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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)

Definitions

• the invention relates to methods and compositions for generating, modifying, adapting, and optimizing polynucleotide sequences that encode proteins having PEPC enzyme activities which are useful for introduction into plant species, and other hosts, and related aspects.
• Phosphoenolpyruvate Carboxylase Phosphoenolpyruvate (PEP) carboxylase (PEPC; EC 4.1.1.31) is a key enzyme of photosynthesis in those plant species exhibiting the C4 or CAM pathway for CO 2 fixation.
• the principal substrate of PEPC is the free form of PEP.
• PEPC catalyzes the conversion of PEP and bicarbonate to oxalacetic acid inorganic phosphate (Pi). This reaction is the first step of a metabolic route known as the C4 dicarboxylic acid pathway, which minimizes losses of energy produced by photorespiration.
• PEPC is present in plants, algae, cyanobacteria, and bacteria; the enzymatic properties differ based on the source.
• PEPC The primary structures of PEPC from E. coli, Anabaena variabilis, and maize, among others, have been deduced from cDNA sequences and are available in the literature and GenBank.
• the homology found in the C-terminal half of the protein are consistent with the C-terminal half containing a catalytic domain, and the sequence between residues 603 to 616 ofthe Zea mays PEPC enzyme (-
• PEPC is a homomultimer, typically a homotetramer or homodimer, and is extrachloroplastic and located in the cytosol ofthe mesophyll leaves of C4 and CAM plants.
• C4-specific PEPC other isozyme forms of the enzyme occur in C3 plants or etiolated C4 leaves.
• PEPC from C4 plants is activated by glucose 6-phosphate (G6P), which induces an increase in Vmax and in substrate affinity for binding PEP.
• G6P glucose 6-phosphate
• a metabolite, L-malate, which is an intermediate product ofthe carboxylation reaction, is an inhibitor of PEPC activity. It shows a cooperative effect and seems to interact with PEPC at different sites, producing noncompetitive or competitive inhibition depending on pH and concentration. G6P produces a decrease in the inhibitory effect of malate.
• oxaloacetate, aspartate, and certain flavenoids have been shown to inhibit PEPC (Pairoba et al. (1996) Biosci. Biotech. Biochem. 60: 779;
• PEPC is a key control point for accomplishing the primary carboxylation of PEP, a major component of CO 2 fixation in C4 and CAM plants, it would be desirable to have a method for producing PEPC encoding sequences and novel PEPC proteins wherein the enzymatic activity of PEPC has (1) an decreased Km for substrate, (2) a decreased Km for activator, (3) a constitutive PEPC activity in the absence of activator which is higher than naturally occurring PEPC in the absence of activator,
• an improved PEPC phenotype e.g., reduced sensitivity to inhibitors (e.g., malate, pH, etc.), reduced dependence on activators (e.g., G6P, serine/threonine), improved catalytic efficiency via increasing Vmax and/or increasing the apparent affinity of substrates for the enzyme, and/or relieving a requirement for allosteric activ
• the present invention provides a method for rapid evolution of polynucleotide sequences encoding a PEPC enzyme, that, when transferred into an appropriate plant cell, or photosynthetic microbial host and expressed therein, confers an enhanced metabolic phenotype to the host to increase carbon fixation ratio and/or rate, or to increase the accumulation or depletion of certain metabolites and energy storage sinks.
• polynucleotide sequence shuffling and phenotype selection such as detection of a parameter of PEPC enzyme activity, is employed recursively to generate polynucleotide sequences which encode novel proteins having desirable PEPC enzymatic catalytic function(s), regulatory function(s), and related enzymatic and physicochemical properties.
• the invention is described principally with reference to the metabolic enzyme activities of plants and/or photosynthetic microbes and/or bacteria, defined as PEPC, or an isozyme thereof, including, respectively, plant and algal as well as bacterial forms.
• PEPC Embodiment - Lowered Km for substrate provides an isolated polynucleotide encoding an enhanced PEPC protein having PEPC catalytic activity wherein the Km for a substrate (PEP, bicarbonate) is significantly lower than in a protein encoded by a parental polynucleotide encoding a naturally-occurring PEPC enzyme.
• the Km for a substrate PEP, bicarbonate
• the isolated polynucleotide encoding an enhanced PEPC protein and in an expressible form can be transferred into a host plant, such as a crop species, wherein suitable expression ofthe polynucleotide in the host plant will result in improved carbon fixation biosynthesis efficiency as compared to the naturally-occurring host plant species, usually under certain conditions.
• the isolated polynucleotide can encode a PEPC, such as a bacterial form, or may encode a PEPC enzyme such as that found in green algae, and higher plants.
• the isolated polynucleotide can comprise a substantially full-length or full-length coding sequence substantially identical to a naturally occurring PEPC gene and/or an isozyme thereof, typically comprising a shuffled PEPC gene.
• the invention provides a polynucleotide comprising: (1) a sequence encoding a shuffled PEPC gene operably linked to a transcriptional regulatory sequence functional in a host cell, and further linked to (2) a selectable marker gene which affords a means of selection when expressed in host cells.
• the invention provides a polynucleotide comprising: (1) a sequence encoding a shuffled PEPC gene having at least 95 percent sequence identity to a PEPC encoding sequence in the genome of a naturally-occurring plant, operably linked to a transcriptional regulatory sequence functional in a host cell, and further linked to (2) a selectable marker gene which affords a means of selection when expressed in host cells.
• the invention provides a polynucleotide comprising: (1) a sequence encoding a shuffled PEPC gene operably linked to a transcriptional regulatory sequence functional in a host cell, (2) a sequence encoding a shuffled Rubisco gene operably linked to a transcriptional regulatory sequence functional in the host cell and, optionally, further linked to (3) a selectable marker gene which affords a means of selection when expressed in host cells.
• the invention provides an isolated polynucleotide encoding an enhanced PEPC protein having PEPC catalytic activity wherein the Km for a substrate is significantly higher than a protein encoded by a parental polynucleotide encoding a naturally-occurring PEPC enzyme.
• the enhanced PEPC protein is often catalytically active in the cytosol of cells of higher plants, particularly plants of agronomic importance.
• the enhanced PEPC protein is at least 90 percent sequence identical to a naturally occurring PEPC protein encoded by a genome of a plant or algae.
• the invention provides an isolated polynucleotide encoding an enhanced PEPC protein having PEPC catalytic activity wherein the Km (Ki) for an inhibitor (e.g., L-malate, aspartate, metabolic effectors), especially at pH levels below 8.0, is significantly higher than a protein encoded by a parental polynucleotide encoding a naturally-occurring PEPC enzyme.
• the concentration of inhibitor required to produce half-maximal inhibition of catalysis is typically at least one-half logarithm unit higher than a parental PEPC, often at least one log unit or more higher.
• the invention provides an isolated polynucleotide encoding an enhanced PEPC protein having PEPC catalytic activity wherein the Km for an activator (e.g., glucose 6-phosphate, G6P; triose phosphate) is significantly lower than in a protein encoded by a parental polynucleotide encoding a naturally- occurring PEPC enzyme.
• the concentration of activator required to produce half-maximal activation of catalysis is typically at least one-half logarithm unit lower than a parental PEPC, often at least one log unit or more lower, in some embodiments at least two log units or more lower.
• the shuffled e.g., glucose 6-phosphate, G6P; triose phosphate
• PEPC protein possesses, in the substantial absence of activator, PEPC catalytic activity approximately equivalent to or greater than that of a naturally-occurring PEPC protein which is maximally stimulated with activator.
• the invention provides an enhanced PEPC protein having PEPC catalytic activity wherein: (1) the Km for substrate is significantly lower than in a protein encoded by a parental polynucleotide encoding a naturally- occurring PEPC enzyme, and (2) the Km for inhibitor is significantly higher than a protein encoded by a parental polynucleotide encoding a naturally-occurring PEPC enzyme, and/or (3) the Km for activator is significantly lower than in a protein encoded by a parental polynucleotide encoding a naturally-occurring PEPC enzyme, and/or (4) the enhanced PEPC protein possesses a catalytic activity in the substantial absence of activator and inhibitor which is at least 25 percent or more greater than a naturally-occurring PEPC that is maximally stimulated with activator in the substantial absence of inhibitor and/or (5) the PEPC activity is desensitized to pH- mediated changes in allosteric control by inhibitors and/or activators; often the naturally-occurring PEPC used for comparison is an
• the invention provides a polynucleotide sequence encoding a shuffled plant or algal PEPC, wherein the shuffled PEPC protein possesses a detectable enzymatic activity wherein: (1) the Km for substrate is significantly lower than in a protein encoded by a parental polynucleotide encoding a naturally-occurring PEPC enzyme, (2) the Km for a PEPC inhibitor is significantly higher than a PEPC protein encoded by a parental polynucleotide encoding a naturally-occurring PEPC enzyme, and/or (3) the Km for an PEPC activator is significantly lower than a protein encoded by a parental polynucleotide encoding a naturally-occurring PEPC enzyme, and/or (4) the Vmax for PEPC catalytic activity is substantially higher than the Vmax for PEPC catalytic activity of naturally-occurring PEPC under equivalent assay conditions (e.g., same concentration(s) of substrates, activators, and inhibitors,
• the shuffled PEPC sequences encode proteins that have an altered binding to, or allosteric interaction with, a protein kinase or protein phosphatase, such that the binding constant for an inhibitor or activator on the PEPC protein may be substantially unchanged, however the shuffled PEPC, when modified by the protein kinase or phosphorylase, results in formation of a PEPC which has: (1) reduced sensitivity to inhibitors (e.g., malate) and/or (2) enhanced sensitivity to activators
• PEPC activity which is insensitive to activator and possesses at least one PEPC catalytic activity (e.g., substrate Km or Vmax) which is at least 25 percent greater than that of a naturally-occurring PEPC that is maximally stimulated with activator in the substantial absence of inhibitor; often the naturally-occurring PEPC used for comparison is a PEPC species which has a polypeptide that has the greatest percentage sequence identity, among the collection of then known PEPC sequences, to the shuffled PEPC polypeptide.
• PEPC catalytic activity e.g., substrate Km or Vmax
• the binding constant for an inhibitor, activator, and/or substrate will be at least one-half log unit higher or lower than an equivalent naturally occurring PEPC of greatest sequence homology (percent sequence identity) to the shufflant.
• the invention provides an improved PEPC, or shufflant thereof, and a polynucleotide encoding same.
• the polynucleotide will be operably linked to a transcription regulation sequence forming an expression construct, which may be linked to a selectable marker gene.
• such a PEPC polynucleotide is present as an integrated transgene in a plant chromosome in a format for expression and processing ofthe enzyme.
• the transferred shuffled PEPC gene sequence is derived by shuffling a pool of parental sequences, at least one of which encodes a bacterial PEPC.
• the transcription control sequences comprise tissue-specific or conditional promoters to overcome possible detrimental effects of constitutive expression.
• the invention provides an improved PEPC, or shufflant thereof, wherein the improved PEPC has at least 80 sequence identity to the polypeptide sequence of a naturally-occurring plant PEPC, and which has an enhanced PEPC enzymatic phenotype; and a polynucleotide encoding same.
• the polynucleotide will be operably linked to a transcription regulation sequence forming an expression construct, which may be linked to a selectable marker gene.
• such a PEPC polynucleotide is present as an integrated transgene in a plant chromosome and may be accompanied, in linked or unlinked configuration, with a Rubisco encoding polynucleotide and/or an ADPGPP encoding polynucleotide; often such Rubisco and/or ADPGPP polynucleotides encode an optimized, shuffled enzyme.
• the invention provides a hybrid PEPC composed of a shufflant comprising a sequence of at least 25 contiguous nucleotides at least 95 percent identical to a plant PEPC gene and a sequence of at least 25 contiguous nucleotides at least 95 percent identical to a bacterial or algal PEPC gene, and a polynucleotide encoding same, and typically encoding a substantially full-length
• PEPC protein usually comprising at least 90 percent ofthe coding sequence length, but not necessarily sequence identity, of a naturally occurring PEPC protein.
• the polynucleotide will be operably linked to a transcription regulation sequence forming an expression construct, which may be linked to a selectable marker gene.
• such a polynucleotide is present as an integrated transgene in a plant chromosome. It can be desirable for such a polynucleotide transgene to be transmissible via germline transmission in a plant.
• the invention provides expression constructs, including bacterial plasmids, shuttle vectors, and plant transgenes, wherein the expression construct comprises a transcriptional regulatory sequence functional in plants operably linked to a polynucleotide encoding an enhanced PEPC protein.
• the expression construct comprises a transcriptional regulatory sequence functional in plants operably linked to a polynucleotide encoding an enhanced PEPC protein.
• polynucleotide sequences encoding PEPC proteins it is generally desirable to express such encoding sequences in plant cells with the expression constructs containing the necessary sequences for appropriate transcription, translation, and processing.
• the invention further provides plants and plant germplasm comprising said expression constructs, typically in stably integrated or other replicable form which segregates and can be stably maintained in the host organism, although in some embodiments it is desirable for commercial reasons that the expression sequence not be in the germline of sexually repoducible plants.
• the invention provides a method for obtaining an isolated polynucleotide encoding an enhanced PEPC protein having PEPC catalytic activity wherein the Km for substrate is significantly lower than a protein encoded by a parental polynucleotide encoding a naturally-occurring PEPC enzyme, the method comprising: (1) recombining sequences of a plurality of parental polynucleotide species encoding at least one PEPC sequence under conditions suitable for sequence shuffling to form a resultant library of sequence-shuffled PEPC polynucleotides, (2) transferring said library into a plurality of host cells forming a library of transformants wherein sequence-shuffled PEPC polynucleotides are expressed, (3) assaying individual or pooled transformants for PEPC catalytic activity to determine the relative or absolute Km for substrate and identifying at least one enhanced transformant that expresses a PEPC activity which has a significantly lower Km for substrate than the PEPC activity encoded by the parental sequence(s), (4) recovering the
• the recovered sequence-shuffled PEPC polynucleotide encoding an enhanced PEPC is recursively shuffled and selected by repeating steps 1 through 4, wherein the recovered sequence-shuffled PEPC polynucleotide is used as at least one parental sequence for subsequent shuffling.
• step 3 comprises assaying individual or pooled transformants for PEPC catalytic activity to determine the relative or absolute Km for the inhibitor and identifying at least one enhanced transformant that expresses a PEPC activity which has a significantly higher Km for inhibitor than the PEPC activity encoded by the parental sequence(s).
• step 3 comprises assaying individual or pooled transformants for PEPC catalytic activity to determine the relative or absolute Km for activator, and identifying at least one enhanced transformant that expresses a
• PEPC activity which has a significantly lower Km for activator than the PEPC activity encoded by the parental sequence(s).
• the PEPC gene sequence(s) is/are obtained as an isolated polynucleotide and is shuffled by any suitable shuffling method known in the art, such as DNA fragmentation and PCR, error-prone PCR, and the like, preferably with one or more additional parental polynucleotides encoding all or a part of another PEPC species.
• the population of sequence-shuffled PEPC polynucleotides are each operably linked to an expression sequence and transferred into host cells, preferably host cells substantially lacking endogenous PEPC activity, wherein the sequence- shuffled PEPC polynucleotides are expressed, forming a library of sequence-shuffled
• PEPC transformants A sample of individual transformants and/or their clonal progeny are isolated into discrete reaction vessels for PEPC activity assay, or are assayed in situ in certain embodiments. For samples assayed in reaction vessels, aliquots ofthe samples are separated into a plurality of reaction vessels containing an approximately equimolar amount of PEPC or total protein, and each vessel is assayed for PEPC activity in the presence of a predetermined concentration of substrate which ranges from about 0.0001 times the predetermined Km for substrate ofthe PEPC encoded by the parental polynucleotide(s) to about 10,000 times the predetermined Km for substrate ofthe PEPC encoded by the parental polynucleotide(s); the plurality of reaction vessels for each shufflant sample may also contain a fixed or variable concentration of activator and/or inhibitor, or neither.
• a Km value and/or Vmax is calculated by conventional art-known means for the sequence-shuffled PEPC of each transformant; typically the Km and Vmax values for a specific inhibitor or activator are determined.
• Sequence-shuffled polynucleotides encoding PEPC proteins that have significantly decreased Km and/or Vmax values for substrate, and/or significantly increased Km values of inhibitor, and/or significantly decreased Km values for activator are selected and used as parental sequences for at least one additional round of sequence shuffling by any suitable method and selection for further optimization ofthe desired PEPC phenotype.
• the shuffling and selection process is performed iteratively until sequence shuffled polynucleotides encoding at least one PEPC enzyme having a desired PEPC enzymatic phenotype is obtained, or until the optimization to reduce the relevant Km (or increase Vmax) has plateaued and no further improvement is seen in subsequent rounds of shuffling and selection.
• the sequence-shuffled polynucleotides operably linked to an expression sequence is also linked, in polynucleotide linkage, to an expression cassette encoding a selectable marker gene. Transformants are propagated on a selective medium to ensure that transformants which are assayed for PEPC activity contain a sequence-shuffled PEPC encoding sequence in expressible form.
• the above-described method is modified such that PEPC activity is assayed in the presence of varying concentrations of inhibitor and the Km for inhibitor is determined.
• Each vessel containing an aliquot of a transformant is assayed for PEPC activity in the presence of a predetermined concentration of inhibitor which ranges from about 0.0001 times the predetermined Km for inhibitor of the PEPC encoded by the parental polynucleotide(s) to about 10,000 times the predetermined Km for inhibitor ofthe PEPC encoded by the parental polynucleotide(s).
• a Km value is calculated by conventional art-known means for the sequence-shuffled PEPC of each transformant.
• Sequence-shuffled polynucleotides encoding PEPC proteins that have significantly increased Km values for inhibitor are selected and used as parental sequences for at least one additional round of sequence shuffling by any suitable method and selection for increased Km values for inhibitor.
• the shuffling and selection process is performed iteratively until sequence shuffled polynucleotides encoding at least one PEPC enzyme having a desired Km value is obtained, or until the optimization to increase the Km has plateaued and no further improvement is seen in subsequent rounds of shuffling and selection.
• the above-described method is modified such that PEPC activity is assayed in the presence of varying concentrations of activator and the Km for activator is determined.
• Each vessel containing an aliquot of a transformant is assayed for PEPC activity in the presence of a predetermined concentration of activator which ranges from about 0.0001 times the predetermined Km for activator ofthe PEPC encoded by the parental polynucleotide(s) to about 10,000 times the predetermined Km for activator ofthe PEPC encoded by the parental polynucleotide(s).
• a Km value is calculated by conventional art-known means for the sequence-shuffled PEPC of each transformant.
• Sequence-shuffled polynucleotides encoding PEPC proteins that have significantly decreased Km values for activator are selected and used as parental sequences for at least one additional round of sequence shuffling by any suitable method and selection for decreased Km values for activator.
• the shuffling and selection process is performed iteratively until sequence shuffled polynucleotides encoding at least one PEPC enzyme having a desired Km value is obtained, or until the optimization to increase the Km has plateaued and no further improvement is seen in subsequent rounds of shuffling and selection.
• the method comprises conducting biochemical assays on sample aliquots of transformants to determine PEPC enzyme activity so as to establish the ratio ofthe Km for activator to the Km for inhibitor for individual transformants.
• Sequence-shuffled polynucleotides encoding PEPC are obtained from transformants exhibiting a decrease in said ratio as compared to the ratio in PEPC produced from the parental encoding polynucleotide(s) to provide selected sequence- shuffled PEPC polynucleotides which can be used as parental sequences for at least one additional round of sequence shuffling by any suitable method and selection for a decreased ratio of Km(activator) to Km(inhibitor).
• the shuffling and selection process is performed iteratively until sequence shuffled polynucleotides encoding at least one PEPC enzyme having a desired Km ratio is obtained, or until the optimization to decrease the Km ratio has plateaued and no further improvement is seen in subsequent rounds of shuffling and selection.
• the method comprises conducting biochemical assays on sample aliquots of transformants to determine the pH profile of PEPC enzyme activity and the pH sensitivity of activator and inhibitor effects.
• a pH desensitized PEPC exhibits PEPC activity such that an increase in pH from approximately 7.0 to 8.0 produces: (1) a decrease in the Ki of malate or other inhibitor of less than one half of the decrease seen in parental PEPC enzyme under identical conditions, and/or (2) an increase in Km of activator of less than one half of the increase seen in parental PEPC enzyme under identical conditions.
• Sequence-shuffled polynucleotides encoding PEPC are obtained from transformants exhibiting a decrease in pH effect as compared to the produced from the parental encoding polynucleotide(s) to provide selected sequence-shuffled PEPC polynucleotides which can be used as parental sequences for at least one additional round of sequence shuffling by any suitable method and selection for a decreased ratio of Km( activator) to Km(inhibitor).
• the shuffling and selection process is performed iteratively until sequence shuffled polynucleotides encoding at least one PEPC enzyme having a desired Km ratio is obtained, or until the optimization to decrease the Km ratio has plateaued and no further improvement is seen in subsequent rounds of shuffling and selection.
• the host cell for transformation with sequence-shuffled polynucleotides encoding PEPC is a bacterial mutant which lacks a functional PEPC protein, such as E. coli mutant or an equivalent.
• polynucleotides encoding naturally- occurring PEPC protein sequences of a plurality of species of photosynthetic prokaryotes and/or algae and/or higher plants are shuffled by a suitable shuffling method to generate a shuffled PEPC polynucleotide library, wherein each shuffled PEPC encoding sequence is operably linked to an expression sequence, and which may optionally comprise a linked selectable marker gene cassette.
• Said library is transformed into a host cell population to form a transformed host cell library.
• the transformed host cell library is propagated on growth medium, which may contain a selection agent to ensure retention of a linked selectable marker gene.
• Transformed host cells which are screened for under the most stringent conditions are isolated individually or in pools, and the sequence-shuffled polynucleotide sequences encoding PEPC are recovered, and optionally subjected to at least one subsequent iteration of shuffling and selection on growth medium and PEPC activity screening.
• transformants are assayed for inhibitor-resistant PEPC activity and/or high activity PEPC in absence of activator.
• the recovered sequence- shuffled PEPC polynucleotide(s) encode(s) an enhanced PEPC protein.
• the invention provides a plant cell protoplast and clonal progeny thereof containing a sequence-shuffled polynucleotide encoding a PEPC which is not encoded by the naturally occurring genome ofthe plant cell protoplast.
• the invention also provides a collection of plant cell protoplasts transformed with a library of sequence-shuffled PEPC polynucleotides in expressible form.
• the invention also provides a regenerated plant containing at least one species of replicable or integrated polynucleotide comprising a sequence-shuffled portion and encoding a PEPC polypeptide.
• the invention provides a method variation wherein at least one round of phenotype selection is performed on regenerated plants derived from protoplasts transformed with sequence-shuffled PEPC library members.
• the phenotype selection comprises a determination, either directly or by proxy, of carbon fixation via the PEPC reaction.
• the invention provides species-specific PEPC shuffling, wherein a transformed plant cell or adult plant or reproductive structure comprises a polynucleotide encoding a shuffled PEPC that is at least 95 percent sequence identical to the corresponding PEPC encoded by an untransformed naturally-occurring genome ofthe same taxonomic species of plant cell or adult plant.
• the shuffled PEPC results from shuffling of one or more alleles encoding the PEPC in the taxonomic species genome, optionally including mutagenesis in one or more ofthe iterative shuffling and selection cycles.
• the species-specific PEPC shuffling may include shuffling a polynucleotide encoding a full-length PEPC of a first taxonomic species under cond'tions whereby PEPC sequences of a second taxonomic species (or collection of species) are shuffled in at a low prevalence, such that the resultant population of shufflant polynucleotides contains, on average, shuffled polynucleotides composed of at least about 95 percent sequence encoding the first taxonomic species PEPC and less than about 5 percent sequence encoding the second taxonomic species (or collection of species) PEPC.
• the species-specific shufflants are thus highly biased towards identity with the first taxonomic species and shufflants which are selected for the desired PEPC phenotype are transferred back into the first taxonoic species for expression and regeneration of adult plants and germplasm.
• selected shufflants are backcrossed against the naturally occurring PEPC encoding sequences ofthe first taxonomic species to remove non-essential sequence alterations and harmonize the final shufflant sequence to the naturally-occurring PEPC sequence ofthe first taxonomic species.
• a variation ofthe method includes adapting a bacterial or algal PEPC for optimal function in a plant cell, or adult vegetative plant. This variation comprises recursive shuffling and selection of a library of bacterial or algal PEPC encoding sequences in a plant cell ofthe taxonomic species of plant for which the bacterial or algal PEPC is being adapted to function in an adult plant.
• This variation can include not only selecting for a desired PEPC enzymatic phenotype, but also selecting for appropriate function of a operably linked transcriptional control sequence in conjunction with PEPC function.
• This variation can employ host cells which are regenerable post-transformation, and selection of adult plants for enhanced carbon fixation via PEPC; recovery ofthe encoding PEPC shufflants (and optionally the linked transcriptional control sequences), and at least one cycle of recursive shuffling and selection to evolve a bacterial or algal PEPC, and optionally a transcriptional control sequence, optimized for function in the desired plant taxonomic species or closely related taxonomic categories.
• An object ofthe invention is the production of higher plants which express one or more PEPC enzyme which confer an enhanced carbon fixation conversion ratio to the plants.
• the invention is described principally with respect to the use of genetic sequence shuffling to generate enhanced PEPC coding sequences, the invention also provides for the introduction of PEPC coding sequences obtained from organisms having PEPC with desirable enzymatic phenotypes, such as inhibitor-resistant PEPC from bacterial mutants, into higher plants.
• the invention provides a method comprising the step of introducing into a higher plant
• An aspect ofthe invention provides C4 land plants comprising a polynucleotide sequence encoding a bacterial or algal PEPC composed in an expression cassette suitable for expression in a C4 land plant; optionally an expression cassette encoding a PEPC operably linked to regulatory sequences for expression in the nucleus ofthe C4 plant, e.g., in tissue such as mesophyll cells, additionally is transferred into the nucleus ofthe C4 plant.
• a C3 plant may be used in place of a C4 plant if desired.
• a specific embodiment comprises a regenerable protoplast of Glycine max, Nicotiana tabacum, or Zea mays (or other agricultural crop species amenable to regeneration from protoplasts) having a nuclear genome containing an expressible shuffled PEPC gene that is obtained from a bacterium or algae, and typically is at least 90 percent up to 99 percent sequence identical to a PEPC gene in the genome of said bacterium or algae, but is mutated in at least one codon as compared to the parental sequence.
• the invention also provides adult plants, cultivars, seeds, vegetative bodies, fruits, germplasm, and reproductive cells obtained from regeneration of such transformed protoplasts.
• the invention provides a kit for obtaining a polynucleotide encoding a PEPC protein having a predetermined enzymatic phenotype, the kit comprising a cell line suitable for forming transformable host cells and a collection sequence-shuffled polynucleotides formed by in vitro sequence shuffling.
• the kit often further comprises a transformation enhancing agent (e.g., lipofection agent, PEG, etc.) and/or a transformation device (e.g., a biolistics gene gun) and/or a plant viral vector which can infect plant cells or protoplasts thereof.
• a transformation enhancing agent e.g., lipofection agent, PEG, etc.
• a transformation device e.g., a biolistics gene gun
• the disclosed method for providing an agricultural organism having an improved PEPC enzymatic phenotype by iterative gene shuffling and phenotype selection is a pioneering method which enables a broad range of novel and advantageous agricultural compositions, methods, kits, uses, plant cultivars, and apparatus which will be apparent to those skilled in the art in view ofthe present disclosure.
• Panel A shows a diagrammatic representation of PEPC activity as a function of activator concentration for a parental wild-type PEPC (solid line), a shufflant which is partially desensitized (dotted line), and a shufflant which is fully desensitized (dashed line) to activator.
• Panel B shows a diagrammatic representation of PEPC activity as a function of inhibitor concentration for a parental wild-type PEPC (solid line), a shufflant which is partially desensitized (dotted line), and a shufflant which is fully desensitized (dashed line) to inhibitor.
• Panel A shows a diagrammatic representation of PEPC activity as a function of substrate concentration for a parental wild-type PEPC (solid line), and a shufflant which is optimized for substrate usage(dashed line); Km for the wildtype Km(wt) and optimized enzyme Km(opt), and Vmax for the wildtype Vmax(wt) and optimized Vmax(opt) are shown.
• Panel B shows a diagrammatic representation of PEPC activity as a function of inhibitor concentration for a parental wild-type PEPC (solid line), and a shufflant which is optimized for substrate usage(dashed line); Km for the wildtype Km(wt) and optimized enzyme Km(opt), and
• Vmax for the wildtype Vmax(wt) and optimized Vmax(opt) are shown.
• DNA shuffling may involve crossover via nonhomologous recombination, such as via cre/lox and/or flp/frt systems and the like, such that recombination need not require substantially homologous polynucleotide sequences.
• nonhomologous recombination such as via cre/lox and/or flp/frt systems and the like
• recombination need not require substantially homologous polynucleotide sequences.
• silico and oligonucleotide mediated approaches also do not require similarity/homology.
• Homologous and non- homologous recombination formats can be used, and, in some embodiments, can generate molecular chimeras and/or molecular hybrids of substantially dissimilar sequences.
• Viral recombination systems such as template-switching and the like can also be used to generate molecular chimeras and recombined genes, or portions thereof.
• chimeric polynucleotide means that the polynucleotide comprises regions which are wild-type and regions which are mutated. It may also mean that the polynucleotide comprises wild-type regions from one polynucleotide and wild-type regions from another related polynucleotide.
• cleaving means digesting the polynucleotide with enzymes or breaking the polynucleotide (e.g., by chemical or physical means), or generating partial length copies of a parent sequence(s) via partial PCR extension, PCR stuttering, differential fragment amplification, or other means of producing partial length copies of one or more parental sequences.
• population means a collection of components such as polynucleotides, nucleic acid fragments or proteins.
• a “mixed population” means a collection of components which belong to the same family of nucleic acids or proteins (i.e. are related) but which differ in their sequence (i.e. are not identical) and hence in their biological activity.
• mutants means changes in the sequence of a parent nucleic acid sequence (e.g., a gene or a microbial genome, transferable element, or episome) or changes in the sequence of a parent polypeptide. Such mutations may be point mutations such as transitions or transversions. The mutations may be deletions, insertions or duplications.
• recursive sequence recombination refers to a method whereby a population of polynucleotide sequences are recombined with each other by any suitable recombination means (e.g., sexual PCR, homologous recombination, site-specific recombination, etc.) to generate a library of sequence- recombined species which is then screened or subjected to selection to obtain those sequence-recombined species having a desired property; the selected species are then subjected to at least one additional cycle of recombination with themselves and/or with other polynucleotide species and at subsequent selection or screening for the desired property.
• amplification means that the number of copies of a nucleic acid fragment is increased.
• naturally-occurring refers to the fact that an object can be found in nature.
• a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
• laboratory strains and established cultivars of plants which may have been selectively bred according to classical genetics are considered naturally-occurring.
• naturally-occurring polynucleotide and polypeptide sequences are those sequences, including natural variants thereof, which can be found in a source in nature, or which are sufficiently similar to known natural sequences that a skilled artisan would recognize that the sequence could have arisen by natural mutation and recombination processes.
• predetermined means that the cell type, non-human animal, or virus may be selected at the discretion of the practitioner on the basis of a known phenotype.
• linked means in polynucleotide linkage (i.e., phosphodiester linkage).
• Unlinked means not linked to another polynucleotide sequence; hence, two sequences are unlinked if each sequence has a free 5' terminus and a free 3' terminus.
• operably linked refers to a linkage of polynucleotide elements in a functional relationship.
• a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription ofthe coding sequence.
• Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
• enhancers generally function when separated from the promoter by severpl kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
• a structural gene (e.g., a PEPC gene) which is operably linked to a polynucleotide sequence corresponding to a transcriptional regulatory sequence of an endogenous gene is generally expressed in substantially the same temporal and cell type-specific pattern as is the naturally-occurring gene.
• an expression cassette refers to a polynucleotide comprising a promoter sequence and, optionally, an enhancer and/or silencer element(s), operably linked to a structural sequence, such as a cDNA sequence or genomic DNA sequence.
• an expression cassette may also include polyadenylation site sequences to ensure polyadenylation of transcripts.
• an expression cassette comprises: (1) a promoter, such as a CaMV 35 S promoter, a NOS promoter or a rbcS promoter, or other suitable promoter known in the art, (2) a cloned polynucleotide sequence, such as a cDNA or genomic fragment ligated to the promoter in sense orientation so that transcription from the promoter will produce a RNA that encodes a functional protein, and (3) a polyadenylation sequence.
• a promoter such as a CaMV 35 S promoter, a NOS promoter or a rbcS promoter, or other suitable promoter known in the art
• a cloned polynucleotide sequence such as a cDNA or genomic fragment ligated to the promoter in sense orientation so that transcription from the promoter will produce a RNA that encodes a functional protein
• a polyadenylation sequence such as a cDNA or genomic fragment ligated to the promoter in sense orientation so that transcription from the promote
• transcriptional unit or “transcriptional complex” refers to a polynucleotide sequence that comprises a structural gene (exons), a cis-acting linked promoter and other cis-acting sequences necessary for efficient transcription ofthe structural sequences, distal regulatory elements necessary for appropriate tissue-specific and developmental transcription ofthe structural sequences, and additional cis sequences important for efficient transcription and translation (e.g., polyadenylation site, mRNA stability controlling sequences).
• transcription regulatory region refers to a DNA sequence comprising a functional promoter and any associated transcription elements (e.g., enhancer, CCAAT box, TATA box, LRE, ethanol-inducible element, etc.) that are essential for transcription of a polynucleotide sequence that is operably linked to the transcription regulatory region.
• transcription elements e.g., enhancer, CCAAT box, TATA box, LRE, ethanol-inducible element, etc.
• xenogeneic is defined in relation to a recipient genome, host cell, or organism and means that an amino acid sequence or polynucleotide sequence is not encoded by or present in, respectively, the naturally- occurring genome ofthe recipient genome, host cell, or organism. Xenogenic DNA sequences are foreign DNA sequences.
• nucleic acid sequence that has been substantially mutated is xenogeneic with respect to the genome from which the sequence was originally derived, if the mutated sequence does not naturally occur in the genome.
• nucleotide sequence “5'- TAT AC” corresponds to a reference sequence "5'-TATAC” and is complementary to a reference sequence "5'-GTATA”.
• reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length viral gene or virus genome. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
• two polynucleotides may each comprise (1) a sequence (i.e., a portion ofthe complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
• sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences ofthe two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
• a “comparison window”, as used herein, refers to a conceptual segment of at least 25 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 25 contiguous nucleotides and wherein the portion ofthe polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which for comparative pu ⁇ oses in this manner does not comprise additions or deletions) for optimal alignment ofthe two sequences.
• Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol.
• sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
• percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 89 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, optionally over a window of at least 30-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence that may include deletions or additions which total 20 percent or less ofthe reference sequence over the window of comparison.
• the reference sequence may be a subset of a larger sequence.
• Specific hybridization is defined herein as the formation, by hydrogen bonding or nucleotide (or nucleobase) bases, of hybrids between a probe polynucleotide (e.g., a polynucleotide ofthe invention and a specific target polynucleotide, wherein the probe preferentially hybridizes to the specific target such that, for example, a single band corresponding to, e.g., one or more ofthe RNA species ofthe gene (or specifically cleaved or processed RNA species) can be identified on a Northern blot of RNA prepared from a suitable source.
• a probe polynucleotide e.g., a polynucleotide ofthe invention
• a specific target polynucleotide wherein the probe preferentially hybridizes to the specific target such that, for example, a single band corresponding to, e.g., one or more ofthe RNA species ofthe gene (or specifically cleaved or processed RNA
• Polynucleotides ofthe invention which specifically hybridize to viral genome sequences may be prepared on the basis ofthe sequence data provided herein and available in the patent applications inco ⁇ orated herein and scientific and patent publications noted above, and according to methods and thermodynamic principles known in the art and described in Sambrooke et al. et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., (1989), Cold Spring Harbor, N.Y.; Berger and Kimmel, Methods in Enzymology. Volume 152. Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, CA; Goodspeed et al. ( 1989) Gene 76: 1 ; Dunn et al. ( 1989) J. Biol. Chem.
• Physiological conditions refers to temperature, pH, ionic strength, viscosity, and like biochemical parameters that are compatible with a viable plant organism or agricultural microorganism (e.g., Rhizobium,
• in vitro physiological conditions can comprise 50-200 mM NaCl or KC1, pH 6.5-8.5, 20- 45EC and 0.001-10 mM divalent cation (e.g., Mg ⁇ , Ca ++ ); preferably about 150 mM NaCl or KC1, pH 7.2-7.6, 5 mM divalent cation, and often include 0.01-1.0 percent nonspecific protein (e.g., BSA).
• BSA nonspecific protein
• a non-ionic detergent (Tween, NP-40, Triton X- 100) can often be present, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).
• Particular aqueous conditions may be selected by the practitioner according to conventional methods. For general guidance, the following buffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HCI, pH 5-8, with optional addition of divalent cation(s), metal chelators, nonionic detergents, membrane fractions, antifoam agents, and/or scintillants.
• label refers to inco ⁇ oration of a detectable marker, e.g., a radiolabeled amino acid or a recoverable label (e.g. biotinyl moieties that can be recovered by avidin or streptavidin).
• Recoverable labels can include covalently linked polynucleobase sequences that can be recovered by hybridization to a complementary sequence polynucleotide.
• a detectable marker e.g., a radiolabeled amino acid or a recoverable label (e.g. biotinyl moieties that can be recovered by avidin or streptavidin).
• recoverable labels can include covalently linked polynucleobase sequences that can be recovered by hybridization to a complementary sequence polynucleotide.
• Various methods of labeling polypeptides, PNAs, and polynucleotides are known in the art and may be used.
• labels include, but are not limited to, the following: radioisotopes (e.g., 3 H, 14 C, 35 S, 125 1, 131 I), fluorescent or phosphorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, ⁇ - galactosidase, luciferase, alkaline phosphatase), biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for antibodies, transcriptional activator polypeptide, metal binding domains, epitope tags).
• labels are attached by spacer arms of various lengths, e.g., to reduce potential steric hindrance.
• statically significant means a result (i.e., an assay readout) that generally is at least two standard deviations above or below the mean of at least three separate determinations of a control assay readout and/or that is statistically significant as determined by Student's t-test or other art-accepted measure of statistical significance.
• transcriptional modulation is used herein to refer to the capacity to either enhance transcription or inhibit transcription of a structural sequence linked in cis; such enhancement or inhibition may be contingent on the occurrence of a specific event, such as stimulation with an inducer and/or may only be manifest in certain cell types.
• agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues. Agents are evaluated for potential activity as PEPC inhibitors or allosteric effectors by inclusion in screening assays described hereinbelow.
• substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual macromolecular species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules ( ⁇ 500 Daltons), and elemental ion species are not considered macromolecular species.
• the term "optimized” is used to mean substantially improved in a desired structure or function relative to an initial starting condition, not necessarily the optimal structure or function which could be obtained if all possible combinatorial variants could be made and evaluated, a condition which is typically impractical due to the number of possible combinations and permutations in polynucleotide sequences of significant length (e.g., a complete plant gene or genome).
• PEPC enzymatic phenotype means an observable or otherwise detectable phenotype that can be discriminative based on PEPC function.
• a PEPC enzymatic phenotype can comprise an enzyme Km for a substrate, Km for an inhibitor (K T) , Km for an activator (Ka), Vmax, a turnover rate, an inhibition coefficient (Ki), or an observable or otherwise detectable trait that reports PEPC function in a cell or clonal progeny thereof, including an adult plant or organ thereof, which otherwise lack said trait in the absence of significant
• the present invention provides methods, reagents, genetically modified plants, plant cells and protoplasts thereof, microbes, and polynucleotides, and compositions relating to the forced evolution of PEPC sequences to improve an enzymatic property of a PEPC protein.
• the invention provides a shuffled PEPC which is catalytically active and which exhibits an improved enzymatic profile, such as an increased Km for inhibitor, decreased Km for activator, and or a decreased Km for substrate, increased Vmax, reduced pH sensitivity, or the like.
• the invention is based, in part, on a method for shuffling polynucleotide sequences that encode a PEPC enzyme.
• the method comprises the step of selecting at least one polynucleotide sequence that encodes a PEPC having an enhanced enzymatic phenotype and subjecting said selected polynucleotide sequence to at least one subsequent round of mutagenesis and/or sequence shuffling, and selection for the enhanced phenotype.
• the method is performed recursively on a collection of selected polynucleotide sequences encoding the PEPC to iteratively provide polynucleotide sequences encoding PEPC species having the desired enhanced enzymatic phenotype.
• the invention provides shuffled PEPC encoding sequences, wherein said shuffled encoding sequences comprise at least 21 contiguous nucleotides, preferably at least 30 contiguous nucleotides, or more, of a first naturally occurring PEPC gene sequence and at least 21 contiguous nucleotides, preferably at least 30 contiguous nucleotides, or more, of a second naturally occurring PEPC sequence, operably linked in reading frame to encode a PEPC which has PEPC activity and which has an enhanced PEPC enzymatic phenotype.
• the invention provides shuffled PEPC encoding sequences, wherein the shuffled sequences comprise portions of a first parental PEPC encoding sequence which comprises at least one mutation in the encoding sequence as compared to the collection of predetermined naturally occurring PEPC sequences.
• Oligonucleotides can be synthesized on an Applied Bio Systems oligonucleotide synthesizer according to specifications provided by the manufacturer. Methods for PCR amplification are described in the art (PCR
• Leaf PCR is suitable for genotype analysis of transgenote plants
• the invention relates in part to a method for generating novel or improved PEPC genetic sequences and improved starch production phenotypes which do not naturally occur or would be anticipated to occur at a substantial frequency in nature.
• a broad aspect ofthe method employs recursive nucleotide sequence recombination, termed "sequence shuffling", which enables the rapid generation of a collection of broadly diverse phenotypes that can be selectively bred for a broader range of novel phenotypes or more extreme phenotypes than would otherwise occur by natural evolution in the same time period.
• a basic variation ofthe method is a recursive process comprising: (1) sequence shuffling of a plurality of species of a genetic sequence, which species may differ by as little as a single nucleotide difference or may be substantially different yet retain sufficient regions of sequence similarity or site-specific recombination junction sites to support shuffling recombination, (2) selection ofthe resultant shuffled genetic sequence to isolate or enrich a plurality of shuffled genetic sequences having a desired phenotype(s), and (3) repeating steps (1) and (2) on the plurality of shuffled genetic sequences having the desired phenotype(s) until one or more variant genetic sequences encoding a sufficiently optimized desired phenotype is obtained.
• the method facilitates the "forced evolution" of a novel or improved genetic sequence to encode a desired PEPC enzymatic phenotype which natural selection and evolution has heretofore not generated in the reference agricultural organism.
• a plurality of PEPC genetic sequences are shuffled and selected by the present method.
• the method can be used with a plurality of alleles, homologs, or cognate genes of a gentic locus, or even with a plurality or genetic sequences from related organisms, and in some instances with unrelated genetic sequences or portions thereof which have recombinogenic portions (either naturally or generated via genetic engineering).
• the method can be used to evolve a heterologous PEPC sequence (e.g., a non-naturally occurring mutant gene from another species) to optimize its function and/or in a particular host cell.
• PEPC Coding sequences for various species are disclosed in the literature and Genbank, among other public sources, and may be obtained by cloning, PCR, or from deposited materials.
• PEPC shufflants are generated by any suitable shuffling method from one or more parental sequences, optionally including mutagenesis, and the resultant shufflants are introduced into a suitable host cell, typically in the form of expression cassettes wherein the shuffled polynucleotide sequence encoding the PEPC is operably linked to a transcriptional regulatory sequence and any necessary sequences for ensuring transcription, translation, and processing ofthe encoded PEPC protein.
• Each such expression cassette or its shuffled PEPC encoding sequence can be referred to as a "library member" composing a library of shuffled PEPC sequences.
• the library is introduced into a population of host cells, such that individual host cells receive substantially one or a few species of library member(s), to form a population of shufflant host cells expressing a library of shuffled PEPC species.
• the population of shufflant host cells is screened so as to isolate or segregate host cells and/or their progeny which express PEPC having the desired enhanced phenotype.
• the shuffled PEPC encoding sequence(s) is/are recovered from the isolated or segregated shufflant host cells, and typically subjected to at least one subsequent round of mutagenesis and/or sequence shuffling, introduced into suitable host cells, and selected for the desired enhanced enzymatic phenotype; this cycle is generally performed iteratively until the shufflant host cells express a PEPC having the desired level or enzymatic phenotype or until the rate of improvement in the desired enzymatic phenotype produced by shuffling has substantially plateaued.
• the shufflant PEPC polynucleotides expressed in the host cells following the iterative process of shuffling and selection encode PEPC specie(s) having the desired enhanced phenotype.
• examples of a desired PEPC enzymatic phenotype can include increased substrate usage rate at a given substrate concentration, decreased inhibition by a PEPC inhibitor (desensitization), increased Km for inhibitor (desensitization), increased activation by an activator
• Patents by the inventors and their co-workers including: United States Patent 5,605,793 to Stemmer (February 25, 1997), “METHODS FOR IN VITRO RECOMBINATION;” United States Patent 5,811,238 to Stemmer et al. (September 22, 1998) "METHODS FOR GENERATING POLYNUCLEOTIDES HAVING DESIRED CHARACTERISTICS BY ITERATIVE SELECTION AND
• ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION by del Cardyre et al. filed July 15, 1998 (USSN 09/166,188), and July 15, 1999 (USSN 09/354,922); "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri et al., filed February 5, 1999 (USSN 60/118,813) and filed June 24, 1999 (USSN 60/141,049) and filed September 28, 1999 (USSN 09/408,392, Attorney
• any of these methods can be adapted to the present invention to evolve PEPC coding nucleic acids or homologues to produce new enzymes with improved properties. Both the methods of making such enzymes and the enzymes or enzyme coding libraries produced by these methods are a feature ofthe invention.
• nucleic acids can be recombined in vitro by any of a variety of techniques discussed in the references above, including e.g.,
• nucleic acids can be recursively recombined in vivo, e.g., by allowing recombination to occur between nucleic acids in cells.
• whole cell genome recombination methods can be used in which whole genomes of cells are recombined, optionally including spiking ofthe genomic or chloroplast recombination mixtures with desired library components such as PEPC encoding nucleic acids.
• oligonucleotides corresponding to different PEPC homologues are synthesized and reassembled in PCR or ligation reactions which include oligonucleotides which correspond to more than one parental nucleic acid, thereby generating new recombined nucleic acids.
• Oligonucleotides can be made by standard nucleotide addition methods, or can be made, e.g., by tri-nucleotide synthetic approaches.
• Fifth, in silico methods of recombination can be effected in which genetic algorithms are used in a computer to recombine sequence strings which correspond to PEPC homologues.
• the resulting recombined sequence strings are optionally converted into nucleic acids by synthesis of nucleic acids which correspond to the recombined sequences, e.g., in concert with oligonucleotide synthesis/ gene reassembly techniques.
• Any of the preceding general recombination formats can be practiced in a reiterative fashion to generate a more diverse set of recombinant nucleic acids.
• the above references provide these and other basic recombination formats as well as many modifications of these formats.
• nucleic acids ofthe invention can be recombined (with each other or with related (or even unrelated) nucleic acids to produce a diverse set of recombinant nucleic acids, including homologous nucleic acids.
• any nucleic acids which are produced can be selected for a desired activity.
• a variety of related (or even unrelated) properties can be assayed for, using any available assay.
• sequence shuffling in broad application, consists of a method for generating a selected polynucleotide sequence or population of selected polynucleotide sequences, typically in the form of amplified and/or cloned polynucleotides, whereby the selected polynucleotide sequence(s) possess or encode a desired phenotypic characteristic (e.g., encode a polypeptide, promote transcription of linked polynucleotides, modify transformation efficiency, bind a protein, and the like) which can be selected for.
• a desired phenotypic characteristic e.g., encode a polypeptide, promote transcription of linked polynucleotides, modify transformation efficiency, bind a protein, and the like
• One method of identifying polypeptides that possess a desired structure or functional property involves the screening of a large library of polynucleotides for individual library members which possess or encode the desired structure or functional property conferred by the polynucleotide sequence.
• the invention provides a method, termed "sequence shuffling", for generating libraries of recombinant polynucleotides having a desired PEPC enzyme characteristic which can be selected or screened for.
• Libraries of recombinant polynucleotides are generated from a population of related-sequence polynucleotides which comprise sequence regions which have substantial sequence identity and can be homologously recombined in vitro or m vivo.
• at least two species ofthe related-sequence polynucleotides are combined in a recombination system suitable for generating sequence-recombined polynucleotides, wherein said sequence-recombined polynucleotides comprise a portion of at least one first species of a related-sequence polynucleotide with at least one adjacent portion of at least one second species of a related-sequence polynucleotide.
• Recombination systems suitable for generating sequence-recombined polynucleotides can be either:
• the population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method.
• the selected sequence- recombined polynucleotides which are typically related-sequence polynucleotides, can then be subjected to at least one recursive cycle wherein at least one selected sequence-recombined polynucleotide is combined with at least one distinct species of related-sequence polynucleotide (which may itself be a selected sequence-recombined polynucleotide) in a recombination system suitable for generating sequence- recombined polynucleotides, such that additional generations of sequence- recombined polynucleotide sequences are generated from the selected sequence- recombined polynucleotides obtained by the selection or screening method employed.
• recursive sequence recombination generates library members which are sequence-recombined polynucleotides possessing desired characteristics.
• characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property.
• Nucleic acid sequence shuffling is a method for recursive in vitro or in vivo homologous or nonhomologous recombination of pools of nucleic acid fragments or polynucleotides (e.g., genes from agricultural organisms or portions thereof).
• Mixtures of related nucleic acid sequences or polynucleotides are randomly or pseudo randomly fragmented, and reassembled to yield a library or mixed population of recombinant nucleic acid molecules or polynucleotides.
• the present invention is directed to a method for generating a selected polynucleotide sequence (e.g., a plant PEPC gene or microbe PEPC gene, or combinations thereof) or population of selected polynucleotide sequences, typically in the form of amplified and/or cloned polynucleotides, whereby the selected polynucleotide sequence(s) possess a desired phenotypic characteristic of PEPC enzymes which can be selected for, and whereby the selected polynucleotide sequences are genetic sequences having a desired functionality and/or conferring a desired phenotypic property to an agricultural organism in which the polynucleotide has been transferred into.
• a selected polynucleotide sequence e.g., a plant PEPC gene or microbe PEPC gene, or combinations thereof
• population of selected polynucleotide sequences typically in the form of amplified and/or cloned polynucleotides
• the invention provides a method, called “sequence shuffling", for generating libraries of recombinant polynucleotides having a subpopulation of library members which encode an enhanced or improved PEPC protein.
• Libraries of recombinant polynucleotides are generated from a population of related-sequence PEPC polynucleotides which comprise sequence regions which have substantial sequence identity and can be homologously recombined m vitro or in vivo.
• At least two species ofthe related-sequence PEPC polynucleotides are combined in a recombination system suitable for generating sequence-recombined polynucleotides, wherein said sequence-recombined polynucleotides comprise a portion of at least one first species of a related-sequence PEPC polynucleotide with at least one adjacent portion of at least one second species of a related-sequence PEPC polynucleotide.
• Recombination systems suitable for generating sequence-recombined polynucleotides can be either: (1) in vitro systems for homologous recombination or sequence shuffling via amplification or other formats described herein, or (2) in vivo systems for homologous recombination or site-specific recombination as described herein, or template-switching of a retroviral genome replication event.
• the population of sequence-recombined polynucleotides comprises a subpopulation of PEPC polynucleotides which possess desired or advantageous enzymatic characteristics and which can be selected by a suitable selection or screening method.
• the selected sequence-recombined PEPC polynucleotides which are typically related-sequence polynucleotides, can then be subjected to at least one recursive cycle wherein at least one selected sequence-recombined PEPC polynucleotide is combined with at least one distinct species of related-sequence PEPC polynucleotide (which may itself be a selected sequence-recombined polynucleotide) in a recombination system suitable for generating sequence-recombined PEPC polynucleotides, such that additional generations of sequence-recombined polynucleotide sequences are generated from the selected sequence-recombined polynucleotides obtained by the selection or screening method employed.
• recursive sequence recombination generates library members which are sequence-recombined polynucleotides possessing desired PEPC enzymatic characteristics.
• Such characteristics can be any property or attribute capable of being selected for or detected in a screening system.
• Screening/selection produces a subpopulation of genetic sequences (or cells) expressing recombinant forms of PEPC gene(s) that have evolved toward acquisition of a desired enzymatic property. These recombinant forms can then be subjected to further rounds of recombination and screening/selection in any order. For example, a second round of screening/selection can be performed analogous to the first resulting in greater enrichment for genes having evolved toward acquisition ofthe desired enzymatic property.
• the stringency of selection can be increased between rounds (e.g., if selecting for drug resistance, the concentration of drug in the media can be increased).
• the first plurality of selected library members is fragmented and homologously recombined by PCR in vitro.
• Fragment generation is by nuclease digestion, partial extension PCR amplification, PCR stuttering, or other suitable fragmenting means, such as described herein and in WO95/22625 published
• Stuttering is fragmentation by incomplete polymerase extension of templates.
• a recombination format based on very short PCR extension times can be employed to create partial PCR products, which continue to extend off a different template in the next (and subsequent) cycle(s), and effect de facto fragmentation.
• Template-switching and other formats which accomplish sequence shuffling between a plurality of sequence-related polynucleotides can be used. Such alternative formats will be apparent to those skilled in the art.
• the first plurality of selected library members is fragmented in vitro, the resultant fragments transferred into a host cell or organism and homologously recombined to form shuffled library members in vivo.
• the first plurality of selected library members is cloned or amplified on episomally replicable vectors, a multiplicity of said vectors is transferred into a cell and homologously recombined to form shuffled library members in vivo.
• the first plurality of selected library members is not fragmented, but is cloned or amplified on an episomally replicable vector as a direct repeat or indirect (or inverted) repeat, which each repeat comprising a distinct species of selected library member sequence, said vector is transferred into a cell and homologously recombined by intra-vector or inter-vector recombination to form shuffled library members in vivo.
• combinations of in vitro and in vivo shuffling are provided to enhance combinatorial diversity.
• the recombination cycles can be performed in any order desired by the practitioner.
• the first plurality of selected library members is fragmented and homologously recombined by PCR in vitro. Fragment generation is by nuclease digestion, partial extension PCR amplification, PCR stuttering, or other suitable fragmenting means, such as described herein and in the documents inco ⁇ orated herein by reference. Stuttering is fragmentation by incomplete polymerase extension of templates.
• the first plurality of selected library members is fragmented in vitro, the resultant fragments transferred into a host cell or organism and homologously recombined to form shuffled library members in vivo.
• the host cell is a plant cell which has been engineered to contain enhanced recombination systems, such as an enhanced system for general homologous recombination (e.g., a plant expressing a recA protein or a plant recombinase from a transgene or plant virus) or a site-specific recombination system (e.g., a cre/LOX or frt/FLP system encoded on a transgene or plant virus).
• enhanced recombination systems such as an enhanced system for general homologous recombination (e.g., a plant expressing a recA protein or a plant recombinase from a transgene or plant virus) or a site-specific recombination system (e.g., a cre/
• the first plurality of selected library members is cloned or amplified on episomally replicable vectors, a multiplicity of said vectors is transferred into a cell and homologously recombined to form shuffled library members in vivo in a plant cell, algae cell, or bacterial cell.
• Other cell types may be used, if desired.
• the first plurality of selected library members is not fragmented, but is cloned or amplified on an episomally replicable vector as a direct repeat or indirect (or inverted) repeat, which each repeat comprising a distinct species of selected library member sequence, said vector is transferred into a cell and homologously recombined by intra-vector or inter-vector recombination to form shuffled library members in vivo in a plant cell, algae cell, or microorganism.
• combinations of in vitro and in vivo shuffling are provided to enhance combinatorial diversity.
• the first, referred to as "in silico" shuffling utilizes computer algorithms to perform “virtual” shuffling using genetic operators in a computer.
• PEPC sequence strings are recombined in a computer system and desirable products are made, e.g., by reassembly PCR or ligation of synthetic oligonucleotides, or other available techniques.
• In silico shuffling is described in detail in Selifonov and Stemmer in "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" filed 02/01/1999,
• genetic operators are used to model recombinational or mutational events which can occur in one or more nucleic acid, e.g., by aligning nucleic acid sequence strings (using standard alignment software, or by manual inspection and alignment) and predicting recombinational outcomes based upon selected genetic algorithms (mutation, recombination, etc.).
• the predicted recombinational outcomes are used to produce corresponding molecules, e.g., by oligonucleotide synthesis and reassembly PCR.
• PEPC nucleic acids are aligned and recombined in silico, using any desired genetic operator, to produce character strings which are then generated synthetically for subsequent screening.
• oligonucleotide mediated shuffling in which oligonucleotides corresponding to a family of related homologous nucleic acids (e.g., as applied to the present invention, families of PEPC variants) which are recombined to produce selectable nucleic acids.
• This format is described in detail in Crameri et al. "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" filed February 5, 1999, USSN 60/118,813, Crameri et al. "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” filed
• oligonucleotides corresponding to multiple homologous parental nucleic acids are synthesized, ligated and elongated (typically in a recursive format), typically either in a polymerase or ligase-mediated elongation reaction, to produce full-length PEPC nucleic acids.
• the technique can be used to recombine homologous or even non-homologous PEPC nucleic acid sequences.
• One advantage of oligonucleotide-mediated recombination is the ability to recombine homologous nucleic acids with low sequence similarity, or even non-homologous nucleic acids.
• one or more set of fragmented nucleic acids are recombined, e.g., with a set of crossover family diversity oligonucleotides.
• Each of these crossover oligonucleotides have a plurality of sequence diversity domains corresponding to a plurality of sequence diversity domains from homologous or non-homologous nucleic acids with low sequence similarity.
• the fragmented oligonucleotides which are derived by comparison to one or more homologous or non-homologous nucleic acids, can hybridize to one or more region ofthe crossover oligos, facilitating recombination.
• sets of overlapping family gene shuffling oligonucleotides (which are derived by comparison of homologous nucleic acids, by synthesis of corresponding oligonucleotides) are hybridized and elongated (e.g., by reassembly PCR or ligation), providing a population of recombined nucleic acids, which can be selected for a desired trait or property.
• the set of overlapping family shuffling gene oligonucleotides includes a plurality of oligonucleotide member types which have consensus region subsequences derived from a plurality of homologous target nucleic acids.
• family gene shuffling oligonucleotides which include one or more PEPC nucleic acid(s) are provided by aligning homologous nucleic acid sequences to select conserved regions of sequence identity and regions of sequence diversity.
• a plurality of family gene shuffling oligonucleotides are synthesized (serially or in parallel) which correspond to at least one region of sequence diversity.
• Sets of fragments, or subsets of fragments used in oligonucleotide shuffling approaches can be provided by cleaving one or more homologous nucleic acids (e.g., with a DNASE), or, more commonly, by synthesizing a set of oligonucleotides corresponding to a plurality of regions of at least one nucleic acid (typically oligonucleotides corresponding to a full-length nucleic acid are provided as members of a set of nucleic acid fragments).
• these cleavage fragments can be used in conjunction with family gene shuffling oligonucleotides, e.g., in one or more recombination reaction to produce recombinant PEPC nucleic acid(s).
• one way of generating diversity in a set of nucleic acids to be shuffled is to provide codon-altered nucleic acids which can be shuffled to provide access to sequence space not present in naturally occurring sequences.
• PEPC nucleic acids By synthesizing nucleic acids in which the codons which encode polypeptides are altered, it is possible to access a completely different mutational spectrum upon subsequent mutation ofthe nucleic acid.
• Codon modification procedures can be used to modify any PEPC nucleic acid or shuffled nucleic acid, e.g., prior to performing DNA shuffling.
• oligonucleotide sets comprising codon variations are synthesized and reassembled into full-length nucleic acids.
• the full length nucleic acids can themselves be shuffled (e.g., where the oligonucleotides to be reassembled provide sequence diversity at selected sites), and/or the full-length sequences can be shuffled by any available procedure to produce diverse sets of PEPC nucleic acids.
• the present invention provides methods, compositions, and uses related to creating novel or improved plants, plant cells, algal cells, soil microbes, plant pathogens, commensal microbes, or other plant- related organisms having art-recognized importance to the agricultural, horticultural, and argonomic areas (collectively, "agricultural organisms").
• any plant, plant cell, algal cell, etc. can be transduced with a shuffled nucleic acid produced according to the present invention.
• agronomically and horticulturally important plant species can be transduced.
• Such species include, but are not restricted to, members ofthe families: Graminae (including corn, rye, triticale, barley, millet, rice, wheat, oats, etc.); Leguminosae (including pea, beans, lentil, peanut, yam bean, cowpeas, velvet beans, soybean, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, and sweetpea); Compositae (the largest family of vascular plants, including at least 1,000 genera, including important commercial crops such as sunflower) and Rosaciae (including raspberry, apricot, almond, peach, rose, etc.), as well as nut plants
• Graminae including corn, rye, triticale, barley, millet, rice, wheat, oats, etc.
• Leguminosae including pea, beans, lentil, peanut, yam bean, cow
• Pennisetum e.g., millet
• Petunia e.g., Pisum
• Phaseolus Phleum
• Poa Prunus
• Ranunculus Raphanus
• Ribes Ricinus
• Rubus Saccharum
• Salpiglossis Secale (e.g., rye)
• Senecio Setaria, Sinapis, Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella, Triticum (e.g., wheat), Vicia, Vigna, Vitis, Zea (e.g., corn), the Olyreae, the Pharoideae and many others.
• common crop plants which are targets ofthe present invention include corn, rice, triticale, rye, cotton, soybean, sorghum, wheat, oats, barley, millet, sunflower, canola, peas, beans, lentils, peanuts, yam beans, cowpeas, velvet beans, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, sweetpea and nut plants (e.g., walnut, pecan, etc).
• corn, rice, triticale, rye, cotton, soybean, sorghum, wheat, oats, barley, millet, sunflower, canola, peas, beans, lentils, peanuts, yam beans, cowpeas, velvet beans, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, sweetpea and nut plants e.g., walnut, pecan, etc.
• naturally occurring m vivo recombination mechanisms of plants, agricultural microorganisms, or vector-host cells for intermediate replication can be used in conjunction with a collection of shuffled polynucleotide sequence variants having a desired phenotypic property to be optimized further; in this way, a natural recombination mechanism can be combined with intelligent selection of variants in an iterative manner to produce optimized variants by "forced evolution", wherein the forced evolved variants are not expected to, nor are observed to, occur in nature, nor are predicted to occur at an appreciable frequency.
• the practitioner may further elect to supplement and/or the mutational drift by introducing intentionally mutated polynucleotide species suitable for shuffling, or portions thereof, into the pool of initial polynucleotide species and/or into the plurality of selected, shuffled polynucleotide species which are to be recombined.
• Mutational drift may also be supplemented by the use of mutagens (e.g., chemical mutagens or mutagenic irradiation), or by employing replication conditions which enhance the mutation rate.
• the invention provides a means to evolve PEPC gene variants and/or suitable host cells, as well as providing a model system for evaluating a library of agents to identify candidate agents that could find use as agricultural reagents for commercial applications.
• agents may exhibit selectivity for inhibition of a naturally occurring PEPC enzyme and may be substantially less effective at inhibiting a shuffled PEPC enzyme which has been evolved to be resistant to the agent.
• PEPC Shuffling Combinations Although the skilled artisan may select alternative shuffling strategies for enhancing PEPC enzyme properties, the following general combinations can be used:
• shuffling a PEPC gene from a first species of bacteria with a PEPC gene from a second species of bacteria may be transformed into bacterial host cells which preferably lack endogenous PEPC activity, algal cells, or plant cells for expression and selection.
• Phenotype selection of shufflants is typically performed by biochemical assay for PEPC, such as according to Gonzalez et al. (19841 J. Plant Phvsiol. 1 16: 425; Devi et al. (1992) J. Plant Biochem. Biotech. 1 : 73; Pairoba et al. (1996) Biosci. Biotech. Biochem.
• Example bacteria for obtaining the PEPC gene(s) include Rhodobacter sphaeroides, Rhodospirrilum rubrum, Escherichia coli, Salmonella typhimurium, and the like.
• a preferred host cell is a strain of bacterium that is transformable and which lacks PEPC activity.
• shuffling a parental plant PEPC encoding sequence with mutagenized variants thereof may be transformed into bacterial host cells which preferably lack endogenous plant-type PEPC activity (e.g., E. coli), algal cells, or plant cells for expression and selection. Phenotype selection of shufflants is typically performed by biochemical assay for PEPC activity or other suitable assay method selected by the artisan.
• shuffling a PEPC from a first species of plant with a PEPC from a non-plant algae or bacterium, cyanobacteria may be transformed into host cells which preferably lack endogenous plant-type PEPC activity (e.g., E. coli), algal cells, or plant cells for expression and selection. Phenotype selection of shufflants is typically performed by biochemical assay for PEPC or other suitable assay method selected by the artisan.
• Example bacteria for the PEPC gene(s) include Rhodobacter sphaeroides (Falcone et al. (1998) J. Bact. 170:
• Example cyanobacteria that can serve as a source of PEPC genes include Synechococcus, Cocochloris peniocystis, and Aphanizomenon flos-aquae.
• Example green algae that can serve as sources of PEPC genes include Euglena gracilis,
• Example plants that can serve as sources for the PEPC genes include corn, rice, maize, potato, wheat, rye, flax, cotton, pea, and the like.
• shuffling a plant PEPC from a first plant taxonomic species with a plant PEPC from a second plant taxonomic species may be transformed into host cells, which can preferably lack endogenous PEPC activity, but which fold and process higher plant PEPC correctly for expression and selection.
• Phenotype selection of shufflants is typically performed by biochemical assay for PEPC or other suitable assay method selected by the artisan.
• Example higher plants that can serve as a source of PEPC genes include, but are not limited to: Zea mays (C4), Amaranthus hybridus (C4), Glycine max (C3), and Nicotiana tabacum (C3), among others.
• a PEPC gene ("parental gene") from a species of C3 or C4 plant is subjected to mutagenesis and shuffling/selection to generate a population of mutagenized shufflants which have substantial sequence identity to the parental gene.
• the population of mutagenized shufflants is transferred into a population of host cells wherein the mutagenized shufflants are expressed and the resultant transformed host cell population is selected or screened for an enhanced PEPC phenotype.
• Phenotype selection of shufflants is typically performed by biochemical assay for PEPC activity or other suitable assay method selected by the artisan.
• Suitable transcriptional regulatory sequences include: cauliflower mosaic virus 19S and 35S promoters, NOS promoter, OCS promoter, rbcS promoter, Brassica heat shock promoter, synthetic promoters, non-plant promoters modified, if necessary, for function in plant cells, substantially any promoter that naturally occurs in a plant genome, promoters of plant viruses or Ti plasmids, tissue-preferential promoters or cis-acting elements, light-responsive promoters or cis-acting elements (e.g., rbcS LRE), hormone-responsive cis-acting elements, developmental stage- specific promoters and cis-acting elements, viral promoters (e.g., from Tobacco Mosaic virus, Brome Mosaic Virus, Cauliflower Mosaic virus, and the like), and the like.
• a transcriptional regulatory sequence from a first plant species is optimized for functionality in a second plant species by application of recursive sequence
• Transcriptional regulatory sequences for expression of shuffled PEPC sequences in chloroplasts is known in the art (Daniell et al. (1998) op.cit; O'Neill et al. (1993) The Plant Journal 3: 729; Maliga P (1993) op.cit). as are homologous recombination vectors.
• PEPC gene shufflants can be expressed in E. coli, as well as higher taxonomic host cells. However, PEPC from higher plants may not always be processed correctly in bacterial host cells, so higher plant PEPC gene shufflants may often be expressed for phenotype screening in plant cells, including mutant plant cell lines wherein an endogenous PEPC encoding gene has been functionally inactivated, preferably in homozygous format, to provide a plant cell substantially lacking endogenous PEPC activity, or the like.
• Transformations may be carried out in essentially any ofthe various ways known to those skilled in the art of plant molecular biology. See, in general, Methods in Enzymology
• t e term transformation means alteration ofthe genotype of a host plant by the introduction of a nucleic acid sequence.
• the nucleic acid sequence need not necessarily originate from a different source, but it will, at some point, have been external to the cell into which it is to be introduced.
• the foreign nucleic acid is mechanically transferred by microinjection directly into plant cells by use of micropipettes.
• the foreign nucleic acid may be transferred into the plant cell by using polyethylene glycol. This forms a precipitation complex with the genetic material that is taken up by the cell (e.g., by incubation of protoplasts with "naked DNA” in the presence of polyethylenelycol)(Paszkowski et al., (1984) EMBO J. 3:2717-22; Baker et al (1985) Plant Genetics, 201-211; Li et al. (1990) Plant Molecular Biology Report 8(4)276-291].
• the introduced gene may be introduced into the plant cells by electroporation (Fromm et al., (1985) "Expression of Genes Transferred into Monocot and Dicot Plant Cells by Electroporation,” Proc. Natl Acad. Sci. USA 82:5824, which is inco ⁇ orated herein by reference).
• plant protoplasts are electroporated in the presence of plasmids or nucleic acids containing the relevant genetic construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction ofthe plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form a plant callus.
• Cauliflower mosaic virus may also be used as a vector for introducing the foreign nucleic acid into plant cells (Hohn et al., (1982) "Molecular Biology of Plant Tumors," Academic Press, New York, pp.549-560; Howell, United States Patent No. 4.407,956).
• CaMV viral DNA genome is inserted into a parent bacterial plasmid creating a recombinant DNA molecule which can be propagated in bacteria. After cloning, the recombinant plasmid again may be cloned and further modified by introduction ofthe desired DNA sequence into the unique restriction site ofthe linker.
• the modified viral portion of the recombinant plasmid is then excised from the parent bacterial plasmid, and used to inoculate the plant cells or plants.
• Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., (1987) Nature 327:70-73). Although typically only a single introduction of a new nucleic acid segment is required, this method particularly provides for multiple introductions.
• a method of introducing the nucleic acid segments into plant cells is to infect a plant cell, an explant, a meristem or a seed with Agrobacterium tumefaciens transformed with the segment.
• the transformed plant cells are grown to form shoots, roots, and develop further into plants.
• the nucleic acid segments can be introduced into appropriate plant cells, for example, by means ofthe Ti plasmid of Agrobacterium tumefaciens.
• the Ti plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens. and is stably integrated into the plant genome (Horsch et al., (1984) "Inheritance of Functional Foreign Genes in Plants," Science. 233:496-498; Fraley et al., (1983) Proc. Natl. Acad. Sci. USA 80:4803).
• Ti plasmids contain two regions essential for the production of transformed cells.
• T DNA transfer DNA
• the transfer DNA region which transfers to the plant genome, can be increased in size by the insertion of the foreign nucleic acid sequence without its transferring ability being affected.
• the modified Ti plasmid can then be used as a vector for the transfer of the gene constructs ofthe invention into an appropriate plant cell, such being a "disabled Ti vector.”
• All plant cells which can be transformed by Agrobacterium and whole plants regenerated from the transformed cells can also be transformed according to the invention so as to produce transformed whole plants which contain the transferred foreign nucleic acid sequence.
• Method (1) uses an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts.
• Method (2) implies (a) that the plant cells or tissues can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
• Method (3) uses micropropagation.
• two plasmids are needed: a T-DNA containing plasmid and a yir plasmid. Any one of a number of T-DNA containing plasmids can be used, the main issue being that one be able to select independently for each ofthe two plasmids.
• those plant cells or plants transformed by the Ti plasmid so that the desired DNA segment is integrated can be selected by an appropriate phenotypic marker.
• These phenotypic markers include, but are not limited to, antibiotic resistance, herbicide resistance or visual observation. Other phenotypic markers are known in the art and may be used in this invention.
• All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred foreign gene.
• Some suitable plants include, for example, species from the genera Fragaria. Lotus, Medicago. Onobrvchis. Trifolium. Trigonella. Vigna. Citrus. Linum. Geranium. Manihot. Daucus. Arabidopsis. Brassica. Raphanus. Sinapis. Atropa. Capsicum. Hyoscyamus. Lycopersicon. Nicotiana. Solanum, Petunia, Digitalis. Majorana. Ciohorium.
• Monocots may also be transformed by techniques or with vectors other than Agrobacterium.
• monocots have been transformed by electroporation (Fromm et al. [1986] Nature 319:791-793; Rhodes et al. Science [1988] 240: 204-207), direct gene transfer (Baker et al. [1985] Plant Genetics 201-211), by using pollen-mediated vectors (EP 0 270 356), and by injection of DNA into floral tillers (de la Pena et al. [1987], Nature
• Additional plant genera that may be transformed by Agrobacterium include Chrysanthemum, Dianthus, Gerbera. Euphorbia. Pelaronium, Ipomoea, Passiflora. Cyclamen, Malus, Prunus. Rosa, Rubus. Populus. Santalum, Allium. Lilium. Narcissus. Ananas, Arachis, Phaseolus and Pisum. Chloroplast Transformation In certain embodiments, it may be desirable for the PEPC enzyme to be present in chloroplasts, possibly in combination with the more conventional cytosolic expression.
• the PEPC enzyme of higher plants may be expressed with a fused chloroplast transit sequence peptide (CTS) to facilitate transloaction ofthe PEPC enzyme into chloroplasts, or it can be advantageous to transform the shufflant PEPC encoding sequences into chloroplasts if the host cells are derived from higher plants.
• CTS chloroplast transit sequence peptide
• Numerous methods are available in the art to accomplish the chloroplast transformation and expression (Daniell et al. (1998) op.cit: O'Neill et al. (1993) The Plant Journal 3: 729; Maliga P (1993) op.cit).
• the expression construct comprises a transcriptional regulatory sequence functional in plants operably linked to a polynucleotide encoding an enhanced PEPC protein.
• the expression cassette comprises the sequences necessary to ensure expression in chloroplasts - typically the encoding sequence is flanked by two regions of homology to the plastid genome so as to effect a homologous recombination with the chloroplastid genome; often a selectable marker gene is also present within the flanking plastid DNA sequences to facilitate selection of genetically stable transformed chloroplasts in the resultant transplastonic plant cells (see Maliga P ( 1993) TIBTECH H : 101 ; Daniell et al. ( 1998) Nature Biotechnology 16: 346, and references cited therein).
• the selected shuffled genetic sequences can be recovered for further shuffling or for direct use by any applicable method, including but not limited to: recovery of DNA, RNA, or cDNA from cells (or PCR-amplified copies thereof) from cells or medium, recovery of sequences from host chromosomal DNA or PCR- amplified copies thereof, recovery of episome (e.g., expression vector) such as a plasmid, cosmid, viral vector, artificial chromosome, and the like, or other suitable recovery method known in the art.
• episome e.g., expression vector
• Any suitable art-known method including RT-PCR or PCR, can be used to obtain the selected shufflant sequence(s) for subsequent manipulation and shuffling.
• Superfluous mutations can be removed by backcrossing, which is shuffling the selected shuffled PEPC gene(s) with one or more parental PEPC gene and/or naturally-occurring PEPC gene(s) (or portions thereof) and selecting the resultant collection of shufflants for those species that retain the desired phenotype.
• a maize PEPC gene can be shuffled and selected for the capacity to substantially function in any Angiosperm plant cells; the resultant selected shufflants can be backcrossed with one or more PEPC genes of a particular plant species and selected for the capacity to retain the capacity to confer the phenotype. After several cycles of such backcrossing, the backcrossing will yield gene(s) which contain the mutations necessary for the desired phenotype, and will otherwise have a genomic sequence substantially identical to the genome(s) ofthe host genome. Isolated components (e.g., genes, regulatory sequences, replication origins, and the like) can be optimized and then backcrossed with parental sequences so as to obtain optimized components which are substantially free of superfluous mutations.
• Isolated components e.g., genes, regulatory sequences, replication origins, and the like
• Transgenic Hosts Transgenes and expression vectors to express shufflant PEPC sequences can be constructed by any suitable method known in the art; by either PCR or RT-PCR amplification from a suitable cell type or by ligating or amplifying a set of overlapping synthetic oligonucleotides; publicly available sequence databases and the literature can be used to select the polynucleotide sequence(s) to encode the specific protein desired, including any mutations, consensus sequence, or mutation kernel desired by the practitioner.
• the coding sequence(s) are operably linked to a transcriptional regulatory sequence and, if desired, an origin of replication.
• Antisense or sense-suppression transgenes and genetic sequences can be optimized or adapted for particular host cells and organisms by the described methods.
• transgene(s) and/or expression vectors are transferred into host cells, protoplasts, pluripotent embryonic plant cells, microbes, or fungi by a suitable method, such as for example lipofection, electroporation, microinjection, biolistics, Agrobacterium tumefaciens transduction of Ti plasmid, calcium phosphate precipitation, PEG-mediated DNA uptake, electroporation, electrofusion, or other method.
• Stable transfectant host cells can be prepared by art-known methods, as can transgenic cell lines.
• plant refers to either a whole plant, a plant part, a plant cell, or a group of plant cells.
• the class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to protoplast transformation techniques, including both monocotyledonous and dicotyledonous plants. It includes plants of a variety of ploidy levels, including polyploid, diploid and haploid, and may employ non-regenerable cells for certain aspects which do not require development of an adult plant for selection or in vivo shuffling.
• PEPC include agronomically and horticulturally important species. Such species include, but are not restricted to members ofthe families: Graminae (including corn, rye, triticale, barley, millet, rice, wheat, oats, etc.); Leguminosae (including pea, beans, lentil, peanut, yam bean, cowpeas, velvet beans, soybean, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, and sweetpea); Compositae (the largest family of vascular plants, including at least 1,000 genera, including important commercial crops such as sunflower) and Rosaciae (including raspberry, apricot, almond, peach, rose, etc.), as well as nut plants (including, walnut, pecan, hazelnut, etc.). Targets for the invention also include plants from the genera: Agrostis,
• Hyoscyamus Ipomoea, Lactuca, Lens, Lilium, Linum, Lolium, Lotus, Lycopersicon, Majorana, Malus, Mangifera, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis, Oryza (e.g., rice), Panicum, Pelargonium, Pennisetum (e.g., millet), Petunia, Pisum, Phaseolus, Phleum, Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus, Saccharum, Salpiglossis, Secale (e.g., rye), Senecio, Setaria, Sinapis, Solanum,
• Sorghum Stenotaphrum, Theobroma, Trifolium, Trigonella, Triticum (e.g., wheat), Vicia, Vigna, Vitis, Zea (e.g., corn), and the Olyreae, the Pharoideae and many others.
• Common crop plants which are targets of the present invention include corn, rice, triticale, rye, cotton, soybean, sorghum, wheat, oats, barley, millet, sunflower, canola, peas, beans, lentils, peanuts, yam beans, cowpeas, velvet beans, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, sweetpea and nut plants
• transgenote refers to the immediate product ofthe transformation process and to resultant whole transgenic plants.
• regeneration means growing a whole plant from a plant cell, a group of plant cells, a plant part or a plant piece (e.g. from a protoplast, callus, or tissue part). Plant regeneration from cultural protoplasts is described in Evans et al., "Protoplasts Isolation and Culture," Handbook of Plant Cell Cultures 1: 124-176 (MacMillan Publishing Co. New York 1983); M.R. Davey, “Recent Developments in the Culture and Regeneration of Plant Protoplasts," Protoplasts. (1983) - Lecture Proceedings, pp.12-29, (Birkhauser, Basal 1983); P.J. Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," Protoplasts (1983) -
• glutamic acid and proline it is sometimes advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa.
• Plants and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history ofthe culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable. Regeneration also occurs from plant callus, explants, organs or parts.
• Transformation can be performed in the context of organ or plant part regeneration. See, Methods in Enzymology, supra; also Methods in Enzymology, Vol. 1 18; and Klee et al., (1987) Annual Review of Plant Physiology. 38:467-486.
• the mature transgenic plants are propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants for trialling, such as testing for production characteristics. Selection of desirable transgenotes is made and new varieties are obtained thereby, and propagated vegetatively for commercial sale.
• the mature transgenic plants are self crossed to produce a homozygous inbred plant.
• the inbred plant produces seed containing the gene for the newly introduced foreign gene activity level. These seeds can be grown to produce plants that would produce the selected phenotype.
• the inbreds according to this invention can be used to develop new hybrids.
• a selected inbred line is crossed with another inbred line to produce the hybrid.
• the offspring resulting from the first experimental crossing of two parents is known in the art as the FI hybrid, or first filial generation.
• the two parents crossed to produce F 1 progeny according to the present invention one or both parents can be transgenic plants.
• Parts obtained from the regenerated plant such as flowers, seeds, leaves, branches, fruit, and the like are covered by the invention, provided that these parts comprise cells which have been so transformed. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention, provided that these parts comprise the introduced DNA sequences. Progeny and variants, and mutants ofthe regenerated plants are also included within the scope of this invention.
• the following example is given to illustrate the invention, but are not to be limiting thereof.
• EXPERIMENTAL EXAMPLE EXAMPLE 1 Shuffling PEP Carboxylase
• C4 plants such as maize and Sorghum, as well as Crassulacean acid metabolism (CAM) plants.
• CAM Crassulacean acid metabolism
• PEPC involved in carbon fixation in C4 and CAM plants have been studied extensively with respect to its catalytic properties and regulation (Andero CS et al. ( 1987) FEBS Letters 213: 1 ; Chollet R ( 1996) Annu. Rev. Plant Phvsiol. Plant
• cDNA coding for PEPC from various C4 and CAM plants are isolated using primers designed from published sequence in the gene bank (Devi M et al. (1992) op.cit; Chollet R (1996) op.cit and references therein). Complete coding sequence for PEPC can also be synthesized.
• the PEPC genes from various related sources, which have high degree of homology at the nucleotide level are shuffled according to published procedures. Briefly, this procedure involves random fragmentation ofthe genes with DNAse I and selecting nucleotide fragments of 100-300 bp. The fragments are reassembled based on sequence similarity by primerless PCR. Recombination as well as variable levels of mutations that are introduced by the PCR reaction generate the diversity. The assembled genes can be cloned into E. coli or an E. coli mutant lacking PEPC. PEPC from C4 plants have been cloned and expressed in both prokaryotes and eukaryotes (Cretin et al.
• Colonies expressing shuffled PEPC genes can be selected and grown in larger amounts in liquid culture and assayed for specific properties.
• the assay procedure for PEPC involves coupling the activity with malic dehydrogenase and determining NADH disappearance spectrophotometrically at 340 nm (Gonzalez et al. (1984) J. Plant Physiology 1 16: 425).
• PEPC shufflant genes from those clones expressing one or more ofthe desired properties mentioned above are iteratively shuffled in order to achieve optimization of each one ofthe properties mentioned above.
• the optimized PEPC gene after appropriate modification for expression in plants, is used to transform the desired C4 crop in order to deregulate and increase carbon fixation.
• the present invention provides computers, computer readable media and integrated systems comprising character strings corresponding to shuffled PEPC enzymes and corresponding enzyme-encoding nucleic acids. These sequences can be manipulated by in silico shuffling methods, or by standard sequence alignment or word processing software.
• BLAST is described in Altschul et al, J. Mol. Biol. 215:403-410 (1990).
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
• This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word ofthe same length in a database sequence.
• HSPs high scoring sequence pairs
• T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
• Extension ofthe word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment.
• the BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of
• PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sha ⁇ ,
• the program can align, e.g., up to 300 sequences of a maximum length of 5,000 letters.
• the multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two aligned sequences. This cluster can then be aligned to the next most related sequence or cluster of aligned sequences.
• Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
• the program can also be used to plot a dendogram or tree representation of clustering relationships. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison.
• the shuffled enzymes ofthe invention are optinally sequenced and the sequences aligned to provide structure- function information. For example, the alignment of shuffled sequences which are selected for conversion activity against the same target provides an indication of which residues are relevant for conversion ofthe target (i.e., conserved residues are likely more important for activity than non-conserved residues).
• Standard desktop applications such as word processing software (e.g., Microsoft WordTM or Corel WordPerfectTM) and database software (e.g., spreadsheet software such as Microsoft ExcelTM, Corel Quattro ProTM, or database programs such as Microsoft AccessTM or ParadoxTM) can be adapted to the present invention by inputting character strings corresponding to shuffled PEPC enzymes (or corresponding coding nucleic acids), e.g., shuffled by the methods herein.
• the integrated systems can include the foregoing software having the appropriate character string information, e.g., used in conjunction with a user interface (e.g., a GUI in a standard operating system such as a Windows, Macintosh or LINUX system) to manipulate strings of characters.
• specialized alignment programs such as BLAST or PILEUP can also be inco ⁇ orated into the systems ofthe invention for alignment of nucleic acids or proteins (or corresponding character strings).
• Integrated systems for analysis in the present invention typically include a digital computer with software for aligning or manipulating sequences, as well as data sets entered into the software system comprising any ofthe sequences herein.
• the computer can be, e.g., a PC (Intel x86 or Pentium chip- compatible DOSTM, OS2TM WINDOWSTM WINDOWS NTTM, WINDOWS95TM, WINDOWS98TM LINUX based machine, a MACINTOSHTM, Power PC, or a UNIX based (e.g., SUNTM work station) machine) or other commercially common computer which is known to one of skill.
• Software for aligning or otherwise manipulating sequences is available, or can easily be constructed by one of skill using a standard programming language such as Visual basic, Fortran, Basic, Java, or the like.
• Any controller or computer optionally includes a monitor which is often a cathode ray tube ("CRT") display, a flat panel display (e.g., active matrix liquid crystal display, liquid crystal display), or others.
• Computer circuitry is often placed in a box which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others.
• the box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements.
• Inputting devices such as a keyboard or mouse optionally provide for input from a user and for user selection of sequences to be compared or otherwise manipulated in the relevant computer system.
• the computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations.
• the software then converts these instructions to appropriate language for instructing the system to carry out any desired operation.
• the computer system is used to perform "in silico" shuffling of character strings.
• a variety of such methods are set forth in "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES &
• Multi-dimensional analysis to optimize sequences can be also be performed in the computer system, e.g., as described in the '375 application.
• a digital system can also instruct an oligonucleotide synthesizer to synthesize oligonucleotides, e.g., used for gene reconstruction or recombination, or to order oligonucleotides from commercial sources (e.g., by printing appropriate order forms or by linking to an order form on the internet).
• the digital system can also include output elements for controlling nucleic acid synthesis (e.g., based upon a sequence or an alignment of a shuffled enzyme as herein), i.e., an integrated system ofthe invention optionally includes an oligonucleotide synthesizer or an oligonucleotide synthesis controller.
• the system can include other operations which occur downstream from an alignment or other operation performed using a character string corresponding to a sequence herein, e.g., as noted above with reference to assays.
• One aspect ofthe present invention is the combinatorial shuffling of PEPC with other enzymes that affect carbon fixation.
• one aspect ofthe present invention involves separately or simultaneously shuffling PEPC in combination with carbon fixation enzymes such as ribulose 1,5-bisphosphate carboxylase/oxygenase ("Rubisco”; EC 4.1.1.39), or with any Calvin cycle enzyme or Krebs cycle enzyme.
• carbon fixation enzymes such as ribulose 1,5-bisphosphate carboxylase/oxygenase (“Rubisco”; EC 4.1.1.39)
• Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase
• shuffled Rubisco and shuffled ADP-glucose pyrophosphorylase (“ADPGPP”; EC 2.7.7.27; an enzyme involved in starch biosynthesis, e.g., in plants) can be expressed together in cells or plants to increase carbon fixation or to improve starch biosynthesis.
• ADPGPP ADP-glucose pyrophosphorylase gene shuffling.
• U.S. Patent Application U.S.S.N. 60/107,782 entitled “MODIFIED ADP-GLUCOSE PYROPHOSPHORYLASE FOR IMPROVEMENT AND OPTIMIZATION OF PLANT PHENOTYPES” filed on 10 November 1998 (Attorney docket number
• the present invention provides for the use of any apparatus, apparatus component, composition or kit herein, for the practice of any method or assay herein, and/or for the use of any apparatus or kit to practice any assay or method herein.

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