CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional

Application No. 61/603,397 filed February 27, 2012, the contents of which are incorporated herein by reference in their entirety.

FEDERAL FUNDING

[002] This invention was made with government support under EEC-0540879 awarded by National Science Foundation and under DE-0000079 awarded by the U.S. Department of Energy. The U.S. government has certain rights in the invention.

SEQUENCE LISTING

[003] The instant application contains a Sequence Listing which has been submitted in

ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on February 21, 2013, is named 002806-072491-PCT_SL.txt and is 92,283 bytes in size.

FIELD

[004] The present invention relates to molecular genetics and biology, biochemistry, and photosynthetic metabolism. More specifically, the present invention provides for the introduction of heterologous genetic constructs into plants and microbes for more efficient use of fixed carbons, primarily through more efficient recycling of the products of photorespiration, for example, by recycling glycolate, glyoxylate, and related molecules. The invention also provides for genetically engineered organisms with superior growth properties.

BACKGROUND

[005] Photosynthesis plays a fundamental role in the capture of solar energy and enabling the growth of plants and photosynthetic bacteria. Agriculture and the capture of carbon dioxide to balance human-derived carbon dioxide emissions are two examples of the many reasons why photosynthesis is important. Photosynthesis in its most abundant form is limited, however, by several inefficient biochemical processes. Specifically, the type of photosynthesis used by green plants and photosynthetic bacteria involves light capture by Photosystems I and II, and carbon capture by the Calvin cycle. The central enzyme of the Calvin cycle, ribulose bisphosphate carboxylase/oxygenase, is a notoriously slow enzyme that carries out a wasteful reaction with oxygen. To wit, it is thought that 30% or more of the light energy captured by plants is wasted on inefficient cycling of photorespiration products. Because plant growth provides much of the food for the world, there is a need in the art for molecules and genetic engineering methods that reduce the inefficiencies of photorespiration, as well as the production of plants that deal with photorespiration more efficiently.

[006] There is also a need in the art for novel biological methods for production of useful chemicals and metabolic intermediates. For example, erythromycin is an antibiotic with a complex and somewhat inefficient metabolic synthesis pathway. More broadly, there is a need in the art for methods of production of intracellular propionyl-CoA, which is an important intermediate in synthesis of erythromycin and other metabolites.

SUMMARY

[007] The present invention provides for compositions and methods for genetically engineering more efficient carbon utilization in plants and bacteria.

[008] In one general embodiment, the invention provides recombinant organisms, such as photosynthetic organisms expressing the Calvin cycle, in which a portion of the 3- hydroxypropionate pathway is expressed by genetic engineering. A "recombinant" organism is an organism that has been genetically engineered. The organisms provided by the invention are organisms that do not naturally express the 3-hydroxypropionate pathway, but have been engineered to express some part of this pathway.

[009] In a preferred embodiment, the invention provides a recombinant organism that expresses both the malonyl-CoA reductase (MCR) and propionyl-CoA synthase (PCS) enzymes.

[0010] In another preferred embodiment, the invention provides a recombinant organism engineered to express functional, heterologous (S)-malyl-CoA/p methylmalyl-CoA/(S)- citramalyl-CoA (MMC lyase) and mesaconyl-Cl-CoA hydratase (β methmalyl-CoA- dehydratase).

[0011] In a more preferred embodiment, the invention provides a recombinant organism engineered to express functional, heterologous (S)-malyl-CoA/p methylmalyl-CoA/(S)- citramalyl-CoA (MMC lyase), mesaconyl-Cl-CoA hydratase (β methmalyl-CoA-dehydratase), and mesaconyl-CoA C1-C4 CoA transferase.

[0012] In a more preferred embodiment, the invention provides a recombinant organism engineered to express functional, heterologous (S)-malyl-CoA/p methylmalyl-CoA/(S)- citramalyl-CoA (MMC lyase), mesaconyl-Cl-CoA hydratase (β methmalyl-CoA-dehydratase), and mesaconyl-Cl CoA hydratase. [0013] In a more preferred embodiment, the invention provides a recombinant organism engineered to express functional, heterologous (S)-malyl-CoA/p methylmalyl-CoA/(S)- citramalyl-CoA (MMC lyase), mesaconyl-Cl-CoA hydratase (β methmalyl-CoA-dehydratase), mesaconyl-CoA C1-C4 CoA transferase, and and mesaconyl-Cl CoA hydratase.

[0014] In another preferred embodiment, the invention provides a recombinant organism expressing MMC lyase and a functional subunit of a nicotinic cofactor-dependent glycolate dehydrogenase. A nicotinic cofactor can be either NAD or NADP. The nicotinic cofactor- dependent glycolate dehydrogenase may be produced from an endogenous gene or an engineered gene.

[0015] In another preferred embodiment, the invention provides a recombinant organism expressing MMC lyase and a functional subunit of a nicotinic cofactor-dependent glycolate dehydrogenase, and also expresses mesaconyl-CoA CI C4 transferase.

[0016] In another preferred embodiment, the invention provides a recombinant organism expressing MMC lyase and a functional subunit of a nicotinic cofactor-dependent glycolate dehydrogenase, and also expresses mesaconyl-CoA C1-C4 CoA transferase.

[0017] In another preferred embodiment, the invention provides the invention provides a recombinant organism expressing a functional subunit of a nicotinic cofactor-dependent glycolate dehydrogenase, and also is engineered to express at least two of the following enzymes: malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA- dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase.

[0018] In another preferred embodiment, the invention provides the invention provides a recombinant organism that is engineered to express at least two of the following enzymes:

malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase; and is also engineered to over-express at least one of the following enzymes: pyruvate kinase, enolase, phosphoglycerate mutase and/or 3-phosphoglycerate kinase.

[0019] In another preferred embodiment, the invention provides a recombinant organism that is engineered to express malonyl-CoA reductase and MMC lyase. In a more preferred embodiment, the invention provides a recombinant organism that is engineered to express malonyl-CoA reductase, MMC lyase, and at least one of the following enzymes: methmalyl-

CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase.

[0020] In another preferred embodiment, the invention provides a recombinant organism that is engineered to express malonyl-CoA reductase, propionyl-CoA synthase, and MMC lyase.

In a more preferred embodiment, the invention provides a recombinant organism that is engineered to express malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, and at least one of the following enzymes: methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase.

[0021] In a distinct class of preferred embodiments, the invention provides a

photosynthetic bacterium that does not naturally express one of the following enzymes:

malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase; but is engineered to do so. In a more preferred embodiment, the invention provides a cyanobacterium that does not naturally express one of the following enzymes: malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase; but is engineered to do so.

[0022] In another distinct class of preferred embodiments, the invention provides a photosynthetic eukaryote that does not naturally express one of the following enzymes:

malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase; but is engineered to do so. In a more preferred embodiment, the invention provides a multicellular plant that does not naturally express one of the following enzymes: malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase; but is engineered to do so. In an even more preferred embodiment, the invention provides a multicellular plant that does not naturally express one of the following enzymes: malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase; but includes a chloroplast genome that is engineered to express one or more of these enzymes. In an alternative even more preferred embodiment, the invention provides a multicellular plant that does not naturally express one of the following enzymes: malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase; but includes a nuclear genome that is engineered to express one or more of these enzymes.

[0023] In an additional more preferred embodiment, the invention provides a plant that is engineered to express a subunit of a glycolate dehydrogenase, and also at least one of the following enzymes: malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-

CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase.

[0024] In another distinct class of preferred embodiments, the invention provides a non- photosynthetic organism that does not naturally express one of the following enzymes: malonyl-

CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA-dehydratase, mesaconyl-

CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase; but is engineered to do so. In a more preferred embodiment, the invention provides a gamma-proteobacterium that does not naturally express one of the following enzymes: malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase; but is engineered to do so. In an even more preferred embodiment, the gamma-proteobacterium is E. coli.

[0025] In a more preferred embodiment, the invention provides an Agrobacterium that does not naturally express one of the following enzymes: malonyl-CoA reductase, propionyl- CoA synthase, MMC lyase, methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase; but is engineered to do so.

[0026] The invention also provides methods of improving the growth or yield of an organism, in which the organism is genetically engineered to express one of the following enzymes: malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methmalyl-CoA- dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-4-CoA hydratase.

DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a scheme showing the 3-hydroxypropionate (3HOP) pathway.

Numbers in circles and ovals indicate the step number for each enzymatic reaction of the pathway. The enzymes for each of these steps are: (1) acetyl-CoA carboxylase; (2) malonyl-CoA reductase (MCR); (3) propionyl-CoA synthase (PCS); (4) propionyl-CoA carboxylase; (5) methylmalonyl-CoA epimerase; (6) methylmalonyl-CoA mutase; (7) succinyl-CoA(S)-malate-

CoA transferase; (8) succinate dehydrogenase; (9) fumarate hydratase; (10a, 10b, 10c) (S)- malyl-CoA/p-methylmalyl-CoA/(S)-citramalyl-CoA (MMC lyase); (11) mesaconyl-Cl-CoA hydratase (β-methmalyl-CoA-dehydratase); (12) mesaconyl-CoA C1-C4 CoA transferase; (13) mesaconyl-C4-CoA hydratase. Abbreviations: CoA, Co-Enzyme A; Ac-CoA, acetyl-CoA;

3HOP, 3-hydroxypropionate; Prop-CoA, propionyl-CoA; MCI -CoA, mesaconyl-Cl-CoA;

MC4-CoA, mesaconyl-C4-CoA; CM-CoA, citramalyl-CoA. Dotted lines represent multiple steps found in intermediary metabolism. Pyruvate is the ultimate product of the pathway.

[0028] Figure 2 is a schematic of partial pathways of the 3HOP pathway. Abbreviations are the same as for Figure 1. Figure 2A: the "Middle Pathway", in which acetyl-CoA is converted to propionyl-CoA. Figure 2B: the "Right-Side Pathway", in which propionyl-CoA and glyoxylate are converted into pyruvate and acetyl-CoA. Figure 2C: The combined "Middle plus

Right-Side Pathways" in which glyoxylate is incorporated into a cycle that produces pyruvate.

[0029] Figure 3 diagrams the use of the Middle Pathway in combination with DEBS genes to produce 6-deoxyerythronolide B, the macrolide core of erythromycin. DEBSl, DEBS2, and DEBS3 are 6-deoxyerythronolide B synthases. [0030] Figure 4 shows a bacterial pathway for recycling glycolate produced during photorespiration.

[0031] Figure 5 shows a eukaryotic pathway for recycling glycolate produced

during photorespiration.

[0032] Figure 6 depicts a cell of an engineered eukaryotic photosynthetic organism, with an engineered pathway for recycling glycolate produced during photorespiration.

[0033] Figure 7 is data from the quantification of flux through middle pathway (MP), which makes propionate. All cell points were measured from E. coli strain J33G

(BL21(DE3)A(sbm-ygfDGHI) harboring pPro33gfp, a plasmid containing a propionate reporter system). Open circles, E. coli J33G harboring empty vector p62xt, exposed to various concentrations of propionate in the medium (1 mM to 20 mM). A small amount (1 mM) of propionate is required to repress background expression. Filled squares, E. coli J33G harboring p62-MP, which expresses mcr and pes; induced and uninduced by 1 mM IPTG as marked.

Uninduced and induced cells correspond to media concentrations of 7.6 mM and 12.2 mM propionate in the medium, respectively. The actual intracellular levels of propionyl-CoA, the direct inducer, were not measured.

[0034] Figure 8 shows the amino acid sequence of Chloroflexus malonyl-CoA reductase.

[0035] Figure 9 shows the nucleotide sequence of a DNA molecule encoding

Chloroflexus malonyl-CoA reductase (mcr), codon optimized for E. coli expression.

[0036] Figure 10 is the amino acid sequence of Chloroflexus propionyl-CoA synthase.

[0037] Figure 11 is the nucleotide sequence of a DNA molecule that encodes

Chloroflexus propionyl-CoA synthase (pes), codon optimized for E. coli expression.

[0038] Figure 12 is the amino acid sequence of Chloroflexus (S)-malyl-CoA/p- methylmalyl-CoA/(S)-citramalyl-CoA (MMC) lyase.

[0039] Figure 13 is the nucleotide sequence of a DNA encoding Chloroflexus (S)-malyl-

CoA/p-methylmalyl-CoA/(S)-citramalyl-CoA (MMC) lyase (mcl), codon optimized for E. coli expression.

[0040] Figure 14 shows the amino acid sequence of Chloroflexus mesaconyl-Cl-CoA hydratase (β-methylmalyl-CoA dehydratase). This construct includes the myc epitope tag (underlined).

[0041] Figure 15 is the nucleotide sequence of a DNA encoding Chloroflexus mesaconyl-Cl-CoA hydratase (β-methylmalyl-CoA dehydratase) (mch), codon optimized for E. coli and including a myc epitope tag (underlined).

[0042] Figure 16 shows the amino acid sequence for Chloroflexus mesaconyl-CoA Cl-

C4 CoA transferase. [0043] Figure 17 is the nucleotide sequence of a DNA that encodes Chloroflexus mesaconyl-CoA C1-C4 CoA transferase (met), codon optimized for expression in E. coli.

[0044] Figure 18 is the amino acid sequence for Chloroflexus mesaconyl-C4-CoA hydratase (mesaconyl-C4-CoA (enoyl-CoA) hydratase).

[0045] Figure 19 is the nucleotide sequence of a DNA encoding Chloroflexus mesaconyl-C4-CoA hydratase (mesaconyl-C4-CoA (enoyl-CoA) hydratase) (meh), codon optimized for E. coli expression.

[0046] Figure 20 shows the amino acid sequence for E. coli glycolate dehydrogenase subunit D. This subunit contains an additional N-terminal plastid localization tag (underlined).

[0047] Figure 21 is the nucleotide sequence of a DNA molecule encoding E. coli glycolate dehydrogenase subunit D (glcD). This molecule contains an added 5-prime plastid localization signal (underlined).

[0048] Figure 22 is the amino acid sequence of the E. coli glycolate dehydrogenase subunit E. This subunit contains an additional N-terminal plastid localization tag (underlined).

[0049] Figure 23 shows the nucleotide sequence of a DNA molecule that encodes E. coli glycolate dehydrogenase subunit E (glcE). This sequence contains an added 5-prime plastid localization signal (underlined).

[0050] Figure 24 is the amino acid sequence of E. coli glycolate dehydrogenase subunit F. This subunit contains an additional N-terminal plastid localization tag (underlined).

[0051] Figure 25 is the nucleotide sequence of a DNA molecule that encodes E. coli glycolate dehydrogenase subunit F (glcF). This sequence contains an added 5-prime plastid localization signal (underlined).

[0052] Figure 26 is a diagram of pPlasmid p62-MP, a broad-host range plasmid expressing the mcr and pes genes of the "middle pathway" of 3HOP.

[0053] Figure 27 is a diagram of plasmid pi 9-RS, an E. coli plasmid expressing genes

MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase and mesaconyl-C4-CoA hydratase, the "right-side pathway" of 3HOP.

[0054] Figure 28 is a scheme showing a configuration of an E. coli strain with activity for "middle" and "right pathways" using medium (p62-MP) and high (pi 9-RS) plasmids and deletions of prpC (2-methylcitrate synthase), gel (glycolate carboligase), aceBAK (glyoxylate shunt).

[0055] Figure 29 is a diagram depicting a configuration of an E. coli strain for activity for "middle" and "right pathways" as integrated into phage attachment sites and appropriate deletions of prpC (2-methylcitrate synthase), gel (glycolate carboligase), aceBAK (glyoxylate shunt). The strain also contains the lambda-DE3 prophage, which encodes a T7 polymerase for transcription from the T7 promoters.

[0056] Figure 30 is a scheme showing the insertion of the "middle pathway" and "right- side pathways" into a cyanobacterial genome. It is contemplated herein that the "middle pathway" and/or "right pathway" can be similarly integrated into plant or algal chromosomes.

[0057] Figure 31 shows growth of a cyanobacterium engineered to express portions of the 3-hydroxypropionate pathway. The X axis represents time in hours. The Y axis represents the optical density of each culture normalized to the optical density at 0 hours. Triangles represent growth of the parental Synechococcus elongatus 7942 strain. Squares represent S. elongatus 7942 engineered to express the right-side pathway (enzymes 10, 11, 12, and 13). Circles represent S. elongatus 7942 engineered to express the right-side pathway (enzymes 10, 11, 12, and 13) and the middle pathway (enzymes 2 and 3). Solid lines represent growth in BG 11 medium (a minimal cyanobacterial medium). Dotted lines represent growth in the presence of 0.4% glycolate.

DETAILED DESCRIPTION

[0058] The present invention focuses primarily on the use of elements of the 3- hydroxypropionate (3HOP) pathway for metabolic engineering in heterologous hosts. More specifically, the 3HOP pathway is a carbon fixation pathway found in the bacterium

Chloroflexus aurantiacus, related bacteria, and possibly certain archaeabacteria. This pathway contains a number of enzymatic activities that, according to the invention, can be introduced into host plants and bacteria to produce useful metabolic intermediates or to recycle metabolic byproducts in a particularly efficient manner.

[0059] The elucidation of the 3HOP pathway has been completed recently, and is schematically shown in Figure 1. Zarzycki et al., 106 PNAS 21317 (2009). The pathway involves fifteen enzymatic reactions of which nine are endogenous only in organisms that use either the 3HOP pathway of carbon fixation or the related 3-hydroxypropionate/4- hydroxybutyrate (3HOP/4HOB) pathway for carbon fixation. The enzymes identified for this pathway are: (1) acetyl-CoA carboxylase; (2) malonyl-CoA reductase (MCR); (3) propionyl- CoA synthase (PCS) ; (4) propionyl-CoA carboxylase; (5) methylmalonyl-CoA epimerase; (6) methylmalonyl-CoA mutase; (7) succinyl-CoA(S)-malate-CoA transferase; (8) succinate dehydrogenase; (9) fumarate hydratase; (10a, 10b, 10c) (S)-malyl-CoA/p-methylmalyl-CoA/(S)- citramalyl-CoA (MMC lyase); (11) mesaconyl-Cl-CoA hydratase (β-methmalyl-CoA- dehydratase; (12) mesaconyl-CoA C1-C4 CoA transferase; and (13) mesaconyl-C4-CoA hydratase. [0060] As shown in Figure 1, five of the six enzymes work in the "middle-pathway" and

"right-side pathway." One enzyme, MMC lyase, catalyzes three reactions in the 3HOP carbon fixation pathway. The enzymes of the "middle pathway" are (1) acetyl-CoA carboxylase;

(2) MCR; and (3) PCS. The enzymes on the "right-side pathway" are (10b, 10c) MMC lyase; (11) mesaconyl-Cl-CoA hydratase (β-methmalyl-CoA-dehydratase; (12) mesaconyl-CoA Cl- C4 CoA transferase; (13) mesaconyl-C4-CoA hydratase.

[0061] As depicted in Figure 1, the 3HOP pathway starts with acetyl-CoA, which is carboxylated to form malonyl-CoA in a reaction that also initiates fatty acid synthesis and that is found in essentially all organisms. The second and third steps are carried out by the enzymes MCR and PCS, which are unique to the 3HOP and 3HOP/4HOB pathways. These three reactions define the "middle pathway" in Figure 1, and are also shown in Figure 2A. The "leftside pathway" of Figure 1 reflects a combination of enzymes that are found in propionate metabolism and in the tricarboxylic acid cycle; i.e., known enzymes found in diverse organisms. Two activities are specific to the 3HOP pathway: One activity is a CoA transferase; a second is MMC lyase, an enzyme having multiple related activities that is used in the "right-side pathway" as well.

[0062] As used herein, "MCR" or "malonyl-CoA reductase" refers to an enzyme which can reduce malonyl-CoA to 3HOP. In some embodiments, MCR can be a polypeptide comprising the sequence of SEQ ID NO: 1. In some embodiments, MCR can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the Chloroflexus MCR, e.g. at least 85% homology to SEQ ID NO: 01 and functional fragments and variants thereof. In some embodiments, MCR can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the polypeptide encoded by SEQ ID NO: 02 and functional fragments and variants thereof. In some embodiments, MCR can be a homolog of Chloroflexus aurantiacus MCR, e.g. MCR from other organisms which comprise a 3HOP pathway, e.g. other Chloroflexus spp.; other green non-sulfur bacteria; Sulfolobaceae spp.; and or members of Creanarchaeota or functional fragments and variants thereof.

[0063] As used herein, "PCS" or "propionyl-CoA synthase" refers to an enzyme which can convert 3HOP to propionyl-CoA. In some embodiments, PCS can be a polypeptide comprising the sequence of SEQ ID NO: 03. In some embodiments, PCS can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the Chloroflexus PCS, e.g. at least 85% homology to SEQ ID NO: 03 and functional fragments and variants thereof. In some embodiments, PCS can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the polypeptide encoded by SEQ ID NO: 04 and functional fragments and variants thereof. In some embodiments, PCS can be a homolog of Chloroflexus aurantiacus PCS, e.g. PCS from other organisms which comprise a 3HOP pathway, e.g. other Chloroflexus spp.; other green non-sulfur bacteria; Sulfolobaceae spp.; and or members of Creanarchaeota or functional fragments and variants thereof.

[0064] As used herein, "MMC lyase," "(S)-malyl-CoA/p-methylmalyl-CoA/(S)- citramalyl-CoA lyase," or "malyl/methylmalyl/citramalyl-coA lyase" refers to an enzyme which can convert propionyl-CoA to methylmalyl-CoA by the addition of a glyoxylate molecule, can convert malyl-CoA to acetyl-CoA plus glyoxylate, and can convert citramalyl-CoA to acetyl-

CoA and pyruvate. MMC lyase is not presently known to have a function outside of the context of the metabolic pathways described herein. Other bacterial genomes comprise MMC lyase homologs (see, e.g. Zarzycki and Fuchs. AEM 2011 77:6181-8; which is incorporated by reference here in its entirety). In some embodiments, MMC lyase can be a polypeptide comprising the sequence of SEQ ID NO: 05. In some embodiments, MMC lyase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least

98% homology) to the Chloroflexus MMC lyase, e.g. at least 85% homology to SEQ ID NO: 05 and functional fragments and variants thereof. In some embodiments, MMC lyase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least

98% homology) to the polypeptide encoded by SEQ ID NO: 06 and functional fragments and variants thereof. In some embodiments, MMC lyase can be a homolog of Chloroflexus aurantiacus MMC lyase, e.g. MMC lyase from other organisms which comprise a 3HOP pathway, e.g. other Chloroflexus spp.; other green non- sulfur bacteria; Sulfolobaceae spp.; and or members of Creanarchaeota or functional fragments and variants thereof.

[0065] As used herein, "mesaconyl-Cl-CoA hydratase", "mch," methylmalyl-CoA dehydratase," or β-methmalyl-CoA-dehydratase" refers to an enzyme which can convert methylmalyl-CoA to mesaconyl-Cl-CoA. In some embodiments, mesaconyl-Cl-CoA hydratase can be a polypeptide comprising the sequence of SEQ ID NO: 07, either with or without the myc epitope tag as described in Figure 14. In some embodiments, mesaconyl-Cl-CoA hydratase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the Chloroflexus mesaconyl-Cl-CoA hydratase, e.g. at least 85% homology to SEQ ID NO: 07 and functional fragments and variants thereof. In some embodiments, mesaconyl-Cl-CoA hydratase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the polypeptide encoded by SEQ ID NO: 08 and functional fragments and variants thereof, either with or without the myc epitope tag as described in Figure 15. In some embodiments, mesaconyl-Cl-CoA hydratase can be a homolog of Chloroflexus aurantiacus mesaconyl-Cl- CoA hydratase, e.g. mesaconyl-Cl-CoA hydratase from other organisms which comprise a 3HOP pathway, e.g. other Chloroflexus spp.; other green non-sulfur bacteria; Sulfolobaceae spp.; and or members of Creanarchaeota or functional fragments and variants thereof.

[0066] As used herein, "mesaconyl-CoA C1-C4 transferase" or "MCT" refers to an enzyme which can convert mesaconyl-Cl-CoA to mesaconyl-C4-CoA. In some embodiments, mesaconyl-CoA C1-C4 transferase can be a polypeptide comprising the sequence of SEQ ID NO: 09. In some embodiments, mesaconyl-CoA C1-C4 transferase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the Chloroflexus mesaconyl-CoA C1-C4 transferase, e.g. at least 85% homology to SEQ ID NO: 09 and functional fragments and variants thereof. In some embodiments, mesaconyl-CoA Cl- C4 transferase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the polypeptide encoded by SEQ ID NO: 10 and functional fragments and variants thereof. In some embodiments, mesaconyl-CoA C1-C4 transferasecan be a homolog of Chloroflexus aurantiacus mesaconyl-CoA C1-C4 transferase, e.g. mesaconyl-CoA C1-C4 transferase from other organisms which comprise a 3HOP pathway, e.g. other Chloroflexus spp.; other green non- sulfur bacteria; Sulfolobaceae spp.; and or members of Creanarchaeota or functional fragments and variants thereof.

[0067] As used herein, "mesaconyl-C4-CoA hydratase", "mesaconyl-C4-CoA (enoyl-

CoA) hydratase" or "MEH" refers to an enzyme which can convert mesaconyl-C4-CoA to citramalyl-CoA. In some embodiments, mesaconyl-C4-CoA hydratase can be a polypeptide comprising the sequence of SEQ ID NO: 11. In some embodiments, mesaconyl-C4- CoA hydratase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the Chloroflexus mesaconyl-C4-CoA hydratase, e.g. at least 85% homology to SEQ ID NO: 11 and functional fragments and variants thereof. In some embodiments, mesaconyl-C4-CoA hydratase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the polypeptide encoded by SEQ ID NO: 12 and functional fragments and variants thereof. In some embodiments, mesaconyl-C4-CoA hydratase can be a homolog of Chloroflexus aurantiacus mesaconyl-C4-CoA hydratase, e.g. mesaconyl-C4-CoA hydratase from other organisms which comprise a 3HOP pathway, e.g. other Chloroflexus spp.; other green non-sulfur bacteria;

Sulfolobaceae spp.; and or members of Creanarchaeota or functional fragments and variants thereof.

[0068] The "right-side pathway" of Figure 1 consists of five enzymatic steps, also shown in Figure 2B. This pathway begins with the addition of a glyoxylate molecule to propionyl-CoA by the MMC lyase to form methylmalyl-CoA (in step 10b). This five-carbon molecule is dehydrated to mesaconyl-Cl-CoA (step 11), rearranged to mesaconyl-C4-CoA (step 12), further rearranged to citramalyl-CoA (step 13), and then split into acetyl-CoA and pyruvate by MMC lyase (step 10c).

[0069] Propionyl-CoA production. An embodiment of the present invention provides for production of propionyl-CoA: an important intermediate in the production of polyketides, which are a commercially significant class of molecules that includes erythromycin and tetracycline. Propionate can be added exogenously or synthesized from other known pathways. According to the invention, however, expression of MCR and PCS in a heterologous host such as E. coli or yeast is sufficient to catalyze production of propionyl-CoA (Figure 2A, Figure 3). Example 1 describes expression of these enzymes in E. coli in further detail, along with accompanying synthesis of propionyl-CoA in E. coli.

[0070] The genes encoding MCR and PCS are large - about 3.5 kilobases and 5.5 kilobases, respectively. In part because of this large size, it is often convenient to express these genes in bacteria from a single promoter such that they form an operon. Similarly, it is often convenient to express these genes together from a single promoter in bacterially derived organelles such as a chloroplast, peroxisome or mitochondrion.

[0071] Expression of MCR and PCS to produce propionyl-CoA may be engineered in an organism that has also been engineered to produce polyketides, for example, according to known methods in which the DEBS genes are expressed heterologously in E. coli to produce the macrolide core of erythromycin (Figure 3). See Pfeifer et al., 291 Science 1790 (2001).

[0072] Glyoxylate utilization. An embodiment of the present invention combines the actions of steps 2, 3, 10b, 11, 12, 13, and 10c in a heterologous host, wherein they function to capture glyoxylate (Figure 2C). This is of particular importance in organisms that use the Calvin cycle in their growth. Such organisms include green plants, eukaryotic algae and cyanobacteria, but also include non-photosynthetic organisms that use electrons from other sources besides the water- splitting reaction, such Ralstonia eutropha that uses hydrogen, Nitrosomonas species that use reduced nitrogen, and Acidothiobacillus ferrooxidans, Siderooxidans species and Geobacter species that can take up electrons from incompletely oxidized inorganic deposits by electrical conduction.

[0073] Glyoxylate is an intermediate in a metabolic process termed photorespiration

(although, as noted, photorespiration can occur in non-photosynthetic systems). The carbon- fixing enzyme Rubisco normally reacts with carbon-dioxide and ribulose 1,5 -diphosphate to produce two molecules of 3-phosphoglycerate. Oxygen can replace carbon dioxide in this reaction, however, such that one 3-phosphoglycerate and one 2-phosphoglycolate are produced. The carbon in 2-phosphoglycolate must be converted to a form that can be used in metabolism or else this precious fixed carbon will be lost. There are two well-studied pathways for recycling of phosphoglycolate. Both begin with dephosphorylation to form glycolate. The first pathway (Figure 4) naturally occurs in bacteria and involves the oxidation of glycolate to glyoxylate by glycolate dehydrogenase; condensation of two gloxylate molecules to a 3-carbon molecule, tartronate semialdehyde, with a loss of carbon dioxide; and conversion of tartronate

semialdehyde into 3-phosphoglycerate in two steps.

[0074] The second pathway (Figure 5) naturally occurs in plants and involves the oxidation of glycolate to glyoxylate; conversion of glyoxylate to glycine by addition of ammonia; condensation of two glycines to form serine with loss of carbon dioxide and ammonia; and the conversion of the 3-carbon serine into three phosphoglycerate in several steps. It is an insight of the invention that both of these pathways involve the condensation of two 2- carbon molecules into a 3-carbon molecule with loss of carbon dioxide that must be re-fixed. It is a further insight of the invention that the 2-carbon glyoxylate can be converted to a 3-carbon molecule by a carbon-fixation pathway, and that the combination of the middle pathway and right-side pathway constitutes such a pathway. The use of direct carbon fixation instead of carbon dioxide loss and subsequent fixation results in energetic and kinetic advantages that increase the growth and yield of organisms that metabolize glyoxylate, such as organisms in which the Calvin cycle is operational.

[0075] The glyoxylate-recycling pathway of the invention would, for example, be employed as follows: In general, MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase (β- methmalyl-CoA-dehydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-C4-CoA hydratase of the 3HOP pathway are expressed in the recombinant organism of interest (Figure 6). It may be useful to express additional enzymes in the organism of interest, depending on what enzymes are already expressed in the organism, as well as the levels of expression of such enzymes and their compartmentalization.

[0076] In an organism in which phosphoglycolate is produced, for example by the

Rubisco oxygenase reaction, the phosphoglycolate is first dephosphorylated, typically by an enzyme endogenous to the organism. The glycolate product is then converted to glyoxylate by the action of a glycolate dehydrogenase or glycolate oxidase. The use of glycolate

dehydrogenase is generally preferred, because this enzyme catalyzes the concomitant conversion of NAD+ to NADH, which conserves reducing equivalents. In contrast, glycolate oxidase converts oxygen (02) to hydrogen peroxide (H202), from which reducing equivalents are generally not recovered. Expression of glycolate dehydrogenase may be engineered into the organism by standard genetic engineering techniques if this enzyme is not already present. For example, the heterologous expression of a bacterial glycolate oxidase in a green plant has been reported. Kebeish et al., 25 Nature Biotech. 593 (2007).

[0077] The resulting glyoxylate is then acted upon by MMC lyase of the 3HOP pathway, whose expression is engineered into the organism according to the present invention.

Specifically, MMC lyase catalyzes the condensation of propionyl-CoA and glyoxylate to form methylmalyl-CoA. This methylmalyl-CoA is then converted to mesaconyl-Cl-CoA by mesaconyl-Cl-CoA hydratase, which mesaconyl-Cl-CoA is then converted to mesaconyl-C4- CoA by mesaconyl-CoA C1-C4 CoA transferase, which mesaconyl-C4-CoA is then converted to citramalyl-CoAby mesaconyl-C4-CoA hydratase, which citramalyl-CoA is then converted to pyruvate and acetyl-CoA by MMC lyase.

[0078] The acetyl-CoA is then carboxylated by acetyl-CoA carboxylase to malonyl-

CoA, which is then converted by MCR to 3-hydroxypropionate, which is then converted by PCS to propionyl-CoA. In this way, the propionyl-CoA is recreated to complete the cycle.

[0079] Pyruvate is the net product of these reactions. Pyruvate is a fundamental molecule of intermediary metabolism and can often be used by other enzymes naturally present in the engineered cell. In some cellular environments, such as the chloroplast in a green plant, there may not be adequate levels of pyruvate-metabolizing enzymes present in the chloroplast, so it is sometimes useful to express pyruvate decarboxylase or pyruvate dehydrogenase to enhance production of acetyl-CoA. In addition, it is sometimes useful to express pyruvate kinase, enolase, and phosphoglycerate mutase to enhance production of 3-phosphoglycerate for re-entry into the Calvin cycle.

[0080] As used herein, "enolase" refers to an enzyme which can convert 2- phosphoglycerate to phosphoenolpyruvate. In some embodiments, enolase can be a polypeptide comprising the sequence of SEQ ID NO: 19. In some embodiments, enolase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least

98% homology) to E. coli enolase, e.g. at least 85% homology to SEQ ID NO: 19 and functional fragments and variants thereof. In some embodiments, enolase can be a homolog of E. coli enolase, e.g. enolase from other organisms or functional fragments and variants thereof.

[0081] As used herein, "phosphoglycerate mutase" refers to an enzyme which can catalyze the transfer of a phosphate group from C-3 to C-2, converting 3-phosphoglycerate

(3PG) to 2-phosphoglycerate (2PG). In some embodiments, phosphoglycerate mutase can be a polypeptide comprising the sequence of SEQ ID NO: 20. In some embodiments,

phosphoglycerate mutase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to E. coli phosphoglycerate mutase, e.g. at least 85% homology to SEQ ID NO: 20 and functional fragments and variants thereof. In some embodiments, phosphoglycerate mutase can be a homolog of E. coli phosphoglycerate mutase, e.g. phosphoglycerate mutase from other organisms or functional fragments and variants thereof.

[0082] As used herein, "pyruvate kinase" refers to an enzyme which can catalyze the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding pyruvate and

ATP. Sequences encoding this enzyme can be obtained from the organism that is being engineered, since this enzyme is expressed by essentially all organisms. Alternatively, sequences encoding the enzyme can be obtained from a bacterium such as E. coli or a cyanobacterium such as Synechococcus elongatus 7942. In some embodiments, pyruvate kinase can be a polypeptide comprising the sequence of SEQ ID NO: 21. In some embodiments, pyruvate kinase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least

90%, at least 95%, at least 98% homology) to E. coli pyruvate kinase, e.g. at least 85% homology to SEQ ID NO: 21 and functional fragments and variants thereof. In some

embodiments, pyruvate kinase can be a homolog of E. coli pyruvate kinase, e.g. pyruvate kinase from other organisms or functional fragments and variants thereof.

[0083] As used herein, "3-phosphoglycerate kinase" or "PGK" or "3-phospho-D- glycerate 1 -phosphotransferase" refers to an enzyme which can catalyze the reversible transfer of a phosphate group from ATP to glycerate-3-P, yielding ADP and glycerate-l,3-P ₂ . This enzyme, along with enolase, phosphoglycerate mutase, and pyruvate kinase either individually or in various combinations, are in some cases useful in enhancing conversion of pyruvate to 3- phosphoglycerate, which is the product of the enzyme Rubisco. Sequences encoding this enzyme can be obtained from the organism that is being engineered, since this enzyme is expressed by essentially all organisms. Alternatively, sequences encoding the enzyme can be obtained from a bacterium such as E. coli or a cyanobacterium such as Synechococcus elongatus

7942. In some embodiments, 3-phosphoglycerate kinase can be a polypeptide comprising the sequence of SEQ ID NO: 22. In some embodiments, 3-phosphoglycerate kinase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least

98% homology) to E. coli 3-phosphoglycerate kinase, e.g. at least 85% homology to SEQ ID

NO: 22 and functional fragments and variants thereof. In some embodiments, 3- phosphoglycerate kinase can be a homolog of E. coli 3-phosphoglycerate, e.g. 3- phosphoglycerate kinase from other organisms or functional fragments and variants thereof.

[0084] Production of useful organic molecules and intermediates. The 3HOP pathway produces several unusual molecules as conjugates with coenzyme A. Such molecules include methylmalate, mesaconate, and citramalate. Expression of subsets of enzymes in the 3HOP pathway is useful to generate CoA derivatives, from which the CoA moiety can then be removed chemically or enzymatically to create the methylmalate, mesaconate, or citramalate product. To produce methylmalate, an organism is engineered to express MMC lyase and is fed with propionate and glyoxylate. Methylmalyl-CoA is isolated from the organism and, for example, chemically converted to methylmalate and CoA. Spontaneous hydrolysis in aqueous solution is one method of chemical conversion, as the thioester bond is rather labile. Propionate is naturally converted to propionyl-CoA by many microbes, but if the relevant enzyme (propionyl-CoA ligase, encoded for example by the prpE genes of E. coli K12 and Salmonella typhimurium LT2) is not present, its expression can be engineered into the organism. To produce mesaconate, an organism is engineered to express MMC lyase and mesaconyl-Cl-CoA hydratase, and is fed with propionate and glyoxylate. Mesaconyl-Cl-CoA is isolated from the organism and, for example, chemically converted to mesaconate and CoA. To produce citramalate, an organism is engineered to express MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-C4-CoA hydratase, and is fed with propionate and glyoxylate.

Citramalyl-CoA is isolated from the organism and, for example, chemically converted to citramalate and CoA.

[0085] Functional expression of elements of the 3HOP pathway in bacteria. To illustrate the general principles for engineering the expression of 3HOP enzymes in bacteria, expression in E. coli and cyanobacteria is described. The functional expression of 3HOP enzymes MCR and PCS is achieved by standard molecular biology techniques, optionally including the synthesis of genes that are codon-optimized for expression in E. coli, placement of one or both genes downstream of a promoter to form an expression construct, and insertion of the expression construct into a plasmid or the bacterial chromosome. A specific illustration of expression of MCR and PCS from a multicopy plasmid is given in Example 1.

[0086] Nucleic acids encoding MCR or PCS, or both, are inserted into a bacterial chromosome such as the E. coli chromosome via recombineering. Thomason et al., CU .

PROTOCOLS MOLE. BIOL. 1.16.1 (John Wiley & Sons, Inc., 2005). In this approach, the gene of interest such as the gene for PCS, is placed downstream of a promoter and linked to an antibiotic resistance gene or other selectable marker by standard techniques such as 'joining PCR' or plasmid construction using restriction enzymes or Gibson assembly. Once a DNA, such as one encoding MCR, has been so assembled, it is amplified with PCR primers that are typically about 70 bases long and typically contain at their 3' ends about 20 bases that are identical to the ends of the MCR-encoding DNA and about 50 bases at the 5' ends that correspond to a region of the bacterial chromosome into which insertion is desired. The recipient bacteria are first engineered to express the phage lambda Red proteins that enhance recombination, such that the

Red proteins are transiently expressed and then the linear, PCR-amplified DNA is introduced by electroporation. Because MCR and PCS are generally used together in the present invention, it is often convenient to configure the corresponding genes into a single operon. Because of the large size of these coding sequences, however, it may be convenient to insert them into a bacterial chromosome in separate steps. Using such methods, sequences encoding MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-

C4-CoA hydratase are introduced into the bacterial chromosome.

[0087] To express at least one of MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase or mesaconyl-C4-CoA hydratase, in certain other bacterial strains for which genetic engineering techniques are more limited, other techniques may be used. For example, it is possible to integrate genes into so-called 'neutral sites' in the chromosomes of cyanobacteria and other bacteria using the endogenous host recombination system and regions of homology from about 500 to 1500 bases. For example,

Golden et al. (Clerico et al., 362 Meths. Mol. Biol. 155 (2007)), and Niederholtmeyer et al. (76

Appl. Environ. Microbiol. 6023 (2010)), have designed 'neutral site vectors' that allow integration of transgenes into the chromosome of the cyanobacterium Synechococcus elongatus

7942. The neutral sites are defined as chromosomal sites into which insertions can be made without causing a deleterious phenotype. In relatively uncharacterized bacteria it is possible to identify neutral sites by first obtaining a genomic sequence of the organism (which can be performed by a contractor such as Genewiz® DNA sequencing services, Cambridge, MA), and then identifying selectively neutral regions such as cryptic prophage remnants or pseudogenes

(i.e., 'junk DNA') into which transgenes can be inserted. From such a region, two adjacent sequence segments are obtained and placed into a vector, and the transgene of interest such as sequences functionally encoding at least one of MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase or mesaconyl-C4-CoA hydratase, along with a selectable marker, is placed between the two chromosomal sequence segments. A linear DNA fragment that includes sequences encoding the enzyme(s) to be expressed with appropriate regulatory elements, a selectable marker, and flanking chromosomal segments is introduced into the target bacterium by an appropriate transformation method. For example, such DNA is introduced into Synechococcus elongatus 7942 by simply mixing DNA with the cells, as this bacterium is naturally transformable. Clerico et al., 362 Meth. Mol. Biol. 155 (2007).

Alternatively, for many bacteria, electroporation may be used to introduce such DNA.

[0088] Actual function of the 3HOP enzymes MCR, PCS, MMC lyase, mesaconyl-Cl-

CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase and mesaconyl-C4-CoA hydratase, in the engineered cells is assayed in a manner that reflects the goals of the user and the particular product or phenotype in question. For example, when MCR and PCS are expressed, synthesis of propionyl-CoA will generally result. This can be detected using an in vivo propionyl-CoA- dependent transcriptional reporter. When MCR and PCS are expressed for the purpose of generating propionyl-CoA as an intermediate to another product such as erythromycin, the rate of production and yield of the product is measured.

[0089] When the goal of expression of the 3HOP enzymes MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase and mesaconyl-C4-CoA hydratase, is enhancement of recycling of the products of photorespiration. For example, in a cyanobacterium, the functional expression of all of these enzymes to form a glyoxylate recycling system may be assayed in a mutant that lacks glyoxylate carboligase and optionally other endogenous glyoxylate-metabolizing enzymes. In the absence of 3HOP transgenes, such mutants grows poorly or not at all in atmospheric levels of C0 ₂ , and requires elevated C0 ₂ levels (such as 2%) for robust growth. Expression of at least one of the 3HOP enzymes MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase or mesaconyl-C4-CoA hydratase, enhances the growth of such a mutant. It may be generally preferable to use all of enzymes MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase and mesaconyl-C4-CoA hydratase for this purpose, although in some situations a subset of enzymes are adequate.

[0090] Rescue of a growth defect of a mutant is useful for determining whether 3HOP enzymes MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase and mesaconyl-C4-CoA hydratase are, in fact, functional and can be used for troubleshooting of enzyme expression. When expression of the transgenes is optimized, an actual growth improvement is observed as a result. Without wishing to be bound by theory, this growth enhancement is due to more efficient recycling of glyoxylate.

[0091] It should be noted that cyanobacteria and many other bacteria already possess ancillary enzymes that may be useful in recycling of photorespiration products. For example, essentially all organisms that use the Calvin cycle also express a phosphoglycolate phosphatase, and many bacteria (but not plants) also express glycolate dehydrogenase. In addition, bacteria generally express enzymes that can metabolize pyruvate. To optimally take advantage of the

3HOP enzymes MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4

CoA transferase and mesaconyl-C4-CoA hydratase, it is often useful to introduce mutations that block the utilization of common intermediates. Specifically, it is useful to eliminate the PrpC gene that encodes the first enzyme in an alternative pathway for propionyl-CoA utilization. It is also sometimes useful to mutate genes encoding enzymes that use glyoxylate as a substrate, such as malate synthase, glyoxylate carboligase, and transaminases that convert glyoxylate to glycine.

[0092] As used herein, "glycolate dehydrogenase" refers to an enzyme which can catalyze the conversion of glycolate to glyoxylate by reducing an acceptor molecule. In some embodiments, the glycolate dehydrogenase can be dependent upon organic cofactors (e.g.

nicotinic cofactors), e.g. mitochondrial and prokaryotic glycolate dehydrogenases. In some embodiments, the glycolate dehydrogenase can be a nicotinic cofactor-dependent glycolate dehydrogenase. In some embodiments, glycolate dehydrogenase can comprise subunits, e.g. E. coli glycolate dehydrogenase requires at least glcD, glcE and glcF to form an active enzyme. In some embodiments, a subunit of glycolate dehydrogenase can be a polypeptide comprising the sequence of any of SEQ ID NOs: 13, 15, or 17, without or without the localization tags indicated in Figures 20, 22, and 24, respectively. In some embodiments, glycolate dehydrogenase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to E. coli glycolate dehydrogenase, e.g. at least 85% homology to any of the subunits of SEQ ID NO: 13, 15, or 17 and functional fragments and variants thereof. In some embodiments, a subunit of glycolate dehydrogenase can be a polypeptide having at least 85% homology (e.g. at least 85%, at least 90%, at least 95%, at least 98% homology) to the polypeptide encoded by any of SEQ ID NOs: 14, 16, and 18, without or without the localization tags indicated in Figures 21, 23, and 25, respectively, and functional fragments and variants thereof. In some embodiments, glycolate dehydrogenase can be a homolog of E. coli glycolate dehydrogenase, e.g. glycolate dehydrogenase from other organisms, or functional fragments and variants thereof. In some embodiments, a plurality of subunits of glycolate dehydrogenase can be comprised by a single operon.

[0093] Functional expression of elements of the 3HOP pathway in plants. To illustrate the general principles for engineering the expression of 3HOP enzymes in a green plant, expression in Arabidopsis is described. Transformation of other plants may also be

accomplished by methods specific for those plants. For example, the biofuel plant Camelina sativa may be transformed by the technique of Kushvinov et al. (U.S. Patent No. 7,910,803) or by Agrobacterium-mediated transformation (Lu et al., 27 Plant Cell Rep. 273 (2005)). Similarly, wheat, rice, or soybeans may be transformed by methods used for those plants, which are known in the art. See, e.g., U.S. Patents No. 8,049,071; No. 8,106,174; No. 8,101,826. Each of the foregoing references is incorporated by reference herein in its entirety.

[0094] Transformation of plants is generally assayed through the use of selectable or screenable markers. For example, in transformation of chloroplasts, a selectable marker such as aadA that confers resistance to streptomycin or spectinomycin may be used (Day et al., 9 Plant

Biotechnol. J. 540 (2011)); selectable markers may also be removed from the transgenic organisms by methods reviewed by Day et al. To express the 3HOP middle and right-side pathways in the chloroplast, the genes encoding enzymes MCR, PCS, MMC lyase, mesaconyl-

Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase or mesaconyl-C4-CoA hydratase may be configured into an operon downstream of a single bacterial promoter, or may be configured into multiple operons and established in a plant by sequential transformation. If a single operon is used, it is often convenient to array the genes in an order from the promoter that corresponds to the desired expression level, with the gene whose expression should be highest coming first. In the case of the 3HOP middle and right-side pathways, the order <promoter- MMC lyase - MCR - PCS - mesaconyl-CoA C1-C4 CoA transferase - mesaconyl-C4-CoA hydratase - mesaconyl-Cl-CoA hydratase > is useful, although other gene orders may also be used. A typical bacterial promoter such as a cyanobacterial promoter or other Gram-negative bacterial promoter may be used, or a chloroplast promoter may be used.

[0095] A particular embodiment of the present invention provides for a recombinant

Camelina that recycles carbon more efficiently, such that the yield of seed oil is increased. More specifically, Camelina sativa (L.) Crantz (Camelina or "false flax"), a member of the mustard family, needs little water or nitrogen to flourish, and can grow on marginal agricultural lands that do not compete with food crops. It may also be used as a rotation crop for wheat, to increase the health of the soil. It has been traditionally cultivated as an oilseed crop to produce vegetable oil and animal feed, being particularly high in omega-3 fatty acids and tocopherols (Vitamin E). Because its seeds are about 40% oil, and because it thrives in temperate climates, Camelina is also a current plant of interest for the production of biodiesel oil. Camelina is also self- pollinating, reducing concerns of contamination in open-field cultivation. The recombinant Camelina of the present invention may product more than twice the energy per acre of corn- derived ethanol. When a different mode of glycolate pathway was introduced into Arabidopsis, growth of the plants increased by ~2.3-fold. Kebeish et al., 2007. In addition to recombinant expression of at least one enzyme of the 3HOP pathway, as described herein, in Camelina, inefficient competing endogenous enzymes of the plant metabolism are down-regulated genetically.

[0096] For convenience, the meaning of some terms and phrases used in the

specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. 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. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail. [0097] The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. In some embodiments, "reduce,"

"reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, "reduction" or "inhibition" does not encompass a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0098] The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms

"increased", "increase", "enhance", or "activate" can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a "increase" is a statistically significant increase in such level.

[0099] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. [00100] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid,

deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single- stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double- stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.

[00101] As used herein, an enzymatic polypeptide is said to be "functional" or expressed as a "functional" polypeptide if the polypeptide can catalyze a detectable level of at least one chemical reaction that it naturally catalyzes in the source organism. The reactions catalyzed by the enzymatic polypeptides relating to the present invention are described elsewhere herein. One of skill in the art can readily detect increases in reaction products and/or detect decreases in reaction substrates, e.g. by mass spectroscopy (MS, including, e.g., MADLI/TOF, SELDI/TOF, LC-MS, GC-MS, HPLC-MS, etc., among others). A level is "detectable" if it is greater, by a stastistically significant amount, than a negative control, e.g. a water control, non-enzymatic control, or deactivated enzyme control.

[00102] As used herein, a "functional fragment" refers to a portion of a polypeptide that retains at least a detectable level of the activity of the native polypeptide from which it is derived. The reactions catalyzed by the enzymatic polypeptides relating to the present invention, as well as methods of detecting such activities are described elsewhere herein. In some embodiments, a functional fragment can retain at least 50% of the activity of the native polypeptide, e.g. 50% or more of the activity, 60% or more of the activity, 75% or more of the activity, or 90% or more of the activity of the native polypeptide.

[00103] A "variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or note, has more than 100% of the activity of a wildtype or native polypeptide, e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.

[00104] One method of identifying amino acid residues which can be substituted is to align, for example, Chloroflexus aurantiacus MCR to a MCR from one or more other species, e.g. from a Sulfolobaceae spp. Alignment can provide guidance regarding not only residues likely to be necessary for function but also, conversely, those residues likely to tolerate change.

Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely that that site is important functionally. Where, conversely, alignment shows residues in corresponding positions to differ significantly in size, charge, hydrophobicity, etc., it is more likely that that site can tolerate variation in a functional polypeptide. Such alignments are readily created by one of ordinary skill in the art, e.g. created using the default settings of the alignment tool of the BLASTP program, freely available on the world wide web at http://blast.ncbi.nlm.nih.gov/. Furthermore, homologs of any given polypeptide or nucleic acid sequence can be found using BLAST programs, e.g. by searching freely available databases of sequence for homologous sequences, or by querying those databases for annotations indicating a homolog (e.g. search strings that comprise a gene name or describe the activity of a gene). Such databases can be found, e.g. on the world wide web at http://ncbi.nlm.nih.gov/.

The variant amino acid or DNA sequence can be at least 90%, at least 95%, at least 96%, at least

97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an "original" sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp

(available on the world wide web at http://blast.ncbi.nlm.nih.gov), with default parameters set.

[00105] In some embodiments, the variant is a conservative substitution variant. Variants can be obtained by mutations of native nucleotide sequences, for example. A "variant," as referred to herein, is a polypeptide substantially homologous to a native or reference

polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide - encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or

3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or note, has more than 100% of the activity of the wildtype enzyme, e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.

[00106] One method of identifying amino acid residues which can be substituted is to align, for example, homologs from one or more non-human species. Alignment can provide guidance regarding not only residues likely to be necessary for function but also, conversely, those residues likely to tolerate change. Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely that that site is important functionally. Where, conversely, alignment shows residues in corresponding positions to differ significantly in size, charge, hydrophobicity, etc., it is more likely that that site can tolerate variation in a functional polypeptide. Similarly, alignment with a related polypeptide from the same species, which does not show the same activity, can also provide guidance with respect to regions or structures required for activity. Alignments are readily generated by one of skill in the art using freely available programs. The variant amino acid or DNA sequence can be at least

90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence, e.g. SEQ ID NOs: l-22. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an

"original" sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp (available on the world wide web at http://blast.ncbi.nlm.nih.gov), with default parameters set. [00107] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired apoptotic activity of a native or reference polypeptide is retained.

Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure. Typically

conservative substitutions for one another include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[00108] As used herein, a polypeptide sequence and/or nucleic acid sequence is

"heterologous" to a host cell and/or organism if the sequence is foreign to the host

cell/organism, e.g. it is not found in the genome, transcriptome, and/or proteome of the host species. In some embodiments, the heterologous sequence is not homologous to a sequence found in the genome, transcriptome, and/or proteome of the host species.

[00109] As used herein, the term "recombinant" refers to a cell, tissue or organism that has undergone transformation with a new combination of nucleic acid, e.g. genes or DNA. When used in reference to nucleic acid molecules, "recombinant" refers to a combination of nucleic acid molecules that are joined together using recombinant DNA technology into a progeny nucleic acid molecule. When used in reference to cells and organisms, the terms "recombinant," "transformed," and "transgenic" refer to a host cell or organism into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Recombinant cells and organisms are understood to encompass not only the end product of a transformation process, but also recombinant progeny thereof. A "non-transformed," "non- transgenic," or "non-recombinant" host refers to a wild type cell or organism that does not contain the heterologous nucleic acid molecule.

[00110] As used herein, "photosynthesis" refers to the process in green plants and certain other organisms by which carbohydrates are synthesized from carbon dioxide and water using light as an energy source. Most forms of photosynthesis release oxygen as a byproduct. As is well known in the art, the photosynthetie process includes several independent reactions, including reactions that are conducted in the presence of and utilizing light energy as well as reactions that can be conducted in the dark or without light energy, in which carbon dioxide and water are converted into organic compounds, e.g., carbohydrates and others, by bacteria, algae and plants in the presence of a pigment, e.g. chlorophyll. As used herein, the term "non- p otosynthetic" refers to a cell or organism which does not have a natural ability to perform photosynthesis.

[00111] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[00112] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±\%.

[00113] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[00114] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00115] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[00116] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." [00117] Definitions of common terms in cell biology and molecular biology can be found in The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569- 8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[00118] Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), which are all incorporated by reference herein in their entireties.

[00119] Other terms are defined herein within the description of the various aspects of the invention.

[00120] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00121] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[00122] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[00123] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.

[00124] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

1. A recombinant organism engineered to express functional, heterologous malonyl-CoA reductase (MCR) and propionyl-CoA synthase (PCS) enzymes.

2. A recombinant organism engineered to express functional, heterologous (S)-malyl- CoA/p-methylmalyl-CoA/(S)-citramalyl-CoA (MMC lyase) and mesaconyl-Cl-CoA hydratase (β-methmalyl-Co A-dehydratase) .

3. The organism of paragraph 2, further engineered to express functional mesaconyl-CoA C1-C4 transferase.

4. The organism of any of paragraphs 2-3, further engineered to express functional mesaconyl-C4-CoA hydratase.

5. The organism of any of paragraphs 2-4, further engineered to express functional mesaconyl-CoA C1-C4 transferase and mesaconyl-C4-CoA hydratase.

6. The organism of any of paragraphs 2-5, further engineered to express a functional subunit of a nicotinic cofactor-dependent glycolate dehydrogenase.

7. The organism of any of paragraphs 2-6, further engineered to express pyruvate kinase, enolase, phosphoglycerate mutase, or 3-phosphoglycerate kinase.

8. The organism of any of paragraphs 2-7, further engineered to express a functional malonyl-CoA reductase.

9. The organism of any of paragraphs 2-8, further engineered to express a functional propionyl-CoA synthase. 10. The organism of any one of paragraphs 1-9, wherein said organism is a cyanobacterium.

11. The organism of any one of paragraphs 1-9, wherein said organism is a plant.

12. The organism of any one of paragraphs 1-9, wherein said organism is a non- photosynthetic organism.

13. A method of improving growth or yield of an organism, comprising introducing the functional expression in said organism of a heterologous protein selected from the group consisting of malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methylmalyl-CoA dehydratase, mesaconyl-CoA C1-C4 transferase, and mesaconyl-CoA hydratase.

14. A recombinant plant that expresses at least one functional, heterologous protein selected from the group consisting of malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methylmalyl-CoA dehydratase, mesaconyl-CoA C1-C4 transferase, and mesaconyl-C4-

CoA hydratase.

15. The recombinant plant of paragraph 14, wherein said heterologous protein is encoded in the chloroplast.

16. The recombinant plant of paragraph 14, wherein said heterologous protein is encoded in the nucleus.

17. The recombinant plant of any of paragraphs 14-16, wherein said plant is further engineered to express a glycolate dehydrogenase.

18. A recombinant cyanobacterium that expresses at least one functional, heterologous protein selected from the group consisting of malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methylmalyl-CoA dehydratase, mesaconyl-CoA C1-C4 transferase, and

mesaconyl- C4-CoA hydratase.

19. A recombinant Agrobacterium containing DNA sequences encoding a protein selected from the group consisting of malonyl-CoA reductase, propionyl-CoA synthase, MMC lyase, methylmalyl-CoA dehydratase, mesaconyl-CoA C1-C4 transferase, and mesaconyl- C4-CoA hydratase.

EXAMPLES

Example 1. Functional expression of the mcr and pes genes to produce propionyl-CoA in a heterologous organism

[00125] As depicted in Figure 1, the 3HOP pathway can be considered to begin with carboxylation of acetyl-CoA by acetyl-CoA carboxylase to produce malonyl-CoA. This reaction initiates fatty acid synthesis and is found in essentially all organisms. The second and third steps of the 3HOP pathway are catalyzed by MCR (mcr) and PCS (pes), enzymes that are not widely distributed in nature and which are found primarily in organisms that use the 3HOP pathway or a related pathway for carbon fixation.

[00126] The mcr and pes genes from Chloroflexus aurantiacus were expressed in modified form in E. coli as follows. Versions of these genes that were codon-optimized for expression in E. coli were ordered from Genscript (Piscataway, NJ), a custom DNA synthesis company. The optimized coding sequences are depicted in Figures 9 and 11. An operon was constructed by standard recombinant DNA techniques that included, in order, the lac promoter, the mcr gene and the pes gene; placed into a multicopy plasmid, termed p62-MP (Figure 26). E. coli was transformed with this plasmid. Protein expression was verified by SDS-PAGE of total E. coli proteins, comparing strains with and without the plasmid. Because of the large size of these proteins, their presence was easily identified; the band corresponding to the PCS protein was the slowest-migrating, and the band corresponding to the MCR protein was also near the top of the gel.

[00127] To demonstrate in vivo, coordinated function of the MCR and PCS proteins, a regulatory system of Lee and Keasling (71 Applied Environ. Microbiol. 6856 (2005)) was used to detect synthesis of propionyl-CoA, the product of the combined action of MCR and PCS. Briefly, the system of Palacios et al. consists of a regulatory protein, PrpR, that responds to propionyl-CoA, and a promoter that responds to this regulatory factor. Palacios et al., 185 J. Bact. 2802 (2003). To create a reporter system for propionyl-CoA, a sequence encoding the Green Fluorescent Protein was placed downstream of the prpB promoter in the plasmid pPro33 of Lee and Keasling, and the resulting DNA segment consisting of the prpR regulatory factor, the prpB promoter, in a plasmid with a pl5a-derived origin of replication that is compatible with the origin of pUC-type plasmids such as the plasmid encoding MCR and PCS. The two plasmids were placed in the same strain of E. coli and GFP fluorescence was measured and compared with a strain carrying the propionyl-CoA-inducible GFP construction alone. The strain expressing MCR and PCS showed a level of GFP fluorescence significantly greater than that of the strain lacking MCR and PCS in the absence of added propionate, and similar to that of the strain lacking MCR and PCS in the presence of about 10 mM propionate. Figure 7 illustrates typical results.

[00128] Taken together, these results indicate that the MCR and PCS proteins can be co- expressed in a heterologous organism, and will be enzymatically active and jointly able to produce propionyl-CoA, a key intermediate in the 3HOP pathway.

Example 2. Functional expression of MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-

CoA C1-C4 Co A transferase, mesaconyl-C4-CoA hydratase of the 3HOP pathway [00129] As depicted in Figure 1 and Figure 2B, the "right side" of the 3HOP cycle consists of five reactions: condensation of glyoxylate and propionyl-CoA into the 5-carbon methylmalyl-CoA, rearrangements into mesaconyl-Cl-CoA, mesaconyl-C4-CoA, and citramalyl-CoA, and then cleavage into pyruvate and acetyl-CoA. The first and last steps are carried out by MMC lyase, while the other steps are carried out by distinct enzymes.

Functionality of this pathway was measured.

[00130] Certain strains of E. coli are sensitive to high levels of propionate, which is used as a preservative. An operon consisting of codon-optimized genes encoding enzymes MMC lyase, mesaconyl-CoA C1-C4 CoA transferase, mesaconyl-C4-CoA hydratase and mesaconyl- Cl-CoA hydratase (in that order from the promoter) was constructed, and the corresponding plasmid was placed in E. coli strain BW25113. In addition, a similar plasmid containing only the gene for enzyme MMC lyase was placed in the same strain. The strains were tested for growth in M9 minimal medium with 0.14% glucose and 0.26% mM sodium propionate. The results were as follows:

[00131] The interpretation of these results showed that the parent strain is sensitive to propionate. E. coli normally converts propionate into propionyl-CoA, which is a toxic intermediate. When enzyme 10 is expressed, some of the propionyl-CoA is converted to methylmalyl-CoA. This reaction also uses glyoxylate as a co-substrate, which is present because of the glyoxylate shunt. The reaction catalyzed by MMC lyase is reversible,however, and the equilibrium favors glyoxylate + propionyl-CoA over methylmalyl-CoA, so the impact on intracellular levels of propionyl-CoA is small and the growth enhancement from expression of only MMC lyase is also small. When the remainder of the enzymes are also functionally expressed, however, the entire right side of the pathway is functional and propionyl-CoA is depleted.

Example 3. Functional expression of MCR and PCS in combination with MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, mesaconyl-C4-CoA hydratase of the 3HOP pathway for metabolism of the byproducts of photorespiration

[00132] Photorespiration is the reaction in which the carbon-fixing enzyme Rubisco

(ribulose bisphosphate carboxylase/oxygenase) reacts with oxygen instead of carbon dioxide. The result of this reaction is the production of phosphoglycolate and 3-phosphoglycerate instead of the two molecules of 3-phosphoglycerate that result from action of Rubisco on C02 and ribulose bisphosphate. The resulting phosphoglycolate represents fixed carbon that must be recycled to avoid waste.

[00133] There are two commonly used pathways for recycling of phosphoglycolate. The first, used in bacteria such as E. coli as well as photosynthetic microbes, involves the

dephosphorylation of 2PG to glycolate via phosphoglycolate phosphatase, NAD+-coupled oxidation of glycolate to glyoxylate via glycolate dehydrogenase, ligation of two glyoxylate molecules to produce tartronate semialdehyde via glyoxylate carboligase, reduction of tartronate semialdehyde to glycerate, and phosphorylation of glycerate to generate 3-phosphoglycerate. This pathway is somewhat inefficient in that a fixed C02 is released in the glyoxylate carboligase reaction.

[00134] The second pathway is used by multicellular green plants. In this pathway, the dephosphorylation of phosphoglycolate occurs by the same type of enzyme, but then glycolate is metabolized by a complex set of reactions involving a shuttling through multiple compartments in the cell. The plant pathway is less energetically efficient than the microbial pathway.

[00135] It is an insight of the invention that the central and right-side subpathways of the

3HOP pathway offer a particularly efficient mechanism for the recycling of glyoxylate, as compared to either the typical bacterial pathway or the plant pathway. The action of the middle and right-side pathways is to absorb one glyoxylate molecule (two carbons), fix one carbon dioxide, and generate pyruvate (three carbons). This contrasts with the existing microbial and multicellular pathways that convert two molecules of glyoxylate into one three-carbon molecule with the loss of a carbon dioxide.

[00136] Plasmids (or integrated constructions) encoding the middle pathway and the right-side pathway are incorporated into E. coli, Salmonella, or another non-C02 fixing microbe by standard techniques, such as the techniques described herein for incorporating these sub- pathways separately into E. coli. To demonstrate the function of the pathways in glycolate and glyoxylate utilization, mutations in central carbon metabolism are introduced.

[00137] For example, mutations such as deletions in the prpC, gel, and aceABK genes were introduced into the BW25113 strain of E. coli. Datsenko & Wanner, 97 PNAS 6640 (2000). The essential process for repeated introduction of defined deletions is described elsewhere (Datsenko & Wanner, 2000; Baba et al., 2 Mol. Syst. Biol. 0008 (2006); Thomason, Curr. Prot. Mol. Biol. 1.16.1 (2005)), and includes introduction of, for example, a kanamycin- resistance element flanked by site- specific recombination elements such as FRT sites (an

'antibiotic resistance cassette') into the genome of an organism such that one or more coding sequences is replaced by the antibiotic -resistance cassette. The antibiotic resistance gene itself is removed by transient expression of a site-specific recombination protein, and then a second antibiotic resistance cassette in another site is introduced. Such cassettes may be introduced by PI transduction, transformation with amplified DNA, an Hfr cross, or other methods. Using such methods, the prpC gel aceABK triple deletion strain was constructed, using prpC::KanR and gcl::KanR elements obtained from the E. coli Genetic Stock Center, and an aceABK: :KanR element which the adjacent isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase genes were jointly deleted.

[00138] The resulting prpC gel aceABK strain is unable to grow on glycolate as sole carbon source and formate as an energy source. The strain does grow on glycolate plus formate, however, when genes encoding MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, mesaconyl-C4-CoA hydratase of the 3HOP pathway are expressed, along with an NAD or NADP-dependent formate dehydrogenase. Without wishing to be bound by theory, growth is enabled by the following mechanism: First, the prpC gel aceABK strain is able to convert glycolate to glyoxylate, but is unable to grow on glycolate because it cannot metabolize glyoxylate any further. Formic acid is normally metabolized by E. coli using formate dehydrogenases that generate C02, reduced electron acceptors, and energy in the form of a proton gradient. In the presence of a heterologous NAD or NADP-dependent formate dehydrogenase, usable reducing equivalents can also be generated. Incorporation of carbon from formic acid does not occur. Thus, in the presence of an NAD or NADP-dependent formate dehydrogenase, addition of formic acid to bacterial medium can provide energy and reducing equivalents, and thus serves as a useful surrogate for the action of Photosystems I and II in the presence of light. When genes encoding MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, mesaconyl-C4-CoA hydratase of the 3HOP pathway and an NAD or NADP-dependent formate dehydrogenase are expressed, glycolate is converted to glyoxylate by the endogenous glycolate dehyrogenase, and then glyoxylate is incorporated into the cycle diagrammed in Figure 6. Specifically, glyoxylate is joined with propionyl-CoA by the enzyme MMC lyase, converted to pyruvate and acetyl-CoA by the sequential actions of enzymes mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, mesaconyl-C4-CoA hydratase, and MMC lyase, and then the acetyl-CoA is carboxylated to produce malonyl-CoA that is then reduced to regenerate propionyl-CoA.

Example 4. Functional expression of MCR and PCS in combination with MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, mesaconyl-C4-CoA hydratase in cyanobacteria. [00139] Using techniques similar to those of Example 3, genes encoding the middle pathway and right-side pathway were incorporated into cyanobacteria (Figure 30). Specifically, the genes of the middle pathway were integrated into neutral site 3 in the genome of

Synechococcus elongatus 7942 (NS3; Neiderholtmeyer et al. Appl Env Micro [2010]

76(11):3462-3466) using a chloramphenicol-resistance marker. The right-side pathway genes were integrated into neutral site 1 (NS1; Clerico et al., 362 Meths. Mol. Biol. 155 (2007)) using spectinomycin resistance as a selectable marker, according to standard procedures described in these references.

[00140] Function of the combination of middle and right-side pathways was observed as follows. Forms of S. elongatus 7942 that either contained no transgenes, the right-side pathway only, or the middle and right-side pathway. Cells were grown in BG11 medium in the absence or presence of 0.4% glycolate for a 144-hour period, with a starting optical density in the range of 0.2 to 0.4. Typical results are shown in Figure 31. In the absence of glycolate, cells engineered to express either the middle pathway or the middle plus right-side pathway showed more rapid growth that the wild-type parent. In the presence of glycolate, cells engineered to express the middle plus right-side pathway showed the most rapid growth and best survival; cells with only the middle pathway showing an intermediate level of growth and survival; and the wild-type parent showing the least rapid growth and poorest survival. These results indicate that expression of 3HOP subpathways such as the middle pathway and right-side pathway can improve the growth and biomass productivity of a photosynthetic organism. Based on these data, glycolate inhibits cyanobacterial growth. Without wishing to be bound by theory, glycolate may be converted to glyoxylate in cyanobacteria, which may be toxic because of the aldehyde group. Expression of 3HOP pathways may relieve this toxicity, as well as allowing more efficient entry of this source of carbon into central metabolism.

[00141] To further demonstrate the function of these sub-pathways, a mutation in tartronate semialdehyde reductase (tsr) was introduced into a cyanobacterium. This mutation blocks the major pathway for glyoxylate utilization and recycling of glycolate produced by photorespiration. The tsr mutant of a cyanobacterium is thus growth-inhibited at atmospheric 02/C02 ratios and will grow well only at elevated C02 levels. For example, a tsr mutant of the cyanobacterium Synechococcus elongatus 7942 was constructed. Portions of the tsr gene of S. elongatus are obtained from this organism by PCR amplification.

[00142] An antibiotic resistance element was inserted into the chromosomal S. elongatus tsr gene by placing the resistance element between the two amplified fragments of the gene, and then transforming the cyanobacteria with the linear DNA fragment and selecting for antibiotic resistance on BG11 plates according to standard procedures. All incubations were performed in a 2% C02 environment. Because S. elongatus and certain other cyanobacteria contain multiple, independently replicating chromosomes, an initially transformed cell was a merozygote with both wild-type and mutant copies of the gene to be knocked out. Therefore the resulting strain is subcultured in the presence of antibiotics, plated for single colonies, and the single colonies are tested by PCR for the absence of the wild-type tsr gene. Homozygous mutant clones are identified, and are found to grow very poorly or not at all in normal atmosphere, and to only grow in high C02.

[00143] The genes for the "middle" and" right-side pathways" of the 3HOP pathway are introduced into a tsr mutant as follows. The entire set of genes encoding enzymes MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-C4-CoA hydratase 3 are introduced using a neutral- site vector. Niederholtmeyer et al., 76 Appl. Environ. Microbiol. 3462 (2010); Clerico et al., 362 Meths. Mol. Biol. 155 (2007). Alternatively, because of the large size of such a construction, the genes may be introduced in two or more neutral site vectors. The genes are expressed from a foreign promoter such as the lac promoter of E. coli, or using an endogenous promoter. For example, most but not all promoters of cyanobacteria are regulated to be active during the day and off at night. A day-on, night-off promoter is ideal for expression of the 3HOP genes in cyanobacteria. When the genes encoding enzymes MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA Cl- C4 CoA transferase and mesaconyl-C4-CoA hydratase are introduced into a cyanobacterium lacking tsr, the resulting cyanobacterium is able to grow on low levels of C02, such as natural atmospheric levels of C02.

[00144] In some strains of cyanobacteria, when genes encoding enzymes MCR, PCS,

MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase and mesaconyl-C4-CoA hydratase are introduced (e.g., into a tsr- strain), low C02 levels do not permit rapid growth. Without wishing to be bound by theory, it is often the case that the relative expression levels of the 3HOP transgenes are not optimized, which may occur if ribosome binding sites are not correctly tuned. In such cases, directed evolution is used to optimize growth. For example, the strain is constructed and grown in high C02 such as 2% C02, and then the C02 concentration is reduced gradually.

[00145] When the genes encoding MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase and mesaconyl-C4-CoA hydratase are correctly introduced into cyanobacteria, the resulting engineered organisms actually grow more quickly than wild- type organisms. Without wishing to be bound by theory, this is because the glycolate recycling mechanism that occurs via these genes is faster and more energetically efficient than the natural mechanism. In both mechanisms, phosphoglycolate is dephosphorylated to glycolated, which is then oxidized to glyoxylate. In the natural cyanobacterial mechanism (Figure 4), two glyoxylate molecules are condensed into tartronate semialdehyde, releasing one C02 that must be re-fixed.

[00146] In the engineered mechanism (Figure 6), one glyoxylate molecule enters a cycle that includes steps 2, 3, 10b, 11, 12, 13 and 10c of Figure 1. As part of this cycle, ATP and NADPH generated by the light reactions of photosynthesis are used to fix a C0 ₂ molecule, and pyruvate is produced. The overall energetics of the engineered mechanism is superior to that of the natural mechanism because less energy in the form of NADPH and ATP is used to produce equivalent molecules. In addition, the overall rate of the engineered mechanism is superior to natural mechanism because it involved C0 ₂ fixation using acetyl-CoA carboxylase, which is much faster than Rubisco.

Example 5. Functional expression MCR and PCS in combination with MMC lyase, mesaconyl- Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-C4-CoA hydratase of the 3HOP pathway for metabolism of the byproducts of photorespiration in plants

[00147] The cycle that includes 3HOP enzymes MCR, PCS, MMC lyase, mesaconyl-Cl-

CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-C4-CoA hydratase for recycling products of photorespiration is introduced into multicellular plants, such as Camelina sativa, as follows. Because the purpose of this cycle is to recycle molecules that are generated in the chloroplast, it is ideal to express these enzymes in the chloroplast, although expression in other compartments, such as the cytoplasm, is also useful.

[00148] Two general approaches may be used to express foreign proteins in the chloroplast. According to the first approach, DNA is directly introduced into the chloroplast, typically via a "gene gun". See, e.g., Rao et al., 27 Biotech. Adv. 753 (2009). The DNA so introduced will recombine into the chloroplast genome at some frequency provided that appropriate regions of homology are available. When this approach is used, the genes may be arrayed into a single operon using a single bacterial-type promoter, or may be arrayed into multiple operons. An antibiotic resistance gene or a screenable marker is used to identify successfully transformed plants.

[00149] The other technique is to introduce foreign genes into the nuclear genome of the plant. The genes may also be introduced using a gene gun (particle bombardment), or via Agrobacterium transformation, or using a combination of the two. See, e.g., Rao et al., 2009. If this strategy is used, it is often valuable to place a eukaryotic promoter element in the vicinity of each coding sequence to be expressed, and also to place a chloroplast targeting sequence upstream at the 5' end of the coding sequence, such that the amino acids of the chloroplast targeting sequence are found at the N-terminus of the primary translation product.

[00150] Given that the introduction of foreign genes into plants is established in the art, the key insights of the invention with respect to functional introduction of MCR and PCS in combination with MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 Co A transferase, and mesaconyl-C4-CoA hydratase of the 3HOP pathway are the following. The goal of introducing these genes is to enhance the recycling of products of photorespiration. To achieve this, it is useful to express a microbial glycolate dehydrogenase in the plant chloroplast in addition to 3HOP enzymes MCR, PCS, MMC lyase, mesaconyl-Cl-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, and mesaconyl-C4-CoA hydratase. This is performed, for example, by inclusion of genes encoding the three subunits of glycolate dehydrogenase from E. coli or a cyanobacterium in addition to the genes from the 3HOP pathway. In addition, in certain plants such as certain C3 plants, the rate of conversion of pyruvate into 3- phosphoglycerate may not be adequate to support re-entry of carbon in pyruvate into the Calvin cycle. In such plants, the enzymes phosphoglycerate isomerase, enolase, and pyruvate kinase are expressed in the chloroplast by introduce of transgenes encoding these enzymes.

[00151] Although the invention has been described with reference to example

embodiments, those skilled in the art will appreciate that certain substitutions, alterations, and omissions may be made to the embodiments without departing from the spirit of the invention. Accordingly, the foregoing description is meant to be exemplary only, and should not limit the scope of the invention.