promoter is suitable for the expression of the nucleotide sequences of the invention in transgenic plants, especially monocotyledons. Further, the promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for the expression of the nucleotide sequences of the invention and are particularly suitable for use in monocotyledonous hosts.

In some embodiments, tissue specific/tissue preferred promoters can be used for expression of a heterologous polynucleotide in a plant cell. Tissue specific or preferred expression patterns include, but are not limited to, green tissue specific or preferred, root specific or preferred, stem specific or preferred, and flower specific or preferred. Promoters suitable for expression in green tissue include many that regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. In one embodiment, a promoter useful with the invention is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:579-589 (1989)). Non-limiting examples of tissue-specific promoters include those associated with genes encoding the seed storage proteins (such as β-conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g., Kridl et al. (1991) Seed Sci. Res. 1:209-219; as well as EP Patent No. 255378). Tissue-specific or tissue-preferential promoters useful for the expression of the nucleotide sequences of the invention in plants, particularly maize, include but are not limited to those that direct expression in root, pith, leaf or pollen. Such promoters are disclosed, for example, in WO 93/07278, herein incorporated by reference in its entirety. Other non-limiting examples of tissue specific or tissue preferred promoters useful with the invention the cotton rubisco promoter disclosed in U.S. Pat. No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Pat. No. 5,604,121; the root specific promoter described by de Framond (FEBS 290:103-106 (1991); EP 0 452 269 to Ciba-Geigy); the stem specific promoter described in U.S. Pat. No. 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene; and the cestrum yellow leaf curling virus promoter disclosed in WO 01/73087.

Additional examples of plant tissue-specific/tissue preferred promoters include, but are not limited to, the root hair-specific cis-elements (RHEs) (Kim et al. The Plant Cell 18:2958-2970 (2006)), the root-specific promoters RCc3 (Jeong et al. Plant Physiol. 153:185-197 (2010)) and RB7 (U.S. Pat. No. 5,459,252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), S-adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8):1108-1115), corn light harvesting complex promoter (Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658), corn heat shock protein promoter (O'Dell et al. (1985) EMBO J. 5:451-458; and Rochester et al. (1986) EMBO J. 5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore, “Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase” pp. 29-39 In: Genetic Engineering of Plants (Hollaender ed., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid nopaline synthase promoter (Langridge et al. (1989), supra), petunia chalcone isomerase promoter (van Tunen et al. (1988) EMBO J. 7:1257-1263), bean glycine rich protein 1 promoter (Keller et al. (1989) Genes Dev. 3:1639-1646), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature 313:810-812), potato patatin promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res. 18:7449), maize zein promoter (Kriz et al. (1987) Mol. Gen. Genet. 207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina et al. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic Acids Res. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872), α-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet. 215:431-440), PEPCase promoter (Hudspeth & Grula (1989) Plant Mol. Biol. 12:579-589), R gene complex-associated promoters (Chandler et al. (1989) Plant Cell 1:1175-1183), and chalcone synthase promoters (Franken et al. (1991) EMBO J. 10:2605-2612).

Particularly useful for seed-specific expression is the pea vicilin promoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; as well as the seed-specific promoters disclosed in U.S. Pat. No. 5,625,136. Useful promoters for expression in mature leaves are those that are switched at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al. (1995) Science 270:1986-1988).

In addition, promoters functional in chloroplasts can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5′ UTR and other promoters disclosed in U.S. Pat. No. 7,579,516. Other promoters useful with the invention include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

In some embodiments of the invention, inducible promoters can be used. Thus, for example, chemical-regulated promoters can be used to modulate the expression of a gene in an organism through the application of an exogenous chemical regulator. Regulation of the expression of nucleotide sequences of the invention via promoters that are chemically regulated enables the polypeptides of the invention to be synthesized only when, for example, a crop of plants are treated with the inducing chemicals. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of a chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.

Chemical inducible promoters useful with plants are known in the art and include, but are not limited to, the maize Int-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid (e.g., the PR1a system), steroid-responsive promoters (see, e.g., the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227, 229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156, Lac repressor system promoters, copper-inducible system promoters, salicylate-inducible system promoters (e.g., the PR1a system), glucocorticoid-inducible promoters (Aoyama et al. (1997) Plant J. 11:605-612), and ecdysone-inducible system promoters.

Other non-limiting examples of inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoid glycosyl-transferase promoter (Ralston et al. (1988) Genetics 119:185-197), the MPI proteinase inhibitor promoter (Cordero et al. (1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphate dehydrogenase promoter (Kohler et al. (1995) Plant Mol. Biol. 29:1293-1298; Martinez et al. (1989)J. Mol. Biol. 208:551-565; and Quigley et al. (1989) J. Mol. Evol. 29:412-421). Also included are the benzene sulphonamide-inducible (U.S. Pat. No. 5,364,780) and alcohol-inducible (Int'l Patent Application Publication Nos. WO 97/06269 and WO 97/06268) systems and glutathione S-transferase promoters. Likewise, one can use any of the inducible promoters described in Gatz (1996) Current Opinion Biotechnol. 7:168-172 and Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108. Other chemically inducible promoters useful for directing the expression of the nucleotide sequences of this invention in plants are disclosed in U.S. Pat. No. 5,614,395 herein incorporated by reference in its entirety. Chemical induction of gene expression is also detailed in the published application EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. In some embodiments, a promoter for chemical induction can be the tobacco PR-1a promoter.

In particular embodiments, promoters useful with algae include, but are not limited to, the promoter of the RubisCo small subunit gene 1 (PrbcS1), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdcal) (See, Walker et al. Plant Cell Rep. 23:727-735 (2005); Li et al. Gene 403:132-142 (2007); Li et al. Mol Biol. Rep. 37:1143-1154 (2010)), the promoter of the σ⁷⁰-type plastid rRNA gene (Prrn), the promoter of the psbA gene (encoding the photosystem-II reaction center protein D1) (PpsbA), the promoter of the psbD gene (encoding the photosystem-II reaction center protein D2) (PpsbD), the promoter of the psaA gene (encoding an apoprotein of photosystem I) (PpsaA), the promoter of the ATPase alpha subunit gene (PatpA), and promoter of the RuBisCo large subunit gene (PrbcL), and any combination thereof (See, e.g., De Cosa et al. Nat. Biotechnol. 19:71-74 (2001); Daniell et al. BMC Biotechnol. 9:33 (2009); Muto et al. BMC Biotechnol. 9:26 (2009); Surzycki et al. Biologicals 37:133-138 (2009)).

In some embodiments, the heterologous polynucleotides of the invention (e.g., the biological carbon sequestration/crTCA cycle polypeptides described herein) can be transformed into the nucleus or into, for example, the chloroplast using standard techniques known in the art of plant transformation.

Thus, in some embodiments, heterologous polynucleotides encoding (1) a succinyl CoA synthetase polypeptide, (2) a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (3) a 2-oxoglutarate carboxylase polypeptide, (4) an oxalosuccinate reductase polypeptide, or (5) an isocitrate lyase polypeptide, or (6) a succinyl CoA synthetase polypeptide and a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (7) a 2-oxoglutarate carboxylase polypeptide and an oxalosuccinate reductase polypeptide, or (8) a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and an isocitrate lyase polypeptide can be transformed into and expressed in the nucleus and the polypeptides produced remain in the cytosol. In other embodiments, heterologous polynucleotides encoding (1) a succinyl CoA synthetase polypeptide, (2) a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (3) a 2-oxoglutarate carboxylase polypeptide, (4) an oxalosuccinate reductase polypeptide, or (5) an isocitrate lyase polypeptide, or (6) a succinyl CoA synthetase polypeptide and a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (7) a 2-oxoglutarate carboxylase polypeptide and an oxalosuccinate reductase polypeptide, or (8) a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and an isocitrate lyase polypeptide can be transformed into and expressed in the nucleus, wherein one or more of the polypeptides can be targeted to the chloroplast. Thus, in particular embodiments, the one or more heterologous polynucleotides encoding (1) a succinyl CoA synthetase polypeptide, (2) a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (3) a 2-oxoglutarate carboxylase polypeptide, (4) an oxalosuccinate reductase polypeptide, or (5) an isocitrate lyase polypeptide, or (6) a succinyl CoA synthetase polypeptide and a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (7) a 2-oxoglutarate carboxylase polypeptide and an oxalosuccinate reductase polypeptide, or (8) a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and an isocitrate lyase polypeptide can be operably associated with at least one targeting nucleotide sequence encoding a signal peptide that targets the polypeptides to the chloroplast.

A signal sequence may be operably linked at the N- or C-terminus of a heterologous nucleotide sequence or nucleic acid molecule. Signal peptides (and the targeting nucleotide sequences encoding them) are well known in the art and can be found in public databases such as the “Signal Peptide Website: An Information Platform for Signal Sequences and Signal Peptides.” (www.signalpeptide.de); the “Signal Peptide Database” (proline.bic.nus.edu.sg/spdb/index.html) (Choo et al., BMC Bioinformatics 6:249 (2005)(available on www.biomedcentral.com/1471-2105/6/249/abstract); ChloroP (www.cbs.dtu.dk/services/ChloroP/; predicts the presence of chloroplast transit peptides (cTP) in protein sequences and the location of potential cTP cleavage sites); LipoP (www.cbs.dtu.dk/services/LipoP/; predicts lipoproteins and signal peptides in Gram negative bacteria); MITOPROT (ihg2.helmholtz-muenchen.de/ihg/mitoprot.html; predicts mitochondrial targeting sequences); PlasMit (gecco.org.chemie.uni-frankfurt.de/plasmit/index.html; predicts mitochondrial transit peptides in Plasmodium falciparum); Predotar (urgi.versailles.inra.fr/predotar/predotar.html; predicts mitochondrial and plastid targeting sequences); PTS1 (mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictorjsp; predicts peroxisomal targeting signal 1 containing proteins); SignalP (www.cbs.dtu.dk/services/SignalP/; predicts the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms: Gram-positive prokaryotes, Gram-negative prokaryotes, and eukaryotes). The SignalP method incorporates a prediction of cleavage sites and a signal peptide/non-signal peptide prediction based on a combination of several artificial neural networks and hidden Markov models; and TargetP (www.cbs.dtu.dk/services/TargetP/); predicts the subcellular location of eukaryotic proteins—the location assignment is based on the predicted presence of any of the N-terminal presequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP)). (See also, von Heijne, G., Eur J Biochem 133 (1) 17-21 (1983); Martoglio et al. Trends Cell Biol 8 (10):410-5 (1998); Hegde et al. Trends Biochem Sci 31(10):563-71 (2006); Dultz et al. J Biol Chem 283(15):9966-76 (2008); Emanuelsson et al. Nature Protocols 2(4) 953-971(2007); Zuegge et al. 280(1-2):19-26 (2001); Neuberger et al. J Mol Biol. 328(3):567-79 (2003); and Neuberger et al. J Mol Biol. 328(3):581-92 (2003)).

Exemplary signal peptides include, but are not limited to those provided in Table 1.

TABLE 1
Amino acid sequences of representative signal peptides.
Source	Sequence	Target
Rubisco small	MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASF	chloroplast
subunit (tobacco)	PVSRKQNLDITSIASNGGRVQC (SEQ ID NO: 72)
Arabidopsis	MRILPKSGGGALCLLFVFALCSVAHS (SEQ ID	cell
proline-rich	NO: 73)	wall/secretory
protein 2		pathway
(AT2G21140)
PTS-2 (conserved in	RLX5HL (SEQ ID NO: 74)	peroxisome
eukaryotes)	MRLSIHAEHL (SEQ ID NO: 75)
	SKL
Arabidopsis	MLRTVSCLASRSSSSLFFRFFRQFPRSYMSLTSSTAAL	mitochondria
presequence	RVPSRNLRRISSPSVAGRRLLLRRGLRIPSAAVRSVN	and chloroplast
proteasel	GQFSRLSVRA (SEQ ID NO: 76)
(AT3G19170)
Chlamydomonas	MALVARPVLSARVAASRPRVAARKAVRVSAKYGEN	chloroplast
reinhardtii-(Stroma-	(SEQ ID NO: 77)
targeting cTPs:	MQALSSRVNIAAKPQRAQRLVVRAEEVKA (SEQ ID
photosystem I (PSI)	NO: 78)
subunits P28, P30,	MQTLASRPSLRASARVAPRRAPRVAVVTKAALDPQ
P35 and P37,	(SEQ ID NO: 79)
respectively)	MQALATRPSAIRPTKAARRSSVVVRADGFIG (SEQ
	ID NO: 80)
C. reinhardtii -	MAFALASRKALQVTCKATGKKTAAKAAAPKSSGVE	chloroplast
chlorophyll a/b	FYGPNRAKWLGPYSEN (SEQ ID NO: 81)
protein (cabII-1)
C. reinhardtii -	MAAVIAKSSVSAAVARPARSSVRPMAALKPAVKAA	chloroplast
Rubisco small	PVAAPAQANQMMVWT (SEQ ID NO: 82)
subunit
C. reinhardtii -	MAAMLASKQGAFMGRSSFAPAPKGVASRGSLQVVA	chloroplast
ATPase-γ	GLKEV (SEQ ID NO: 83)
Arabidopsis thaliana	CVVQ (SEQ ID NO: 84)	membrane
abscisic acid
receptor PYL 10
X5 means any five amino acids can be present in the sequence to target the protein to the peroxisome (e.g. RLAVAVAHL, SEQ ID NO: 85).

Thus, in representative embodiments of the invention, a heterologous polynucleotide encoding (a) succinyl CoA synthetase, (b) 2-oxoglutarate:ferredoxin oxidoreductase, (c) 2-oxoglutarate carboxylase, (d) oxalosuccinate reductase, and/or (e) isocitrate lyase to be expressed in a plant, plant cell, plant part can be operably linked to a chloroplast targeting sequence encoding a chloroplast signal peptide, optionally wherein said chloroplast signal peptide is encoded by an amino acid sequence that includes, but is not limited to, the amino acid sequence of SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82,or SEQ ID NO:83.

Targeting to a membrane is similar to targeting to an organelle. Thus, specific sequences on a protein (targeting sequences or motifs) can be recognized by a transporter, which then imports the protein into an organelle or in the case of membrane association, the transporter can guide the protein to and associate it with a membrane. Thus, for example, a specific cysteine residue on a C-terminal motif of a protein can be modified posttranslation where an enzyme (prenyltransferases) then attaches a hydrophobic molecule (geranylgeranyl or farnesyl) (See, e.g., Running et al. Proc Natl Acad Sci USA 101: 7815-7820 (2004); Maurer-Stroh et al. Genome Biology 4:212 (2003)). This hydrophobic addition guides and associates the protein to a membrane (in case of the cytosol, the membrane would be the plasma membrane or the cytosolic site of the nuclear membrane (Polychronidou et al. Molecular Biology of the Cell 21: 3409-3420 (2010)). More specifically, in representative embodiments, a protein prenyltransferase can catalyze the covalent attachment of a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid to C-terminal cysteines of selected proteins carrying a CaaX motif where C=cysteine; a=aliphatic amino acid; x=any amino acid. For plants, this motif most often is CVVQ (SEQ ID NO:84). The addition of prenyl groups facilitates membrane association and protein—protein interactions of the prenylated proteins.

In still other embodiments of the invention, a signal peptide can direct a polypeptide of the invention to more than one organelle (e.g., dual targeting). Thus, in some embodiments, a signal peptide that can target a polypeptide of the invention to more than one organelle is encoded by an amino acid sequence that includes, but is not limited to, the amino acid sequence of SEQ ID NO:76.

In addition to promoters operably linked to a heterologous polynucleotide of the invention, an expression cassette also can include other regulatory sequences. As used herein, “regulatory sequences” means nucleotide sequences located upstream (5′ non-coding sequences), within or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, enhancers, introns, translation leader sequences, translation termination sequences, and polyadenylation signal sequences, as described herein.

Thus, in some embodiments of the present invention, the expression cassettes can include at least one intron. An intron useful with this invention can be an intron identified in and isolated from a plant to be transformed and then inserted into the expression cassette to be used in transformation of the plant. As would be understood by those of skill in the art, the introns as used herein comprise the sequences required for self excision and are incorporated into the nucleic acid constructs in frame. An intron can be used either as a spacer to separate multiple protein-coding sequences in one nucleic acid construct, or an intron can be used inside one protein-coding sequence to stabilize the mRNA. If they are used within a protein-coding sequence, they are inserted “in-frame” with the excision sites included.

Non-limiting examples of introns useful with the present invention can be introns from the RuBisCO small subunit (rbcS) gene, the RuBisCO large subunit (rbcL) gene, the actin gene, the nitrate reductase gene (nr), the duplicated carbonic anhydrase gene 1 (Tdca1), the psbA gene, the atpA gene, or any combination thereof.

In some embodiments of the invention, an expression cassette can comprise an enhancer sequence. Enhancer sequences can be derived from, for example, any intron from any highly expressed gene. In particular embodiments, an enhancer sequence usable with this invention includes, but is not limited to, the nucleotide sequence of ggagg (e.g., ribosome binding site).

An expression cassette also can optionally include a transcriptional and/or translational termination region (i.e., termination region) that is functional in plants, yeast or bacteria. A variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous polynucleotide of interest and correct mRNA polyadenylation. The termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host cell, or may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the host cell, or any combination thereof). Non-limiting examples of transcriptional terminators useful for plants can be a CAMV 35S terminator, a tml terminator, a nopaline synthase terminator and/or a pea rbcs E9 terminator, a RubisCo small subunit gene 1 (TrbcS1) terminator, an actin gene (Tactin) terminator, a nitrate reductase gene (Tnr) terminator, and/or aa duplicated carbonic anhydrase gene 1 (Tdca1) terminator.

Further non-limiting examples of terminators useful with this invention for expression of the heterologous polynucleotides of the invention or other heterologous polynucleotides of interest in algae include a terminator of the psbA gene (TpsbA), a terminator of the psaA gene (encoding an apoprotein of photosystem I) (TpsaA), a terminator of the psbD gene (TpsbD), a RuBisCo large subunit terminator (TrbcL), a terminator of the σ⁷⁰-type plastid rRNA gene (Trrn), and/or a terminator of the ATPase alpha subunit gene (TatpA).

An expression cassette of the invention also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed plant, plant part and/or plant cell. As used herein, “selectable marker” means a nucleotide sequence that when expressed imparts a distinct phenotype to a plant, plant part and/or plant cell expressing the marker and thus allows such a transformed plant, plant part, and/or plant cell to be distinguished from that which does not have the marker. Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like), or whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., the R-locus trait). Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

Examples of selectable markers include, but are not limited to, a nucleotide sequence encoding aadA (i.e., spectinomycin and streptomycin resistance), a nucleotide sequence encoding neo (i.e., kanamycin resistance), a nucleotide sequence encoding aphA6 (i.e., kanamycin resistance), a nucleotide sequence encoding nptII (i.e., kanamycin resistance), a nucleotide sequence encoding bar (i.e., phosphinothricin resistance), a nucleotide sequence encoding cat (i.e., chloramphenicol resistance), a nucleotide sequence encoding badh (i.e., betaine aldehyde resistance), a nucleotide sequence encoding egfp, (i.e., enhanced green fluorescence protein), a nucleotide sequence encoding gfp, (i.e., green fluorescent protein), a nucleotide sequence encoding luc (i.e., luciferase), a nucleotide sequence encoding ble (bleomycin resistance), a nucleotide sequence encoding ereA (erythromycin resistance), and any combination thereof.

Further examples of selectable markers useful with the invention include, but are not limited to, a nucleotide sequence encoding an altered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which confers resistance to glyphosate (Hinchee et al. (1988) Biotech. 6:915-922); a nucleotide sequence encoding a nitrilase such as bxn from Klebsiella ozaenae that confers resistance to bromoxynil (Stalker et al. (1988) Science 242:419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP Patent Application No. 154204); a nucleotide sequence encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. 263:12500-12508); a nucleotide sequence encoding a dalapon dehalogenase that confers resistance to dalapon; a nucleotide sequence encoding a mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI)) that confers an ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5-methyl tryptophan; and/or a nucleotide sequence encoding hph that confers resistance to hygromycin.

Additional selectable markers include, but are not limited to, a nucleotide sequence encoding β-glucuronidase or uidA (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus nucleotide sequence that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., “Molecular cloning of the maize R-nj allele by transposon-tagging with Ac” 263-282 In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); a nucleotide sequence encoding β-lactamase, an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737-3741); a nucleotide sequence encoding xylE that encodes a catechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding β-galactosidase, an enzyme for which there are chromogenic substrates; a nucleotide sequence encoding luciferase (lux) that allows for bioluminescence detection (Ow et al. (1986) Science 234:856-859); a nucleotide sequence encoding Bla that confers ampicillin resistance; or a nucleotide sequence encoding aequorin which may be employed in calcium-sensitive bioluminescence detection (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126:1259-1268), and/or any combination thereof. One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette of this invention.

An expression cassette comprising a heterologous polynucleotide of the invention (e.g., polynucleotide(s) encoding biological carbon sequestration polypeptides of the invention), also can optionally include additional polynucleotides that encode other desired traits. Such desired traits can be, for example, polynucleotides which confer high light tolerance, increased drought tolerance, increased flooding tolerance, increased tolerance to soil contaminants, increased yield, modified fatty acid composition of the lipids, increased oil production in seed, increased and modified starch production in seeds, increased and modified protein production in seeds, modified tolerance to herbicides and pesticides, production of terpenes, increased seed number, and/or other desirable traits for agriculture or biotechnology.

Such polynucleotides can be stacked with any combination of nucleotide sequences to create plants, plant parts and/or plant cells having the desired phenotype. Stacked combinations can be created by any method including, but not limited to, any conventional methodology (e.g., cross breeding for plants), or by genetic transformation. If stacked by genetic transformation, nucleotide sequences encoding additional desired traits can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The additional nucleotide sequences can be introduced simultaneously in a co-transformation protocol with a nucleotide sequence, nucleic acid molecule, nucleic acid construct, and/or other composition of the invention, provided by any combination of expression cassettes. For example, if two nucleotide sequences will be introduced, they can be incorporated in separate cassettes (trans) or can be incorporated on the same cassette (cis). Expression of the nucleotide sequences can be driven by the same promoter or by different promoters. It is further recognized that nucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, e.g., International Patent Application Publication Nos. WO 99/25821; WO 99/25854; WO 99/25840; WO 99/25855 and WO 99/25853.

Any nucleotide sequence to be transformed into a plant, plant part and/or plant cell can be modified for codon usage bias using species specific codon usage tables. The codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest. When the nucleotide sequences are to be expressed in the nucleus, the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest. The modifications for the nucleotide sequences for selection are determined by comparing the species specific codon usage table with the codons present in the native polynucleotide sequences. In those embodiments in which each of codons in native polynucleotide sequence for selection are sufficiently used, then no modifications are needed (e.g., a frequency of more than 30% for a codon used for a specific amino acid in that species would indicate no need for modification). In other embodiments, wherein up to 3 nucleotides have to be modified in the polynucleotide sequence, site-directed mutagenesis can be used according to methods known in the art (Zheng et al. Nucleic Acids Res. 32:e115 (2004); Dammai, Meth. Mol. Biol 634:111-126 (2010); Davis and Vierstra. Plant Mol. Biol. 36(4): 521-528 (1998)). In still other embodiments, wherein more than three nucleotide changes are necessary, a synthetic nucleotide sequence can be generated using the same codon usage as the highly expressed genes that were used to develop the codon usage table.

A heterologous polynucleotide encoding (a) a succinyl CoA synthetase polypeptide, (b) an 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (c) a 2-oxoglutarate carboxylase polypeptide, (d) an oxalosuccinate reductase polypeptide, and/or (e) an isocitrate lyase polypeptide as described herein; and/or functional fragments thereof (e.g., a functional fragment of the nucleotide sequences of SEQ ID NOs:3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, and/or any combination thereof or the amino acid sequences of SEQ ID NOs:1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, and/or any combination thereof) can be introduced into a cell of a plant by any method known to those of skill in the art. In some embodiments of the invention, transformation of a cell comprises nuclear transformation. In other embodiments, transformation of a cell comprises plastid transformation (e.g., chloroplast transformation).

Procedures for transforming plants are well known and routine in the art and are described throughout the literature. Non-limiting examples of transformation methods include transformation via bacterial-mediated nucleic acid delivery (e.g., via Agrobacteria), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium-phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, as well as any other electrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of nucleic acid into the plant cell, including any combination thereof. General guides to various plant transformation methods known in the art include Miki et al. (“Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)). General guides to the transformation of yeast include Guthrie and Fink (1991) (Guide to yeast genetics and molecular biology. In Methods in Enzymology, (Academic Press, San Diego) 194:1-932) and guides to methods related to the transformation of bacteria include Aune and Aachmann (Appl. Microbiol Biotechnol 85:1301-1313 (2010)).

A polynucleotide therefore can be introduced into a plant, plant part, plant cell in any number of ways that are well known in the art. The methods of the invention do not depend on a particular method for introducing one or more nucleotide sequences into a plant, only that they gain access to the interior the cell. Where more than polynucleotide is to be introduced, they can be assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, the polynucleotide can be introduced into the cell of interest in a single transformation event, or in separate transformation events, or, alternatively, a polynucleotide can be incorporated into a plant as part of a breeding protocol.

In some embodiments, when a plant part or plant cell is stably transformed, it can then be used to regenerate a stably transformed plant comprising a heterologous polynucleotide encoding (1) a succinyl CoA synthetase polypeptide, (2) a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (3) a 2-oxoglutarate carboxylase polypeptide, (4) an oxalosuccinate reductase polypeptide, or (5) an isocitrate lyase polypeptide, or (6) a succinyl CoA synthetase polypeptide and a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (7) a 2-oxoglutarate carboxylase polypeptide and an oxalosuccinate reductase polypeptide, or (8) a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and an isocitrate lyase polypeptide as described herein. Means for regeneration can vary from plant species to plant species, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently root. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and plant hormones, such as auxin and cytokinins. It may also be advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.

The regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner. The plants are grown and harvested using conventional procedures.

The particular conditions for transformation, selection and regeneration of a plant can be optimized by those of skill in the art. Factors that affect the efficiency of transformation include the species of plant, the target tissue or cell, composition of the culture media, selectable marker genes, kinds of vectors, and light/dark conditions. Therefore, these and other factors may be varied to determine an optimal transformation protocol for any particular plant species. It is recognized that not every species will react in the same manner to the transformation conditions and may require a slightly different modification of the protocols disclosed herein. However, by altering each of the variables, an optimum protocol can be derived for any plant species.

Further, the genetic properties engineered into the transgenic seeds and plants, plant parts, and/or plant cells of the present invention described herein can be passed on by sexual reproduction or vegetative growth and therefore can be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as harvesting, sowing or tilling.

Accordingly, in some aspects of the invention, a stably transformed plant, plant part and/or plant cell, or a stably transformed photosynthetic bacterium is provided, which comprises in its genome one or more recombinant nucleic acid molecules/heterologous polynucleotides of the invention and has increased carbon fixation and/or increased biomass production. Thus, in some embodiments, the invention provides a stably transformed plant, plant part and/or plant cell, or a stably transformed photosynthetic bacterium comprising in its genome at least one heterologous polynucleotide encoding (1) a succinyl CoA synthetase polypeptide, (2) a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (3) a 2-oxoglutarate carboxylase polypeptide, (4) an oxalosuccinate reductase polypeptide, or (5) an isocitrate lyase polypeptide, or (6) a succinyl CoA synthetase polypeptide and a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (7) a 2-oxoglutarate carboxylase polypeptide and an oxalosuccinate reductase polypeptide, or (8) a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and an isocitrate lyase polypeptide, which when expressed results in the stably transformed plant, plant part or plant cell, or the stably transformed photosynthetic bacterium having increased carbon fixation and/or increased biomass production. In representative embodiments, the at least one heterologous polynucleotide encoding (1) a succinyl CoA synthetase polypeptide, (2) a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (3) a 2-oxoglutarate carboxylase polypeptide, (4) an oxalosuccinate reductase polypeptide, or (5) an isocitrate lyase polypeptide, or (6) a succinyl CoA synthetase polypeptide and a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (7) a 2-oxoglutarate carboxylase polypeptide and an oxalosuccinate reductase polypeptide, or (8) a 2-oxoglutarate carboxylase polypeptide, an oxalosuccinate reductase polypeptide and an isocitrate lyase polypeptide when expressed in a plant, plant part, and/or plant cell may be expressed in the nucleus and targeted to the chloroplast and/or may be expressed in the chloroplast.

Additionally provided herein are seeds produced from the stably transformed plants of the invention, wherein said seeds comprise in their genome (1) a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, (2) a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (3) a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, (4) a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide, or (5) a heterologous polynucleotide encoding an isocitrate lyase polypeptide, or (6) a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, (7) a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide, or (8) a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and a heterologous polynucleotide encoding an isocitrate lyase polypeptide, wherein said heterologous polynucleotides are from a bacterial or archaeal species.

The present invention further provides products produced from the stably transformed plant, plant cell or plant part of the invention. In some embodiments, the product produced can include but is not limited to biofuel, food, drink, animal feed, fiber, and/or pharmaceuticals.

The following examples are not intended to be a detailed catalog of all the different ways in which the present invention may be implemented or of all the features that may be added to the present invention. Persons skilled in the art will appreciate that numerous variations and additions to the various embodiments may be made without departing from the present invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

EXAMPLES

Example 1. The Synthetic crTCA Pathway Enzymes

Increasing the productivity of a C₃ plant such as camelina to levels seen for C₄ plants (e.g. corn) requires improving photosynthetic carbon fixation. One limiting factor is the oxygenase activity of the CO₂-fixing Ribulose 1,5 bisphosphate Carboxylase/Oxygenase (RUBISCO) that reduces the photosynthetic productivity by up to 30%. The present invention provides methods and compositions for improving carbon fixation in plants by introducing a synthetic carbon fixation pathway that is independent of RUBISCO but works in concert with the existing Calvin Benson cycle.

Specifically, this invention provides a “condensed reverse TCA (crTCA) cycle,” that employs a (1) succinyl-CoA synthetase for catalyzing the conversion of succinate to succinyl-CoA, (2) a 2-oxoglutarate:ferredoxin oxidoreductase for converting succinyl-CoA to 2-oxoglutarate (i.e., 2-ketoglutarate), (3) a 2-oxoglutarate carboxylase for converting 2-oxoglutarate to oxalosuccinate, (4) an oxalosuccinate reductase for converting oxalosuccinate to isocitrate, and (5) an isocitrate lyase for cleaving isocitrate into succinate and glyoxylate (FIG. 1).

For generation of the synthetic crTCA cycle, specific enzymes were chosen from source bacteria based on the following criteria: (1) experimentally determined function of the enzyme, (2) target enzymes having the fewest subunits, and (3) in cases in which enzyme activity is unavailable, enzyme choice based on highest homology levels to characterized enzymes having the desired activity.

Candidate Enzymes

For the succinyl CoA synthetase enzyme activity, the characterized Escherichia coli version of this enzyme can be used (e.g., SucC and SucD, NCBI Accession Nos: NC_000913.2 (762,237 . . . 763,403), NC_000913.2 (763,403 . . . 764,272),NP_415256.1 and NP_415257.1) (Buck et al. J Gen Microbiol 132:1753-62 (1986)). Additional succinyl CoA synthetase versions that can also be used include those from Azotobacter vinelandii DJ, (NCBI Accession Nos. NC_012560.1 (3,074,152 . . . 3,075,321), NC_012560.1 (3,073,268 . . . 3,074,155, YP_002800115.1 and YP_002800114.1; Bradyrhizobium sp. BTAi1, (NCBI Accession Nos. NC_009485.1 (393,292 . . . 394,488), NC_009485.1 (394,545 . . . 395,429),YP_001236586.1 and YP_001236587.1); and/or Azospirillum sp. B510, (NCBI Accession Nos. NC_013854.1 (2,941,010 . . . 2,942,206), NC_013854.1 (2,942,208 . . . 2,943,083), YP_003449758.1 and YP_003449759.1) (See, e.g., the nucleotide sequences of SEQ ID NOs:3, 6, 9 and/or 12; the amino acid sequences of SEQ ID NOs:1, 2, 4, 5, 7, 8, 10 and/or 11).

Oxoglutarate:ferredoxin oxidoreductase (OOR) enables the crTCA cycle to function in the reverse direction (Buchanan and Arnon Photosynth Res 24:47-53 (1990). There are two types of OORs, a two subunit version expressed in the anaerobic phototrophic bacterium Chlorobium limicola (Buchanan and Arnon Photosynth Res 24:47-53 (1990)) and the aerobic halophile Halobacterium salinarum (Kerscher and Oesterhelt Eur J Biochem 116:587-94(1981)) and a four subunit version expressed in anaerobic sulfur reducing bacteria such as Sulfurimonas denitrificans (Hugler et al. J. Bacteriol 187:3020-7 (2005)). Because the crTCA cycle is meant to function in plants using oxygenic photosynthesis and limiting enzyme subunits can simplify the generation of the transgenic plant lines, the two subunit version of OOR from an aerobic bacterium can be used. Based on homology to the biochemically characterized H. salinarum OOR, a two subunit OOR was selected with good identity from the aerobic bacterium Paenibacillus larvae subsp. larvae B-3650 ((NCBI Accession Nos. PlarlB_020100012680 and PlarlB_020100012675, NZ_ADZY02000226.1 (7,939 . . . 9,687), NZ_ADZY02000226.1 (7,085 . . . 7,951), ZP_09070120.1 and ZP_09070119.1). Additional versions of OOR that could be used include the following: Halobacterium sp. NRC-1 korA, korB, (NCBI Accession Nos. NC_002607.1 (856,660 . . . 858,582), NC_002607.1 (855,719 . . . 856,657), AAG19514.1 and AAG19513.1, NP_280034.1 and NP_280033.1); Hydrogenobacter thermophilus TK-6 korA, korB, ((NCBI Accession Nos. NC_013799.1 (997,525 . . . 999,348), NC_013799.1 (996,624 . . . 997,511), YP_003432752.1 and YP_003432751.1; Bacillus sp. M3-13 Bm3-1 010100005806, Bm3-1_010100005801, NZ_ACPC01000013.1 (932Dz,668), NZ_ACPC01000013.1 (65 . . . 931), ZP_07708142.1 and ZP_07708141.1); Haladaptatus paucihalophilus DX253 (NCBI Accession Nos. ZOD2009_10775, ZOD2009-10770, contig00009, whole genome shotgun sequence NZ_AEMG01000009.1 (157,678DZ59,432), NZ_AEMG01000009.1 (156,818 . . . 157,681), ZP_08044530.1 and ZP_08044529.1); and/or Magnetococcus sp. (NCBI Accession Nos. MC-1 Mmc1_1749, Mmc1_1750, NC_008576.1 (2,161,258 . . . 2,162,979), NC_008576.1 (2,162,976 . . . 2,163,854), YP_865663.1 and YP_865664.1). (See, e.g., the nucleotide sequences of SEQ ID NOs:15, 18, 21, 24, 27 and/or 30; or the amino acid sequences of SEQ ID NOs: 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28 and/or 29).

A 2-oxoglutarate carboxylase characterized from the thermophilic chemoautotrophic bacterium Hydrogenobacter thermophilus TK-6, optimally functions at 80° C. (Aoshima and Igarashi Mol Microbiol 62:748-59(2006)). Homology analysis using the H. thermophilus korA; and korB subunit sequences identified subunits from a nitrite-oxidizing bacterium Candidatus Nitrospira defluvii having high identity (pycA, and pycB; NCBI Accession Nos. NC_014355.1 (1,174,721DZ,176,652), NC_014355.1 (1,176,781DZ,178,199), YP_003796887.1 and YP_003796888.1). These genes are identified as subunits of pyruvate carboxylase in the N. defluvii genome; however, protein modeling analysis determined that the N. defluvii carboxylase has high specificity for oxoglutarate. Additional versions of 2-oxoglutarate carboxylase that could be used include, for example, Hydrogenobacter thermophilus TK-6 cfiA, cfiB, (NCBI Accession Nos. NC_013799.1 (1,271,487 . . . 1,273,445), NC_013799.1 (1,273,469DZ,274,887), YP_003433044.1 and YP_003433045.1 and its modified version (see, e.g., SEQ ID NOs:37-42)); Thiocystis violascens DSM198 (NCBI Accession Nos. ThiviDRAFT 1483, ThiviDRAFT 1486, whole genome shotgun sequence, ctg263, NZ_AGFC01000013.1 (61,879 . . . 63,297) and (63,889 . . . 65,718), ZP_08925050.1 and ZP_08925052.1); Mariprofundus ferrooxydans PV-1 (NCBI Accession Nos. SPV1_07811, SPV1_07816, NZ_AATS01000007.1 whole genome shotgun sequence, 1099921033908 (81,967 . . . 83,385) and (83,475 . . . 85,328), ZP_01452577.1 AND ZP_01452578.1); and/or Pseudomonas stutzeri ATCC14405 (NCBI Accession Nos. PstZobell_14412 and PstZobell_14407, CCUG 16156 contig00098, whole genome shotgun sequence AGSL01000085.1 (52,350 . . . 53,765) and (50,522 . . . 52,339), EHY78621.1 and EHY78620.1). (See, e.g., the nucleotide sequences of SEQ ID NOs: 33, 36, 39, 42, 45, 48 and/or 51; or the amino acid sequences of SEQ ID NOs: 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49 and/or 50).

An oxalosuccinate reductase has also been characterized from H. thermophilus (Aoshima and Igarashi Mol Microbiol 62:748-59(2006)). We identified a further oxalosuccinate reductase from the soil bacterium Acinetobacter baumannii (NCBI Accession Nos. ACICU_02687, NC_010611.1 (2,855,563 . . . 2,856,819) YP_001847346.1), which has high homology to oxalosuccinate reductase from H. thermophilus. Additional versions of oxalosuccinate reductase that also could be used include the following: Chlorobium limicola DSM 245 Cl-idh, (NCBI Accession Nos. AB076021.1, BAC00856.1); Kosmotoga olearia TBF 19.5.1 (NCBI Accession Nos. Kole 1227, NC_012785.1 (1,303,493DZ,304,695), YP_002940928.1); Marine gamma proteobacterium HTCC2080 (NCBI Accession Nos. MGP2080_11238, 1100755000543, whole genome shotgun sequence NZ_AAVV01000002.1 (123,681 . . . 124,934), ZP_01625318.1); and/or Nitrosococcus halophilus Nc4 (NCBI Accession Nos. NhaI_2539, NC_013960.1 (2,610,547Dz,611,815), YP_003528006.1). (See, e.g., the nucleotide sequences of SEQ ID NOs: 53, 55, 57, 59 and/or 61; or the amino acid sequences of SEQ ID NOs: 52, 54, 56, 58 and/or 60).

For isocitrate lyase, the biochemically characterized version from Corynebacterium glutamicum ((NCBI Accession Nos. NCgl2248, NC_003450.3 (2,470,741 . . . 2,472,039) NP_601531.1) can be used (Reinscheid et al. J Bacteriol 176:474-83 (1994)). Additional versions of isocitrate lyase that could be used include the following: Gordonia alkanivorans NBRC 16433 aceA (locus tag=GOALK_050_00390), contig: GOALK050, whole genome shotgun sequence (NCBI Accession Nos. NZ_BACI01000050.1 (37,665 . . . 38,960), ZP_08765259.1); Nocardia farcinica IFM 10152 aceA (locus tag=nfa52300), NC_006361.1 (5,525,226 . . . 5,526,515) YP_121446.1; Rhodococcus pyridinivorans AK37 (NCBI Accession Nos. AK37 18248, contig53, whole genome shotgun sequence NZ_AHBW01000053.1 (20,169 . . . 21,458), ZP_09310682.1); and/or Rhodococcus jostii RHA1 (NCBI Accession Nos. RHA1_ro02122, NC_008268.1 (2,230,309Dz,231,598), YP_702087.1). (See, e.g., the nucleotide sequences of SEQ ID NOs: 63, 65, 67, 69 and/or 71; or the amino acid sequences of SEQ ID NOs: 62, 64, 66, 68 and/or 70).

Example 2. In Vitro Function of the Individual crTCA Cycle Enzymes and Demonstration of the Full Cycle Function Using LC-MS Analysis

C₃ plants do not grow efficiently in hot and/or dry areas because, as the temperature increases, Rubisco incorporates more oxygen. To help overcome this limitation, two sets of bacterial enzymes with carbon fixing activities have been cloned into plant expression constructs for use in the C₃ oilseed producing plant Camelina sativa. The first carbon fixing construct known as C1 contains sequences for: (1) a 2-oxoglutarate carboxylase that converts 2-oxoglutarate to oxalosuccinate, (2) a oxalosuccinate reductase (ICDH/OSR) that converts oxalosuccinate to isocitrate, and (3) a isocitrate lyase that cleaves isocitrate into succinate and glyoxylate. The second carbon fixing construct known as C2 contains sequences for: (1) succinyl-CoA synthetase that catalyzes the conversion of succinate to succinyl-CoA and (2) a 2-oxoglutarate:ferredoxin oxidoreductase that converts succinyl-CoA to 2-oxoglutarate (a.k.a 2-ketoglutarate). A 2-oxoglutarate carboxylase utilizes ATP to catalyze the carboxylation of 2-oxoglutarate into oxalosuccinate. Transformed into the chloroplast, this reaction can provide additional carbon assimilation for the formation of substrate for nitrogen assimilation or incorporation in other biomass. Bacterial oxalosuccinate reductase catalyzes the NAD-dependent conversion of oxalosuccinate to isocitrate which can further be exported from the chloroplast into the mitochondria for further energy production or conversion into amino acids or other components. Individually expressed in chloroplasts, this enzyme can provide a bypass for feedback inhibition of carbon assimilation. Thus, these bacterial enzymes will scavenge carbon dioxide and bicarbonate from the chloroplast and generate metabolites that can feed into existing pathways present in chloroplasts, thereby enhancing the ability of plant cells to fix carbon, which can increase plant productivity.

Further carbon fixing constructs include those designed to express (1) a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide; (2) a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide; (3) a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase; (4) a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide; (5) a heterologous polynucleotide encoding an isocitrate lyase polypeptide; or (6) a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide.

The present invention demonstrates in vitro function of the complete crTCA cycle using a ¹³C-labeled biocarbonate containing reaction and LC-MS analysis.

Purified crTCA cycle enzymes were evaluated individually using appropriate spectrophotometric-based activity assays and their specific activities determined. To demonstrate the in vitro function of the crTCA cycle, we conducted triplicate LC-MS experiments, and followed the production of crTCA cycle intermediates using ¹³C-labeled bicarbonate. The LC-MS reaction samples including all of the five crTCA cycle enzymes (see Table 1 for quantities and identity of enzymes used). In addition to the crTCA cycle enzymes, the reaction mixtures contained 50 mM Tris-HCl, pH 8.0, 4 mM NADH, 5 mM ATP, 5 mM MgCl2, 20 mM NaH13CO3, 0.5 mM CoA, 100 μM succinyl-CoA, 1 mM 2-oxoglutarate, and 70 μg HyTh ferredoxin, in a 700 μL volume. The reactions were initiated by addition of succinyl CoA and incubated for 0, 15, 30, 60, 90, and 120 minutes at 30° C. and were stopped by adding 50 μL of methanol. The ¹³C-labeled crTCA cycle intermediates were analyzed by LC-MS, and quantification of the different labeled species of the crTCA cycle intermediates was performed using standards analyzed alongside the experimental samples.

TABLE 2
crTCA Cycle Enzymes Used for LC-MS Analysis
		Amount of
		Enzyme
Cycle Enzyme	Source Organism	Used
(1) Succinyl CoA	Bradyrhizobium sp.BTAi1	6 μg
Synthetase (SCS)
(2) 2-Oxoglutarate:ferredoxin	Hydrogenobacter	9.6 μg
oxidoreductase (KOR)	thermophilus TK-6
(3) 2-Oxoglutarate	Mariprofundus	66 μg
carboxylase (OGC)	ferrooxydans PV-1
(4) Oxalosuccinate	Nitrosococcus halophilus Nc4	9.6 μg
reductase (OSR, ICDH)
(5) Isocitrate lyase	Nocardia farcinica	9.2 μg
(ICL)	IFM 10152

In FIG. 6, the averaged amounts of glyoxylate from three separate reaction replicates (unlabeled, 1-¹³C-glyoxylate and 2-¹³C-glyoxylate) are shown in response to incubation time. As seen in FIG. 6, 1-C and 2-C ¹³C-glyoxylate species are reproducibly observed, which indicates that the crTCA cycle successfully functioned in replicate reactions as a cycle under the conditions tested.

Quantitation of the ¹³C-labeled succinate and 2-oxoglutarate was also completed and the results are shown in FIG. 7 and FIG. 8, respectively.

The averages of the highest observed quantities for crTCA cycle intermediates for the three reaction replicates (including all unlabeled and labeled species detected) for the reactions conducted at 30° C. under anaerobic conditions are presented in Table 3.

TABLE 3
LC-MS Quantitation of crTCA Cycle Metabolites
	Glyoxylate	non-labeled	0	290 ± 30
		one 13C labeled	0	182 ± 10
		two 13C labeled	0	21 ± 4
	Succinate and	non-labeled	100	N/A
	succinyl-CoA	one 13C labeled	0	180 ± 4
		two 13C labeled	0	43 ± 1
	2-Oxoglutarate	non-labeled	1000	N/A
		one 13C labeled	0	120 ± 10
		two 13C labeled	0	40 ± 10
		three 13C labeled	0	12 ± 3

The step 3 intermediate, oxalosuccinate, could not be detected by LC-MS because of its lability, and the step 4 intermediate, isocitrate, was not detected in quantifiable amounts, most likely because the isocitrate lyase step (enzyme step 5) is so efficient in converting isocitrate to succinate and glyoxylate that detectable pools of isocitrate do not accumulate. From the quantification of the crTCA cycle intermediates it is clear that the cycle is functional as the glyoxylate produced in the reaction (493 μM total) is approximately 49% of the starting concentration of 2-oxoglutarate (1 mM).

Example 3. Western Analysis and Activity for Transient Expression of crTCA Cycle Enzymes in Tobacco

Each of the cycle genes was used to transform tobacco for transient expression. A strategy similar to the one used for Camelina transformation was used to design these constructs. The synthesized crTCA cycle nucleotide sequences also include a chloroplast localization (ctp from tobacco) sequence and a 6×-his tag to aid in confirmation of expression and affinity-purification for activity assays. Synthesized elements were cloned into pCAMBIA2300_EGFP_BAR (containing the eGFP fluorescent marker and a Basta resistance gene) using HindIII and BamHI restriction sites. Each crTCA nucleotide sequence includes its own 35S promoter and NOS terminator. We have obtained RT-PCR, Western blot and enzymatic assay data for the transient expression for four of the five crTCA cycle enzymes (SCS, KOR, OSR, and ICL) in tobacco. However, we have not yet confirmed the expression of the M. ferrooxydans OGC (MaFe OGC) in tobacco by Western analysis.

TABLE 4
Summary of Western Analysis and Activity for Transient Expression of crTCA
Cycle Enzymes in Tobacco
			Detection
	crTCA cycle	Gene variant and	by Western	Enzyme Activity
Construct Name	enzyme	Source Organism	Analysis	(nmole/min/mg)
Tob1-SCS-BrBT-C	#1 - Succinyl CoA	BrBT	Yes	133 ± 7.2 (EV)
	Synthetase	Bradyrhizobium		170 ± 5 (BrBt-C)
		sp.BTAi1
Tob2-KOR-BaM3-C	#2 - 2-	BaM3	Yes	0.44 ± 0.26 (EV)
	Oxoglutarate:	Bacillus sp. M3-13		3.67 ± 0.6 (BaM3-C)
	ferredoxin
	Oxidoreductase
Tob3-OGC-MaFe-N	#3 - 2-Oxoglutarate	MaFe	Not	15 ± 9 (EV)
	Carboxylase	Mariprofundus	confirmed	20 ± 10 (MaFe-N)
		ferrooxydans PV-1
Tob4-ICDH-NiHa-N	#4 - Oxalosuccinate	NiHa	Yes	2.7 ± 0.45 (EV)
	Reductase/ICDH	Nitrosococcus		8.95 ± 0.44 (NiHa-N)
		halophilus Nc4
Tob5-ICL-NoFa-N	#5 - Isocitrate	NoFa	Yes	1.94 ± 0.3 (EV)
	Lyase	Nocardia farcinica		4.66 ± 0.25 (NoFa-N)
		IFM 10152

Example 4. Western Blot Analysis Demonstrating crTCA Cycle Enzyme Expression in Camelina Transformed with Either crTCA Cycle Construct #1 or #2

Camelina plants transformed with either Construct 1 (OGC/OSR (or ICDH)/ICL; FIG. 4) or Construct 2 (SCS/KOR; FIG. 5) were evaluated by western blot analysis to detect expression of crTCA cycle enzymes. Leaf samples used for the western analysis came from plants which were confirmed for gene expression by RT-PCR analysis. The membrane was probed with peptide antibodies designed to detect crTCA cycle enzymes. Probing with peptide antibodies designed to detect crTCA cycle enzymes #1 or #2, peptide antibodies designed to detect crTCA cycle enzymes #3 or #4 and with peptide antibodies designed to detect crTCA cycle enzyme #5, isocitrate lyase showed that each of enzymes was produced (see, Table 4). The WT sample was used as a control in a western blot analysis of Camelina transformed with Construct #1 and verified by RT-PCR analysis to express crTCA cycle enzyme #5 (ICL).

Example 5. crTCA Expressing Camelina Plants

Transgene-positive T1 plants for C1 (OCG+OSR (ICDH)+ICL) or C2 (SCS+KOR) have yielded T2 plants that were confirmed by genotyping and analyzed for expression. Crosses between C1 and C2 T2 plants were made in November and December 2014. F1 seeds were harvested and analyzed for mCherry and eGFP fluorescence. 71 mCherry-positive F1 seeds were harvested from 11 crosses with a C2 female parent. This suggests that the integration of both constructs was complete following those crosses. Another 373 mCherry-positive F1 seeds were harvested from C1 female parents. Observation of eGFP fluorescence in those seedlings confirmed the presence of the C2 construct in F1 tissue.

Forty-eight F1 plants showing mCherry and eGFP fluorescence were genotyped and those confirmed by genotyping to contain C1 and C2 constructs were planted in soil. At 30 days after planting (DAP), the F1 plants (FIG. 9 and FIG. 10, panel B) had an average height of 42 cm, compared to 23 cm for the wild-type (FIG. 9 and FIG. 10, panel A). The parental C1 and C2 (T3) were planted at the same time to perform phenotypic analysis and comparison of the F1 crosses and the parental lines. All the C1 and C2 T3 plants carried all the elements of their corresponding construct. At 30 DAP, the C1 and C2 T3 plants (FIG. 9, FIG. 10, panel C and panel D) had an average height of 41.6 cm and 34 cm, respectively. Additionally, on Mar. 5, 2015, 24 C1 T3 seedlings, 24 C2 T3 seedlings and 48 C1×C2 F1 seedlings were transplanted and genotyped. The differences in development and height continued as shown in FIG. 11.

Example 6

C1, C2 and C3 are contained in three different pCambia 2300 plasmids.

C1 carries the genes coding for the OGC small and large subunits, ICDH and ICL, C2 carries the genes coding for the small and large subunits of SCS and KOR, and C3 carries sequences coding for a biotin ligase and a ferredoxin which will support crTCA function. A modified version of the C2 construct holding different versions of the KOR elements was also introduced. C1 and C2 plasmids hold the sequences for a different fluorescent selectable marker while C3 carries the Bar gene sequence. crTCA Expression in Camelina was confirmed by RT-PCR and Western Blot for all crTCA enzymes (FIG. 12).

C1 T3, C2 T3 and C1×C2 F1 plants sets have been growing in the greenhouse or Phytotron and are being or have been harvested (T4 and F2 seeds). C3 (C1 background) T1, C2-mod (C1 background) T1 as well as C2-mod (w-t background) T2 seeds have been harvested. Those seeds have been plated on selection medium and have generated T1/T2 seedlings, which are now growing in the greenhouse (aged 14 to 21 DAP). Finally, a set of C3 (C1, C2 and w-t background) T1 plants is at the maturing stage. These lines were crossed and are currently grown out for evaluation. Data showing the number filling pods per total crosses in parent lines containing the F1 generation of the crossed lines is provided in Table 5 below.

TABLE 5
Number filling pods/total crosses in parent lines.
	Female	Male	# pods/total crosses
	C2MT3	C3C1T2	53/72
	C3C1T2	C2MT3	48/75
	C2MC1T2	C3T2	16/28
	C3T2	C2MC1T2	16/27

Analysis of Metabolites in the Generated Camelina Lines.

Leaf tissue was collected from plants that were about 4 week-old, ground in liquid nitrogen and lyophilized for analysis. The results show an increase in metabolites associated with respiration. Potential partial pathways function in shifting plant metabolism in the partial crTCA expressing lines (FIG. 13). Compared to WT tissue, transgenic lines showed quantitative differences in the steady-state levels of several metabolites involved in the crTCA cycle or in the Krebs cycle (FIG. 14). Compared to WT tissue, transgenic lines showed quantitative differences in the steady-state levels of the Benson Calvin cycle, but there were no statistically significant differences in sugar levels (FIG. 15).

The ratio of serine/glycine indicates shifts in photorespiration when CO2 fixation is limiting. We did not observe any significant shift in the ser/gly ratio (FIG. 16) and an accumulation of pyruvate could indicate that flux through glycosysis for pyruvate synthesis is faster than metabolic reactions (or transport) that require pyruvate or other down-stream metabolites as substrates as shown in FIG. 17. See also, FIG. 18.

The data in FIG. 19 suggest that the activity of the synthetic cycle enzymes is affected by light and availability of CO₂. Thus, it may be that field growth would provide higher light intensities.

Example 7

This example presents data for the fully integrated Synthetic CO2 fixation cycle genes. All genes are present. The plants were generated by crossing homozygeous partial cycle plants (C1, C2+C3) as parents and are therefore, heterozygeous for all transgenes. independent crosses (X13, X32, X36, x50) were generated and analyzed plants for their physiology, genomics and agronomic traits.

The growth phenotype and height of Camelina wild type (wt) plants and C1 and C2 parent plants are shown in FIG. 20. The constructs are as follows

C1:

2-Oxoglutarate carboxylase: OGC (small/large subunits)

Isocitrate Dehydrogenase=Oxalosuccinate reductase ICDH

Isocitrate Lyase ICL

mCherry

C2:

Succinyl-CoA Synthase SCS (large/small subunits)

2-Oxoglutarate:ferredoxin oxidoreductase=Ketoglutarate Oxidoreductase KOR (large/small subunits)

eGFP

C3:

Biotin ligase (BirA): Coenzyme for OGC

Ferredoxin (Fdx)

BAR

While variation in the height of the parent plants was observed, the wt plants are fairly uniform. The tallest wt plant (WT3) was chosen as a visual comparison in the images with the integrated crosses (FIG. 21A-21C).

FIGS. 21A-21C shows the growth phenotype of full cycle (C1, C2, C3) integrated lines The x13 plants were clearly taller when compared with wt, while 2 of the 3 x36 crosses were taller than wt. All of the x50 crosses were the same height or taller than wt.

A functional CO2 fixation cycle would be expected to show increased CO₂ fixation rates during photosynthesis. Photosynthetic CO₂ fixation rates were measured at either 10 am in the morning or 10 pm at night from 3 independent leaves from 3 independent plants per genotype at either ambient (400 ppm) (FIG. 22A) or elevated (1600 ppm) (FIG. 22B) CO2 atmosphere.

As shown in FIGS. 22A-22C, photosynthetic CO2 fixation rates were significantly higher in the integrated crosses (x13, x36, x50) compared to wt or parent lines when measured under ambient CO2 (400 ppm) (FIG. 22A). When the CO2 fixation rates were measured under elevated CO2 (1600 ppm) (FIG. 22B), the rates were significantly elevated in all genotypes with exception of C2. Interestingly, the transgenic lines only containing construct C1 were performing better under elevated CO2 compared to wt, and similar to the integrated crosses. Dark respiration (FIG. 22C) was significantly higher in C1 and integrated crosses compared to wt and C2. This suggests that the elevated CO2 fixation rate resulted in increased carbon storage (e.g. starch or sucrose or lipids) in the leaf during the day which enabled increased respiration and therefore growth (see FIG. 20 and FIGS. 21A-21C) during the night.

TABLE 6
The ratio of photosynthetic (light) CO2 assimilation rates
at elevated CO2 (1600 ppm) to ambient CO2 (400 ppm)
	WT	C1	C2	×13	×36	×50
ΔPSR (1600-400 ppm	4.7	6.5	1.3	4.5	2.3	5.2

To evaluate the actual expression levels of the transgenes in the parent lines and crosses, transcript (qRT-PCR) and protein levels were measured.

FIG. 23 shows transcript abundance of C1 transgenes in the C1 parent lines and the integrated crosses. Transgene specific primers were used and expression was normalized to standard endogenous genes. While we were able to detect transcript for all transgenes in all genotypes, the relative abundance levels varied dramatically. Our analysis showed that the transcript abundances were not due to the different promoters used in the constructs.

FIG. 24 shows transcript abundance of C2 transgenes in the C2 parent lines and the integrated crosses. Transgene specific primers were used and expression was normalized to standard endogenous genes. While we were able to detect transcript for all transgenes in all genotypes, the relative abundance levels varied dramatically. Our analysis showed that the transcript abundances were not due to the different promoters used in the constructs.

FIG. 25 shows transcript abundance of C3 transgenes in the integrated crosses. Transgene specific primers were used and expression was normalized to standard endogenous genes. While we were able to detect transcript for all transgenes in all genotypes, the relative abundance levels varied dramatically. Our analysis showed that the transcript abundances were not due to the different promoters used in the constructs.

Despite the large differences in the transcript abundances (FIGS. 23-25), the respective protein levels (western blots) (FIGS. 26A-26C) did not show the same variation. Instead, fairly similar quantities of transgenic proteins were observed that did not correlate with transcript abundance differences. Without being bound by theory, this may suggest that the proteins were more stable than the transcripts and post-transcriptional regulation may be responsible for the discrepancy between transcript and protein levels for each transgene.

Further, despite the increases in CO2 fixation, seed yield did not correlate with height increases or CO2 fixation rates. As shown in FIGS. 27A-27B, seed yield (g seed/plant) was not significantly different between wt, parent and integrated, crossed lines. Further, average seed weight was lower in the integrated lines compared to parent and wt plants. This indicates that the parent and wt plants made relatively fewer but heavier seeds per plant compared to the integrated crosses which may make more seeds having lower per seed weight. The statistical significance here is relatively low due to the low number of plants for each line. Experiments with larger plant numbers are currently under way and field trials planned for fully homozygous integrated lines.

The above examples clearly illustrate the advantages of the invention. Although the present invention has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.

Claims

1. A method for increasing carbon fixation and/or increasing biomass production in a plant, comprising: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide, and/or a heterologous polynucleotide encoding an isocitrate lyase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide, and the heterologous polynucleotide encoding an isocitrate lyase polypeptide are from a bacterial and/or an archaeal species.

2-5. (canceled)

6. The method of claim 1, comprising: introducing into a plant, plant part, and/or plant cell (a) a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide or (b) a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide are from a bacterial and/or an archaeal species.

7. (canceled)

8. A method for producing a plant having increased carbon fixation and/or increased biomass production, comprising: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and/or a heterologous polynucleotide encoding an isocitrate lyase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide, and the heterologous polynucleotide encoding an isocitrate lyase polypeptide are from a bacterial and/or an archaeal species.

9. the method for producing a plant of claim 8, comprising: introducing into a plant, plant part, and/or plant cell (a) a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide or (b) a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide are from a bacterial and/or an archaeal species.

10-11. (canceled)

12. The method of claim 1, wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and/or the heterologous polynucleotide encoding an isocitrate lyase polypeptide is/are operably linked to a promoter.

13. The method of claim 6, wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and/or the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide is/are operably linked to a promoter.

14. The method of claim 8, wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and/or the heterologous polynucleotide encoding an isocitrate lyase polypeptide is/are operably linked to a promoter.

15. The method of claim 9, wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and/or the heterologopolynucleotide encoding an oxalosuccinate reductase polypeptide is/are operably linked to a promoter.

16. (canceled)

17. The method of claim 1, wherein the heterologous polynucleotide encoding a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and/or the heterologous polynucleotide encoding an isocitrate lyase polypeptide are introduced into a nucleus and/or a chloroplast of said plant, plant part, and/or plant cell.

18. The method of claim 1, wherein the heterologous polynucleotide encoding a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and/or the heterologous polynucleotide encoding an isocitrate lyase polypeptide are introduced into a nucleus of said plant, plant part, and/or plant cell and are operably linked to an amino acid sequence that targets said polypeptides to the chloroplast.

19. The method of claim 1, wherein the heterologous polynucleotide encoding the succinyl CoA synthetase polypeptide is from Escherichia coli, Azotobacter vinelandii, Bradyrhizobium sp., Azospirillum sp., or any combination thereof, the heterologous polynucleotide encoding the 2-oxoglutarate:ferredoxin oxidoreductase polypeptide is from Paenibacillus sp Halobacterium sp., Hydrogenobacter thermophilus, Bacillus sp, Paenibacillus larvae subsp. larvae, Haladaptus paucihalophilus, Magnetococcus sp., or any combination thereof, the heterologous polynucleotide encoding the 2-oxoglutarate carboxylase polypeptide is from Candidatus Nitrospira defluvii, Hydrogenobacter thermophilus, Thiocystis violascens, Mariprofundus ferroxydans, Pseudomonas stutzeri, or any combination thereof, the heterologous polynucleotide encoding the oxalosuccinate reductase polypeptide is from Acinetobacter baumannii, Chlorobium limicola, Kosmotoga olearia, Marine gamma proteobacterium, or any combination thereof, and/or the heterologous polynucleotide encoding the isocitrate lyase is from Corynebacterium glutamicum, Gordonia alkanivorans, Nocardia farcinica, Rhodococcus pyridinivorans, Rhodococcus jostii, or any combination thereof.

20. The method of claim 6, wherein-the heterologous polynucleotide encoding the succinyl CoA synthetase polypeptide is from Escherichia coli, Azotobacter vinelandii, Bradyrhizobium sp., Azospirillum sp., or any combination thereof, the heterologous polynucleotide encoding the 2-oxoglutarate:ferredoxin oxidoreductase polypeptide is from Paenibacillus sp Halobacterium sp., Hydrogenobacter thermophilus, Bacillus sp, Paenibacillus larvae subsp. larvae, Haladaptus paucihalophilus, Magnetococcus sp., or any combination thereof, the heterologous polynucleotide encoding the 2-oxoglutarate carboxylase polypeptide is from Candidatus Nitrospira defluvii, Hydrogenobacter thermophilus, Thiocystis violascens, Mariprofundus ferroxydans, Pseudomonas stutzeri, or any combination thereof, and/or the heterologous polynucleotide encoding the oxalosuccinate reductase polypeptide is from Acinetobacter baumannii, Chlorobium limicola, Kosmotoga olearia, Marine gamma proteobacterium, or any combination thereof.

21. The method of claim 8, wherein the heterologous polynucleotide encoding the succinyl CoA synthetase polypeptide is from Escherichia coli, Azotobacter vinelandii, Bradyrhizobium sp., Azospirillum sp., or any combination thereof, the heterologous polynucleotide encoding the 2-oxoglutarate:ferredoxin oxidoreductase polypeptide is from Paenibacillus sp Halobacterium sp., Hydrogenobacter thermophilus, Bacillus sp, Paenibacillus larvae subsp. larvae, Haladaptus paucihalophilus, Magnetococcus sp., or any combination thereof, the heterologous polynucleotide encoding the 2-oxoglutarate carboxylase polypeptide is from Candidatus Nitrospira defluvii, Hydrogenobacter thermophilus, Thiocystis violascens, Mariprofundus ferroxydans, Pseudomonas stutzeri, or any combination thereof, the heterologous polynucleotide encoding the oxalosuccinate reductase polypeptide is from Acinetobacter baumannii, Chlorobium limicola, Kosmotoga olearia, Marine gamma proteobacterium, or any combination thereof, and/or the heterologous polynucleotide encoding the isocitrate lyase is from Corynebacterium glutamicum, Gordonia alkanivorans, Nocardia farcinica, Rhodococcus pyridinivorans, Rhodococcus jostii, or any combination thereof.

22. The method of claim 9, wherein-the heterologous polynucleotide encoding the succinyl CoA synthetase polypeptide is from Escherichia coli, Azotobacter vinelandii, Bradyrhizobium sp., Azospirillum sp., or any combination thereof, the heterologous polynucleotide encoding the 2-oxoglutarate:ferredoxin oxidoreductase polypeptide is from Paenibacillus sp Halobacterium sp., Hydrogenobacter thermophilus, Bacillus sp, Paenibacillus larvae subsp. larvae, Haladaptus paucihalophilus, Magnetococcus sp., or any combination thereof, the heterologous polynucleotide encoding the 2-oxoglutarate carboxylase polypeptide is from Candidatus Nitrospira defluvii, Hydrogenobacter thermophilus, Thiocystis violascens, Mariprofundus ferroxydans, Pseudomonas stutzeri, or any combination thereof, and/or the heterologous polynucleotide encoding the oxalosuccinate reductase polypeptide is from Acinetobacter baumannii, Chlorobium limicola, Kosmotoga olearia, Marine gamma proteobacterium, or any combination thereof.

23. (canceled)

24. A stably transformed plant, plant part or plant cell produced by the method of claim 1.

25. A stably transformed plant, plant part or plant cell comprising a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and/or the heterologous polynucleotide encoding an isocitrate lyase polypeptide, wherein said heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide, the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide and/or the heterologous polynucleotide encoding an isocitrate lyase polypeptide is/are from a bacterial and/or an archaeal species.

26-29. (canceled)

30. the stably transformed plant, plant part or plant cell of claim 25, comprising (a) a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide or (b) a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide, wherein then heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide are from a bacterial and/or an archaeal species.

31-37. (canceled)

38. A seed of the stably transformed plant of claim 25, wherein the seed comprises in its genome at least one heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, encoding a 2-oxoglutarate carboxylase polypeptide, encoding an oxalosuccinate reductase polypeptide and/or encoding an isocitrate lyase polypeptide, wherein said heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, encoding a 2-oxoglutarate carboxylase polypeptide, encoding an oxalosuccinate reductase polypeptide and/or encoding an isocitrate lyase polypeptide is/are from a bacterial and/or an archaeal species.

39-42. (canceled)

43. A seed of the stably transformed plant of claim 30, wherein the seed comprises in its genome (a) a heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide and a heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, or (b) a heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and a heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide wherein the heterologous polynucleotide encoding a succinyl CoA synthetase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate:ferredoxin oxidoreductase polypeptide, the heterologous polynucleotide encoding a 2-oxoglutarate carboxylase polypeptide and the heterologous polynucleotide encoding an oxalosuccinate reductase polypeptide are from a bacterial and/or an archaeal species.

44-45. (canceled)

46. A product produced from the stably transformed plant, plant part or plant cell of claim 8.

47. The product of claim 46, wherein the product is a food, drink, animal feed, fiber, oil, pharmaceutical and/or biofuel.