ENHANCED CARBON FIXATION IN PHOTOSYNTHETIC HOSTS
United States Patent Application
This invention provides genetically modified photosynthetic organisms and methods and constructs for enhancing inorganic carbon fixation. A photosynthetic organism of the present invention comprises a RUBISCO fusion protein operatively coupled to a protein-protein interaction domain to enable the functional association of RUBISCO and carbonic anhydrase.
Inventors:
Sayre, Richard T. (Webster Groves, MO, US)
Application Number:
Publication Date:
06/06/2013
Filing Date:
04/25/2011
Export Citation:
DONALD DANFORTH PLANT SCIENCE CENTER (St. Louis, MO, US)
Primary Class:
Other Classes:
800/317.2,
800/317.3,
800/317.4,
800/320.2,
800/320.3,
800/323.1,
800/323.2,
International Classes:
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Related US Applications:
May, 2008Hashimoto et al.January, 2002Franken et al.June, 2006Wu et al.September, 2003Frazer et al.September, 2006Gleissner et al.August, 2009Burdett et al.July, 2009Amouyel et al.September, 2009WebsterDecember, 2008Spangenberg et al.January, 2008Widholm et al.March, 2010Surritte et al.
Other References:
Demirevska-Kepova et al., 1999, Bulg. J. Plant Physiol. 25: 31-44.
Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory.
Tabita et al., 2008, Journal of Experimental Botany 59: .
Guo et al., 2004, Proceedings of the National Academy of Sciences USA 101: .
Iwaki et al., 2006, Photosynthesis Research 88: 287-297.
Fabre et al., 2007, Plant Cell and Environment 30: 617-629.
Sharma et al., 2011, Cellular Physiology and Biochemistry 28: 407-422.
1. 1-52. (canceled)
A genetically modified photosynthetic organism having increased carbon fixation comprising a heterologous polynucleotide sequence which encodes a fusion protein of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) and a protein-protein interaction domain operably linked to a promoter sequence.
The photosynthetic organism of claim 53 wherein said RuBisCO sequence further comprises: (a) a polynucleotide of SEQ ID NO:82; (b) a polynucleotide having at least 90% sequence identity across the entire sequence to SEQ ID NO:82; (c) a polynucleotide amplified from a nucleic acid library using primers which selectively hybridize, under stringent hybridization conditions, to a sequence within a polynucleotide of SEQ ID NO:82; or (d) a polynucleotide which is a full length complement of a polynucleotide of (a) (b), or (c).
The photosynthetic organism of claim 53 wherein said protein-protein interaction domain of said fusion protein is a STAS domain.
The photosynthetic organism of claim 53 further comprising a second heterologous polynucleotide sequence which encodes a high activity carbonic anhydrase operably linked to a promoter sequence.
The photosynthetic organism of claim 53 wherein said heterologous polynucleotide sequence further comprises a sequence that encodes a high activity carbonic anhydrase operably linked to a promoter sequence.
The photosynthetic organism of claim 56 wherein said second recombinant polynucleotide construct further encodes a protein-protein interaction domain that forms a protein-protein interaction pair with the protein-protein interaction domain of the RuBisCO fusion protein.
The photosynthetic organism of claim 557 wherein said high activity carbonic anhydrase comprises a human carbonic anhydrase II.
The photosynthetic organism of claim 57 wherein said high activity carbonic anhydrase comprises a polynucleotide having at least 90% sequence identity across the entire sequence to SEQ ID NO:1.
The photosynthetic organism of claim 53 wherein said RuBisCO is a large subunit RuBisCO.
The photosynthetic organism of claim 53 wherein said RuBisCO is a small subunit RuBisCO.
The photosynthetic organism of claim 60 further comprising a heterologous polynucleotide sequence that encodes a RuBisCO large subunit and a heterologous polynucleotide sequence that encodes a high activity carbonic anhydrase.
The photosynthetic organism of claim 63 wherein the heterologous polynucleotide sequence encoding at least two of said small subunit RuBisCO, said large subunit RuBisCO. and said carbonic anhydrase also encodes a protein-protein interaction domain.
The photosynthetic organism of claim 64 wherein the protein-protein interaction domain encoded by the heterologous polynucleotide sequence encoding at least two of said small subunit RuBisCO, said large subunit RuBisCO, and said carbonic anhydrase is a STAS domain.
The photosynthetic organism of claim 63 wherein said small subunit RuBisCO, said large subunit RuBisCO, and said carbonic anhydrase are encoded by the same heterologous polynucleotide.
The photosynthetic organism of claim 53 wherein said promoter sequence is a chloroplast promoter.
A plant part or tissue of the photosynthetic organism of claim 53.
A method for increasing carbon fixation in a photosynthetic organism comprising: introducing into a photosynthetic organism an expression cassette comprising a heterologous polynucleotide sequence which encodes a fusion protein of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) and a protein-protein interaction domain operably linked to a promoter sequence.
The method of claim 69 wherein said RuBisCO sequence further comprises: (a) a polynucleotide of SEQ ID NO:82; (b) a polynucleotide having at least 90% sequence identity across the entire sequence to SEQ ID NO:82; (c) a polynucleotide amplified from a nucleic acid library using primers which selectively hybridize, under stringent hybridization conditions, to a sequence within a polynucleotide of SEQ ID NO:82; or (d) a polynucleotide which is a full length complement of a polynucleotide of (a), (h), or (c).
The method of claim 69 wherein said protein-protein interaction domain of said fusion protein is a STAS domain.
The method of claim 69 further comprising introducing a heterologous polynucleotide sequence that encodes a high activity carbonic anhydrase operably linked to a promoter sequence.
The method of claim 72 wherein said second recombinant polynucleotide construct that encodes a high activity carbonic anhydrase further encodes protein-protein interaction domain that forms a protein-protein interaction pair with the protein-protein interaction domain of the RuBisCO fusion protein.
The method of claim 72 wherein said high activity carbonic anhydrase comprises a human carbonic anhydrase II.
The method of claim 72 wherein said high activity carbonic anhydrase comprises a polynucleotide having at least 90% sequence identity across the entire sequence to SEQ NO:1
The method of claim 69 wherein said RuBisCO is a large subunit RuBisCO.
The method of claim 69 wherein said RuBisCO is a small subunit RuBisCO.
The method of claim 77 further comprising introducing a heterologous polynucleotide sequence that encodes a RuBisCO large subunit and a heterologous polynucleotide sequence that encodes a high activity carbonic anhydrase.
The method of claim 78 wherein the heterologous polynucleotide sequence encoding at least two of said small subunit RuBisCO, said large subunit RuBisCO, and said carbonic anhydrase also encodes a protein-protein interaction domain.
The method of claim 79 wherein the protein-protein interaction domain encoded by the heterologous polynucleotide sequence encoding at least two of said small subunit RuBisCO, said large subunit RuBisCO, and said carbonic anhydrase is a STAS domain.
The method of claim 77 wherein said small subunit RuBisCO, said large subunit RuBisCO, and said carbonic anhydrase are encoded by the same expression cassette.
The method of claim 69 wherein said promoter sequence is a chloroplast promoter.
The method of claim 69, wherein the expression cassette is introduced by a method selected from one of the following: electroporation, micro-projectile bombardment and Agrobacterium-mediated transfer.
An isolated polynucleotide comprising a nucleotide sequence encoding a fusion protein of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) and a protein-protein interaction domain.
The isolated polynucleotide of claim 84 wherein said RuBisCO sequence further comprises: (a) a polynucleotide of SEQ ID NO:82; (b) a polynucleotide having at least 90% sequence identity across the entire sequence to SEQ ID NO:82; (c) a polynucleotide amplified from a nucleic acid library using primers which selectively hybridize, under stringent hybridization conditions, to a sequence within a polynucleotide of SEQ ID NO:82; or (d) a polynucleotide which is a full length complement of a polynucleotide of (a), (b), or (c).
The photosynthetic organism of claim 84 wherein said protein-protein interaction domain of said fusion protein is a STAS domain.
The photosynthetic organism of claim 84 further comprising a second heterologous polynucleotide sequence which encodes a high activity carbonic anhydrase operably linked to a promoter sequence.
The photosynthetic organism of claim 84 wherein said heterologous polynucleotide sequence further comprises a sequence that encodes a high activity carbonic anhydrase operably linked to a promoter sequence.
The photosynthetic organism of claim 86 wherein said second recombinant polynucleotide construct further encodes a protein-protein interaction domain that forms a protein-protein interaction pair with the protein-protein interaction domain of the RuBisCO fusion protein.
The photosynthetic organism of claim 87 wherein said high activity carbonic anhydrase comprises a human carbonic anhydrase II.
The photosynthetic organism of claim 87 wherein said high activity carbonic anhydrase comprises a polynucleotide having at least 90% sequence identity across the entire sequence to SEQ ID NO:1.
The photosynthetic organism. of claim 84 wherein said RuBisCO is a large subunit RuBisCO.
The photosynthetic organism of claim 84 wherein said RuBisCO is a small subunit RuBisCO.
The photosynthetic organism of claim 92 further comprising a heterologous polynucleotide sequence that encodes a RuBisCO large subunit and a heterologous polynucleotide sequence that encodes a high activity carbonic anhydrase.
The photosynthetic organism of claim 94 wherein the heterologous polynucleotide sequence encoding at least two of said small subunit RuBisCO, said large subunit RuBisCO, and said carbonic anhydrase also encodes a protein-protein interaction domain.
The photosynthetic organism of claim 96 wherein the protein-protein interaction domain encoded by the heterologous polynucleotide sequence encoding at least two of said small subunit RuBisCO, said large subunit RuBisCO, and said carbonic anhydrase is a STAS domain.
The photosynthetic organism of claim 95 wherein said small subunit RuBisCO, said large subunit RuBisCO, and said carbonic anhydrase are encoded by the same heterologous polynucleotide.
The photosynthetic organism of claim 84 wherein said promoter sequence is a chloroplast promoter.
A plant part or tissue of the photosynthetic organism of claim 84.
Description:
CROSS REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of U.S. provisional patent application No. 61/327,717 filed on Apr. 25, 2010, the entire contents of which are incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with US government support. The government has certain rights in the invention.TECHNICAL FIELDThe present invention relates generally to methods and constructs for enhancing inorganic carbon fixation in photosynthetic organisms.BACKGROUND OF THE INVENTIONOne of the major constraints limiting photosynthetic efficiency in algae and many crop plants is the competitive inhibition of CO2 fixation by oxygen at the active site of Ribulose-1,5-bisphosphate carboxylase oxygenase (RubisCO). In plants such as these (“C3” plants), RubisCO catalyzes the primary fixation of CO2 in the Calvin cycle leading to the production of two molecules of the 3-carbon product 3-phosphoglycerate (3-PGA). However in such C3 plants when oxygen is present, RubisCO can also accept oxygen producing 2-phosphoglycolate and 3-PGA. 2-phosphoglycolate is subsequently metabolized by the photorespiratory pathway leading to the loss of one previously fixed carbon as CO2 and the generation of one molecule of 3-phosphoglycerate from two molecules of phosphoglycolate. Moreover the photorespiratory pathway not only losses previously fixed carbon as CO2 it also reduces the regeneration of ribulose-1,5-bisphosphate (RuBP), the substrate for RubisCO. Overall, the competitive inhibition of CO2 fixation by oxygen and the associated photorespiratory pathway reduce carbon fixation efficiency by 30% or more in C3 plants.One way to reduce the competition of O2 for CO2 fixation is to increase the CO2 concentration at the active site of RubisCO. Certain plants (“C4 plants”) effectively do this by pumping CO2 into bundle sheath chloroplast. CO2 is initially fixed by the cytoplasmic enzyme PEP carboxylase localized in the outer mesophyll cells and the resulting 4-carbon dicarboxylic acids are shunted to the bundle sheath cells where they are decarboxylated. Importantly, PEP carboxylase does not fix oxygen and has a higher Kcat for CO2 than RubisCO. The CO2 resulting from C4 acid decarboxylation elevates the CO2 concentration around RubisCO (localized in bundle sheath cell chloroplasts) by 10-fold inhibiting the oxygenase reaction and photorespiration pathway.Similarly, Cyanobacteria concentrate CO2 near RubisCO to inhibit the RubisCO oxygenase reaction. In Cyanobacteria, bicarbonate, the non-gaseous hydrated form of CO2 is pumped into the cell and concentrated in an energy-dependent manner. In the carboxysomes, which is a protein assemblage of carbonic anhydrase (CA), RubisCO activase and RubisCO, CA accelerates the conversion of bicarbonate to CO2, the substrate for RubisCO. The close association of CA with RubisCO reduces the distance over which CO2 must diffuse before contacting RubisCO, and effectively elevates the local CO2 concentration around RubisCO inhibiting photorespiration. In some eukaryotic algae, a structure similar to the carboxysome, the chloroplastic pyrenoid body, carries out a similar function. Eukaryotic algae also pump and concentrate bicarbonate into the cell/chloroplast where it is fixed by RubisCO (reviewed by Spalding, (2008) J. Exp. Bot. 59(7): ).Carbonic anhydrases also play an important role in CO2 fixation during photosynthesis, particularly in plants where a substantial portion of the dissolve inorganic carbon dioxide in cells is present as bicarbonate. This is attributable to the fact that under physiological conditions (i.e. at pH 8.0 and 25° C.), the spontaneous rate of conversion of bicarbonate into CO2 is significantly slower than the rate of photosynthetic carbon fixation.In fact it has been calculated that the spontaneous rate of conversion of bicarbonate to CO2 is approximately 10,000 times slower (0.5×μM CO2 s-1) than the rate of photosynthetic CO2 fixation (2.8 mM CO2 s-1) (Badger and Price, (1994) Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 369-92). Accordingly to enhance physiological rates of CO2 fixation significantly more rapid rates of CO2 production from bicarbonate are required.Consistent with this conclusion, in C4 plants and algae, the presence of carbonic anhydrases has been demonstrated to have a substantial stimulatory effect on photosynthetic carbon fixation. This is due, at least in part to the fact that bicarbonate represents a substantial fraction of the total inorganic carbon in these cells. By comparison, in C3 plants, which do not pump bicarbonate or elevate internal CO2 or bicarbonate concentrations, the expression of carbonic anhydrases alone would be predicted to have only a relatively slight impact on the overall rate of carbon fixation. CA (Badger and Price, (1994) Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 369-92).The two different mechanisms of concentrating CO2 that have evolved in C4 plants and Cyanobacteria, suggests that this approach to improving photosynthetic efficiency provides a significant selective advantage. Accordingly these well-studied photosynthetic systems have led researchers to consider the usefulness of such approaches in other species that lack these CO2 concentrating mechanisms.For example, currently there is a large effort to improve the yield of C3 plants such as rice by redesigning these plants at the cellular level to include C4 photosynthetic pathway and Kranz anatomy (See for example, Sage and Sage (2009) Plant and Cell Physiol. 50 (4):756-772; Zhu et al., (2010) J. Interg. Plant Biol. 52 (8):762-770; Furbank et al., (2009) Funct. Plant Biol. 36 (11):845-856; Weber and von Caemmerer (2010) Cum Opin. Plant Biol. 13 (3):257-265).Additionally other strategies to improve carbon fixation rates include the use of directed evolution strategies to improve the kinetic properties of RubisCO by improving the rate of catalysis (Kcat) and/or the affinity for CO2 (lower Km), as described by Stemmer et al. (US
A1).Another strategy has been to overexpress a carbonic anhydrase, an enzyme that catalyzes the conversion of bicarbonate to CO2, as described by Edgerton et al. (US
A1), or to fuse carbonic anhydrase to a RubisCO-binding protein in order to increase the local concentration of CO2 at the active site of RubisCO, as described by Houtz (US
A1).Another strategy has been to express a bicarbonate transporter to raise levels of intracellular bicarbonate, as described by Kaplan et al. (US
A1) and Edgerton et al. (US
A1).While these strategies have been to some extend effective, there remains the need for simple and reliable methods to increase improve carbon fixation rates across all photosynthetic organisms. The present invention, by exploiting the use of protein-protein interaction domains fused to RuBisCO, enables the formation of a functional complex between RubisCO and carbonic anhydrase. Surprisingly, the RubisCO fusion protein can still functionally associate with other large and small RuBisCO subunits to form a fully functional complex which is capable of high efficiency carbon fixation. Furthermore co-expression of a high activity carbonic anhydrase enables the local concentration of carbon dioxide in the immediate vicinity of RubisCO to be significantly increased, thereby decreasing competitive inhibition of CO2 fixation by oxygen. As a result, the overall rate of carbon fixation is significantly increased.SUMMARY OF THE INVENTIONOne embodiment includes a method of increasing the efficiency of carbon dioxide fixation in a photosynthetic organism, comprising the steps of:
i) providing a carbonic anhydrase enzyme which either a) inherently comprises a first protein-protein interaction domain partner, or b) is fused in frame to a first heterologous protein-prii) providing a fusion protein comprising a RubisCO protein subunit fused in frame to a second protein-protein wherein the first protein-protein interaction partner and said second protein-protein interaction partner, or the first heterologous protein-protein domain partner and the second protein-protein interaction partner can associate to fo and iii) expressing the carbonic anhydrase enzyme and the fusion protein in a chloroplast within the photosynthetic organism.
In some embodiments, the carbonic anhydrase enzyme comprises a sequence selected from Tables D2 to D5. In some embodiments, the second protein interaction domain partner is a STAS domain. In some embodiments, the carbonic anhydrase enzyme has a Kcat/Km of from about 1×107 M-1s-1 to about 1.5×108 M-1s-1. In some embodiments, the carbonic anhydrase is codon optimized for the photosynthetic organism. In some embodiments, the carbonic anhydrase is a human carbonic anhydrase II. In some embodiments, the carbonic anhydrase comprises SEQ. ID. No. 1. In some embodiments, the RubisCO protein subunit is the large subunit of RubisCO. In some embodiments, the RubisCO protein subunit is the small subunit of RubisCO.In some embodiments, the second fusion protein comprises a RubisCO large protein subunit fused in frame to a STAS wherein the method further includes a third fusion protein comprising a RubisCO small protein subunit fused in frame to a STAS and wherein the method further comprises the step of expressing the first fusion protein, the second fusion protein, and the third fusion protein in a chloroplast within the photosynthetic organism.Another embodiment includes a transgenic organism comprising:
i) a first nucleic acid sequence comprising a first heterologous polynucleotide sequence encoding a carbonic anhydrase enzyme which either a) inherently comprises a first protein-protein interaction domain partner, or b) is fused in frame to a first heterologous protein-prii) a second nucleic acid sequence comprising a second heterologous polynucleotide sequence encoding a RubisCO protein subunit operatively coupled to a second protein-protein wherein the first protein-protein interaction partner and said second protein-protein interaction partner, or the first heterologous protein-protein domain partner and the second protein-protein interaction partner can associate to form a protein complex.
In some embodiments, the carbonic anhydrase enzyme has a Kcat/Km of from about 1×107 M-1s-1 to about 1.5×108 M-1s-1. In some embodiments, the carbonic anhydrase is codon optimized for the photosynthetic organism. In some embodiments, the carbonic anhydrase is a human carbonic anhydrase II. In some embodiments, the carbonic anhydrase enzyme comprises a sequence selected from Tables D2 to D5. In some embodiments, the second protein interaction domain partner is a STAS domain. In some embodiments, the carbonic anhydrase comprises SEQ. ID. No. 1. In some embodiments, the first heterologous polynucleotide sequence is operatively coupled to a leaf specific promoter. In some embodiments, the first heterologous polynucleotide sequence is operatively coupled to a CAB1 promoter. In some embodiments, the second heterologous polynucleotide sequence is operatively coupled to a leaf specific promoter. In some embodiments, the second heterologous polynucleotide sequence is operatively coupled to a Cab1 promoter. In some embodiments, the RubisCO protein subunit is the large subunit of RubisCO. In some embodiments, the RubisCO protein subunit is the small subunit of RubisCO.In some embodiments, the transg a) a second nucleic acid sequence comprising a second heterologous polynucleotide sequence encoding a RubisCO large protein subunit fused in frame to a STAS domain, and b) a third nucleic acid sequence comprising a third heterologous polynucleotide sequence encoding a RubisCO small protein subunit fused in frame to a STAS domain.In some embodiments, the transgenic plant is a C3 plant. In some embodiments, the transgenic plant is selected from the from the group c cereals including wheat, beans including mung bean, starch-storing plants including potato, cass oil-storing plants including soybean, rape, sunflo vegetables including tomato, cucumber, eggplant, carrot, hot pepper, Chinese cabbage, radish, water melon, cucumber, melon, crown daisy, spinach, ca garden plants including chrysanthemum, rose, carnation and petunia and Arabidopsis, and trees.In some embodiments, the transgenic organism is an eukaryotic alga. In some embodiments, the transgenic plant is a C4 plant.In some embodiments, the transgenic organism exhibits an increased growth rate and/or biomass of at least about any of: 10%, 12%, and 15%, as compared to a control host. In some embodiments, the transgenic organism exhibits an increased growth rate and/or biomass of at least about any of: 10%, 20%, 25%, 50%, 100%, and 200%, as compared to a control host.In some embodiments, the transgenic organism exhibits a decrease in oxygenase activity catalyzed by RubisCO of at least about any of: 10%, 20%, 25%, 50%, 100%, and 200% as compared to a control host. In some embodiments, the transgenic organism exhibits an increase in carboxylase activity catalyzed by RubisCO of at least about any of: 10%, 20%, 25%, 50%, 100%, and 200%, as compared to a control host. In some embodiments, the transgenic organism exhibits an increase in the rate of carbon fixation of at least about any of: 10%, 20%, 25%, 50%, 100%, and 200%, as compared to a control host. In some embodiments, the transgenic organism exhibits an increase in the rate of oxygen evolution of at least about any of: 10%, 20%, 25%, 50%, 100%, and 200%, as compared to a control host. In some embodiments, the transgenic organism exhibits an increase in ATP levels of at least about any of: 10%, 20%, 25%, 50%, 100%, and 200%, as compared to a control host.Another embodiment includes an expression vector comprising:
i) a first nucleic acid sequence comprising a first heterologous polynucleotide sequence encoding a carbonic anhydrase enzyme which either a) inherently comprises a first protein-protein interaction domain partner, or b) is fused in frame to a first heterologous protein-prii) a second nucleic acid sequence comprising a second heterologous polynucleotide sequence encoding a RubisCO protein subunit operatively coupled to a second protein-protein wherein the first protein-protein interaction partner and said second protein-protein interaction partner, or the first heterologous protein-protein domain partner and the second protein-protein interaction partner can associate to form a protein complex.
In some embodiments, the carbonic anhydrase is codon optimized for the photosynthetic organism. In some embodiments, the carbonic anhydrase is a human carbonic anhydrase II. In some embodiments, the carbonic anhydrase enzyme comprises a sequence selected from Tables D2 to D5. In some embodiments, the second protein interaction domain partner is a STAS domain. In some embodiments, the carbonic anhydrase comprises SEQ. ID. No. 1. In some embodiments, the first heterologous polynucleotide sequence is operatively coupled to a leaf specific promoter. In some embodiments, the first heterologous polynucleotide sequence is operatively coupled to a CAB1 promoter. In some embodiments the second heterologous polynucleotide sequence is operatively coupled to a leaf specific promoter. In some embodiments, the second heterologous polynucleotide sequence is operatively coupled to a CAB1 promoter. In some embodiments, the RubisCO protein subunit is the large subunit of RubisCO. In some embodiments, the RubisCO protein subunit is the small subunit of RubisCO.Another embodiment includes method of producing a product from biomass from a photosynthetic organism comprising the steps of:
i) expressing a first nucleic acid sequence comprising a first heterologous polynucleotide sequence encoding a carbonic anhydrase enzyme which either a) inherently comprises a first protein-protein interaction domain partner, or b) is fused in frame to a first heterologous protein-prii) expressing a second nucleic acid sequence comprising a second heterologous polynucleotide sequence encoding a RubisCO protein subunit operatively coupled to a second protein-proteinwherein the first protein-protein interaction partner and said second protein-protein interaction partner, or the first heterologous protein-protein domain partner and the second protein-protein interaction partner can associate to foiii) growing the andiv) harvesting the biomass.
In some embodiments, the product is selected from the group consisting of starches, oils, lipids, fatty acids, cellulose, carbohydrates, alcohols, sugars, nutraceuticals, pharmaceuticals and organic acids. In some embodiments, the transgenic organism is an eukaryotic algae. In some embodiments, the transgenic organism is a C3 plant. In some embodiments, the transgenic organism is a C4 plant.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 Shows an exemplary vector for creating an rbcL deletion host.FIG. 2 Shows an exemplary expression vector for expressing a codon optimized human carbonic anhydrase (hs CAII) in the stroma of a chloroplast.FIG. 3 Shows the nucleic acid, and translated amino acid sequence for an exemplary CA expression cassette for expression of a codon optimized human CA for expression in Chlamydomonas cells with ATP promoter and Rbc terminator.FIG. 4 Shows the Relative colony growth of transgenic Chlamydomonas cells expressing Human CA-II and wild-type cells (—CA).FIG. 5 Shows the Relative colony growth of transgenic Chlamydomonas cells expressing Human CA-II and wild-type cells (—CA) when grown at pH 8.5.FIG. 6 depicts oxygen evolution from a photosynthetic host transformed with a CA and a control host.FIG. 7 shows an exemplary RubisCO (RbcL) large subunit-STAS fusion protein construct.FIG. 8 an exemplary expression vector for expressing a codon optimized human carbonic anhydrase (hs CAII) and RubisCO-STAS fusion proteins in the stroma of a chloroplast.DETAILED DESCRIPTION OF THE INVENTIONIn order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. As used herein and in the appended claims, the singular forms “a,” “an,” and “the,” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a molecule” includes one or more of such molecules, “a reagent” includes one or more of such different reagents, reference to “an antibody” includes one or more of such different antibodies, and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.The terms “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or 2 standard deviations, from the mean value. Alternatively, “about” can mean plus or minus a range of up to 20%, preferably up to 10%, more preferably up to 5%.As used herein, the terms “cell,” “cells,” “cell line,” “host cell,” and “host cells,” are used interchangeably and, encompass animal cells and include plant, invertebrate, non-mammalian vertebrate, insect, algal, and mammalian cells. All such designations include cell populations and progeny. Thus, the terms “transformants” and “transfectants” include the primary subject cell and cell lines derived therefrom without regard for the number of transfers.The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag).Examples of amino acid groups defined in this manner include: a “charged/polar group,” consisting of Glu, Asp, Asn, Gln, Lys, Arg and H an “aromatic, or cyclic group,” consisting of Pro, Phe, Tyr and T and an “aliphatic group” consisting of Gly, Ala, Val, Leu, Ile, Met, Ser, Thr and Cys.Within each group, subgroups can also be identified, for example, the group of charged/polar amino acids can be sub-divided into the sub-groups consisting of the “positively-charged sub-group,” consisting of Lys, Arg and H the negatively-charged sub-group,” consisting of Glu and Asp, and the “polar sub-group” consisting of Asn and Gln. The aromatic or cyclic group can be sub-divided into the sub-groups consisting of the “nitrogen ring sub-group,” consisting of Pro, His and T and the “phenyl sub-group” consisting of Phe and Tyr. The aliphatic group can be sub-divided into the sub-groups consisting of the “large aliphatic non-polar sub-group,” consisting of Val, Leu and I the “aliphatic slightly-polar sub-group,” consisting of Met, Ser, Thr and C and the “small-residue sub-group,” consisting of Gly and Ala.Examples of conservative mutations include substitutions of amino acids within the sub-groups above, for example, Lys for Arg and vice versa such that a positive char Glu for Asp and vice versa such that a negative char Ser for Thr such that a free —OH and Gln for Asn such that a free —NH2 can be maintained.The term “expression” as used herein refers to transcription and/or translation of a nucleotide sequence within a host cell. The level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired polypeptide encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantified by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to cellular RNA or by PCR. Proteins encoded by a selected sequence can be quantified by various methods including, but not limited to, e.g., ELISA, Western blotting, radioimmunoassays, immunoprecipitation, assaying for the biological activity of the protein, or by immunostaining of the protein followed by FACS analysis.“Expression control sequences” are regulatory sequences of nucleic acids, such as promoters, leaders, transit peptide sequences, enhancers, introns, recognition motifs for RNA, or DNA binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES) and the like, that have the ability to affect the transcription, targeting, or translation of a coding sequence in a host cell. Exemplary expression control sequences are described in G Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).A “gene” is a sequence of nucleotides which code for a functional gene product. Generally, a gene product is a functional protein. However, a gene product can also be another type of molecule in a cell, such as RNA (e.g., a tRNA or an rRNA). A gene may also comprise expression control sequences (i.e., non-coding) sequences as well as coding sequences and introns. The transcribed region of the gene may also include untranslated regions including introns, a 5′-untranslated region (5′-UTR) and a 3′-untranslated region (3′-UTR).The term “heterologous” refers to a nucleic acid or protein which has been introduced into an organism (such as a plant, animal, or prokaryotic cell), or a nucleic acid molecule (such as chromosome, vector, or nucleic acid), which are derived from another source, or which are from the same source, but are located in a different (i.e. non native) context.The term “homology” describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs. The nucleic acid and protein sequences of the present invention can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members, related sequences or homologs. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention.To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and BLAST) can be used.The term “homologous” refers to the relationship between two proteins that possess a “common evolutionary origin”, including proteins from superfamilies (e.g., the immunoglobulin superfamily) in the same species of animal, as well as homologous proteins from different species of animal (for example, myosin light chain polypeptide, etc.; see Reeck et al., (1987) Cell, 50:667). Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.As used herein, the term “increase” or the related terms “increased”, “enhance” or “enhanced” refers to a statistically significant increase. For the avoidance of doubt, the terms generally refer to at least a 10% increase in a given parameter, and can encompass at least a 20% increase, 30% increase, 40% increase, 50% increase, 60% increase, 70% increase, 80% increase, 90% increase, 95% increase, 97% increase, 99% or even a 100% increase over the control value.The term “isolated,” when used to describe a protein or nucleic acid, means that the material has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with research, diagnostic or therapeutic uses for the protein or nucleic acid, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the protein or nucleic acid will be purified to at least 95% homogeneity as assessed by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated protein includes protein in situ within recombinant cells, since at least one component of the protein of interest's natural environment will not be present. Ordinarily, however, isolated proteins and nucleic acids will be prepared by at least one purification step.As used herein, “identity” means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: ). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs.Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, Calif., United States of America), or by visual inspection. See generally, (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25:
(1997)).One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in (Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; & Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair
0) and N (penalty score for
0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the -27 cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W. T. and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix.In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in one embodiment less than about 0.1, in another embodiment less than about 0.01, and in still another embodiment less than about 0.001.The terms “operably linked”, “operatively linked,” or “operatively coupled” as used interchangeably herein, refer to the positioning of two or more nucleotide sequences or sequence elements in a manner which permits them to function in their intended manner. In some embodiments, a nucleic acid molecule according to the invention includes one or more DNA elements capable of opening chromatin and/or maintaining chromatin in an open state operably linked to a nucleotide sequence encoding a recombinant protein. In other embodiments, a nucleic acid molecule may additionally include one or more DNA or RNA nucleotide sequences chosen from: (a) a nucleotide sequence capable of in (b) a nucleotide sequence capable of increasing secretion of the recombinant pr (c) a nucleotide sequence capable of increasing the mRNA stability, and (d) a nucleotide sequence capable of binding a trans-acting factor to modulate transcription or translation, where such nucleotide sequences are operatively linked to a nucleotide sequence encoding a recombinant protein. Generally, but not necessarily, the nucleotide sequences that are operably linked are contiguous and, where necessary, in reading frame. However, although an operably linked DNA element capable of opening chromatin and/or maintaining chromatin in an open state is generally located upstream of a nucleotide sequence encoding a it is not necessarily contiguous with it. Operable linking of various nucleotide sequences is accomplished by recombinant methods well known in the art, e.g. using PCR methodology, by ligation at suitable restrictions sites or by annealing. Synthetic oligonucleotide linkers or adaptors can be used in accord with conventional practice if suitable restriction sites are not present.The terms “polynucleotide,” “nucleotide sequence” and “nucleic acid” are used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. In addition, a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer. A nucleic acid molecule can take many different forms, e.g., a gene or gene fragment, one or more exons, one or more introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches. As used herein, a polynucleotide includes not only naturally occurring bases such as A, T, U, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides.A “promoter” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. As used herein, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. A transcription initiation site (conveniently defined by mapping with nuclease S1) can be found within a promoter sequence, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.A large number of promoters, including constitutive, inducible and repressible promoters, from a variety of different sources are well known in the art. Representative sources include for example, viral, mammalian, insect, plant, yeast, and bacterial cell types, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available on line or, for example, from depositories such as the ATCC as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3′ or 5′ direction). Non-limiting examples of promoters active in plants include, for example nopaline synthase (nos) promoter and octopine synthase (ocs) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens and the caulimovirus promoters such as the Cauliflower Mosaic Virus (CaMV) 19S or 35S promoter (U.S. Pat. No. 5,352,605), CaMV 35S promoter with a duplicated enhancer (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938; 5,359,142; and 5,424,200), the Figwort Mosaic Virus (FMV) 35S promoter (U.S. Pat. No. 5,378,619), and the cassaya vein mosaic virus promoter (U.S. Pat. No. 7,601,885). These promoters and numerous others have been used in the creation of constructs for transgene expression in plants or plant cells. Other useful promoters are described, for example, in U.S. Pat. Nos. 5,391,725; 5,428,147; 5,447,858; 5,608,144; 5,614,399; 5,633,441; 6,232,526; and 5,633,435, all of which are incorporated herein by reference.The term “purified” as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell. Methods for purification are well-known in the art. As used herein, the term “substantially free” is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% more preferably, at least 75% pure, and more preferably still at least 95% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art. The term “substantially pure” indicates the highest degree of purity, which can be achieved using conventional purification techniques known in the art.The term “sequence similarity” refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin. However, in common usage and in the instant application, the term “homologous”, when modified with an adverb such as “highly”, may refer to sequence similarity and may or may not relate to a common evolutionary origin.In specific embodiments, two nucleic acid sequences are “substantially homologous” or “substantially similar” when at least about 85%, and more preferably at least about 90% or at least about 95% of the nucleotides match over a defined length of the nucleic acid sequences, as determined by a sequence comparison algorithm known such as BLAST, FASTA, DNA Strider, CLUSTAL, etc. An example of such a sequence is an allelic or species variant of the specific genes of the present invention. Sequences that are substantially homologous may also be identified by hybridization, e.g., in a Southern hybridization experiment under, e.g., stringent conditions as defined for that particular system.In particular embodiments of the invention, two amino acid sequences are “substantially homologous” or “substantially similar” when greater than 90% of the amino acid residues are identical. Two sequences are functionally identical when greater than about 95% of the amino acid residues are similar. Preferably the similar or homologous polypeptide sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Version 7, Madison, Wis.) pileup program, or using any of the programs and algorithms described above. The program may use the local homology algorithm of Smith and Waterman with the default values: Gap creation penalty=-(1+1/k), k being the gap extension number, Average match=1, Average mismatch=-0.333.As used herein, a “transgenic plant” is one whose genome has been altered by the incorporation of heterologous genetic material, e.g. by transformation as described herein. The term “transgenic plant” is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a transgenic plant, so long as the progeny contains the heterologous genetic material in its genome.The term “transformation” or “transfection” refers to the transfer of one or more nucleic acid molecules into a host cell or organism. Methods of introducing nucleic acid molecules into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, scrape loading, ballistic introduction, or infection with viruses or other infectious agents.“Transformed”, “transduced”, or “transgenic”, in the context of a cell, refers to a host cell or organism into which a recombinant or heterologous nucleic acid molecule (e.g., one or more DNA constructs or RNA, or siRNA counterparts) has been introduced. The nucleic acid molecule can be stably expressed (i.e. maintained in a functional form in the cell for longer than about three months) or non-stably maintained in a functional form in the cell for less than three months i.e. is transiently expressed. For example, “transformed,” “transformant,” and “transgenic” cells have been through the transformation process and contain foreign nucleic acid. The term “untransformed” refers to cells that have not been through the transformation process.The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory P Ausubel, F. M. et al. (1995 and
Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons J. M. Polak and James O′D. McGee, 1990, In Situ Hybridization: Principles and P Oxford University P M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl P D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic P Buchanan et al., Biochemistry and Molecular Biology of Plants, Courier Companies, USA, 2000; Mild and Iyer, Plant Metabolism, 2nd Ed. D. T. Dennis, D H Turpin, D D Lefebrve, D G Layzell (eds) Addison Wesly, Langgmans Ltd. London (1997); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-. Each of these general texts is herein incorporated by reference.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods, compositions, reagents, cells, similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are described herein.The publications discussed above are provided solely for their disclosure before the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and reference.I. OverviewThe present invention relates to transgenic strategies for enhancing carbon fixation in a photosynthetic organism by concentrating CO2 in the microenvironment of RubisCO. As detailed herein, the co-expression of Carbonic anhydrase with RubisCo within the chloroplasts of plants results in an increase in the carboxylase activity and/or decrease in oxygenase activity of RubisCO.In certain embodiments, the RubsiCO is fused to a protein-protein interaction domain that mediated the formation of a complex of RubisCO and carbonic anhydrase that results in a significant enhance in carbon dioxide fixation rate and biomass yield.II. Carbonic AnhydraseCarbonic anhydrases (CA) are zinc-containing metalo-enzymes found ubiquitously throughout nature in prokaryotes and eukaryotes. Carbonic anhydrases catalyses the reversible hydration of CO2 to bicarbonate and play a central role in controlling pH balance and inorganic carbon sequestration and flux in many organisms. The carbonic anhydrases are a diverse group of proteins but can be divided into four evolution the α-CAs (found in vertebrates, bacteria, algae and cytoplasm of green plants); β-CAs (found in bacteria, algae and chloroplasts); —CAs (found in archaea and bacteria); and δ-CAs (found in marine diatoms). (Supuran, (2008) Curr. Pharma. Des. 14: 603-614).There are approximately 16 different classes of α-CAs found in mammals (See Table D1), and these, as well as any of the homologous genes from other organisms are potentially suitable for use in any of the claimed methods, DNA constructs, and transgenic plants.TABLE D1Kcat/KcatKmKmKiSubcellularTissue/organIsoenzyme(s-1)(mM)(M-1s-1)(nM)localizationlocalizationhCAI
2 × 1054.05.0 × 107250cytosolE, GIhCAII1.4 × 1069.31.5 × 10812cytosolE, eye, GI, BO,K, L, T, BhCAIII1.0 × 10433.33.0 × 1052 × 105cytosolSM, AhCAIV1.0 × 10621.55.1 × 10774membraneK, L, P, B, C, HhCAVA2.9 × 10510.02.9 × 10763mitochondriaLihCAVB9.5 × 1059.79.8 × 10754mitochondriaH, SM, P, K,SC, GIhCAVI3.4 × 1056.94.9 × 10711secretedGhCAVII9.5 × 10511.48.3 × 1072.5cytosolCNShCAVIIIcytosolCNShCAIX3.8 × 1056.95.5 × 10725transmembraneTU, GIhCAXcytosolCNShCAXIcytosolCNShCAXII4.2 × 10512.03.5 × 1075.7transmembraneR, I, RE, eye,TUhCAXIII1.5 × 10513.81.1 × 10716cytosolK, B, L, GI, REhCAXIV3.1 × 1057.93.9 × 10741transmembraneK, B, LhCAXV4.7 × 10514.23.3 × 10772membraneKH = H M = M hCAVIII, X, and XI are devoid of catalytic activity. E = E GI = GI BO = B K = kidney, L = L T = B = SM = A = A P = C = H = Li = SC = G = saliva R = I = TU = tumors, RE = Reproductive In any of these methods, DNA constructs, and transgenic organisms, the terms “CA” or “carbonic anhydrase” refers to all naturally-occurring and synthetic genes encoding carbonic anhydrase. In one aspect, the carbonic anhydrase gene is from a plant. In one aspect the carbonic anhydrase is from a mammal. In one aspect, the carbonic anhydrase is from a human. In one aspect the carbonic anhydrase can bind to a STAS domain. In one aspect the carbonic anhydrase is naturally expressed within the cytosol or is secreted. In one aspect the carbonic anhydrase has a Kcat/Km of greater than about 1×107 M-1s-1. In one aspect the carbonic anhydrase has a Kcat/Km of greater than about 2×107 M-1s-1. In one aspect the carbonic anhydrase has a Kcat/Km of greater than about 5×107 M-1s-1. In one aspect the carbonic anhydrase has a Kcat/Km of greater than about 1×108 M-1s-1. Representative species, Gene bank accession numbers, and amino acid sequences for various species of suitable CA genes are listed below in Tables D2-D4.TABLE D2 Exemplary Type II Carbonic AnhydrasesAccessionSEQ. IDOrganismSequenceNumberNOHumanMSHHWGYGKH NGPEHWHKDF PIAKGERQSPNP_SEQ. ID.VDIDTHTAKY DPSLKPLSVS YDQATSLRILNO. 1NNGHAFNVEF DDSQDKAVLK GGPLDGTYRLIQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVGSAKPGLQKVV DVLDSIKTKG KSADFTNFDPRGLLPESLDY WTYPGSLTTP PLLECVTWIVLKEPISVSSE QVLKFRKLNF NGEGEPEELMVDNWRPAQPL KNRQIKASFK MacacaMSHHWGYGKH NGPEHWHKDF PIAKGQRQSPBAE91302.1SEQ. ID.fascicularisVDIDTHTAKY DPSLKPLSVS YDQATSLRILNO. 2(crab-eatingNNGHSFNVEF DDSQDKAVIK GGPLDGTYRLmacaque)IQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVGSAKPGLQKVV DVLDSIKTKG KSADFTNFDPRGLLPESLDY WTYPGSLTTP PLLECVTWIVLKEPISVSSE QMSKFRKLNF NGEGEPEELMVDNWRPAQPL KNRQIKASFK Pan troglodytesMSHHWGYGKH NGPEHWHKDF PIAKGERQSPNP_SEQ. ID.VDIDTHTAKY DPSLKPLSVS YGQATSLRILNO.3NNGHAFNVEF DDSQDKAVLK GGPLDGTYRLIQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVGSAKPGLQKVV DVLDSIKTKG KSADFTNFDPHGLLPESLDY WTYPGSLTTP PLLECVTWIVLKEPISVSSE QMLKFRKLNF NGEGEPEELMVDNWRPAQPL KNRQIKASFK Macaca mulattaMSHHWGYGKH NGPEHWHKDF PIAKGQRQSPNP_SEQ. ID.VDINTHTAKY DPSLKPLSVS YDQATSLRILNO. 4NNGHSFNVEF DDSQDKAVIK GGPLDGTYRLIQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVGSAKPGLQKVV DVLDSIKTKG KSADFTNFDPRGLLPESLDY WTYPGSLTTP PLLECVTWIVLKEPISVSSE QMSKFRKLNF NGEGEPEELMVDNWRPAQPL KNRQIKASFK Pongo abeliiMSHHWGYGKH NGPEHWHKDF PIAKGERQSPXP_SEQ. ID.VDIDTHTAKY DPSLKPLSVC YDQATSLRILNO. 5NNGHSFNVEF DDSQDKAVLK GGPLDGTYRLIQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVGSAKPGLQKVV DVLDSIKTKG KCADFTNFDPRGLLPASLDY WTYPGSLTTP PLLECVTWIVLKEPISVSSE QMLKFRKLNF NGEGEPEELMVDNWRPAQPL KKRQIKASFK CallithrixMSHHWGYGKH NGPEHWHKDF PIAKGERQSPXP_SEQ. ID.jacchusVDIDTHTAKY DPSLKPLSVS YDQATSWRILNO. 6NNGHSFNVEF DDSQDKAVLK GGPLDGTYRLIQFHFHWGST DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAAQQPDGL AVLGIFLKVGSAKPGLQKVV DVLDSIKTKG KSADFTNFDPRGLLPESLDY WTYPGSLTTP PLLESVTWIVLKEPISVSSE QILKFRKLNF SGEGEPEELMVDNWRPAQPL KNRQIKASFK Lemur cattaMSHHWGYGKH NGPEHWHKDF PIAKGERQSPADD83028SEQ. ID.VDINTGAAKH DPSLKPLSVY YEQATSRRILNO. 7NNGHSFNVEF DDSQDKAVLK GGPLDGTYRLIQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVGSAKPGLQKVV DVLDSIKTKG KSADFTNFDPRGLLPESLDY WTYLGSLTTP PLLECVTWIVLKEPISVSSE QMMKFRKLSF SGEGEPEELMVDNWRPAQPL KNRQIKASFK AiluropodaMAHHWGYGKH NGPEHWYKDF PIAKGQRQSPXP_SEQ. ID.melanoleucaVDIDTKAAIH DPALKALCPT YEQAVSQRVINO. 8NNGHSFNVEF DDSQDNAVLK GGPLTGTYRLIQFHFHWGSS DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKIGDARPGLQKVL DALDSIKTKG KSADFTNFDPRGLLPESLDY WTYPGSLTTP PLLECVTWIVLKEPISVSSE QMLKFRRLNF NKEGEPEELMVDNWRPAQPL HNRQINASFK Equus caballusMSHHWGYGQH NGPKHWHKDF PIAKGQRQSPXP_SEQ. ID.VDIDTKAAVH DAALKPLAVH YEQATSRRIVNO. 9NNGHSFNVEF DDSQDKAVLQ GGPLTGTYRLIQFHFHWGSS DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVVGVFLKVGGAKPGLQKVL DVLDSIKTKG KSADFTNFDPRGLLPESLDY WTYPGSLTTP PLLECVTWIVLREPISVSSE QLLKFRSLNF NAEGKPEDPMVDNWRPAQPL NSRQIRASFK Canis lupusMAHHWGYAKH NGPEHWHKDF PIAKGERQSPNP_SEQ. ID.familiarisVDIDTKAAVH DPALKSLCPC YDQAVSQRIINO. 10NNGHSFNVEF DDSQDKTVLK GGPLTGTYRLIQFHFHWGSS DGQGSEHTVD KKKYAAELHLVHWNTKYGEF GKAVQQPDGL AVLGIFLKIGGANPGLQKIL DALDSIKTKG KSADFTNFDPRGLLPESLDY WTYPGSLTTP PLLECVTWIVLKEPISVSSE QMLKFRKLNF NKEGEPEELMMDNWRPAQPL HSRQINASFK OryctolagusMSHHWGYGKH NGPEHWHKDF PIANGERQSPNP_SEQ. ID.cuniculusIDIDTNAAKH DPSLKPLRVC YEHPISRRIINO. 11NNGHSFNVEF DDSHDKTVLK EGPLEGTYRLIQFHFHWGSS DGQGSEHTVN KKKYAAELHLVHWNTKYGDF GKAVKHPDGL AVLGIFLKIGSATPGLQKVV DTLSSIKTKG KSVDFTDFDPRGLLPESLDY WTYPGSLTTP PLLECVTWIVLKEPITVSSE QMLKFRNLNF NKEAEPEEPMVDNWRPTQPL KGRQVKASFV AiluropodaGPEHWYKDFP IAKGQRQSPV DIDTKAAIHDEFB24165SEQ. ID.melanoleucaPALKALCPTY EQAVSQRVIN NGHSFNVEFDNO. 12DSQDNAVLKG GPLTGTYRLI QFHFHWGSSDGQGSEHTVDK KKYAAELHLV HWNTKYGDFGKAVQQPDGLA VLGIFLKIGD ARPGLQKVLDALDSIKTKGK SADFTNFDPR GLLPESLDYWTYPGSLTTPP LLECVTWIVL KEPISVSSEQMLKFRRLNFN KEGEPEELMV DNWRPAQPLHNRQINASFK Sus scrofaMSHHWGYDKH NGPEHWHKDF PIAKGDRQSPXP_SEQ. ID.VDINTSTAVH DPALKPLSLC YEQATSQRIVNO. 13NNGHSFNVEF DSSQDKGVLE GGPLAGTYRLIQFHFHWGSS DGQGSEHTVD KKKYAAELHLVHWNTKYKDF GEAAQQPDGL AVLGVFLKIGNAQPGLQKIV DVLDSIKTKG KSVEFTGFDPRDLLPGSLDY WTYPGSLTTP PLLESVTWIVLREPISVSSG QMMKFRTLNF NKEGEPEHPMVDNWRPTQPL KNRQIRASFQ CallithrixMSHHWGYGKH NGPEHWHKDF PIAKGERQSPXP_SEQ. ID.jacchusVDIDTHTAKY DPSLKPLSVS YDQATSWRILNO. 14NNGHSFNVEF DDSQDKAVLK GGPLDGTYRLIQLHLVHWNT KYGDFGKAAQ QPDGLAVLGIFLKVGSAKPG LQKVVDVLDS IKTKGKSADFTNFDPRGLLP ESLDYWTYPG SLTTPPLLESVTWIVLKEPI SVSSEQILKF RKLNFSGEGEPEELMVDNWR PAQPLKNRQI KASFK Mus musculusMSHHWGYSKH NGPENWHKDF PIANGDRQSPNP_033931SEQ. ID.VDIDTATAQH DPALQPLLIS YDKAASKSIVNO. 15NNGHSFNVEF DDSQDNAVLK GGPLSDSYRLIQFHFHWGSS DGQGSEHTVN KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKIGPASQGLQKVL EALHSIKTKG KRAAFANFDPCSLLPGNLDY WTYPGSLTTP PLLECVTWIVLREPITVSSE QMSHFRTLNF NEEGDAEEAMVDNWRPAQPL KNRKIKASFK Bos taurusMSHHWGYGKH NGPEHWHKDF PIANGERQSPNP_848667SEQ. ID.VDIDTKAVVQ DPALKPLALV YGEATSRRMVNO. 16NNGHSFNVEY DDSQDKAVLK DGPLTGTYRLVQFHFHWGSS DDQGSEHTVD RKKYAAELHLVHWNTKYGDF GTAAQQPDGL AVVGVFLKVGDANPALQKVL DALDSIKTKG KSTDFPNFDPGSLLPNVLDY WTYPGSLTTP PLLESVTWIVLKEPISVSSQ QMLKFRTLNF NAEGEPELLMLANWRPAQPL KNRQV