# Patent

## Title
Ribulose 1,5-bisphosphate carboxylase/oxygenase polypeptides and related polynucleotides

## Abstract

The present invention relates to novel ribulose-1,5-bisphosphate carboxylase/oxygenase polypeptides and the polynucleotides that encode them. The invention also provides related host cells and methods.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Ser. No. 60/375,910, filed Apr. 26, 2002, which is incorporated herein in its entirety.


COPYRIGHT NOTIFICATION
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
The present invention relates to novel ribulose-1,5-bisphosphate carboxylase/oxygenase polypeptides and the polynucleotides that encode them.
BACKGROUND OF THE INVENTION
Carbon fixation, or the conversion of CO2 to reduced forms amenable to cellular biochemistry, occurs by several metabolic pathways in diverse organisms. The most familiar of these is the Calvin Cycle (or “Calvin-Benson” cycle), which is present in cyanobacteria and their plastid derivatives (i.e., chloroplasts), as well as in proteobacteria. The Calvin cycle in these organisms utilizes the enzyme, ribulose-1,5-bisphosphate carboxylate/oxygenase (“Rubisco”). See, e.g., the world wide web at blc.Arizona.edu/courses/181gh/rick/photosynthesis/Calvin.html; Raven, et al. (1981) The Biology of Plants, 3rd Edition, Worth Publishers, Inc., NY, N.Y. Rubisco exists in at least two forms: Form I Rubisco, which is found in proteobacteria, cyanobacteria, and plastids; and Form II Rubisco, which is found in proteobacteria. Form I Rubisco is encoded by two genes encoding large and small subunits (rbcL and rbcS), and may exist as an octo-dimer composed of eight large subunits (rbcL) and eight small subunits (rbcS). Form II Rubisco is a dimeric form of the enzyme. Form II Rubisco has clear similarities to the large subunit of Form I Rubisco, and is encoded by a single gene, also referred to as rbcL. The evolutionary origin of the small subunit of Form I Rubisco remains uncertain; it is less highly conserved than the large subunit, and may have cryptic homology to a portion of the Form II protein.
All photosynthetic organisms catalyze the fixation of atmospheric CO2 by the bifunctional enzyme Rubisco. Significant variations in kinetic properties of this enzyme are found among various phylogenetic groups. Because of the abundance and fundamental importance of Rubisco, the enzyme has been extensively studied. Well over 1,000 different Rubisco homologues are available in the public literature and the crystal structure of Rubisco has been solved for several variants of the protein.
Rubisco contains two competing enzymatic activities: an oxygenase and a carboxylase activity. The oxygenation reaction catalyzed by Rubisco is considered a “wasteful” process because it competes with, and significantly reduces the net amount of carbon fixed by an organism. The Rubisco enzyme species encoded in various photosynthetic organisms have been selected by natural evolution to provide higher plants with a Rubisco enzyme that is substantially more efficient at carboxylation in the presence of atmospheric oxygen.
The creation of plants and other photosynthetic organisms having improved Rubisco biosynthetic pathways can provide increased yields of certain types of foodstuffs, enhanced biomass energy sources, and may alter the types and amounts of nutrients present in certain foodstuffs, among other desirable phenotypes. The development of technologies for effective biological fixation of CO2 on a global scale can mitigate the effects of atmospheric greenhouse gas emission. Cyanobacterial aquaculture (“cyanofarming”) offers one of the most productive solutions for global greenhouse gas control, as compared to other biological alternatives aimed at CO2 abatement technology for global use. However, it would be desirable to improve biomass productivity of cyanofarming by 10 to 20 fold over current production levels. Thus, a need exists for improved Rubisco enzymes.
BRIEF SUMMARY OF THE INVENTION
The present invention provides novel ribulose 1,5-bisphosphate carboxylase/oxygenase (“Rubisco”) polypeptides, including the large and small subunits. In particular, the present invention provides an isolated or recombinant Rubisco large subunit polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence that is at least 99% identical to SEQ ID NO: 5;
(b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 8;
(c) an amino acid sequence that is at least 97% identical to SEQ ID NO: 35; and
(d) an amino acid sequence that is at least 99% identical to SEQ ID NO: 11.
Specific Rubisco large subunit polypeptides of the present invention comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 50, and SEQ ID NO: 53.
Certain large subunit Rubisco polynucleotides encode large subunit Rubisco polypeptides having at least one amino residue from the set of (a)-(bd) residues listed below. The amino acid residue positions refer to the position in the encoded amino acid sequence when it is optimally aligned with reference sequence SEQ ID NO: 5, 8, 35, or 11. The present invention further provides Rubisco large subunit polypeptides that have at least one amino acid residue selected from the group consisting of: : (a) V at position 84; (b) D at position 92; (c) F at position 93; (d) L at position 113; (e) L at position 116; (f) L at position 117; (g) L at position 127; (h) A at position 129; (i) V at position 137; (j) I at position 139; (k) Y at position 141; (l) L at position 142; (m) S at position 149; (n) G at position 154; (o) K at position 158; (p) L at position 166; (q) M at position 209; (r) Q at position 219; (s) E at position 220; (t) E at position 223; (u) A at psition 225 (v) T at position 232; (w) Q at position 246; (x) E at position 249; (y) A at position 252; (z) I at position 257; (aa)T at position 259; (ab) G at position 269; (ac) S at postion 276; (ad) Y at position 280; (ae) L at position 286; (af) A at position 297; (ag) K at position 303; (ah) T at position 304; (ai) M at position 317; (aj) Q at position 322; (ak) T at position 325; (al) R at position 336; (am) Q at position 337; (an) T at position 338; (ao) I at position 343; (ap) Q at position 345; (aq) L at position 346; (ar) S at position 349; (as) F at position 350; (at)P at position 352; (au) E at position 353; (av) N or T at position 356; (aw) N at position 359; (ax) D at position 362; (ay) G at position 366; (az) F at position 372; (ba) A at position 373; (bb) A at position 389; (bc) I at position 415; (bd) R at position 450; and (be) I at position 454.
The present invention also provides Rubisco small subunit polypeptides that comprise an amino acid sequence corresponding to SEQ ID NO: 3 and having one or more substitutions selected from the group consisting of: (a) D23N; (b) M33T; (c) K66N; (d) S67G; (e) S102G; and (f) P108S. The present invention provides specific Rubisco small subunit polypeptides selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 48, and SEQ ID NO: 54.
The present invention further provides Rubisco polypeptides having both large and small subunits and that exhibit ribulose 1,5-bisphosphate carboxylase/oxygenase (“Rubisco”) activity,
wherein the polypeptide comprises a large subunit and a small subunit,

    
    
        wherein the large subunit comprises an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence that is at least 99% identical to SEQ ID NO: 5;

(b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 8;

(c) an amino acid sequence that is at least 97% identical to SEQ ID NO: 35;

(d) an amino acid sequence that is at least 99% identical to SEQ ID NO: 11; and

(e) an amino acid sequence corresponding to SEQ ID NO: 2; and

        wherein the small subunit comprises an amino acid sequence selected from the group consisting of:

(f) SEQ ID NO:3; and

(g) SEQ ID NO: 3 having one or more substitutions selected from the group consisting of: (i) D23N; (ii) M33T; (iii) K66N; (iv) S67G; (v) S103G; and (vi) P108S; and

    
    


wherein the polypeptide does not comprise (e) and (f) together.
The present invention also provides additional Rubisco polypeptides, as well as the Rubisco polynucleotides that encode them, related vectors, host cells, and methods, all of which are provided in more detail below.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts the Calvin-Benson Cycle
FIG. 2 depicts Vector pCK110700-I-Bla.
FIG. 3 depicts Vector pGR-1.
FIG. 4 provides a schematic description of the Whole Cell CO2 fixation assay described in Example 6.



DETAILED DESCRIPTION
The present invention provides novel ribulose 1,5-bisphosphate carboxylase/oxygenase polypeptides and the polynucleotides that encode them. As used herein, the terms “ribulose 1,5-bisphosphate carboxylase/oxygenase” and “Rubisco” are used interchangeably herein to refer to a polypeptide that, in nature, is made up of two subunits, a large subunit and a small subunit. The large subunit of Rubisco is also referred to as “rbcL” and the small subunit of Rubisco is also referred to as “rbcS”. Both subunits together are referred to herein as “rbcLS.” The term “Rubisco activity” refers herein to the ability to catalyze the conversion of ribulose 1,5-bisphosphate (“RuBP”) to 3-phosphoglycerate (“PG”) in the presence of carbon dioxide. This reaction takes place as part of the Calvin-Benson cycle, and is depicted as step “A” of FIG. 1.
The present invention provides Rubisco large subunit polypeptides and polynucleotides, Rubisco small subunit polypeptides and polynucleotides, as well as Rubisco large/small subunit (i.e., having both large and small subunits together in a single polypeptide) polypeptides and polynucleotides (collectively referred to herein as “Rubisco polypeptides” and “Rubisco polynucleotides”). The terms “Rubisco large subunit polypeptide” and “Rubisco rbcL polypeptide” are used interchangeably herein to refer to a polypeptide corresponding to the large subunit of Rubisco. The terms “Rubisco small subunit polypeptide” and “Rubisco rbcS polypeptide” are used interchangeably herein to refer to a polypeptide corresponding to the small subunit of Rubisco. The terms “Rubisco large/small subunit polypeptide” and “Rubisco rbcLS polypeptide” are used interchangeably herein to refer herein to a polypeptide that corresponds to both large and small subunits of Rubisco. Similarly, the terms “Rubisco large subunit polynucleotide” and “Rubisco rbcL polynucleotide” are used interechangeably herein to refer to a polynucleotide that encodes a Rubisco large subunit polypeptide. The terms “Rubisco small subunit polynucleotide” and “Rubisco rbcS polynucleotide” are used interchangeably herein to refer to a polynucleotide that encodes a Rubisco small subunit polypeptide. As used herein, the terms “Rubisco large and small subunit polypeptide” and “Rubisco rbcLS polynucleotide” are used interchangeably herein to refer to a polynucleotide that encodes both a Rubisco large subunit polypeptide and a Rubisco small subunit polypeptide.
Rubisco Polypeptides
Rubisco polypeptides of the present invention include Rubisco large subunit polypeptides (“rbcL”), Rubisco small subunit polypeptides (“rbcS”), and Rubisco large/small polypeptides (“rbcLS”). The terms “protein” and “polypeptide” are used interchangeably herein to refer to a polymer of amino acids. The term “amino acid sequence” refers to the order of amino residues in the protein or polypeptide. Large and small subunits of the present invention may be combined in different combinations with each other together in a single enzyme having Rubisco specific acitivity. Alternatively, the large and small subunits of the present invention may be combined with the large large and small subunits from a wild type Rubisco polypeptides (i.e., invention Rubisco large subunit combined with wild type Rubisco small subunit, or wild type Rubisco large subunit combined with invention Rubisco small subunit) to form a polypeptide having Rubisco activity.
Rubisco rbcLS polypeptides of the present invention exhibit a detectable level of Rubisco specific activity as measured in the assay described in Example 3.
Rubisco Large Subunit Polypeptides
The present invention provides an isolated or recombinant Rubisco large subunit Rubisco polypeptide that comprises an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence that is at least 99% identical to SEQ ID NO: 5;
(b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 8;
(c) an amino acid sequence that is at least 97% identical to SEQ ID NO: 35; and
(d) an amino acid sequence that is at least 99% identical to SEQ ID NO: 11.
As used herein, the term “isolated” refers to a nucleic acid, polynucleotide, polypeptide, protein, or other component that is partially or completely separated from components with which it is normally associated (other proteins, nucleic acids, cells, synthetic reagents, etc.). A nucleic acid or polypeptide is “recombinant” when it is artificial or engineered, or derived from an artificial or engineered protein or nucleic acid. For example, a polynucleotide that is inserted into a vector or any other heterologous location, e.g, in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide. A protein expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Likewise, a polynucleotide sequence that does not appear in nature, for example a variant of a naturally occurring gene, is recombinant.
Specific Rubisco large subunit polypeptides of the present invention include those selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 50, and SEQ ID NO: 53.
The terms “percent identity,” “% identity,” “percent identical,” and “% identical” are used interchangeably herein to refer to the percent amino acid sequence identity that is obtained by ClustalW analysis (version W 1.8 available form European bioinformatics Institue, Cambridge, UK), counting the number of identical matches in the alignment and dividing such number of identical matches by the length of the reference sequence, and using the following default ClustalW parameters to achieve slow/accurate pairwise alignments—Gap Open Penalty:10; Gap Extension Penalty:0.10; Protein weight matrix:Gonnet series; DNA weight matrix:IUB; Toggle Slow/Fast pairwise alignments=SLOW or FULL Alignment.
When optimally aligned with reference sequence SEQ ID NO: 5, 8, 35, or 11, certain Rubisco large subunit polypeptides of the present invention are characterized by having at least one amino acid residue selected from the group consisting of: (a) V at position 84; (b) D at position 92; (c) F at position 93; (d) L at position 113; (e) L at position 116; (f) L at position 117; (g) L at position 127; (h) A at position 129; (i) V at position 137; (j) I at position 139; (k) Y at position 141; (l) L at position 142; (m) S at position 149; (n) G at position 154; (o) K at position 158; (p) L at position 166; (q) M at position 209; (r) Q at position 219; (s) E at position 220; (t) E at position 223; (u) A at psition 225 (v) T at position 232; (w) Q at position 246; (x) E at position 249; (y) A at position 252; (z) I at position 257; (aa)T at position 259; (ab) G at position 269; (ac) S at postion 276; (ad) Y at position 280; (ae) L at position 286; (af) A at position 297; (ag) K at position 303; (ah) T at position 304; (ai) M at position 317; (aj) Q at position 322; (ak) T at position 325; (al) R at position 336; (am) Q at position 337; (an) T at position 338; (ao) I at position 343; (ap) Q at position 345; (aq) L at position 346; (ar) S at position 349; (as) F at position 350; (at)P at position 352; (au) E at position 353; (av) N or T at position 356; (aw) N at position 359; (ax) D at position 362; (ay) G at position 366; (az) F at position 372; (ba) A at position 373; (bb) A at position 389; (bc) I at position 415; (bd) R at position 450; and (be) I at position 454.
Two sequences are “optimally aligned” when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.b., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest core possible for that pair of sequences. Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art. See e.g., Dayhoff et al. (1978), “A model of evolutionary change in proteins”; “Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (Ed. M. O. Dayhoff), pp. 345-352, Natl. Biomed. Res. Round., Washington, D.C.; Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA, 89:10915-10919. The BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols such as Gapped BLAST 2.0. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acids position of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences so as to arrive at the highest possible score. While optimal alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences, so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer-implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul, et al. (1997) Nucleic Acids Res., 25:3389-3402, and made available to the public at the National Center for Biotechnology Information Website (the world wide web at ncbi.nlm.nih.gov). Optimal alignments, including multiple alignments can be prepared using readily available programs such as PSI-BLAST, which is described by Altschul, et al. (1997) Nucleic Acids Res., 25:3389-3402.
With respect to an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue “corresponds to” the position in the reference sequence with which the residue is paired in the alignment. The “position” is denoted by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. Owing to deletions, insertions, truncations, fusions, etc. that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence is determined by simply counting from the N-terminal will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where there is a deletion in an aligned test sequence, there will be no amino acid that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to any amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.
Rubisco large subunit polypeptides having an amino acid sequence at least 99% identical to SEQ ID NO: 5 typically comprise at least two amino acid residues selected from the group consisting of: I at position 257, T at position 259, M at position 317, A at position 389, and I at position 454.
Rubisco large subunitpolypeptides that have an amino acid sequence at least 95% identical to SEQ ID NO: 8 typically comprise at least two amino acid residues selected from the group consisting of: L at position 113, L at position 117, L at position 127, A at position 129, V at position 137, I at position 139, Y at position 141, L at position 142, Q at position 322, T at position 325, R at position 336, Q at position 337, T at position 338, I at position 343, Q at position 345, L at position 346, S at position 349, F at position 350, P at position 352, E at position 353, T at position 356, N at position 359, D at position 362, G at position 366, F at position 372, and A at position 373.
Rubisco large subunit polypeptides having an amino acid sequence at least 97% identical to SEQ ID NO: 35 typically comprise at least two amino acid residues selected from the group consisting of: S at position 149, M at position 209, Q at position 219, E at position 220, E at position 223, A at position 225, Q at position 246, E at position 249, A at position 252, I at position 257, T at position 259, G at position 269, S at position 276, Y at position 280, L at position 286, K at position 303, T at position 304, and A at position 389.
Rubisco large subunit polypeptides having an amino acid sequence at least 99% identical to SEQ ID NO: 11 typically comprise at least two amino acid residues selected from the group consisting of: V at position 84, K at position 158, L at position 166, M at position 317, and I at position 415.
The present invention also provides an isolated or recombinant Rubisco large subunit polypeptide that comprises an amino acid sequence corresponding to SEQ ID NO: 2 and having one of more substitutions selected from the group consisting of: (a) V at position 84; (b) D at position 92; (c) F at position 93; (d) L at position 113; (e) L at position 116; (f) L at position 117; (g) L at position 127; (h) A at position 129; (i) V at position 137; (j) I at position 139; (k) Y at position 141; (l) L at position 142; (m) S at position 149; (n) G at position 154; (o) K at position 158; (p) L at position 166; (q) M at position 209; (r) Q at position 219; (s) E at position 220; (t) E at position 223; (u) A at psition 225 (v) T at position 232; (w) Q at position 246; (x) E at position 249; (y) A at position 252; (z) I at position 257; (aa)T at position 259; (ab) G at position 269; (ac) S at postion 276; (ad) Y at position 280; (ae) L at position 286; (af) A at position 297; (ag) K at position 303; (ah) T at position 304; (ai) M at position 317; (aj) Q at position 322; (ak) T at position 325; (al) R at position 336; (am) Q at position 337; (an) T at position 338; (ao) I at position 343; (ap) Q at position 345; (aq) L at position 346; (ar) S at position 349; (as) F at position 350; (at)P at position 352; (au) E at position 353; (av) N or T at position 356; (aw) N at position 359; (ax) D at position 362; (ay) G at position 366; (az) F at position 372; (ba) A at position 373; (bb) A at position 389; (bc) I at position 415; (bd) R at position 450; and (be) I at position 454.
The present invention also provides an isolated or recombinant Rubisco large subunit polypeptide that comprises an amino acid sequence encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
(a) a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 5, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 5, comprises at least two amino acid residues selected from the group consisting of: I at position 257, T at position 259, M at position 317, A at position 389, and I at position 454;
(ii) SEQ ID NO: 8, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 8, comprises at least two amino acid residues selected from the group consisting of: L at position 113, L at position 117, L at position 127, A at position 129, V at position 137, I at position 139, Y at position 141, L at position 142, Q at position 322, T at position 325, R at position 336, Q at position 337, T at position 338, I at position 343, Q at position 345, L at position 346, S at position 349, F at position 350, P at position 352, E at position 353, T at position 356, N at position 359, D at position 362, G at position 366, F at position 372, and A at position 373;
(iii) SEQ ID NO: 35, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 35, comprises at least two amino acid residues selected from the group consisting of: : S at position 149, M at position 209, Q at position 219, E at position 220, E at position 223, A at position 225, Q at position 246, E at position 249, A at position 252, I at position 257, T at position 259, G at position 269, S at position 276, Y at position 280, L at position 286, K at position 303, T at position 304, and A at position 389;
(iv) SEQ ID NO: 11, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 11, comprises at least two amino acid residues selected from the group consisting of: V at position 84, K at position 158, L at position 166, M at position 317, and I at position 415; and
(b) a complementary nucleic acid that is complementary to the nucleic acid of (a).
Nucleic acids “hybridize” when they associate, typically in solution. Nucleic acids hybridize due to a variety of well-characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) “Laboratory Techniques in biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes,” Part I, Chapter 2 (Elsevier, N.Y.).
As used herein, the term “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments, such as Southern and northern hybridizations, are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijessen (1993) “Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes,” Part I, Chapter 2 (Elsevier, N.Y.).
For purposes of the present invention, “highly stringent” hybridization and wash conditions are generally selected to be about 5° C. or less lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH (as noted below, highly stringent conditions can also be referred to in comparative terms). The Tm is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe.
The Tm of a nucleic acid duplex indicates the temperature at which the duplex is 50% denatured under the given conditions and it represents a direct measure of the stability of the nucleic acid hybrid. Thus, the Tm corresponds to the temperature corresponding to the midpoint in transition from helix to random coil; it depends on length, nucleotide composition, and ionic strength for long stretches of nucleotides.
After hybridization, unhybridized nucleic acid material can be removed by a series of washes, the stringency of which can be adjusted depending upon the desired results. Low stringency washing conditions (e.g., using higher salt and lower temperature) increase sensitivity, but can produce nonspecific hybridization signals and high background signals. Higher stringency conditions (e.g., using lower salt and higher temperature that is closer to the hybridization temperature) lowers the background signal, typically with only the specific signal remaining. See Rapley, R. and Walker, J. M. Eds., “Molecular Biomethods Handbook” (Humana Press, Inc. 1998).
The Tm of a DNA-DNA duplex can be estimated using Equation 1 as follows:

Tm (° C.)=81.5° C.+16.6(log 10M)+0.41 (%G+C)−0.72(%f)−500/n,

    
    
        where M is the molarity of the monovalent cations (usually Na+), (%G+C) is the percentage of guanosine (G) and cystosine (C) nucleotides, (%f) is the percentage of formalize and n is the number of nucleotide bases (i.e., length) of the hybrid. See id.
    
    


The Tm of an RNA-DNA duplex can be estimated by using Equation 2 as follows:

Tm(° C.)=79.8° C.+18.5(log 10M)+0.58(%G+C)−11.8(%G+C)2−0.56(%f)−820/n,

    
    
        where M is the molarity of the monovalent cations (usually Na+), (%G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (%f) is the percentage of formamide and n is the number of nucleotide bases (i.e., length) of the hybrid. Id.
    
    


Equations 1 and 2 are typically accurate only for hybrid duplexes longer than about 100-200 nucleotides. Id.
The Tm of nucleic acid sequences shorter than 50 nucleotides can be calculated as follows:

Tm (° C.)=4(G+C)+2(A+T),

where A (adenine), C, T (thymine), and G are the numbers of the corresponding nucleotides.
An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42° C., with the hybridization being carried out overnight. An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes (see Sambrook, et al., Molecular Cloning—A Laboratory Manual” (1989) Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y.) for a description of SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal. An example low stringency wash is 2×SSC at 40° C. for 15 minutes.
In general, a signal to noise ratio of 2.5×-5× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Detection of at least stringent hybridization between two sequences in the context of the present invention indicates relatively strong structural similarity or homology to, e.g., the nucleic acids of the present invention provided in the sequence listings herein.
As noted, “highly stringent” conditions are selected to be about 5° C. or less lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Target sequences that are closely related or identical to the nucleotide sequence of interest (e.g., “probe”) can be identified under highly stringent conditions. Lower stringency conditions are appropriate for sequences that are less complementary.
One measure of stringent hybridization is the ability to hybridize to a nucleic acid that encodes an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 35, and SEQ ID NO: 11, or complementary polynucleotide sequence thereof, under highly stringent conditions (or very stringent conditions, or ultra-high stringency hybridization conditions, or ultra-ultra high stringency hybridization conditions). Stringent hybridization (as well as highly stringent, ultra-high stringency, or ultra-ultra high stringency hybridization conditions) and wash conditions can be readily determined empirically for any test nucleic acid. For example, in determining highly stringent hybridization and wash conditions, the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents, such as formalin, in the hybridization or wash), until a selected set of criteria are met. For example, the stringency of hybridization and wash conditions are gradually increased until a probe comprising one or more nucleic acid sequences encoding an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 35, and SEQ ID NO: 11, binds to a perfectly matched complementary target. A test nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least 1⁄2 as well to the probe as to the perfectly matched complementary target, i.e., with a signal to noise ratio at least 1⁄2 as high as hybridization of the probe to the target under conditions in which the perfectly matched probe binds to the perfectly matched complementary target.
Ultra high-stringency hybridization and wash conditions are those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least 10×. A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least 1⁄2 that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.
Similarly, even higher levels of stringency can be determined by gradually increasing the stringency of hybridization and/or wash conditions of the relevant hybridization assay. For example, those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least 10×, 20×, 50×, 100×, or 500×. A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least 1⁄2 that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-ultra-high stringency conditions.
The present invention includes the following target nucleic acids that hybridize under high, ultra-high and ultra-ultra high stringency conditions: (1) target nucleic acids which hybridize to nucleic acids that encode amino acid sequence SEQ ID NO: 5, and which encode an amino acid sequence that comprises at least two amino acid residues selected from the group consisting of: I at position 257, T at position 259, M at position 317, A at position 389, and I at position 454; (2) target nucleic acids which hybridize to nucleic acids that encode SEQ ID NO: 8, and which encode an amino aid sequence that comprises at least two amino acid residues selected from the group consisting of: L at position 113, L at position 117, L at position 127, A at position 129, V at position 137, I at position 139, Y at position 141, L at position 142, Q at position 322, T at position 325, R at position 336, Q at position 337, T at position 338, I at position 343, Q at position 345, L at position 346, S at position 349, F at position 350, P at position 352, E at position 353, T at position 356, N at position 359, D at position 362, G at position 366, F at position 372, and A at position 373; (3) target nucleic acids which hybridize to nucleic acids that encode SEQ ID NO: 35, and which encode an amino acid sequence that comprises at least two amino acid residues selected from the group consisting of: S at position 149, M at position 209, Q at position 219, E at position 220, E at position 223, A at position 225, Q at position 246, E at position 249, A at position 252, I at position 257, T at position 259, G at position 269, S at position 276, Y at position 280, L at position 286, K at position 303, T at position 304, and A at position 389; and (4) target nucleic acids which hybridize to nucleic acids that encode SEQ ID NO: 11, and which encode an amino acid sequence that comprises an amino acid sequence that comprises at least two amino acid residues selected from the group consisting of: V at position 84, K at position 158, L at position 166, M at position 317, and I at position 415; and (5) a complementary nucleic acid that is complementary to any one of (1)-(5).
The present invention also provides Rubisco large subunit polypeptides that comprise at least one of a group of certain specific amino acid residues at positions determined upon optimum alignment with the amino acid sequence corresponding to SEQ ID NO: 5, 8, 35, or 11. These residues are: (a) I at position 454; (b) V at position 84; (c) K at position 158; (d) L at position 166; and (e) M at position 317.
A Rubisco large subunit polypeptide having the amino acid residue I at position 454 of the large subunit appeared to be associated with higher kcat for RuBP as determined by the method described in Example 4. The residues V at position 84, K at position 158, L at position 166, and M at position 317 appeared to confer a lower KM as determined by the method described in Example 4.
Rubisco Small Subunit Polypeptides
The present invention provides an isolated or recombinant small subunit Rubisco polypeptide that comprises an amino acid sequence corresponding to SEQ ID NO: 3, and having one or more substitutions selected from the group consisting of: (a) D23N; (b) M33T; (c) K66N; (d) S67G; (e) S102G; and (f) P108S.
Exemplary Rubisco small subunit polypeptides of the present invention include those having an amino acid sequence corresponding to SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 48, and SEQ ID NO: 54.
The invention further provides Rubisco small subunit polypeptides of the present invention that are encoded by an isolated or recombinant polynucleotide comprising:
(a) a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 12, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 12, comprises at position 23, amino acid residue N;
(ii) SEQ ID NO: 18, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 18, comprises at position 67, amino acid residue G;
(iii) SEQ ID NO: 24, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 24, comprises at position 108, amino acid residue S;
(iv) SEQ ID NO: 27, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 27, comprises at position 66, amino acid residue N;
(v) SEQ ID NO: 30, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 30, comprises at position 102, amino acid residue G; and
(vi) SEQ ID NO: 39, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 39, comprises at position 33, amino acid residue T; or
(b) a complementary nucleic acid that is complementary to the nucleic acid of (a).
The present invention also provides Rubisco small subunit polypeptides that comprise N at position 23, where position 23 is determined by optimum alignment with the amino acid sequence corresponding to SEQ ID NO: 3, 12, 18, 24, 27, 30, or 39. This residue appears to be associated with lower KM.
Rubisco Large and Small Subunit (rbcLS) Polypeptides
The present invention provides an isolated or recombinant polypeptide having Rubisco specific activity (as determined by the method of Example 3),
wherein the polypeptide comprises a large subunit and a small subunit,

    
    
        wherein the large subunit comprises an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence that is at least 99% identical to SEQ ID NO: 5;

(b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 8;

(c) an amino acid sequence that is at least 97% identical to SEQ ID NO: 35;

(d) an amino acid sequence that is at least 99% identical to SEQ ID NO: 11; and

(e) an amino acid sequence corresponding to SEQ ID NO: 2; and

        wherein the small subunit comprises an amino acid sequence selected from the group consisting of:

(f) SEQ ID NO:3; and

(g) SEQ ID NO: 3 having one or more substitutions selected from the group consisting of: (i) D23N; (ii) M33T; (iii) K66N; (iv) S67G; (v) S102G; and (vi) P108S; and

    
    


wherein the polypeptide does not comprise (e) and (f) together.
The present invention also provides an isolated or recombinant Rubisco rbcLS polypeptide having Rubisco specific activity,
wherein the polypeptide comprises a large subunit and a small subunit,

    
    
        wherein the large subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, and SEQ ID NO: 40, and
        wherein the small subunit comprises an amino acid sequence corresponding to SEQ ID NO: 3.
    
    


The present invention further provides an isolated or recombinant Rubisco rbcLS polypeptide having Rubisco specific activity and comprising a combination of large and small subunit amino acid sequences selected from the group consisting of:
(a) SEQ ID NO: 11 and SEQ ID NO: 12;
(b) SEQ ID NO: 29 and SEQ ID NO: 30;
(c) SEQ ID NO: 38 and SEQ ID NO: 39;
(d) SEQ ID NO: 47 and SEQ ID NO: 48; and
(e) SEQ ID NO: 53 and SEQ ID NO: 54.
Rubisco rbcLS polypeptides of the present invention also include an isolated or recombinant polypeptide having ribulose 1,5-bisphosphate carboxylase/oxygenase activity,
wherein the polypeptide comprises a large subunit and a small subunit,

    
    
        wherein the large subunit comprises an amino acid sequence corresponding to SEQ ID NO: 3, and
        wherein the small subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 45.

Rubisco Polypeptide Variants

    
    


Variants of Rubisco large and small subunit polypeptides of the present invention may be generated using methods that are well known to those having ordinary skill in the art. Libraries of these variants may be generated and screened using the methods described in Example 4 hereinbelow to identify those having Rubisco specific activity.
For example, mutagenesis and directed evolution methods are well known in the art. See, e.g., Ling, et al., “Approaches to DNA mutagenesis: an overview,” Anal. Biochem., 254(2):157-78 (1997); Dale, et al., “Oligonucleotide-directed random mutagenesis using the phosphorothioate method,” Methods Mol. Biol., 57:369-74 (1996); Smith, “In vitro mutagenesis,” Ann. Rev. Genet., 19:423-462 (1985); Botstein, et al., “Strategies and applications of in vitro mutagenesis,” Science, 229:1193-1201 (1985); Carter, “Site-directed mutagenesis,” Biochem. J., 237:1-7 (1986); Kramer, et al., “Point Mismatch Repair,” Cell, 38:879-887 (1984); Wells, et al., “Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites,” Gene, 34:315-323 (1985); Minshull, et al., “Protein evolution by molecular breeding,” Current Opinion in Chemical Biology, 3:284-290 (1999); Christians, et al., “Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling,” Nature Biotechnology, 17:259-264 (1999); Crameri, et al., “DNA shuffling of a family of genes from diverse species accelerates directed evolution,” Nature, 391:288-291; Crameri, et al., “Molecular evolution of an arsenate detoxification pathway by DNA shuffling,” Nature Biotechnology, 15:436-438 (1997); Zhang, et al., “Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening,” Proceedings of the National Academy of Sciencess. U.S.A., 94:45-4-4509; Crameri, et al., “Improved green fluorescent protein by molecular evolution using DNA shuffling,” Nature Biotechnology<14:315-319 (1996); Stemmer, “Rapid evolution of a protein in vitro by DNA shuffling,” Nature, 370:389-391 (1994); Stemmer, “DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution,” Proceedings of the National Academy of Sciences, U.S.A., 91:10747-10751 (1994); WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; and WO 01/75767.
Rubisco Polynucleotides
Rubisco Large Subunit Polynucleotides
The present invention provides an isolated or recombinant Rubisco large subunit polynucleotide that comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence encoding an amino acid sequence that is at least 99% identical to SEQ ID NO: 5;
(b) a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO: 8;
(c) a nucleotide sequence encoding an amino acid sequence that is at least 97% identical to SEQ ID NO: 35;
(d) a nucleotide sequence encoding an amino acid sequence that is at least 99% identical to SEQ ID NO: 11; and
(e) a nucleotide sequence that is complementary to any one of (a) through (d).
The terms “polynucleotide” and “nucleic acid” are used interchangeably herein to refer to a polymer of nucleotides (A, C, T, U, G, etc. or naturally occurring or artificial nucleotide analogues), e.g., DNA or RNA, or a representation thereof. The terms “polynucleotide sequence” and “nucleic acid sequence” are used interchangeably herein to refer to the order of nucleotides in the polynucleotide or nucleic acid. A complementary polynucleotide can be readily determined from any specified polynucleotide sequence.
Specific large subunit Rubisco polynucleotides of the present invention comprise a polynucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 50, and SEQ ID NO: 53.
Certain Rubisco large subunit polynucleotides encode Rubisco large subunit polypeptides having at least one amino residue from the set of (a)-(bd) residues listed below. The amino acid residue positions refer to the position in the encoded amino acid sequence when it is optimally aligned with reference sequence SEQ ID NO: 5, 8, 35, or 11. The polypeptides encoded by the large subunit Rubisco polynucleotides typically have at least one amino acid residue selected from the group consisting of: (a) V at position 84; (b) D at position 92; (c) F at position 93; (d) L at position 113; (e) L at position 116; (f) L at position 117; (g) L at position 127; (h) A at position 129; (i) V at position 137; (j) I at position 139; (k) Y at position 141; (l) L at position 142; (m) S at position 149; (n) G at position 154; (o) K at position 158; (p) L at position 166; (q) M at position 209; (r) Q at position 219; (s) E at position 220; (t) E at position 223; (u) A at position 225 (v) T at position 232; (w) Q at position 246; (x) E at position 249; (y) A at position 252; (z) I at position 257; (aa)T at position 259; (ab) G at position 269; (ac) S at postion 276; (ad) Y at position 280; (ae) L at position 286; (af) A at position 297; (ag) K at position 303; (ah) T at position 304; (ai) M at position 317; (aj) Q at position 322; (ak) T at position 325; (al) R at position 336; (am) Q at position 337; (an) T at position 338; (ao) I at position 343; (ap) Q at position 345; (aq) L at position 346; (ar) S at position 349; (as) F at position 350; (at)P at position 352; (au) E at position 353; (av) N or T at position 356; (aw) N at position 359; (ax) D at position 362; (ay) G at position 366; (az) F at position 372; (ba) A at position 373; (bb) A at position 389; (bc) I at position 415; (bd) R at position 450; and (be) I at position 454.
When the amino acid sequence encoded by the Rubisco large subunit polynucleotide is at least 99% identical to SEQ ID NO: 5, it typically comprises at least two amino acid residues selected from the group consisting of: I at position 257, T at position 259, M at position 317, A at position 389, and I at position 454. When the amino acid sequence encoded by the Rubisco large subunit polynucleotide is at least 95% identical to SEQ ID NO: 8, it typically comprises at least two amino acid residues selected from the group consisting of: L at position 113, L at position 117, L at position 127, A at position 129, V at position 137, I at position 139, Y at position 141, L at position 142, Q at position 322, T at position 325, R at position 336, Q at position 337, T at position 338, I at position 343, Q at position 345, L at position 346, S at position 349, F at position 350, P at position 352, E at position 353, T at position 356, N at position 359, D at position 362, G at position 366, F at position 372, and A at position 373. Rubisco large subunit polynucleotides encoding an amino acid sequence that is at least 97% identical to SEQ ID NO: 35 typically encode an amino acid sequence that comprises at least two amino acid residues selected from the group consisting of: S at position 149, M at position 209, Q at position 219, E at position 220, E at position 223, A at position 225, Q at position 246, E at position 249, A at position 252, I at position 257, T at position 259, G at position 269, S at position 276, Y at position 280, L at position 286, K at position 303, T at position 304, and A at position 389. position 450; and (bd) I at position 454. When the amino acid sequence encoded by the Rubisco large subunit polynucleotide is at least 99% identical to SEQ ID NO: 11, it typically comprises at least two amino acid residues selected from the group consisting of: V at position 84, K at position 158, L at position 166, M at position 317, and I at position 415.
The present invention also provides an isolated or recombinant Rubisco large subunit polynucleotides comprising a nucleotide sequence encoding an amino acid sequence corresponding to SEQ ID NO: 2 and having one of more substitutions selected from the group consisting of: (a) V at position 84; (b) D at position 92; (c) F at position 93; (d) L at position 113; (e) L at position 116; (f) L at position 117; (g) L at position 127; (h) A at position 129; (i) V at position 137; (j) I at position 139; (k) Y at position 141; (l) L at position 142; (m) S at position 149; (n) G at position 154; (o) K at position 158; (p) L at position 166; (q) M at position 209; (r) Q at position 219; (s) E at position 220; (t) E at position 223; (u) A at psition 225 (v) T at position 232; (w) Q at position 246; (x) E at position 249; (y) A at position 252; (z) I at position 257; (aa)T at position 259; (ab) G at position 269; (ac) S at postion 276; (ad) Y at position 280; (ae) L at position 286; (af) A at position 297; (ag) K at position 303; (ah) T at position 304; (ai) M at position 317; (aj) Q at position 322; (ak) T at position 325; (al) R at position 336; (am) Q at position 337; (an) T at position 338; (ao) I at position 343; (ap) Q at position 345; (aq) L at position 346; (ar) S at position 349; (as) F at position 350; (at)P at position 352; (au) E at position 353; (av) N or T at position 356; (aw) N at position 359; (ax) D at position 362; (ay) G at position 366; (az) F at position 372; (ba) A at position 373; (bb) A at position 389; (bc) I at position 415; (bd) R at position 450; and (be) I at position 454.
The present invention also provides an isolated or recombinant Rubisco large subunit polynucleotide comprising:
(a) a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 5, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 5, comprises at least two amino acid residues selected from the group consisting of: I at position 257, T at position 259, M at position 317, A at position 389, and I at position 454;
(ii) SEQ ID NO: 8, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 8, comprises at least two amino acid residues selected from the group consisting of: L at position 113, L at position 117, L at position 127, A at position 129, V at position 137, I at position 139, Y at position 141, L at position 142, Q at position 322, T at position 325, R at position 336, Q at position 337, T at position 338, I at position 343, Q at position 345, L at position 346, S at position 349, F at position 350, P at position 352, E at position 353, T at position 356, N at position 359, D at position 362, G at position 366, F at position 372, and A at position 373;
(iii) SEQ ID NO: 35, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 35, comprises at least two amino acid residues selected from the group consisting of: : S at position 149, M at position 209, Q at position 219, E at position 220, E at position 223, A at position 225, Q at position 246, E at position 249, A at position 252, I at position 257, T at position 259, G at position 269, S at position 276, Y at position 280, L at position 286, K at position 303, T at position 304, and A at position 389;
(iv) SEQ ID NO: 11, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 11, comprises at least two amino acid residues selected from the group consisting of: V at position 84, K at position 158, L at position 166, M at position 317, and I at position 415; and
(b) a complementary nucleic acid that is complementary to the nucleic acid of (a).
Specific isolated and recombinant Rubisco large subunit polynucleotides of the present invention correspond in sequence to positions 1 through 1419, inclusive, of a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO 49, and SEQ ID NO: 52, and SEQ ID NO: 55.
Rubisco Small Subunit Polynucleotides
The present invention provides an isolated or recombinant Rubisco small subunit polynucleotide comprising a nucleotide sequence that encodes an amino acid sequence corresponding to SEQ ID NO: 3 that has one or more substitutions selected from the group consisting of: (a) D23N; (b) M33T; (c) K66N; (d) S67G; (e) S102G; and (f) P108S. Specific Rubisco small subunit polynucleotides of the present invention comprise a polynucleotide sequence that encodes an amino acid sequence that is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, and SEQ ID NO: 39.
The present invention also provides an isolated or recombinant Rubisco small subunit polynucleotide comprising a nucleic acid selected from the group consisting of:
(a) a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 12, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 12, comprises at position 23, amino acid residue N;
(ii) SEQ ID NO: 18, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 18, comprises at position 67, amino acid residue G;
(iii) SEQ ID NO: 24, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 24, comprises at position 108, amino acid residue S;
(iv) SEQ ID NO: 27, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 27, comprises at position 66, amino acid residue N;
(v) SEQ ID NO: 30, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 30, comprises at position 102, amino acid residue G; and
(vi) SEQ ID NO: 39, wherein the nucleic acid encodes an amino acid sequence that, when optimally aligned with SEQ ID NO: 39, comprises at position 33, amino acid residue T; or
(b) a complementary nucleic acid that is complementary to the nucleic acid of (a).
Specific Rubisco small subunit polynucleotides of the present invention comprise a polynucleotide sequence corresponding to positions 1510 through 1845 inclusive, of a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 55.
Rubisco Large and Small Subunit Polynucleotides
The present invention provides an isolated or recombinant rbcLS polynucleotide comprising a nucleic acid that encodes a Rubisco large subunit polypeptide and a nucleic acid encoding a Rubisco small subunit polypeptide,
wherein the nucleic acid encoding the Rubisco large subunit polypeptide is selected from the group consisting of:
(a) a nucleotide sequence encoding an amino acid sequence that is at least 99% identical to SEQ ID NO: 5;
(b) a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO: 8;
(c) a nucleotide sequence encoding an amino acid sequence that is at least 97% identical to SEQ ID NO: 35;
(d) a nucleotide sequence encoding an amino acid sequence that is at least 99% identical to SEQ ID NO: 11; and
(e) a nucleotide sequence that is complementary to any one of (a) through (d); and
wherein the nucleic acid encoding the Rubisco small subunit polypeptide encodes an amino acid sequence having a sequence selected from the group consisting of:
(a) SEQ ID NO:3; and
(b) SEQ ID NO: 3 having one or more substitutions selected from the group consisting of: (i) D23N; (ii) M33T; (iii) K66N; (iv) S67G; (v) S103G; and (vi) P108S.
Isolated or recombinant Rubisco polynucleotides comprise a nucleic acid encoding a Rubisco large subunit polypeptide and a nucleic acid encoding a Rubisco small subunit polypeptide,
wherein the nucleic acid encoding the Rubisco large subunit polypeptide has a nucleotide sequence that encodes an amino acid sequence corresponding to SEQ ID NO: 2 and
wherein the nucleic acid encoding the Rubisco small subunit polypeptide encodes an amino acid sequence corresponding to SEQ ID NO: 3 that has one or more substitutions selected from the group consisting of: (a) D23N; (b) M33T; (c) K66N; (d) S67G; (e) S102G; and (f) P108S. Specific Rubisco polynucleotides of the present invention include a polynucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, and SEQ ID NO: 52.
Polynucleotides that encode both large and small subunits of the Rubisco polyptides (rbcLS) of the present invention typically are separated by an intervening, non-coding polynucleotide sequence that operates as a linker. The linker separates the subunit polynucleotide coding sequences, and extends from the 3′ end of the large subunit coding sequence to the 5′ end of the small subunit coding sequence. The specific sequence of the linker is not critical. The linker is generally at least about 30 nucleotides in length, typically at least about 50 nucleotides in length, and usually at least about 80 nucleotides in length, up to about 100 nucleotides in length. The present invention provides isolated or recombinant Rubisco rbcLS polynucleotides having a linker sequence separating Rubisco rbcL and Rubisco rbcS polynucleotide sequences. Exemplary linkers include the polynucleotide sequence extending from position 1420 to position 1509, inclusive, of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, and 52.
Polynucleotide Sequence Variations
Those having ordinary skill in the art will readily appreciate that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding Rubisco polypeptides of the present invention exist. Table I is a Codon Table that provides the synonymous codons for each amino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acids of the invention where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence.







TABLE 1







Codon Table









Amino acids

Codon


















Alanine
Ala
A
GCA
GCC
GCG
GCU




Cysteine
Cys
C
UGC
UGU


Aspartic acid
Asp
D
GAC
GAU


Glutamic acid
Glu
E
GAA
GAG


Phenylalanine
Phe
F
UUC
UUU


Glycine
Gly
G
GGA
GGC
GGG
GGU


Histidine
His
H
CAC
CAU


Isoleucine
Ile
I
AUA
AUC
AUU


Lysine
Lys
K
AAA
AAG


Leucine
Leu
L
UUA
UUG
CUA
CUC
CUG
CUU


Methionine
Met
M
AUG


Asparagine
Asn
N
AAC
AAU


Proline
Pro
P
CCA
CCC
CCG
CCU


Glutamine
Gln
Q
CAA
CAG


Arginine
Arg
R
AGA
AGG
CGA
CGC
CGG
CGU


Serine
Ser
S
AGC
AGU
UCA
UCC
UCG
UCU


Threonine
Thr
T
ACA
ACC
ACG
ACU


Valine
Val
V
GUA
GUC
GUG
GUU


Tryptophan
Trp
W
UGG


Tyrosine
Tyr
Y
UAC
UAU









Such “silent variations” are one species of “conservative” variation. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified by standard techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence. The invention provides each and every possible variation of nucleic acid sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code (set forth in Table 1), as applied to the polynucleotide sequences encoding the Rubisco large subunit, small subunit, and large and small subunit polypeptides of the present invention.
A group of two or more different codons that, when translated in the same context, all encode the same amino acid, are referred to herein as “synonymous codons.”Rubisco polynucleotides of the present invention may be codon optimized for expression in a particular host organism by modifying the polynucleotides to conform with the optimum codon usage of the desired host organism. Those having ordinary skill in the art will recognize that tables and other references providing preference information for a wide range of organisms are readily available See e.g., the world wide web at kazusa.orgjp/codon/. For example, Rubisco polynucleotides may be codon optimized for expression from a blue green algae, such as a Synechocystis sp. An exemplary codon optimized Rubisco polynucleotide sequence of the present invention is provided as SEQ ID NO: 55, in which SEQ ID NO: 40 has been codon optimized for expression from Synechocystis. 
The terms “conservatively modified variations” and “conservative variations” are used interchangeably herein to refer to those nucleic acids that encode identical or or essentially identical amino acid sequences, or in the situation where the nucleic acids are not coding sequences, the term refers to nucleic acids that are identical. One of ordinary skill in the art will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are considered conservatively modified variations where the alterations result in one or more of the following: the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. When more than one amino acid is affected, the percentage is typically less than 5% of amino acid residues over the length of the encoded sequence, and more typically less than 2%. Conservative substitution tables providing amino acids that are considered conservative substitutions for one another are well known in the art. Table 2 provides a list of six conservative substitution groupings of amino acids.







TABLE 2





Conservative Substitution Groups



















1
Alanine (A)
Serine (S)
Threonine (T)



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)









Conservatively substituted variations of the Rubisco polypeptides of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 2%, and often less than 1% of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group. The addition of sequences which do not alter the encoded activity of a Rubisco polynucleotide, such as the addition of a non-functional or non-coding sequence, is considered a conservative variation of the Rubisco polynucleotide.
Vectors, Promoters, and Expression Systems
The present invention also includes recombinant constructs comprising one or more of the nucleic acid sequences as broadly described above. The term “construct” or “nucleic acid construct” refers herein to a nucleic acid, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature. The term “nucleic acid construct” is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
The term “control sequences” refers herein to all the components that are necessary or advantageous for the expression of a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limitd to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
The term “operably linked” refers herein to a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polypeptide.
When used herein, the term “coding sequence” is intended to cover a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon. The coding sequence typically includes a DNA, cDNA, and/or recombinant nucleotide sequence.
As used herein, the term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term “expression vector” refers herein to a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of the invention, and which is operably linked to additional segments that provide for its transcription.
As used herein, the term “host cell” refers to any cell type which is susceptible to transformation with a nucleic acid construct.
Nucleic acid constructs of the present invention comprise a vector, such as, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.
General texts which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other relevant topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) (“Ausubel”). Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Q∃-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), e.g., for the production of the homologous nucleic acids of the invention are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the references cited therein, in which PCR amplicons of up to 40 kb are generated. One of of ordinary skill in the art will readily appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, e.g., Ausubel, Sambrook and Berger, all supra.
The present invention also relates to engineered host cells that are transduced (transformed or transfected) with a vector of the invention (e.g., an invention cloning vector or an invention expression vector), as well as the production of polypeptides of the invention by recombinant techniques. The vector may be, for example, a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the Rubisco polynucleotide. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, e.g., Sambrook, Ausubel and Berger, as well as e.g., Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and the references cited therein.
Rubisco polypeptides of the invention can be produced in non-animal cells such as plants, yeast, fungi, bacteria (e.g., cyanobacteria) and the like. In addition to Sambrook, Berger and Ausubel, details regarding non-animal cell culture can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.
Polynucleotides of the present invention can be incorporated into any one of a variety of expression vectors suitable for expressing a polypeptide. Suitable vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses and many others. Any vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host can be used.
When incorporated into an expression vector, a polynucleotide of the invention is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis. Examples of such transcription control sequences particularly suited for use in transgenic plants include the cauliflower mosaic virus (CaMV) and figwort mosaic virus (FMV). Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses and which can be used in some embodiments of the invention include SV40 promoter, E. coli lac or trp promoter, phage lambda PL promoter. An expression vector optionally contains a ribosome binding site for translation initiation, and a transcription terminator, such as PinII. The vector also optionally includes appropriate sequences for amplifying expression, e.g., an enhancer.
In addition, the expression vectors of the present invention optionally contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Suitable marker genes include those coding for resistance to the antibiotic spectinomycin or streptomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance. Additional selectable marker genes include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance in E. coli. 
Vectors of the present invention can be employed to transform an appropriate host to permit the host to express an invention protein or polypeptide. Examples of appropriate expression hosts include bacterial cells, such as E. coli, B. subtilis, and Streptomyces, cyanobacterial cells such as Synechocystis, Synechococcus, Anabaena, Anacystis, and the like, and plant cells.
In bacterial systems, a number of expression vectors may be selected, such as, for example, multifunctional E. coli cloning and expression vectors. In cyanobacterial systems, vectors such as those described in Example 5 may be used.
In plant cells, expression can be driven from a transgene integrated into a plant chromosome, or cytoplasmically from an episomal or viral nucleic acid. In the case of stably integrated transgenes, it is often desirable to provide sequences capable of driving constitutive or inducible expression of the Rubisco polynucleotides of the invention, for example, using viral, e.g., CaMV, or plant derived regulatory sequences. Numerous plant derived regulatory sequences have been described, including sequences which direct expression in a tissue specific manner, e.g., TobRB7, patatin B33, GRP gene promoters, the rbcS-3A promoter, and the like. Alternatively, high level expression can be achieved by transiently expressing exogenous sequences of a plant viral vector, e.g., TMV, BMV, etc. Typically, transgenic plants constitutively expressing a Rubisco polynucleotide of the invention will be preferred, and the regulatory sequences selected to insure constitutive stable expression of the Rubisco polypeptide.
Typical vectors useful for expression of nucleic acids in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al., Meth. In Enzymol., 153:253-277 (1987). Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et al., Gene, 61:1-11 (1987) and Berger et al., Proc. Natl. Acad. Sci. U.S.A., 86:8402-8406 (1989). Another useful vector herein is plasmid pBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto, Calif.). A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.
In some embodiments of the present invention, a Rubisco polynucleotide construct suitable for transformation of plant cells is prepared. For example, a desired Rubisco polynucleotide can be incorporated into a recombinant expression cassette to facilitate introduction of the gene into a plant and subsequent expression of the encoded polypeptide. An expression cassette will typically comprise a Rubisco polynucleotide, or functional fragment thereof, operably linked to a promoter sequence and other transcriptional and translational initiation regulatory sequences which will direct expression of the sequence in the intended tissues (e.g., entire plant, leaves, seeds) of the transformed plant.
For example, a strongly or weakly constitutive plant promoter can be employed which will direct expression of the Rubisco polypeptide all tissues of a plant. Such promoters are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosiac virus (CaMV) 35S transcription initiationa region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter and other transcription initiation regions from various plant genes known to those of skill. In situations in which overexpression of a Rubisco poynucleotide is detrimental to the plant or otherwise undesirable, one of skill, upon review of this disclosure, will recognize that weak constitutive promoters can be used for low-levels of expression. In those cases where high levels of expression is not harmful to the plant, a strong promoter, e.g., a t-RNA or other pol III promoter, or a strong pol II promoter, such as the cauliflower mosaic virus promoter, can be used.
Alternatively, a plant promoter may be under environmental control. Such promoters are referred to here as “inducible” promoters. Examples of environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light. In particular, examples of inducible promoters are the Adh 1 promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, and the PPDK promoter which is inducible by light. Also useful are promoters which are chemically inducible.
The promoters used in the present invention can be “tissue-specific” and, as such, under developmental control in that the polynucleotide is expressed only in certain tissues, such as leaves, roots, fruit, flowers and seeds. An exemplary promoter is the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051). Examples of seed-preferred promoters include, but are not limited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A., Martinez, M. C., Reina, M., Puigdomenech, P. and Palau, J.; Isolation and sequencing of a 28 kD glutelin-2 gene from maize: Common elements in the 5′ flanking regions among zein and glutelin genes; Plant Sci. 47, 95-102 (1986) and Reina, M., Ponte, I., Guillen, P., Boronat, A. and Palau, J., Sequence analysis of a genomic clone encoding a Zc2 protein from Zea mays W64 A, Nucleic Acids Res. 18 (21), 6426 (1990). See the following site relating to the waxy promoter: Kloesgen, R. B., Gierl, A., Schwarz-Sommer, ZS. and Saedler, H., Molecular analysis of the waxy locus of Zea mays, Mol. Gen. Genet. 203, 237-244 (1986). In embodiments in which one or more nucleic acid sequences endogenous to the plant system are incorporated into the construct, the endogenous promoters (or variants thereof) from these genes can be employed for directing expression of the genes in the transfected plant. Tissue-specific promoters can also be used to direct expression of heterologous polynucleotides.
In general, the particular promoter used in the expression cassette in plants depends on the intended application. Either heterologous or non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. These promoters can also be used, for example, in expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter concentration and/or composition of the proteins of the present invention in a desired tissue. Any of a number of promoters which direct transcription in plant cells are suitable. The promoter can be either constitutive or inducible. In addition to the promoters noted above, promoters of bacterial origin which operate in plants include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids (see, Herrara-Estrella et al. (1983) Nature 303:209-213). Viral promoters include the 35S and 19S RNA promoters of cauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812). Other plant promoters include the ribulose-1,3-bisphosphate carboxylase small subunit promoter and the phaseolin promoter. The promoter sequence from the E8 gene and other genes may also be used. The isolation and sequence of the E8 promoter is described in detail in Deikman and Fischer (1988) EMBO J. 7:3315-3327.
To identify candidate promoters, the 5′ portions of a genomic clone is analyzed for sequences characteristic of promoter sequences. For instance, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site. In plants, further upstream from the TATA box, at positions −80 to −100, there is typically a promoter element with a series of adenines surrounding the trinucleotide G (or T) as described by Messing, et al. (1983) Genetic Engineering in Plants, Kosage, et al. (Eds.), pp. 221-227.
In preparing polynucleotide constructs, vectors, of the invention, sequences other than the promoter and the cojoined polynucleotide can also be employed. The polyadenylation region can be derived, for example, from a variety of plant genes, or from T-DNA. The 3′ end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence can be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates. See e.g., Buchman and Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis, et al., Genes Dev. 1:1183-1200 (1987). Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, “The Maize Handbook,” Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
Specific initiation signals can aid in efficient translation of a Rubisco polynucleotide-encoding sequence of the present invention. These signals can include, e.g., the ATG initiation codon and adjacent sequences. In cases where a Rubisco polypeptide-encoding sequence, its initiation codon and upstream sequences are inserted into an appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence (e.g., a mature protein coding sequence), or a portion thereof, is inserted, exogenous transcriptional control signals including the initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf D et al. (1994) Results Probl Cell Differ 20:125-62; Bittner et al. (1987) Methods in Enzymol 153:516-544).
Secretion/Localization Sequences
Polynucleotides of the invention can also be fused, for example, in-frame to nucleic acids encoding a secretion/localization sequence, to target polypeptide expression to a desired cellular compartment, membrane, or organelle of a cell, or to direct polypeptide secretion to the periplasmic space or into the cell culture media. Such sequences are known to those of skill, and include secretion leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization/anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.
Expression Hosts
In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a eukaryotic cell, such as a plant cell. Alternatively, the host cell can be a prokaryotic cell, such as a bacterial cell, and more typically, a cyanobacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation, or other common techniques (Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in Molecular Biology). Preferred host cells are those having the cellular machinery to carry out photosynthesis.
Expression Conditions
Host cells transformed with a Rubisco polynucleotide are optionally cultured under conditions to optimize carbon fixation by the host cell. The present invention provides a method of fixing carbon in a host cell, the method comprising:
(i) introducing the vector comprising a Rubisco rbcLS polynucleotide into one or more photosynthesizing host cell;
(ii) incubating the host cell to allow expression of a Rubisco rbcLS polynucleotide. Photosynthesizing host cells employed in the practice of the present invention include plant cells and cyanobacterial cells.
Suitable conditions for inducing carbon fixation in a cell capable of photosynthesis include exposure to light in the visible range. Typically, light having a wavelength in the range of from about 380 nm to 780 nm is employed. Transformed host cells are optimally incubated at a pH in the range of from about 7 to 11, and at a temperature in the range of from about 24° C. to about 32° C. Carbon dioxide can be provided in the form of atmospheric air, or with added CO2 in an air/CO2 mixture. Typically up to about 5% CO2 is provided in a CO2/air mixture. For large scale carbon fixation processes, the cells are typically incubated in a vessel that is transparent to light, under low shear agitation.
Fusion Polypeptides for Purification
Rubisco polypeptides of the present invention may also be expressed as part of a fusion polypeptide to facilitate purification of the encoded Rubisco polypeptide. Polynucleotides encoding such fusion polypeptides comprise a nucleic acid sequence corresponding to a Rubisco polynucleotide of the present invention that is fused-in frame to a purification facilitating domain. As used herein, the term “purification facilitating domain” refers to a domain that mediates purification of the polypeptide to which it is fused. Suitable purification domains include metal chelating peptides, histidine-tryptophan modules that allow purification on immobilized metals, a sequence which binds glutathione (e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitope derived from the influenza hemagglutinin protein; Wilson et al. (1984) Cell 37:767), maltose binding protein sequences, the FLAG epitope utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, Wash.), and the like. The inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and the Rubisco polypeptide is useful to facilitate purification. One expression vector contemplated for use in the compositions and methods described herein provides for expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath et al. (1992) Protein Expression and Purification 3:263-281) while the enterokinase cleavage site provides a means for separating the Rubisco polypeptide from the fusion protein. pGEX vectors (Promega; Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.
Production and Recovery of Rubisco Polypeptides
Following transduction of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well known to those skilled in the art.
As noted, many references are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archebacterial origin. See e.g., Sambrook, Ausubel, and Berger (all supra), as well as Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and the references cited therein; Doyle and Griffiths (1997) Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, fourth edition W. H. Freeman and Company; and Ricciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016-1024. For plant cell culture and regeneration, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York); Jones, ed. (1984) Plant Gene Transfer and Expression Protocols, Humana Press, Totowa, N.J. and Plant Molecular Biology (1993) R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6. Cell culture media in general are set forth in Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla. Additional information for cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-LSRCCC”) and, e.g., The Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”). Further details regarding plant cell transformation and transgenic plant production are found below.
Rubisco polypeptides of the present invention can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using any of the tagging systems noted herein), hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as desired, in completing the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed in the final purification steps. In addition to the references noted supra, a variety of purification methods are well known in the art, including, e.g., those set forth in Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; and Bollag et al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3rd Edition Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ.
In some cases it may be desirable to produce the Rubisco polypeptides of the invention in a large scale suitable for industrial and/or commercial applications. In such cases bulk fermentation procedures are employed. Briefly, a Rubisco polynucleotide, is cloned into an expression vector, such as, for example, the vector described in U.S. Pat. No. 5,955,310 to Widner et al. “METHODS FOR PRODUCING A POLYPEPTIDE IN A BACILLUS CELL. After inserting the polynucleotide of interest into a vector, the vector is transformed into a bacterial, e.g., a Bacillus subtilis strain PL1801IIE (amyE, apr, npr, spoIIE::Tn917) host. The introduction of an expression vector into a Bacillus cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen (1979) Molecular General Genetics 168:111), by using competent cells (see, e.g., Young and Spizizin (1961) Journal of Bacteriology 81:823, or Dubnau and Davidoff-Abelson (1971) Journal of Molecular Biology 56:209), by electroporation (see, e.g., Shigekawa and Dower (1988) Biotechniques 6:742), or by conjugation (see, e.g., Koehler and Thome (1987) Journal of Bacteriology 169:5271).
The transformed cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods that are known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). The secreted polypeptide can be recovered directly from the medium.
The resulting polypeptide may be isolated by methods known in the art. For example, the polypeptide may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The isolated polypeptide may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Bollag et al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ; Bollag et al. (1996) Protein Methods 2nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ).
Cell-free transcription/translation systems can also be employed to produce polypeptides using DNAs or RNAs of the present invention. Several such systems are commercially available. A general guide to in vitro transcription and translation protocols is found in Tymms (1995) In vitro Transcription and Translation Protocols: Methods in Molecular Biology Volume 37, Garland Publishing, NY.
The foregoing and other aspects of the invention may be better understood in connection with the following non-limiting examples.
EXAMPLES
Example 1
Expression of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from E. coli 
Transformation:
Rubisco polynucleotides of the present invention that encode both Rubisco large and small subunits were cloned into vector pCK 110700-I-Bla depicted in FIG. 2, then transformed into E. Coli host strain NM522 (Stratagene, La Jolla, Calif.) using heat shock treatment at 42° C.
Cell Growth:
200 μl of cell growth media (32 g casein hydrosylate, 6 g KH2HPO4, 6 g Na2HPO4, and 0.68 g K2SO4) was aliquoted into a Nunc steril 96 well flat bottomed plate. Cultures were inoculated with 160 μl/well cell media containing 1% glucose and 30 μg/ml chloramphenicol. Plates were sealed with Qiagen Air Pore Tape and a sterile Nunc plate lid was placed over the plates. The plates were shaken at 37° C. in a Kuhner Shaker.
Induction:
The next day, 290 μl of cell growth media with 1% glucose and 30 μg/mL chloramphenicol (“inducing media”) was added to each well of a 96 well MegaTitre plate. Cells from the overnight cultures were mixed, then inoculated into the wells (10 μl/well) of the Megatitre plates containing the inducing media. The plates were sealed with Air Pore Tape and shaken at 37° C. in a Kuhner Shaker for 1 to 2 hours until reaching an OD600 of 0.2 to 0.6, after which 30 μl of 1 mM isoprophylthio-β-galactoside (IPTG) was added to each well. The plates were resealed and allowed to incubate on the shaker for 6 hours. The plates were then centrifuged at 3300 rpm for 15 minutes at 4° C. The cell pellets were stored at −20° C. until assayed.
Cell Lysis:
Cells were lysed just prior to assaying. 300 μl of lysis buffer (50 mM HEPES buffer pH 7.5, 300 mM KCl, 20 mM MgCl2, 1 mM DTT, 5% Glycerol, 1 μl ReadyLyse Lysozyme per ml lysis buffer, 20 μl 10 mg/ml PMBS per ml lysis buffer, 1 μl 200 mM PMSF in isopropanol per ml lysis buffer) was added to each well of the plates. The plates were then sealed and shaken until the cells were lysed (30 minutes to 2 hours).
Example 2
Assay for Presence of Rubisco Activity
The following assay was used to ascertain the presence of Rubisco activity. 100 μl cell lysate from Example 1 was transferred into the wells of a 96 well flat bottomed plate. A solution of 14C sodium bicarbonate was prepared by mixing 1 ml of a 14C sodium bicarbonate solution, 1 mCi/1 ml, (Sigma-Aldrich, Inc., St. Louis, Mo.) with 63 ml of 16 mM 12C sodium bicarbonate. A 330 mM stock solution of ribulose 1,5-bisphosphate was prepared by dissolving 100 mg ribulose 1,5-bisphosphate (Sigma-Aldrich, Inc., St. Louis, Mo.) in 1 ml water. The 330 mM ribulose 1,5-bisphosphate stock solution was diluted to make a 6 mM stock solution. 50 μl of a 50:50 6 mM Ribulose 1,5-Bisphosphate: 14C sodium bicarbonate solution was added to each well of the plate. After 1.5 to 2 hours, 100 μl 1N HCl was added to each well. The plates were then placed in a 70° C. oven overnight to dry.
A Nunc nylon transfer membrane was placed into the bottom of a Nunc Omnitray (Nalge Nunc International, Rochester, N.Y.) and 3 μl of cell lysate/Ribulose 1,5-Bisphosphate: 14C sodium bicarbonate mixture from each well of the flat bottomed plate was transferred onto the nylon membrane. The membrane was allowed to dry, after which it was placed in a Molecular Dynamics Phosphorimaging Cassette (Amersham Biosciences, Piscataway, N.J.). The cassette was exposed overnight and the phosphorscreen was removed from the cassette and scanned in a Molecular Dynamics Phosphorimager using standard methods.
14C incorporation at a level greater than a negative control, which was a vector without a Rubisco polynucleotide (rbcLS) insert, indicated the presence of Rubisco activity.
Example 3
Assay to determine Specific Activity of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase
The value that roughly describes the specific activity of Rubisco is CPM/nM Rubisco. The following assay utilizes an active site titration with 2-carboxyarabinitol 1,5-bisphosphate (CABP) along with a time course 14CO2 incorporation assay to roughly determine Rubisco specific activity.
50 μl aliquots of cell lysate from Example 1 were dispensed into polypropylene 96 well plates. Various concentrations of CABP inhibitor were added to the wells. 50 μl of the 50:50 Ribulose 1,5-bisphosphate (“RuBP”): 14C sodium bicarbonate solution was added to each well of the plates. In half the wells the reaction was stopped after 10 minutes by adding 50 μl of 1 N HCl. After 20 minutes, the reaction was stopped in the remaining wells by adding 50 μl of 1 N HCl. The plates were dried overnight at 70° C. The following day, 150 μl of 10 mM HCl was added to each well to resuspend the mixtures. The plates were blotted onto a nylon membrane, then exposed to phosphorscreens prior to phosphorimaging as described in Example 2.
Initial rates were monitored at saturating RuBP concentrations (1 mM) for the carboxylation reaction run for 5 minutes, with samples take at the following timepoints: 5=0, 1 minute, 2 minutes, 3 minutes, 4 minutes, and 5 minutes. The rates were determined as counts incorporated per minute or as density per minute. Rubisco polypeptide sample concentrations were determined using CABP titration as described below, and/or by quantitative western blots.
To compute Rubisco polypeptide sample concentrations from CABP titrations, Intensity vs. time was plotted for each concentration of CABP. From the slope of each plot (i.e., each plot corresponds to a fixed concentration of CABP), the value for CPM (i.e., counts per minute) was determined. A plot of CPM/min vs. concentration of CABP was then made. The x-intercept provided the concentration for Rubisco. The specific activity was computed for each Rubisco polypeptide as CPM/weight Rubisco.
The specific activity values for the enzymes corresponding to Rubisco polypeptides of the present invention are provided in Table 3.







TABLE 3







Rubisco Specific Activity (counts per minute (“cpm”)/min/mg)











Rubisco Specific Activity



Clone Name
(cpm/min/mg Rubisco)














RT24
180



(Encoded by SEQ ID ID



NO: 4)



RT25
300



(Encoded by SEQ ID



NO: 7)



RT28
600



(Encoded by SEQ ID



NO: 10)



RT30
600



(Encoded by SEQ ID



NO: 13)



RT106
270



(Encoded by SEQ ID



NO: 106)



RT108
180



(Encoded by SEQ ID



NO: 19)



RT111
300



(Encoded by SEQ ID



NO: 22)



RT113
480



(Encoded by SEQ ID



NO: 25)



RT115
300



(Encoded by SEQ ID



NO: 28)



RT116
300



(Encoded by SEQ ID



NO: 31)



RT117
300



(Encoded by SEQ ID



NO: 34)



RT118
300



(Encoded by SEQ ID



NO: 37)



Synechococcus PCC 6301
300



(wildtype, encoded by



SEQ ID NO: 1)



F2A-10
1710



(encoded by SEQ ID



NO: 40)



F2A-16
1530



(encoded by SEQ ID



NO: 43)



F2A-20
580



(encoded by SEQ ID



NO: 46)



F2B-2
1280



(encoded by SEQ ID



NO: 49)



F2B-3
1280



(encoded by SEQ ID



NO: 52)










Example 4
Michaelis-Menten Kinetics Characterization of Rubisco Polypeptides
Vmax and KM were determined by Michaelis-Menten kinetics for the Rubisco polypeptides encoded by SEQ ID NO: 1, 10, and 40. 14CO2 incorporation was measured as described in Example 3 at various timepoints. Rates were measured over a range of RuBP concentrations to obtain rate (V) vs. [RuBP (substrate)] plots that provided a best fit to the Michaelis-Menten kinetic equation:




  V
  =
  
    
      V
      max
    
    ⁢
    
      
        [
        
          substrate
          ⁢
          
            
          
          ⁢
          
            (
            RuBP
            )
          
        
        ]
      
      
        
          [
          
            substrate
            ⁢
            
              
            
            ⁢
            
              (
              RuBP
              )
            
          
          ]
        
        +
        
          K
          M
        
      
    
  




Using GraphPad Prizm software, the V. vs. [RuBP] plots were fit to the Michaelis-Menten kinetic equation and Vmax and KM were extracted. Kcat (i.e., Vmax/[Rubisco Polypeptide]) was determined from the previously determined Vmax. Rubisco polypeptide concentration was determined from a quantitative western in accordance with methods known to those having ordinary skill in the art. The kinetic characterization data is provided in Table 4.








TABLE 4







Kinetic Parameters for Rubisco Polypeptides















kcat/KM






normalized to


Rubisco



wildtype


Polypeptide

KM (μM

Synechococcus


(RuBP)
Kcat (s−1)
RuBP)
Kcat/KM
sp. PCC6301














Synechococcus
6.3
78.6
0.08
1


sp. PCC6301


(wildtype


encoded by SEQ


ID NO: 1)


RT28
2.6
20.8
0.13
1.6


(encoded by SEQ


ID NO: 10)


F2A-10
26.5
58.5
0.45
5.7


(encoded by SEQ


ID NO: 40)









Example 5
Transformation of Rubisco Polynucleotides into Snechocystis sp.
Rubisco polypeptides of the present invention were transformed into Synechocystis sp. PCC 6803 using the Gene replacement vector pGR-1 depicted in FIG. 3. The vector contains 1.5 kb of upstream sequence of Synechocystis sp. PCC6803 wildtype Rubisco gene, rbcLS, which contains the wildtype promoter and ribosome binding site. The upstream sequence also provides for homologous recombination to replace the wildtype Rubisco gene in Synechocystis sp. PCC 6803. The vector was designed so that the wildtype rubisco coding sequence is replaced with SEQ ID NO: 10 (clone RT28) via a double crossover in both 5′ and 3′ flanking regions. The vector was a pBluescript II KS (2.96 kb) from Invitrogen, Inc. (Carlsbad, Calif.) with the internal Af1III site destroyed. The kanamycin resistance cassette, nptII, was cloned from pUC4K. The polynucleotide sequence corresponding to SEQ ID NO: 10 was cloned into this gene replacement vector and transformed into Synechocystis 6803 At kanamycin 150 μg/ml, by a PCR check, it was determined to have replaced the wildtype Rubisco gene in about 50% of the clones.
Other vectors were designed for transforming Rubisco polynucleotides into Synechocystis sp. pDNR-1 (BD Biosciences, Clontech, Palo Alto, Calif.) is modified to remove the loxP sites, replace the pUC origin of replication with a p15A origin of replication, and remove the chloramphenicol resistance cassette (CmR). In addition, 5′ and 3′ sequences flanking Synechocystis sp. rbcLS are inserted to create gene replacement vectors, pGR-2a, pGR-2b, and pGR-3a. Vector pGR-2a contains the 5′ and 3′ sequences flanking Synechocystis sp. rbcLS depicted as SEQ ID NOS: 58 and 59, respectively. Vector pGR-2b contains the 5′ sequence flanking Synechocystis sp. rbcLS depicted as SEQ ID NOS: 60, and the same 3′ flanking sequence as in pGR-2a (i.e., SEQ ID NO: 59). Vector pGR-3a contains the same 3′ rbcLS flanking sequence as pGR-2a, and the 3′ rbcLS flanking sequence depicted as SEQ ID NO: 61.
Synechocystis sp. PCC 6803 is transformed with the Rubisco polynucleotides of the present invention. A 20-50 ml PCC 6803 culture are grown on BG11+16 mM NaHCO3 for about 4 to 5 days and cultured until reaching an OD730 of about 1 to 1.5 ( ̃108 cells/ml). All steps are carried out under visible light. 100 μl of cells (clumps broken up by mixing) are transferred into the wells of a sterile 96-well plate. 1-7 μg DNA (plasmid) is added and mixed with the cells. The plate is left uncovered under light at room temperature for about 24 hours. On day 2, all cells are plated directly on selective medium (BG11 agar+10 μg/ml kanamycin+16 mM NaHCO3) and incubated under light at room temperature prior to picking.
Example 6
Whole Cell CO2 Fixation Assay
The whole cell CO2 fixation assay measures the flux through the Calvin cycle in a live-photosynthesizing cell. The difference between this assay and the in vitro assays described above is that RuBP is not added to the cells. The cells have the capability to regenerate RuBP using their endogenous Calvin cycle machinery. A schematic of the assay is provided in FIG. 4.
Synechocystis sp. PCC 6803 was cultured at room temperature, under light, and in BG11, 16 mM bicarbonate. After reaching an OD730 of about 0.45 to 0.7, 600 μl of culture was placed into a short glass vial with cap and placed on a light box for about 15 minutes. Add 50 μl of a 14C sodium bicarbonate solution, 1 mCi/1 ml, (Sigma-Aldrich, Inc., St. Louis, Mo.) to 450 μl of cell. Take 50 μl of the culture/14C-NaHCO3 mixture was quenched in 100 μl of 1 N HCl at various timepoints (e.g., t=0 minutes, 5 minutes, 10 minutes, 15 minutes, and so on) on a NUNC Heat Resistant (96 well) plate. Dry the plate completely overnight in an oven at −70° C. 150 μl of scintillation cocktail was added, and the vials were maintained away from the light. The plates were read by a scintillation counter. Normalized rates (CPM/min) to Rubisco concentrations obtained by quantitative western.
While the above CO2 fixation assay was performed, 150 μl of culture was removed and quickly spun down to remove all supernatant for use in a western blot quantitation assay. The cell pellet was resupended in 32.5 μl of water, 12.5 μl of NP0007 NUPAGE LDS Sample Buffer (4×) (Invitrogen, Carlsbad, (Calif.). The resuspended mixture was boiled for about 10 minutes, after which 10 μl of NP0004 NUPAGE Sample Reducing Agent (10×) reducing agent (Invitrogen, Carlsbad, Calif.) was added. The boiled samples were flash frozen in a mixture of ethanol and dry ice, then stored at −20° C.
A quantitative western blot was done using Spinach Rubisco (Sigma-Adrich, St. Louis, Mo.) as a standard, to quantify the amount of Rubisco polypeptide in the CO2 fixation assay.
All publications, patents, patent applications, and other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.
While preferred embodiments of the invention have been illustrated and described, it will be readily appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

## Claims

1. An isolated or recombinant Rubisco large subunit polypeptide comprising an amino acid sequence that has at least 99% sequence identity-to SEQ ID NO: 11 and at least one of the following residues: (a) an isoleucine at position 454, (b) a valine at position 84, (c) a lysine at position 158, (d) a leucine at position 166, and (e) a methionine at position 317.

2. The isolated or recombinant polypeptide of claim 1, which comprises the amino acid sequence listed in SEQ ID NO: 11 or the amino acid sequence listed in SEQ ID NO:11 having conservative mutations at less than 1% of the positions.

3. The isolated or recombinant polypeptide of claim 1, wherein the amino acid sequence further comprises at least one amino acid residue selected from the group consisting of:
D at position 92; F at position 93; L at position 113; L at position 116; L at position 117; L at position 127; A at position 129; V at position 137; I at position 139; Y at position 141; L at position 142; S at position 149; G at position 154; M at position 209; Q at position 219; E at position 220; E at position 223; A at position 225; T at position 232; Q at position 246; E at position 249; A at position 252; I at position 257; T at position 259; G at position 269; S at position 276; Y at position 280; L at position 286; A at position 297; K at position 303; T at position 304; Q at position 322; T at position 325; R at position 336; Q at position 337; T at position 338; I at position 343; Q at position 345; L at position 346; S at position 349; F at position 350; P at position 352; E at position 353; N or T at position 356; N at position 359; D at position 362; G at position 366; F at position 372; A at position 373; A at position 389; I at position 415; and R at position 450.

4. The isolated or recombinant polypeptide of claim 1, wherein the amino acid sequence comprises at least two amino acid residues selected from the group consisting of: V at position 84, K at position 158, L at position 166, M at position 317, and I at position 415.

5. An isolated polypeptide having Rubisco specific activity comprising the Rubisco large subunit polypeptide of claim 1, wherein the amino acid sequence has an isoleucine at position 454 and wherein the polypeptide is capable of a higher kcat than the wild-type polypeptide encoded by SEQ ID NO:1.

6. An isolated polypeptide having Rubisco specific activity comprising the Rubisco large subunit polypeptide of claim 1, wherein the amino acid sequence has a valine at position 84 and wherein the polypeptide is capable of a lower KM than the wild-type polypeptide encoded by SEQ ID NO:1.

7. An isolated polypeptide having Rubisco specific activity comprising the Rubisco large subunit polypeptide of claim 1, wherein the amino acid sequence has a lysine at position 158 and wherein the polypeptide is capable of a lower KM than the wild-type polypeptide encoded by SEQ ID NO:1.

8. An isolated polypeptide having Rubisco specific activity comprising the Rubisco large subunit polypeptide of claim 1, wherein the amino acid sequence has a leucine at position 166 and wherein the polypeptide is capable of a lower KM than the wild-type polypeptide encoded by SEQ ID NO:1.

9. An isolated polypeptide having Rubisco specific activity comprising the Rubisco large subunit polypeptide of claim 1, wherein the amino acid sequence has a valine at position 84 and wherein the polypeptide is capable of a lower KM than the wild-type polypeptide encoded by SEQ ID NO:1.