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WO2000061740A1 - Production de lipides modifies - Google Patents

Production de lipides modifies Download PDF

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Publication number
WO2000061740A1
WO2000061740A1 PCT/US2000/009285 US0009285W WO0061740A1 WO 2000061740 A1 WO2000061740 A1 WO 2000061740A1 US 0009285 W US0009285 W US 0009285W WO 0061740 A1 WO0061740 A1 WO 0061740A1
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WIPO (PCT)
Prior art keywords
nucleic acid
lipid
lipid biosynthetic
recombinant
synthase
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PCT/US2000/009285
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English (en)
Inventor
Ling Yuan
Sun Ai Raillard
Michael Lassner
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Maxygen, Inc.
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Publication date
Application filed by Maxygen, Inc. filed Critical Maxygen, Inc.
Priority to AU42116/00A priority Critical patent/AU4211600A/en
Publication of WO2000061740A1 publication Critical patent/WO2000061740A1/fr

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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
    • C12N15/8247—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 lipid metabolism, e.g. seed oil composition

Definitions

  • This invention relates to the application of DNA shuffling technologies to nucleic acids coding factors which affect lipid biosynthesis and metabolism.
  • the invention provides strategies for modifying every relevant lipid synthetic gene, coding sequence or promoter, as well as all other relevant DNA sequences involved in fatty acid biosynthetic pathways.
  • Fatty acids are organic acids having a hydrocarbon chain of about 4 to
  • fatty acids which differ from each other in chain length and in the presence, number and position of double or triple bonds.
  • fatty acids typically exist in covalently bound forms, with the carboxyl portion of the fatty acid being referred to as a fatty acyl group.
  • the chain length and degree of saturation of these molecules is often depicted by the formula CX:Y, where "X" indicates number of carbons and "Y” indicates number of double bonds (e.g. oleate, an 18 carbon molecule with 1 double bound, is shown as C 18:1, or more simply, 18:1).
  • Fatty acids are the major components of all edible and industrial oils.
  • the fatty acid composition of an oil determines its physical and chemical properties and thus its uses.
  • the fatty acyl group in a lipid molecule can be covalently bound to another group via a different linkage. It can be linked, e.g., through a thioester bond to an acyl carrier protein (ACP) or a Coenzyme A (CoA) to form an acyl-ACP or acyl-CoA, respectively.
  • ACP acyl carrier protein
  • CoA Coenzyme A
  • it can also be linked through an ester bond to a fatty alcohol to form a wax
  • three acyl groups can be linked to a glycerol molecule to form triacylglycerol (triglyceride).
  • Novel fatty acids can also be produced as a result of the expression of a DNA sequence foreign to other organisms.
  • fungi can be transformed with recombinant DNA and used in fermentation for the production of certain fatty acids.
  • major hurdles to novel fatty acid production exist, due, e.g., to the difficulty of isolating enzymes with desired specificity, poor kinetics, different gene codon usage, incompatibility with the substrate pool and lack of strong promoters with appropriate expression timing and desirable tissue specificity.
  • the present invention provides a strategy for solving each of the problems outlined above, as well as providing a variety of other features which will become apparent upon complete review of the following.
  • the invention provides methods of modifying any or all genes encoding lipid synthesis-related enzymes and all nucleic acid elements involved in controlling the expressions of these genes and the cellular localization of the gene products.
  • this invention also covers the use of these modified DNA sequences in transgenic (recombinant) organisms, including bacteria, fungi, algae, plants and animals. Modification of the fatty acid composition of a transgenic organism is achieved as a result of the introduction of DNA sequence(s) which have been modified, e.g., through DNA shuffling. Accordingly, the invention provides a method of making a nucleic acid encoding a lipid biosynthetic activity.
  • a plurality of parental nucleic acids are recombined to produce one or more recombinant lipid biosynthetic nucleic acids comprising distinct or improved lipid biosynthetic activities.
  • the one or more recombinant lipid biosynthetic nucleic acids are selected for one or more encoded lipid biosynthetic activities or, e.g., for reduced or enhanced encoded polypeptide expression or stability. These selection steps provide a selected shuffled lipid biosynthetic nucleic acid which encodes one or more selected lipid biosynthetic activities.
  • a variety of lipid biosynthetic activities can be selected, separately or in combination, including: modulation of lipid saturation for one or more selected lipids produced by a lipid synthetic pathway comprising activity encoded by the one or more selected shuffled lipid biosynthetic nucleic acids, modulation of fatty acid composition in a transgenic plant, algae, animal, bacteria, fungus or other organism expressing the selected shuffled lipid biosynthetic nucleic acid, modulation of fatty alcohol composition in a transgenic plant, algae, animal, bacteria, fungus or other organism expressing the selected shuffled lipid biosynthetic nucleic acid, modulation of a wax composition in a transgenic plant, algae, animal, bacteria, fungus or other organism expressing the selected shuffled lipid biosynthetic nucleic acid, modification of acyl chain length in a lipid produced by a lipid synthetic pathway comprising activity encoded by the selected shuffled lipid biosynthetic nucleic acid, location
  • the activity of the one or more recombinant lipid biosynthetic nucleic acid or the selected shuffled lipid biosynthetic nucleic acid is selected e.g., by detecting one or more of: a change in a physical property of one or more lipid, fatty acid, wax or oil in the presence of a polypeptide or RNA encoded by the one or more lipid biosynthetic nucleic acid or the selected shuffled lipid biosynthetic nucleic acid, a protein-protein interaction in a two hybrid assay, expression of a reporter gene in a one hybrid assay, growth or survival of a recombinant cell expressing the one or more recombinant lipid biosynthetic nucleic acid or the selected shuffled lipid biosynthetic nucleic acid in an elevated temperature environment, growth or survival of a recombinant cell expressing the one or more recombinant lipid biosynthetic nucleic acid or the selected shuffle
  • a variety of parental nucleic acids are suitable substrates for recombination (a nucleic acid which is to be recombined is a "substrate for recombination"), including nucleic acids which are the same as, or homologous to, a nucleic acid encoding a protein such as any of the following proteins: an Acetyl-CoA carboxylase (an ACCase), a homomeric acetyl-CoA carboxylase, a heteromeric acetyl- CoA carboxylase BC subunit, a heteromeric acetyl-CoA carboxylase, a BCCP subunit, a heteromeric acetyl-CoA carboxylase (alpha)-CT subunit, a heteromeric acetyl-CoA carboxylase (beta)-CT subunit, an acyl carrier protein (ACP) (plastidial isoform or mitochondrial isoform), a malonyl-CoA: ACP transacylase, a
  • one or more of the parental nucleic acids are the same as, or homologous to, a nucleic acid encoding a protein which affects oil yield, such as an ACCase, an sn-2 acyltransferase, an acyltransferase other than sn-2 acyltransferase, a malonyl-CoA:ACP transacylase, an oleosin, a fatty acid binding protein, an Acyl-CoA synthase, or an acyl-ACP synthase.
  • a protein which affects oil yield such as an ACCase, an sn-2 acyltransferase, an acyltransferase other than sn-2 acyltransferase, a malonyl-CoA:ACP transacylase, an oleosin, a fatty acid binding protein, an Acyl-CoA synthase, or an acyl-ACP syntha
  • At least one of the parental nucleic acids can be the same as, or homologous to, a nucleic acid encoding a protein which affects fatty acid acyl chain length or composition, such as a thioseterase or an elongase.
  • at least one of the parental nucleic acids can be the same as, or homologous to, a nucleic acid encoding a protein which affects fatty acid saturation, such as a desaturase, a cis-trans isomerase, or a lipoxygenase (LOX).
  • LOX lipoxygenase
  • the parental nucleic acids can also be the same as, or homologous to, a nucleic acid encoding a protein which affects fatty acid branch structures, such as a reductase, or to a nucleic acid encoding a protein which affects flavor, such as a Lox protein, a desaturase, a beta-oxidation enzyme, or a hydroperoxide lyase.
  • the parental nucleic acid can be the same as, or homologous to, a nucleic acid encoding a protein which affects polyunsaturation, such as a protein in the polyketide synthase-like operon, a desaturase, or an elongase.
  • the parental nucleic acid can be the same as, or homologous to, a nucleic acid encoding a lipase or a DNA binding protein.
  • the parental nucleic acids can be homologous or non-homologous.
  • the parental nucleic acids can encode or not encode a lipid biosynthetic activity.
  • the parental nucleic acids, the one or more the one or more recombinant lipid biosynthetic nucleic acid, or the selected recombinant lipid biosynthetic nucleic acid can be cloned into an expression vector.
  • Recombinant libraries are also an aspect of the invention.
  • the plurality of parental nucleic acids are shuffled to produce a library of recombinant nucleic acids comprising one or more library member nucleic acid encoding one or more lipid biosynthetic activity.
  • the library is optionally selected for one or more lipid biosynthetic activity such as those noted above, providing a second library.
  • the selected library can be recombined with itself or any other nucleic acid and shuffled to produce additional recombinant selected libraries.
  • Any of the libraries herein can be in a variety of different formats, including a phage display library, a library in a cell or cell culture such as E.
  • the libraries can be made in a first cell type and transduced into a second cell type, e.g., the library can first be made in E. coli or cynobacteria and then transduced into a Synechocystis.
  • Another example cell type for library construction is Pseudomonas putida.
  • the parental nucleic acids can be shuffled in any of a plurality of cells e.g., prokaryotes or eukaryotes such as plants, yeast, bacteria, fungal cells, archae cells, or organisms.
  • the parental nucleic acids are shuffled in a plurality of cells and the method further includes recombining DNA from the plurality of cells that display lipid biosynthetic activity with a library of DNA fragments, at least one of which undergoes recombination with a segment in a cellular DNA present in the cells to produce recombined cells, or recombining DNA between the plurality of cells that display lipid biosynthetic activity to produce cells with modified lipid biosynthetic activity.
  • the method includes recombining and screemng the recombined or modified cells to produce further recombined cells that have evolved additionally modified lipid biosynthetic activity.
  • the method includes recombining at least one selected shuffled lipid biosynthetic nucleic acid with a further lipid biosynthetic activity nucleic acid.
  • This further nucleic acid is the same or different from one or more of the plurality of parental nucleic acids.
  • This recombination produces a library of recombinant lipid biosynthetic nucleic acids.
  • the library can be screened to identify at least one further selected distinct or improved recombinant lipid biosynthetic nucleic acid that exhibits a further improvement or distinct property compared to the plurality of parental nucleic acids.
  • the one or more recombinant lipid biosynthetic nucleic acid is present in one or more bacterial, yeast, plant or fungal cells and the method includes pooling multiple separate lipid biosynthetic nucleic acids.
  • the resulting pooled lipid biosynthetic nucleic acids are screened to identify distinct or improved recombinant lipid biosynthetic nucleic acids that exhibit distinct or improved lipid biosynthetic activity compared to a non-recombinant lipid biosynthetic activity nucleic acid.
  • the distinct or improved recombinant nucleic acid is typically cloned, transduced into a target cell or organism, or otherwise manipulated to achieve a desired effect.
  • One preferred recombination format is family gene shuffling.
  • shuffling protocols such as individual gene shuffling, oligonucleotide-mediated gene shuffling, in silico gene shuffling, and whole genome shuffling can be used.
  • diversity generation methods such as mutagenesis can be used to create libraries of divers nucleic acids, separate from, or in conjunction with, shuffling methods.
  • One aspect of the invention is a selected shuffled lipid biosynthetic nucleic acid made by the methods herein.
  • a plant, bacteria or fungus transduced with the selected shuffled lipid biosynthetic nucleic acid are a feature of the invention.
  • plants are a preferred target for incorporation of selected nucleic acids.
  • Preferred plants for transduction include plants in the families Gramineae, Composite, and Leguminosae.
  • Example preferred plants include corn, peanut, barley, millet, rice, soybean, sorghum, wheat, oats, rapeseed, oil palm, sunflower, and nut plants.
  • the plants optionally exhibit a new lipid biosynthetic activity as compared to a wild-type non-transduced plant.
  • the invention also provides DNA shuffling mixtures used e.g., in the methods of the invention.
  • the mixture comprises at least three homologous DNAs, each of which is derived from a nucleic acid encoding a polypeptide or polypeptide fragment which encodes a lipid biosynthetic activity.
  • the at least three homologous DNAs can be present e.g., in cell culture or in vitro.
  • methods of modulating lipid biosynthetic activity in a cell are provided.
  • whole genome shuffling of a plurality of genomic nucleic acids in the cell is performed and one or more lipid biosynthetic activity is selected.
  • the genomic nucleic acids can be from a species or strain the same as or different from the cell, which may be of, e.g., eukaryotic or prokaryotic origin. Any of the lipid biosynthetic activities noted above can be selected.
  • the invention provides methods of obtaining a recombinant lipid biosynthetic nucleic acid which can confer modified lipid production to a plant in which the recombinant lipid biosynthetic nucleic acid is present.
  • a plurality of forms of a selected lipid synthetic nucleic acid are recombined. These forms include segments derived from one or more parental nucleic acid which encode a lipid biosynthetic activity, or which can be shuffled to confer a lipid biosynthetic activity.
  • the plurality of forms of the selected nucleic acid differ from each other in at least one nucleotide. This recombination produces a library of recombinant lipid biosynthetic nucleic acids.
  • the library is screened to identify at least one recombinant lipid biosynthetic nucleic acid that exhibits distinct or improved lipid biosynthetic activity as compared to the parental nucleic acid.
  • one or more parental nucleic acid encodes a lipid biosynthetic enzyme, although the parental nucleic acids can encode other factors which affect lipid synthesis, or which can be shuffled to affect lipid synthesis. Any of the biosynthetic coding sequences and selection procedures noted above are applicable to this method.
  • the libraries produced can be screened by any of a variety of methods, such as selection by growing cells comprising the library in or on a medium comprising a cell membrane disruptive agent.
  • the libraries are optionally selected for one or more additional lipid biosynthetic activity.
  • the step of recombining cells of a library is optionally performed in a plurality of cells.
  • This recombination can include recombining DNA from the plurality of cells that display a selected lipid biosynthetic phenotype with a second library of DNA fragments, at least one of which undergoes recombination with a segment in a nucleic acid present in the cells to produce recombined modified lipid synthetic cells, or recombining DNA between the plurality of cells that display a selected lipid biosynthetic phenotype to produce modified lipid synthetic cells.
  • the library can be recombined and screened to produce further recombined cells that have evolved additionally distinct or improved lipid synthetic activity. These steps can be reiteratively repeated.
  • Fig. 1 is a diagram of fatty acid synthesis in plants.
  • Fig. 2 is a diagram of elongation of the acyl group in the fatty acid synthase cycle.
  • Fig. 3 A-3B is a schematic of a hybrid protein assay.
  • Fig. 4 is a schematic of a LUX assay.
  • a “recombinant” nucleic acid is a nucleic acid produced by recombination between two or more nucleic acids, or any nucleic acid made by an in vitro or artificial process.
  • the term “recombinant” when used with reference to a cell indicates that the cell comprises (and optionally replicates) a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid.
  • Recombinant cells can contain genes that are not found within the native (non-recombinant) parental form of the cell. Recombinant cells can also contain genes found in the native form of the cell where the genes are modified and re-introduced into the cell by artificial means.
  • the term also encompasses cells that contain a nucleic acid endogenous to the cell that has been artificially modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, and related techniques.
  • a "lipid” is a molecule which is fat soluble (e.g., soluble in a non-polar solvent), such as a fatty acid, glyceride, glyceryl ether, phospholipid, sphingolipid, alcohol, wax, oil, terpene, steroid, fat soluble vitamin, or the like.
  • a "recombinant lipid biosynthetic nucleic acid” is a recombinant nucleic acid encoding a protein, RNA (or other active nucleic acid) which produces one or more lipid, in vitro or in vivo or which interacts with one or more additional RNAs (or other active nucleic acids, such as DNAs in the case where the recombinant lipid biosynthetic nucleic acid encodes a transcription factor) or proteins in vitro or in vivo to produce one or more lipid.
  • a lipid biosynthetic activity is any activity which produces or controls production of one or more lipid (including both yield and lipid type).
  • a "plurality of forms" of a selected nucleic acid refers to a plurality of homologs of the nucleic acid.
  • the homologs can be from naturally occurring homologs (e.g., two or more homologous genes) or by artificial synthesis of one or more nucleic acid(s) having related sequences (e.g., as occurs during oligonucleotide-mediated family gene shuffling), or by modification of one or more nucleic acid to produce related nucleic acids.
  • Nucleic acids are homologous when they are derived, naturally or artificially, from a common ancestor sequence. During natural evolution, this occurs when two or more descendent sequences diverge from a parent sequence over time, i.e., due to mutation and natural selection.
  • a given sequence can be artificially recombined with another sequence, as occurs, e.g., during typical cloning, to produce a descendent nucleic acid.
  • a nucleic acid can be synthesized de novo, by synthesizing a nucleic acid which varies in sequence from a given parental nucleic acid sequence.
  • homology is typically inferred by sequence comparison between two sequences. Where two nucleic acid sequences show sequence similarity it is infe ⁇ ed that the two nucleic acids share a common ancestor.
  • sequence similarity varies in the art depending on a variety of factors.
  • two sequences are considered homologous, e.g., where they share sufficient sequence identity to allow recombination to occur between two nucleic acid molecules.
  • nucleic acids require regions of close similarity spaced roughly the same distance apart to permit recombination to occur.
  • regions of at least about 60% sequence identity or higher are optimal for recombination.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of skill) or by visual inspection.
  • substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least about 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • Such "substantially identical" sequences are typically considered to be homologous.
  • the "substantial identity” exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues, or over the full length of the two sequences to be compared.
  • sequence comparison and homology determination typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the 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 of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl Acad. Sci. USA 89:10915).
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff (1989) Proc. Natl Acad. Sci. USA 89:10915.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl Acad. Sci. USA 90:5873-5787).
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • Another indication that two nucleic acid sequences are substantially identical homologous is that the two molecules hybridize to each other under stringent conditions.
  • the phrase "hybridizing specifically to,” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions, including when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Bind(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42 °C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.15M NaCI at 72 °C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65 °C for 15 minutes (see, Sambrook, infra., for a description of SSC buffer).
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • a “subsequence” refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., polypeptide) respectively.
  • genes are used broadly to refer to any segment of nucleic acid (typically DNA, but optionally RNA) associated with expression of a given RNA or protein.
  • genes include sequences encoding expressed RNAs (which can include polypeptide coding sequences) and, often, the regulatory sequences required for their expression.
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • nucleic acid or protein when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid is generic to the terms “gene”, “DNA,” “cDNA”, “oligonucleotide,” “RNA,” “mRNA,” and the like.
  • Nucleic acid derived from a gene refers to a nucleic acid for whose synthesis the gene, or a subsequence thereof, has ultimately served as a template.
  • an mRNA, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc. are all derived from the gene and detection of such derived products is indicative of the presence and/or abundance of the original gene and/or gene transcript in a sample.
  • 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 increases the transcription of the coding sequence.
  • a "recombinant expression cassette” or simply an “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a structural gene in hosts compatible with such sequences.
  • Expression cassettes include at least promoters and optionally, transcription termination signals.
  • the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or helpful in effecting expression may also be used as described herein.
  • an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette.
  • the present invention provides ways of improving or modulating lipid production in cells and whole organisms such as oil crop plants.
  • fatty acid modifying phenotypes include, but are not limited to, an increase or decrease in level of saturation of the fatty acid, the length of the carbon backbone, the position of double or triple bonds, the linkage forms of the fatty acyl groups, the location of fatty acid accumulation, and the quantity (yield) of total triglycerides and other forms of lipid in the organism.
  • DNA sequences that can be shuffled according to the present invention include, but are not limited to, the exons and introns of a gene, promoter sequences controlling expression of genes, transcription factors regulating gene expression, operons (an "operon” is typically a group of contiguous structural genes which are transcribed as a single transcription unit from a common promoter and can be thereby subject to coordinated regulation) involved in a fatty acid production pathway and the like.
  • operons an "operon” is typically a group of contiguous structural genes which are transcribed as a single transcription unit from a common promoter and can be thereby subject to coordinated regulation
  • a complete fatty acid synthetic pathway from plants or other organisms, including bacteria, fungi, algae and animals can be linked together through cloning to result in a polyprotein (Halpin, C. et al. 1999.
  • Self-processing 2A- polyproteins a system for co-ordinate expression of multiple proteins in transgenic plants (Plant J 17(2): 453-459).
  • Such polyprotein genes can be subject to shuffling in the individual gene segment or the entire gene.
  • These polyprotein genes or T-DNAs containing multiple fatty acid-related genes can be introduced into plant chromosomes or plastid genomes by conventional plant gene transformation methods.
  • genes involved in fatty acid synthesis in animal, bacteria or fungi are modified to utilize substrates abundant in the plant pathway.
  • the encoded product can have a modified property, such as increase or decrease of the substrate specificity when compared to the wild-type protein.
  • the enzyme is altered to recognize a substrate other than its natural one, resulting in a novel phenotype in a desired host.
  • fatty acid synthesis mainly occurs in the plastids and most enzymes involved utilize acyl-ACP, but not free fatty acids, as substrates (see Fig. 2).
  • a bacterial enzyme utilizing free fatty acid to produce a desired product is shuffled to recognize acyl-ACP and thus can be transformed into plants to produce a novel phenotype.
  • the production of certain fatty acids are controlled by a gene operon, in which the acyl group is channeled from one gene product to the next. It is often not clear which gene product is rate- limiting. The complete operon can be shuffled as a whole, resulting in a much more efficient system for fatty acid production of one or more fatty acids from any of a variety of substrates.
  • biotechnological approaches to conferring desirable lipid production to crops involves either: (a) altering the gene that codes for a target site in order to confer desirable properties, or (b) engineering a gene into crops that codes for an enzyme with a desirable property.
  • enzymes are discovered either by extensive screening of organisms or by mutagenesis followed by rigorous selection. In spite of this rigorous scheme, selected enzymes may not have the ideal properties to confer crop selectivity or to function effectively in transgenic crops, and the process is, at best, labor intensive.
  • the present invention overcomes these difficulties by applying DNA shuffling and other diversity generation/ selection techniques to gene-families that code for lipid synthesis or metabolizing genes, including those listed herein.
  • genes are optimized by DNA shuffling in order to enhance the rate of metabolism or synthesis of specific lipids or lipid substrates, optionally without altering other parameters, such as affinity for natural substrates, effectors, etc., or, alternately, optionally including altering these additional parameters.
  • DNA shuffling in order to enhance the rate of metabolism or synthesis of specific lipids or lipid substrates, optionally without altering other parameters, such as affinity for natural substrates, effectors, etc., or, alternately, optionally including altering these additional parameters.
  • Fatty acid compositions can be analyzed using several established protocols. For example, plant seed fatty acid composition may be determined by the acid methanolysis method described by Browse et al. (Anal. Biochem. 1986. 152: 141- 145). In other cases where a large number of samples are involved, to identify novel fatty acids produced the shuffled genes or pathways is performed using high throughput assays. Fatty acids, phopholipids and triglycerides are detected, e.g., using ESI (electrospray ionization) or APCI (atmospheric pressure chemical ionization) mass spectrometry (Karlsson, A. A. et al., J. Mass Spectrom..
  • ESI electrospray ionization
  • APCI atmospheric pressure chemical ionization
  • a high throughput method for detecting analyte molecules from a complex biological matrix by electrospray tandem mass spectrometry is taught in "HIGH THROUGHPUT MASS SPECTROMETRY" by Sun Ai Raillard, USSN 60/119,766, filed 02/11/1999, which utilizes off-line parallel sample purifications and fast flow-injection analysis, typically reducing the time of analysis to 30 to 40 seconds per sample. All steps starting from cell picking, cell growth, sample preparation and analysis are automated and can be carried out overnight by various robotic workstations.
  • Tandem mass spectrometry allows for high selectivity and sensitivity and for simultaneous detection of multiple analytes.
  • the analysis by mass spectrometry allows for identification based on mass over charge. Tandem mass spectrometry can potentially distinguish regioisomers as well.
  • UN for conjugation of double bonds
  • capillary electrophoresis separation system with UV or fluorescent detection Akasaka, K. et al. Enantiomer, 1998.
  • Fig. 2 shows elongation of acyl groups in the fatty acid synthase cycle.
  • Enzymes in the pathway include: 1.- Acetyl-CoA carboxylase
  • ACCases la.- Homomeric acetyl-CoA carboxylase (EC 6.4.1.2); lb.- Heteromeric acetyl-CoA carboxylase BC subunit (EC 6.4.1.2); lc- Heteromeric acetyl-CoA carboxylase BCCP subunit (EC 6.4.1.2); Id.- Heteromeric acetyl-CoA carboxylase (alpha)-CT subunit (EC 6.4.1.2); le- Heteromeric acetyl-CoA carboxylase (beta)-CT subunit (EC 6.4.1.2); 2.- Acyl carrier proteins (ACP) plastidial isoforms mitochondrial isoforms; 3.- Malonyl-CoA:ACP transacylase (EC 2.3.1.39); 4.- Ketoacyl-ACP synthase (KAS); 4a.- KAS I (EC 2.3.1.41); 4b.- KAS II (EC 2.3.1.41); 4c- KAS
  • Monogalactosyldiacyl-glycerol synthase (EC2.4.1.46); 20.- Monogalactosyldiacyl- glycerol desaturase(palmitate-specific)(EC 1.14.99.-); 21.- Digalactosyldiacyl-glycerol synthase (EC2.4.1.184); 22.- Sulfolipid biosynthesis protein; 23.- Long-chain acyl-CoA synthetase.
  • ACCase (Reverdatto S. 1999. Plant Phvsiol. 119:961-978 ' ): sn-2 acyltransferase (Knutzon DS et al. 1995 Plant Phyisol. 109 : 999- 1006); other acyltransferases (for example, Lassner MW et al. 1995, Plant Phvsiol. 109:1389-1394); malonyl-CoA:ACP transacylase (Verwoert II. Et al. 1992. J. Bacteriol. 174:2851-2857;
  • Flavor Lox; desaturases; beta-oxidation enzymes (Bojorguez G et al. 1995 Plant
  • DNA shuffling can result in optimization of a desirable property even in the absence of a detailed understanding of the mechanism by which the particular property is mediated (this is especially the case for whole-genome shuffling formats where even the targets for shuffling can be completely unknown).
  • entirely new properties can be obtained upon shuffling of DNAs, i.e., shuffled DNAs can encode polypeptides or RNAs with properties entirely absent in the parental DNAs which are shuffled.
  • Sequence recombination can be achieved in many different formats and permutations of formats, as described in further detail below. These formats share some common principles.
  • the substrates for modification, or "forced evolution,” vary in different applications, as does the property sought to be acquired or improved. Examples of candidate substrates for acquisition of a property or improvement in a property include genes (typically the regulatory elements directing expression of a coding sequence in a cell or in vitro transcription reaction) that encode proteins which have enzymatic or other activities useful forming linkages in lipid molecules, in breaking down lipid molecules, in sequestering lipid molecules and the like.
  • the methods typically use at least two variant forms of a starting substrate.
  • the variant forms of candidate substrates can show substantial sequence or secondary structural similarity with each other, but they should also differ in at least one and preferably at least two positions.
  • the initial diversity between forms can be the result of natural variation, e.g., the different variant forms (homologs) are obtained from different individuals or strains of an organism (including geographic variants) or constitute related sequences from the same organism (e.g., allelic variations), or constitute homologs from different organisms (interspecific variants).
  • Shuffling of such natural variants is one form of "family gene shuffling.”
  • initial diversity can be induced, e.g., the variant forms can be generated by error-prone transcription, such as an error-prone PCR or use of a polymerase which lacks proof-reading activity (see, Liao (1990) Gene 88:107-111), of the first variant form, or, by replication of the first form in a mutator strain (mutator host cells are discussed in further detail below, and are generally well known).
  • error-prone transcription such as an error-prone PCR or use of a polymerase which lacks proof-reading activity (see, Liao (1990) Gene 88:107-111)
  • mutator host cells are discussed in further detail below, and are generally well known.
  • the initial diversity between substrates is greatly augmented in subsequent steps of recombination for library generation.
  • a mutator strain can include any mutants in any organism impaired in the functions of mismatch repair.
  • Impairment can be of the genes noted, or of homologous genes in any organism.
  • properties that can be acquired or improved vary widely, and, of course, depend on the choice of substrate.
  • properties that one can improve include, but are not limited to a change in a physical property of one or more lipid, fatty acid, wax or oil in the presence of a polypeptide or RNA encoded by the one or more lipid biosynthetic nucleic acid or the selected shuffled lipid biosynthetic nucleic acid, a protein-protein interaction in a two hybrid assay, expression of a reporter gene in a one hybrid assay, growth or survival of a recombinant cell expressing the one or more recombinant lipid biosynthetic nucleic acid or the selected shuffled lipid biosynthetic nucleic acid in an elevated temperature environment, growth or survival of a recombinant cell expressing the one or more recombinant lipid biosynthetic nucleic acid or the selected shuffled lipid biosynthetic nucleic acid
  • At least two variant forms of a nucleic acid which can confer lipid synthetic of metabolic activity are recombined to produce a library of recombinant nucleic acids.
  • the library is then screened to identify at least one recombinant nucleic acid that is optimized for the particular property or properties of interest.
  • Recursive sequence recombination can be employed to achieve still further improvements in a desired property, or to bring about new (or "distinct") properties.
  • Recursive sequence recombination entails successive cycles of recombination to generate molecular diversity. That is, one creates a family of nucleic acid molecules showing some sequence identity to each other but differing in the presence of mutations. In any given cycle, recombination can occur in vivo or in vitro, intracellularly or extracellularly.
  • recombination cycle is usually followed by at least one cycle of screening or selection for molecules having a desired property or characteristic.
  • a recombination cycle is performed in vitro, the products of recombination, i.e., recombinant segments, are sometimes introduced into cells before the screening step.
  • Recombinant segments can also be linked to an appropriate vector or other regulatory sequences before screening.
  • products of recombination generated in vitro are sometimes packaged in viruses (e.g., bacteriophage) before screening. If recombination is performed in vivo, recombination products can sometimes be screened in the cells in which recombination occurred.
  • recombinant segments are extracted from the cells, and optionally packaged as viruses, before screening.
  • lipid biosynthetic gene can have many component sequences each having a different intended role (e.g., coding sequence, regulatory sequences, targeting sequences, stability-conferring sequences, and sequences affecting integration). Each of these component sequences can be varied and recombined simultaneously. Screening/selection can then be performed, for example, for recombinant segments that have increased ability to confer lipid synthetic traits to a plant without the need to attribute such improvement to any of the individual component sequences of the vector.
  • initial round(s) of screening can sometimes be performed using bacterial cells due to high transfection efficiencies and ease of culture.
  • Later rounds, and other types of screening which are not amenable to screening in bacterial cells can be performed, e.g., in plant cells to optimize recombinant segments for use in an environment close to that of their intended use.
  • Final rounds of screening can be performed in the precise cell type of intended use (e.g., a cell which is present in a plant), or even in whole plants (e.g., crop tests in the field) or other organisms.
  • a recombinant gene can itself be used as a round of screening. That is, recombinant genes that are successfully taken up and/or expressed by the intended target cells are recovered from those target cells and used to confer traits upon other plants.
  • the recombinant genes that are recovered from the first target cells are enriched for genes that have evolved, i.e., have been modified by recursive sequence recombination, toward improved or new properties or characteristics for specific uptake and integration of the gene, desired lipid levels, stability, and the like.
  • the screening or selection step identifies a subpopulation of recombinant segments that have evolved toward acquisition of a new or improved desired property or properties useful in conferring lipid synthetic activity upon plants.
  • the recombinant segments can be identified as components of cells, components of viruses or in free form. More than one round of screening or selection can be performed after each round of recombination. If further improvement in a property is desired, at least one and usually a collection of recombinant segments surviving a first round of screening/selection are subject to a further round of recombination. These recombinant segments can be recombined with each other or with exogenous segments representing the original substrates or further variants thereof.
  • recombination can proceed in vitro or in vivo.
  • the components can be subjected to further recombination in vivo, or can be subjected to further recombination in vitro, or can be isolated before performing a round of in vitro recombination.
  • the previous screening step identifies desired recombinant segments in naked form or as components of viruses, these segments can be introduced into cells to perform a round of in vivo recombination.
  • the second round of recombination irrespective how performed, generates further recombinant segments which encompass additional diversity than is present in recombinant segments resulting from previous rounds.
  • the second round of recombination can be followed by a further round of screening/selection according to the principles discussed above for the first round.
  • the stringency of screening/selection can be increased between rounds.
  • the nature of the screen and the property being screened for can vary between rounds if improvement in more than one property is desired or if acquiring more than one new property is desired. Additional rounds of recombination and screening can then be performed until the recombinant segments have sufficiently evolved to acquire the desired new or improved property or function.
  • the practice of this invention involves the construction of recombinant nucleic acids and the expression of genes in transfected host cells.
  • Molecular cloning techniques to achieve these ends are known in the art.
  • a wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids such as expression vectors are well-known to persons of skill.
  • General texts which describe molecular biological techniques useful herein, including mutagenesis include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzvmology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol.
  • RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, Ausbel, Sambrook and Berger, all supra.
  • Oligonucleotides for use as probes, e.g., in in vitro amplification methods, for use as gene probes, or as shuffling targets (e.g., synthetic genes or gene segments) are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using an automated synthesizer, as described in Needham-NanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168.
  • Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill.
  • library members e.g., cells, viral plaques, spores or the like
  • solid media e.g., a cell, viral plaques, spores or the like
  • colonies are identified, picked, and 10,000 different mutants inoculated into 96 well microtiter dishes containing two 3 mm glass balls/well.
  • the Q-bot does not pick an entire colony but rather inserts a pin through the center of the colony and exits with a small sampling of cells, (or mycelia) and spores (or viruses in plaque applications).
  • the uniform process of the Q-bot decreases human handling error and increases the rate of establishing cultures (roughly 10,000/4 hours). These cultures are then shaken in a temperature and humidity controlled incubator.
  • the glass balls in the microtiter plates act to promote uniform aeration of cells and the dispersal of mycelial fragments similar to the blades of a fermenter.
  • the ability to detect a subtle increase in the performance of a shuffled library member over that of a parent strain relies on the sensitivity of the assay.
  • the chance of finding the organisms having an improvement is increased by the number of individual mutants that can be screened by the assay.
  • a prescreen that increases the number of mutants processed by 10-fold can be used.
  • the goal of the primary screen is to quickly identify mutants having equal or better product titers than the parent strain(s) and to move only these mutants forward to liquid cell culture for subsequent analysis.
  • An especially preferred high throughput method for detecting analyte molecules from a complex biological matrix is by electrospray tandem mass spectrometry as taught in "HIGH THROUGHPUT MASS SPECTROMETRY" by Sun Ai Raillard, USSN 60/119,766, filed 02/11/1999.
  • methods which utilize off-line parallel sample purification and fast flow-injection analysis, typically reducing the time of analysis to 30 to 40 seconds per sample. All steps starting from cell picking, cell growth, sample preparation and analysis are automated and can be carried out overnight by various robotic workstations.
  • the methods of the invention optionally entail performing recombination ("shuffling") or other sequence diversity generation protocols (e.g., mutation) and screening or selection to "evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes (see also, Stemmer (1995) Bio/Technology 13:549-553, for an introduction to shuffling). Reiterative cycles of diversity generation/ recombination and screening/selection can be performed to further evolve the nucleic acids of interest. Such techniques do not require the extensive analysis and computation required by conventional methods for polypeptide engineering.
  • Shuffling allows the recombination of large numbers of mutations in a minimum number of selection cycles, in contrast to natural pairwise recombination events (e.g., as occur during sexual replication).
  • sequence recombination techniques described herein provide particular advantages in that they provide recombination between mutations in any or all of these, thereby providing a very fast way of exploring the manner in which different combinations of mutations can affect a desired result.
  • structural and/or functional information is available which, although not required for sequence recombination, provides opportunities for modification of the technique.
  • shuffling procedures can be used in conjunction with other diversity generation protocols for generating libraries of diverse
  • a variety of diversity generating protocols including nucleic acid shuffling protocols, including nucleic acid family shuffling protocols and other specific desirable recombination formats are available and described in the art.
  • the following publications describe a variety of recursive recombination procedures and/or methods which can be incorporated into such procedures, as well as other diversity generating protocols: Stemmer, et al., (1999) "Molecular breeding of viruses for targeting and other clinical properties.” Tumor Targeting 4:1-4; Nesset al. (1999) "DNA Shuffling of subgenomic sequences of subtilisin” Nature Biotechnology 17:893-896; Chang et al.
  • nucleic acids can be recombined in vitro by any of a variety of techniques discussed in the references above, including e.g., DNAse digestion of nucleic acids to be recombined followed by ligation and/or PCR reassembly of the nucleic acids.
  • nucleic acids can be recursively recombined in vivo, e.g., by allowing recombination to occur between nucleic acids in cells.
  • whole genome recombination methods can be used in which whole genomes (or significant fractions thereof) of cells or other organisms are recombined, optionally including spiking of the genomic recombination mixtures with selected nucleic acids (e.g., which encode lipid synthesis enzymes or other relevant factors for enhanced lipid biosynthesis, as noted herein).
  • selected nucleic acids e.g., which encode lipid synthesis enzymes or other relevant factors for enhanced lipid biosynthesis, as noted herein.
  • oligonucleotides corresponding to targets of interest are synthesized and reassembled in PCR and/or ligation reactions which include oligonucleotides which correspond to more than one parental nucleic acid (e.g., including one or more lipid biosynthetic 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.
  • silico methods of recombination can be effected in which genetic algorithms are used in a computer to recombine sequence strings which co ⁇ espond to nucleic acid homologues (or even non-homologous) sequences.
  • 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 methods can also be practiced in combination.
  • nucleic acids of the 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, e.g., sets of homologous or non-homologous nucleic acids.
  • any nucleic acids which are produced can be selected for a desired activity.
  • this can include testing for and identifying any activity that can be detected, including in an automatable format, by any of the assays in the art.
  • a variety of related (or even unrelated) properties can be assayed for, using any available assay.
  • DNA shuffling and related techniques provide a robust, widely applicable, means of generating diversity useful for the engineering of proteins, pathways, cells and organisms with improved characteristics.
  • additional techniques for generating diversity In conjunction with (or separately from) recombination-based methods, a variety of other diversity generation methods can be practiced and the results (i.e., diverse populations of nucleic acids) screened for. Additional diversity can be introduced into nucleic acids by methods which result in the alteration of individual nucleotides or groups of contiguous or non-contiguous nucleotides, e.g., mutagenesis methods.
  • Mutagenesis methods include, for example, recombination (PCT/US98/05223; Publ. No. WO98/42727); oligonucleotide-directed mutagenesis (for review see, Smith, Ann. Rev.Genet. 19: 423- 462 (1985)); Botstein and Shortle, Science 229: 1193-1201 (1985); Carter, Biochem. J. 237: 1-7 (1986); Kunkel, "The efficiency of oligonucleotide directed mutagenesis" in Nucleic acids & Molecular Biology. Eckstein and Lilley, eds., Springer Verlag, Berlin (1987)).
  • oligonucleotide-directed mutagenesis Zoller and Smith, Nucl. Acids Res. 10: 6487-6500 (1982), Methods in Enzvmol. 100: 468-500 (1983), and Methods in Enzvmol. 154: 329-350 (1987)) phosphothioate- modified DNA mutagenesis (Taylor et al., Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye and Eckstein, Nucl. Acids Res. 14: 9679-9698 (1986); Sayers et al., Nucl.
  • the invention involves creating recombinant libraries of polynucleotides that are then screened to identify those library members that exhibit a desired property, e.g. , which encode lipid synthetic or metabolic activity.
  • the recombinant libraries can be created using any of the various methods herein, as well as many others which would be apparent to one of skill. Methods for obtaining recombinant polynucleotides and/or for obtaining diversity in nucleic acids used as the substrates for DNA shuffling as described herein include, for example, those references noted in the preceding section, including those related to recombination, mutation and other diversity generation procedures.
  • the recombinant libraries are prepared, at least in part, using DNA shuffling. Reiterative cycles of recombination and screening/selection can be performed to further evolve any nucleic acid(s) of interest. Such techniques do not require the extensive analysis and computation required by conventional methods for polypeptide engineering. Shuffling allows the recombination of large numbers of mutations in a minimum number of selection cycles, in contrast to traditional, pairwise recombination events. Thus, the sequence recombination techniques described herein provide particular advantages in that they provide recombination between mutations in any or all of these, thereby providing a very fast way of exploring the manner in which different combinations of mutations can effect a desired result. In some instances, however, structural and/or functional information is available which, although not required for sequence recombination, provides opportunities for modification of the technique.
  • the breeding procedure starts with at least two substrates that generally show substantial sequence identity to each other (i.e., at least about 30%, 50%, 70%, 80% or 90% sequence identity), but differ from each other at certain positions.
  • the difference can be any type of mutation, for example, substitutions, insertions, deletions or the like.
  • different segments differ from each other in about 5-20 positions.
  • the starting materials typically differ from each other in at least two nucleotide positions. That is, if there are only two substrates, there are usually at least two divergent positions.
  • the starting DNA segments can be natural variants of each other, for example, allelic or species variants.
  • the segments can also be from nonallelic genes showing some degree of structural and usually (though not necessarily) functional relatedness (e.g., different genes within a superfamily).
  • the starting DNA segments can also be induced variants of each other.
  • one DNA segment can be produced by error-prone PCR replication of the other, or by substitution of a mutagenic cassette. Induced mutants can also be prepared by propagating one (or both) of the segments in a mutagenic strain.
  • the second DNA segment is not generally a single segment but a large family of related segments.
  • the different segments forming the starting materials are often the same length or substantially the same length. However, this need not be the case; for example; one segment can be a subsequence of another.
  • the segments can be present as part of larger molecules, such as vectors, or can be in isolated form.
  • the starting DNA segments are recombined by any of the sequence recombination formats provided herein to generate a diverse library of recombinant DNA segments.
  • a library can vary widely in size from having fewer than 10 to more than 10 5 , 10 9 , 10 12 or more members.
  • the starting segments and the recombinant libraries generated will include full-length coding sequences and any essential regulatory sequences, such as a promoter and polyadenylation sequence, required for expression.
  • the recombinant DNA segments in the library can be inserted into a common vector providing sequences necessary for expression before performing screening/selection.
  • restriction enzyme sites in nucleic acids it is advantageous to use restriction enzyme sites in nucleic acids to direct the recombination of mutations in a nucleic acid sequence of interest. These techniques are particularly preferred in the evolution of fragments that cannot readily be shuffled by existing methods due to the presence of repeated DNA or other problematic primary sequence motifs. These situations also include recombination formats in which it is preferred to retain certain sequences unmutated.
  • restriction enzyme sites are also preferred for shuffling large fragments (typically greater than 10 kb), such as gene clusters that cannot be readily shuffled and "PCR-amplified” because of their size.
  • fragments up to 50 kb have been reported to be amplified by PCR (Barnes, Proc. Natl. Acad. Sci. U.S.A. 91:2216-2220 (1994)), it can be problematic for fragments over 10 kb, and thus alternative methods for shuffling in the range of 10 - 50 kb and beyond are preferred.
  • the restriction endonucleases used are of the Class II type (Sambrook, Ausubel and Berger, supra) and of these, preferably those which generate nonpalindromic sticky end overhangs such as Alwn I, Sfi I or BstXl. These enzymes generate nonpalindromic ends that allow for efficient ordered reassembly with DNA ligase.
  • restriction enzyme (or endonuclease) sites are identified by conventional restriction enzyme mapping techniques (Sambrook, Ausubel, and Berger, supra.), by analysis of sequence information for that gene, or by introduction of desired restriction sites into a nucleic acid sequence by synthesis (i.e. by incorporation of silent mutations).
  • the DNA substrate molecules to be digested can either be from in vivo replicated DNA, such as a plasmid preparation, or from PCR amplified nucleic acid fragments harboring the restriction enzyme recognition sites of interest, preferably near the ends of the fragment.
  • at least two variants of a gene of interest, each having one or more mutations are digested with at least one restriction enzyme determined to cut within the nucleic acid sequence of interest.
  • the restriction fragments are then joined with DNA ligase to generate full length genes having shuffled regions. The number of regions shuffled will depend on the number of cuts within the nucleic acid sequence of interest.
  • the shuffled molecules can be introduced into cells as described above and screened or selected for a desired property as described herein. Nucleic acid can then be isolated from pools (libraries), or clones having desired properties and subjected to the same procedure until a desired degree of improvement is obtained.
  • At least one DNA substrate molecule or fragment thereof is isolated and subjected to mutagenesis.
  • the pool or library of religated restriction fragments are subjected to mutagenesis before the digestion-ligation process is repeated.
  • "Mutagenesis" as used herein comprises such techniques known in the art as PCR mutagenesis, oligonucleotide-directed mutagenesis, site-directed mutagenesis, etc., and recursive sequence recombination by any of the techniques described herein. Reassembly PCR
  • a further technique for recombining mutations in a nucleic acid sequence utilizes "reassembly PCR.” This method can be used to assemble multiple segments that have been separately evolved into a full length nucleic acid template such as a gene. This technique is performed when a pool of advantageous mutants is known from previous work or has been identified by screening mutants that may have been created by any mutagenesis technique known in the art, such as PCR mutagenesis, cassette mutagenesis, doped oligo mutagenesis, chemical mutagenesis, or propagation of the DNA template in vivo in mutator strains.
  • Boundaries defining segments of a nucleic acid sequence of interest preferably lie in intergenic regions, introns, or areas of a gene not likely to have mutations of interest.
  • oligonucleotide primers are synthesized for PCR amplification of segments of the nucleic acid sequence of interest, such that the sequences of the oligonucleotides overlap the junctions of two segments.
  • the overlap region is typically about 10 to 100 nucleotides in length.
  • Each of the segments is amplified with a set of such primers.
  • the PCR products are then "reassembled" according to assembly protocols such as those discussed herein to assemble randomly fragmented genes.
  • the PCR products are first purified away from the primers, by, for example, gel electrophoresis or size exclusion chromatography. Purified products are mixed together and subjected to about 1-10 cycles of denaturing, reannealing, and extension in the presence of polymerase and deoxynucleoside triphosphates (dNTP's) and appropriate buffer salts in the absence of additional primers ("self-priming"). Subsequent PCR with primers flanking the gene are used to amplify the yield of the fully reassembled and shuffled genes. In some embodiments, the resulting reassembled genes are subjected to mutagenesis before the process is repeated.
  • dNTP's polymerase and deoxynucleoside triphosphates
  • the PCR primers for amplification of segments of the nucleic acid sequence of interest are used to introduce variation into the gene of interest as follows. Mutations at sites of interest in a nucleic acid sequence are identified by screening or selection, by sequencing homologues of the nucleic acid sequence, and so on. Oligonucleotide PCR primers are then synthesized which encode wild type or mutant information at sites of interest. These primers are then used in PCR mutagenesis to generate libraries of full length genes encoding permutations of wild type and mutant information at the designated positions.
  • sequence information from one or more substrate sequences is added to a given "parental" sequence of interest, with subsequent recombination between rounds of screening or selection.
  • site-directed mutagenesis performed by techniques well known in the art (e.g., Berger, Ausubel and Sambrook, supra.) with one substrate as template and oligonucleotides encoding single or multiple mutations from other substrate sequences, e.g. homologous genes.
  • the selected recombinant(s) can be further evolved using RSR techniques described herein.
  • site-directed mutagenesis can be done again with another collection of oligonucleotides encoding homologue mutations, and the above process repeated until the desired properties are obtained.
  • degenerate oligonucleotides can be used that encode the sequences in both homologues.
  • One oligonucleotide can include many such degenerate codons and still allow one to exhaustively search all permutations over that block of sequence.
  • homologue sequence space When the homologue sequence space is very large, it can be advantageous to restrict the search to certain variants.
  • computer modeling tools (Lathrop et al. (1996) J. Mol. Biol, 255: 641-665) can be used to model each homologue mutation onto the target protein and discard any mutations that are predicted to grossly disrupt structure and function.
  • the initial substrates for recombination are a pool of related sequences, e.g., different, variant forms, as homologs from different individuals, strains, or species of an organism, or related sequences from the same organism, as allelic variations.
  • the sequences can be
  • DNA or RNA can be of various lengths depending on the size of the gene or DNA fragment to be recombined or reassembled.
  • sequences are from about 50 base pairs (bp) to about 50 kilobases (kb).
  • the pool of related substrates are converted into overlapping fragments, e.g., from about 5 bp to 5 kb or more. Often, for example, the size of the fragments is from about 10 bp to 1000 bp, and sometimes the size of the DNA fragments is from about 100 bp to 500 bp.
  • the conversion can be effected by a number of different methods, such as DNase I or RNase digestion, random shearing or partial restriction enzyme digestion. For discussions of protocols for the isolation, manipulation, enzymatic digestion, and the like of nucleic acids, see, for example, Sambrook et al. and Ausubel, both supra.
  • the concentration of nucleic acid fragments of a particular length and sequence is often less than 0.1 % or 1% by weight of the total nucleic acid.
  • the number of different specific nucleic acid fragments in the mixture is usually at least about 100, 500 or 1000.
  • the mixed population of nucleic acid fragments are converted to at least partially single-stranded form using a variety of techniques, including, for example, heating, chemical denaturation, use of DNA binding proteins, and the like. Conversion can be effected by heating to about 80 °C to 100°C, more preferably from 90 °C to 96 °C, to form single-stranded nucleic acid fragments and then reannealing. Conversion can also be effected by treatment with single-stranded DNA binding protein (see Wold (1997) Annu. Rev. Biochem. 66:61-92) or recA protein (see, e.g., Kiianitsa (1997) Proc. Natl. Acad. Sci. USA 94:7837-7840).
  • Single-stranded nucleic acid fragments having regions of sequence identity with other single-stranded nucleic acid fragments can then be reannealed by cooling to 20°C to 75 °C, and preferably from 40°C to 65 °C. Renaturation can be accelerated by the addition of polyethylene glycol (PEG), other volume-excluding reagents or salt.
  • PEG polyethylene glycol
  • the salt concentration is preferably from 0 mM to 200 mM, more preferably the salt concentration is from 10 mM to 100 mM.
  • the salt may be KC1 or NaCI.
  • the concentration of PEG is preferably from 0% to 20%, more preferably from 5% to 10%.
  • the fragments that reanneal can be from different substrates.
  • the annealed nucleic acid fragments are incubated in the presence of a nucleic acid polymerase, such as Taq or Klenow, and dNTP's (i.e. dATP, dCTP, dGTP and dTTP).
  • a nucleic acid polymerase such as Taq or Klenow
  • dNTP's i.e. dATP, dCTP, dGTP and dTTP.
  • Taq polymerase can be used with an annealing temperature of between 45-65 °C.
  • Klenow polymerase can be used with an annealing temperature of between 20-30°C.
  • the polymerase can be added to the random nucleic acid fragments prior to annealing, simultaneously with annealing or after annealing.
  • the process of denaturation, renaturation and incubation in the presence of polymerase of overlapping fragments to generate a collection of polynucleotides containing different permutations of fragments is sometimes referred to as shuffling of the nucleic acid in vitro.
  • This cycle is repeated for a desired number of times. Preferably the cycle is repeated from 2 to 100 times, more preferably the sequence is repeated from 10 to 40 times.
  • the resulting nucleic acids are a family of double- stranded polynucleotides of from about 50 bp to about 100 kb, preferably from 500 bp to 50 kb.
  • the population represents variants of the starting substrates showing substantial sequence identity thereto but also diverging at several positions. The population has many more members than the starting substrates.
  • the population of fragments resulting from shuffling is used to transform host cells, optionally after cloning into a vector.
  • subsequences of recombination substrates can be generated by amplifying the full-length sequences under conditions which produce a substantial fraction, typically at least 20 percent or more, of incompletely extended amplification products.
  • Another embodiment uses random primers to prime the entire template DNA to generate less than full length amplification products.
  • the amplification products, including the incompletely extended amplification products are denatured and subjected to at least one additional cycle of reannealing and amplification.
  • This variation in which at least one cycle of reannealing and amplification provides a substantial fraction of incompletely extended products, is termed "stuttering.”
  • the partially extended (less than full length) products reanneal to and prime extension on different sequence-related template species.
  • the conversion of substrates to fragments can be effected by partial PCR amplification of substrates.
  • a mixture of fragments is spiked with one or more oligonucleotides.
  • the oligonucleotides can be designed to include precharacterized mutations of a wildtype sequence, or sites of natural variations between individuals or species.
  • the oligonucleotides also include sufficient sequence or structural homology flanking such mutations or variations to allow annealing with the wildtype fragments. Annealing temperatures can be adjusted depending on the length of homology.
  • recombination occurs in at least one cycle by template switching, such as when a DNA fragment derived from one template primes on the homologous position of a related but different template.
  • Template switching can be induced by addition of recA (see, Kiianitsa (1997) supra), rad51 (see, Namsaraev (1997) Mol. Cell. Biol. 17:5359-5368), rad55 (see, Clever (1997) EMBOJ. 16:2535-2544), rad57 (see, Sung (1997) Genes Dev. 11:1111-1121) or other polymerases (e.g., viral polymerases, reverse transcriptase) to the amplification mixture.
  • Template switching can also be increased by increasing the DNA template concentration.
  • Another embodiment utilizes at least one cycle of amplification, which can be conducted using a collection of overlapping single-stranded DNA fragments of related sequence, and different lengths. Fragments can be prepared using a single stranded DNA phage, such as Ml 3 (see, Wang (1997) Biochemistry 36:9486-9492). Each fragment can hybridize to and prime polynucleotide chain extension of a second fragment from the collection, thus forming sequence-recombined polynucleotides.
  • ssDNA fragments of variable length can be generated from a single primer by Pfu, Taq, Vent, Deep Vent, UlTma DNA polymerase or other DNA polymerases on a first DNA template (see, Cline (1996) Nucleic Acids Res.
  • shuffled nucleic acids obtained by use of the recursive recombination methods of the invention are put into a cell and/or organism for screening.
  • Shuffled lipid synthetic genes can be introduced into, for example, bacterial cells, yeast cells, or plant cells for initial screening. Bacillus species (such as B. subtilis and E.
  • coli are two examples of suitable bacterial cells into which one can insert and express shuffled genes.
  • the shuffled genes can be introduced into bacterial or yeast cells either by integration into the chromosomal DNA or as plasmids. Shuffled genes can also be introduced into plant cells for screening purposes.
  • a transgene of interest can be modified using the recursive sequence recombination methods of the invention in vitro and reinserted into the cell for in vivolin situ selection for the new or improved property.
  • DNA substrate molecules are introduced into cells, wherein the cellular machinery directs their recombination.
  • a library of mutants is constructed and screened or selected for mutants with improved phenotypes by any of the techniques described herein.
  • the DNA substrate molecules encoding the best candidates are recovered by any of the techniques described herein, then fragmented and used to transfect a plant host and screened or selected for improved function. If further improvement is desired, the DNA substrate molecules are recovered from the plant host cell, such as by PCR, and the process is repeated until a desired level of improvement is obtained.
  • the fragments are denatured and reannealed prior to transfection, coated with recombination stimulating proteins such as recA, or co-transfected with a selectable marker such as Neo to allow the positive selection for cells receiving recombined versions of the gene of interest.
  • recombination stimulating proteins such as recA
  • a selectable marker such as Neo
  • the efficiency of in vivo shuffling can be enhanced by increasing the copy number of a gene of interest in the host cells.
  • the majority of bacterial cells in stationary phase cultures grown in rich media contain two, four or eight genomes. In minimal medium the cells contain one or two genomes.
  • the number of genomes per bacterial cell thus depends on the growth rate of the cell as it enters stationary phase. This is because rapidly growing cells contain multiple replication forks, resulting in several genomes in the cells after termination.
  • the number of genomes is strain dependent, although all strains tested have more than one chromosome in stationary phase.
  • the number of genomes in stationary phase cells decreases with time. This appears to be due to fragmentation and degradation of entire chromosomes, similar to apoptosis in mammalian cells.
  • This fragmentation of genomes in cells containing multiple genome copies results in massive recombination and mutagenesis.
  • the presence of multiple genome copies in such cells results in a higher frequency of homologous recombination in these cells, both between copies of a gene in different genomes within the cell, and between a genome within the cell and a transfected fragment.
  • the increased frequency of recombination allows one to evolve a gene evolved more quickly to acquire optimized characteristics.
  • Modified cells surviving exposure to mutagen are enriched for cells with multiple genome copies.
  • selected cells can be individually analyzed for genome copy number (e.g., by quantitative hybridization with appropriate controls).
  • individual cells can be sorted using a cell sorter for those cells containing more DNA, e.g., using DNA specific fluorescent compounds or sorting for increased size using light dispersion.
  • phage libraries are made and recombined in mutator strains such as cells with mutant or imparied gene products of mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc.
  • the impairment is achieved by genetic mutation, allelic replacement, selective inhibition by an added reagent such as a small compound or an expressed antisense RNA, or other techniques.
  • High multiplicity of infection (MOI) libraries are used to infect the cells to increase recombination frequency.
  • the selection methods herein are utilized in a "whole genome shuffling" format.
  • An extensive guide to the many forms of whole genome shuffling is found in the pioneering application to the inventors and their co-workers, e.g., Del Cardayre et al. "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” WO 9831837 and "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION", by del Cardyre et al. filed July 15, 1999 (USSN 09/354,922).
  • whole genome shuffling makes no presuppositions at all regarding what nucleic acids may confer a desired property. Instead, entire genomes (e.g., from a genomic library, or isolated from an organism) are shuffled in cells and selection protocols applied to the cells.
  • the substrates for recombination can be, e.g., whole genomic libraries, fractions thereof or focused libraries containing variants of gene(s) known or suspected to confer tolerance to one of the above agents. Frequently, library fragments are obtained from a different species to the plant being evolved. Regardless of the precise shuffling methodology used, the selection methods described above for lipid biosynthetic selection, including selection for any of the desirable traits noted herein can be performed.
  • the DNA fragments are introduced into plant tissues, cultured plant cells or plant protoplasts by standard methods including electroporation (From et al., Proc. Natl Acad. Sci. USA 82, 5824 (1985), infection by viral vectors such as cauliflower mosaic virus (CaMN) (Hohn et al., Molecular Biology of Plant Tumors, (Academic Press, New York, 1982) pp.
  • electroporation from et al., Proc. Natl Acad. Sci. USA 82, 5824 (1985)
  • viral vectors such as cauliflower mosaic virus (CaMN) (Hohn et al., Molecular Biology of Plant Tumors, (Academic Press, New York, 1982) pp.
  • the T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome (Horsch et al., Science 233, 496-498 (1984); Fraley et al, Proc. Natl. Acad. Sci. USA 80, 4803 (1983)). Diversity can also be generated by genetic exchange between plant protoplasts. Procedures for formation and fusion of plant protoplasts are described by Takahashi et al., US 4,677,066; Akagi et al., US 5,360,725; Shimamoto et al., Us 5,250,433; Cheney et al., US 5,426,040.
  • the plant cells are assayed for lipid production (e.g., membrane lipid composition), and suitable plant cells are collected. Some or all of these plant cells can be subject to a further round of recombination and screening. Eventually, plant cells having the required degree of lipid expression are obtained. This is especially suitable, e.g., for plant oil accumulation in seeds.
  • lipid production e.g., membrane lipid composition
  • Plant regeneration from cultured protoplasts is described in Evans et al., "Protoplast Isolation and Culture," Handbook of Plant Cell Cultures 1, 124-176 (MacMillan Publishing Co., New York, 1983); Davey, “Recent Developments in the Culture and Regeneration of Plant Protoplasts,” Protoplasts, (1983) pp. 12-29, (Birkhauser, Basal 1983); Dale, “Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops,” Protoplasts (1983) pp. 31-41, (Birkhauser, Basel 1983); Binding, "Regeneration of Plants,” Plant Protoplasts, pp. 21-73, (CRC Press, Boca Raton, 1985) and other references available to persons of skill. Additional details regarding plant regeneration from cells are also found below.
  • one or more preliminary rounds of recombination and screening can be performed in bacterial cells according to the same general strategy as described for plant cells. More rapid evolution can be achieved in bacterial cells due to their greater growth rate and the greater efficiency with which DNA can be introduced into such cells.
  • a DNA fragment library is recovered from bacteria and transformed into the plants.
  • the library can either be a complete library or a focused library.
  • a focused library can be produced by amplification from primers specific for plant sequences, particularly plant sequences known or suspected to have a role in conferring a desirable lipid production or metabolic property.
  • Plant genome shuffling allows recursive cycles to be used for the introduction and recombination of genes or pathways that confer improved properties to desired plant species. Any plant species, including weeds and wild cultivars, showing a desired trait, such as high oil production, can be used as the source of DNA that is introduced into the crop or horticultural host plant species.
  • Genomic DNA prepared from the source plant is fragmented (e.g. by DNasel, restriction enzymes, or mechanically) and cloned into a vector suitable for making plant genomic libraries, such as pGA482 (An. G., 1995, Methods Mol. Biol. 44:47-58).
  • This vector contains the A. tumefaciens left and right borders needed for gene transfer to plant cells and antibiotic markers for selection in E. coli, Agrobacterium, and plant cells.
  • a multicloning site is provided for insertion of the genomic fragments.
  • a cos sequence is present for the efficient packaging of DNA into bacteriophage lambda heads for transfection of the primary library into E. coli.
  • the vector accepts DNA fragments of 25-40 kb.
  • the primary library can also be directly electroporated into an A. tumefaciens or A. rhizogenes strain that is used to infect and transform host plant cells (Main, GD et al., 1995, Methods Mol. Biol. 44:405-412).
  • DNA can be introduced by electroporation or P ⁇ G-mediated uptake into protoplasts of the recipient plant species (Bilang et al. (1994) Plant Mol. Biol Manual. Kluwer Academic Publishers, Al:l-16) or by particle bombardment of cells or tissues (Christou, ibid, A2:l-15). If necessary, antibiotic markers in the T-DNA region can be eliminated, as long as selection for the trait is possible, so that the final plant products contain no antibiotic genes.
  • Stably transformed whole cells acquiring the trait are selected on solid or liquid media. If the trait in question cannot be selected for directly, transformed cells can be selected with antibiotics and allowed to form callus or regenerated to whole plants and then screened for the desired property.
  • the second and further cycles consist of isolating genomic DNA from each transgenic line and introducing it into one or more of the other transgenic lines.
  • transformed cells are selected or screened, typically in an incremental fashion (increasing dosages, etc.).
  • plant regeneration can be eliminated until the last round.
  • Callus tissue generated from the protoplasts or transformed tissues can serve as a source of genomic DNA and new host cells.
  • fertile plants are regenerated and the progeny are selected for homozygosity of the inserted DNAs.
  • a new plant is created that carries multiple inserts which additively or synergistically combine to confer high levels of the desired trait.
  • the introduced DNA that confers the desired trait can be traced because it is flanked by known sequences in the vector. Either PCR or plasmid rescue is used to isolate the sequences and characterize them in more detail. Long PCR (Foord, OS and Rose, EA, 1995, PCR Primer: A Laboratory Manual. CSHL Press, pp 63-77) of the full 25-40 kb insert is achieved with the proper reagents and techniques using as primers the T-DNA border sequences. If the vector is modified to contain the E.
  • a rare cutting restriction enzyme such as Notl or Sfil, that cuts only at the ends of the inserted DNA is used to create fragments containing the source plant DNA that are then self-ligated and transformed into E. coli where they replicate as plasmids.
  • the total DNA or sub fragment of it that is responsible for the transferred trait can be subjected to in vitro evolution by DNA shuffling.
  • the shuffled library is then introduced into host plant cells and screened for improvement of the trait. In this way, single and multigene traits can be transferred from one species to another and optimized for higher expression or activity leading to whole organism improvement.
  • lipid synthetic or metabolic gene sequence strings (or sequence strings corresponding to any other factors which affect lipid biosynthesis or metabolism) are recombined in a computer system and desirable products are made, e.g., by reassembly PCR of synthetic oligonucleotides.
  • oligonucleotide mediated shuffling in which oligonucleotides co ⁇ esponding to a family of related homologous nucleic acids (e.g., as applied to the present invention, interspecific or allelic variants of a lipid metabolic or synthetic nucleic acid) are recombined to produce selectable nucleic acids.
  • nucleic acids shuffled for lipid synthetic or metabolic activity by any of the techniques noted above are used to make transgenic plants, thereby providing transgenic plants.
  • Methods of transducing plant cells with nucleic acids are generally available.
  • useful general references for plant cell cloning, culture and regeneration include Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY (Payne); and Gamborg and Phillips (eds) (1995) Plant Cell. Tissue and Organ Culture: Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York) (Gamborg).
  • recombinant DNA vectors which contain isolated selected shuffled sequences and are suitable for transformation of plant cells are prepared.
  • a DNA sequence coding for the desired nucleic acid for example a cDNA or a genomic sequence encoding a full length protein, is conveniently used to construct a recombinant expression cassette which can be introduced into the desired plant.
  • An expression cassette will typically comprise a selected shuffled nucleic acid sequence operably linked to a promoter sequence and other transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues (e.g., entire plant, leaves, seeds) of the transformed plant.
  • a strongly or weakly constitutive plant promoter can be employed which will direct expression of a shuffled enzyme gene as set forth herein in all tissues of a plant.
  • Such promoters are active under most environmental conditions and states of development or cell differentiation.
  • constitutive promoters include the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill. Where overexpression of a shuffled gene is detrimental to the plant, one of skill, upon review of this disclosure, will recognize that weak constitutive promoters can be used for low-levels of expression.
  • a strong promoter e.g., a t-RNA or other pol III promoter, or a strong pol II promoter, such as the cauliflower mosaic virus promoter
  • a plant promoter may be under environmental control. Such promoters are refened to here as "inducible" promoters. Examples of environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light.
  • the promoters used in the constructs of the invention will be "tissue-specific" and are under developmental control such that the desired gene is expressed only in certain tissues, such as leaves and seeds.
  • the endogenous promoters (or shuffled variants thereof) from lipid synthetic genes are particularly useful for directing expression of these genes to the transfected plant.
  • Tissue-specific promoters can also be used to direct expression of heterologous structural genes, including shuffled nucleic acids as described herein.
  • Examples include genes encoding proteins which ordinarily provide the plant with lipid synthetic activity and genes that encode useful phenotypic characteristics, e.g., which influence heterosis.
  • promoters used in the expression cassette in plants depends on the intended application. Any of a number of promoters which direct transcription in plant cells can be suitable.
  • the promoter can be either constitutive or inducible.
  • promoters of bacterial origin which operate in plants include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids. See, Herrara-Estrella et al. (1983), Nature, 303:209-213.
  • Viral promoters include the 35S and 19S RNA promoters of cauliflower mosaic virus. See, Odell et al. (1985) Nature, 313:810-812.
  • Other plant promoters include the ribulose-l,3-bisphosphate carboxylase small subunit promoter and the phaseolin promoter.
  • the promoter sequence from the E8 gene and other genes may also be used. The isolation and sequence of the E8 promoter is described in detail in Deikman and Fischer, (1988) EMBOJ. 7:3315- 3327.
  • promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site.
  • TATAAT TATA box consensus sequence
  • promoter element with a series of adenines surrounding the trinucleotide G (or T) N G. Messing et al, GENETIC ENGINEERING IN PLANTS, Kosage, et al (eds.), pp. 221-227 (1983).
  • sequences other than the promoter and the shuffled gene are also preferably used. If normal polypeptide expression is desired, a polyadenylation region at the 3 '-end of the shuffled coding region should be included.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the vector comprising the shuffled sequence will typically comprise a marker gene which confers a selectable phenotype on plant cells.
  • the marker may encode biocide tolerance, particularly antibiotic tolerance, such as tolerance to kanamycin, G418, bleomycin, hygromycin, or herbicide tolerance, such as tolerance to chlorosluforon, or phosphinothricin (the active ingredient in the herbicides bialaphos and Basta).
  • antibiotic tolerance such as tolerance to kanamycin, G418, bleomycin, hygromycin
  • herbicide tolerance such as tolerance to chlorosluforon, or phosphinothricin (the active ingredient in the herbicides bialaphos and Basta).
  • DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Payne, Gamborg, Atlas, Sigma-LSRCCC and Sigma-PCCS, all supra, as well as, e.g., Weising, et al, Ann. Rev. Genet. 22:421- 477 (1988).
  • DNAs may be introduced directly into the genomic DNA of a plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
  • the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.
  • Microinjection techniques are known in the art and well described in the scientific and patent literature.
  • Agrobacterium-mQdiated transformation is useful primarily in dicots, however, certain monocots can be transformed by Agrobacterium.
  • Agrobacterium transformation of rice is described by Hiei, et al, Plant J. 6:271-282 (1994); U.S. Patent No. 5,187, 073; U.S. Patent 5,591,616; Li, et al, Science in China 34:54 (1991); and Raineri, et al, Bio/Technology 8:33 (1990).
  • T-inducing (Ti) plasmid of A. tumefaciens the ability of the tumor-inducing (Ti) plasmid of A. tumefaciens to integrate into a plant cell genome is used advantageously to co-transfer a nucleic acid of interest into a recombinant plant cell of this invention.
  • an expression vector is produced wherein the nucleic acid of interest is ligated into an autonomously replicating plasmid which also contains T-DNA sequences.
  • T-DNA sequences typically flank the expression cassette nucleic acid of interest and comprise the integration sequences of the plasmid.
  • T-DNA also typically comprises a marker sequence, e.g., antibiotic tolerance genes.
  • the plasmid with the T-DNA and the expression cassette are then transfected into Agribacterium tumefaciens.
  • the A. tumefaciens bacterium also comprises the necessary vir regions on a native Ti plasmid.
  • both the T-DNA sequences as well as the vtr sequences are on the same plasmid.
  • explants are made of the tissues of desired plants, e.g., leaves.
  • the explants are then incubated in a solution of A. tumefaciens at about 0.8 x 10 9 to about 1.0 x 10 9 cells/mL for a suitable time, typically several seconds.
  • the explants are then grown for approximately 2 to 3 days on suitable medium.
  • Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences.
  • Plant regeneration from cultured protoplasts is described in Evans, et al. , PROTOPLASTS ISOLATION AND CULTURE, HANDBOOK OF PLANT CELL CULTURE, pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, REGENERATION OF PLANTS, PLANT PROTOPLASTS, pp. 21-73, CRC Press, Boca Raton, 1985.
  • Regeneration can also be obtained from plant callus, explants, organs, or parts thereof.
  • the transformants will develop roots in 1 to about 2 weeks and form plantlets. After the plantlets are from about 3 to about 5 cm in height, they should be placed in sterile soil in fiber pots. Those of skill in the art will realize that different acclimation procedures should be used to obtain transformed plants of different species. In a prefe ⁇ ed embodiment, cuttings, as well as somatic embryos of transformed plants, after developing a root and shoot, are transfe ⁇ ed to medium for establishment of plantlets. For a description of selection and regeneration of transformed plants, see, Dodds & Roberts, EXPERIMENTS IN PLANT TISSUE CULTURE, 3RD ED.,Cambridge University Press (1995).
  • the transgenic plants of this invention can be characterized either genotypically or phenotypically to determine the presence of the shuffled gene.
  • Genotypic analysis is the determination of the presence or absence of particular genetic material.
  • Phenotypic analysis is the determination of the presence or absence of a phenotypic trait.
  • a phenotypic trait is a physical characteristic of a plant determined by the genetic material of the plant in concert with environmental factors.
  • the presence of shuffled DNA sequences can be detected as described in the preceding sections on identification of an optimized shuffled nucleic acid, e.g., by PCR amplification of the genomic DNA of a transgenic plant and hybridization of the genomic DNA with specific labeled probes.
  • the survival of plants on exposure to a selected stress where lipid production or type helps cope with the stress can also be used to monitor incorporation of a lipid synthetic shuffled gene into the plant.
  • Plants which are transduced with shuffled nucleic acids as taught herein to achieve desirable lipid production can acquire lipid production by the techniques herein.
  • Some suitable plants for modified lipid biosynthesis include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Anti ⁇ hinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Ze
  • grass family crops such as maize, wheat, barley, oats, alfalfa, rice, millet, rye and the like as well as oil producing crops such as rapeseed, sunflower (and other composite family members).
  • oil producing crops such as rapeseed, sunflower (and other composite family members).
  • Industrially important legume crops such as soybeans are also especially suitable.
  • EXAMPLE MODIFICATION OF THE ACTIVITY AND SUBSTRATE SPECIFICITY OF A FATTY ACID CHAIN-LENGTH DETERMINING ENZYME THROUGH DNA SHUFFLING
  • An acyl-ACP thioesterase hydrolyzes the thioester bond linking the acyl group and ACP, thus releasing a free fatty acid.
  • the lipid composition of the canola oil was altered from mainly 18:1 to 12:0 (laureate, an oil mostly found in tropical plants such as coconuts).
  • the primary substrate of the enzyme is 12:0-ACP; however, it also hydrolyzes 14:0-ACP at a much lower rate (-10%).
  • the hydrolysis of 12:0-ACP by the enzyme was modified by replacing several amino acids, creating a 14:0-ACP thioesterase.
  • the channeling of fatty acids into a fatty aldehyde substrate for the bioluminescence reaction is catalyzed by a multienzyme complex which channels fatty acids through LuxD (a 14:0-ACP thioesterase), LuxE (a synthetase) and LuxC (a reductase).
  • the Lux operon has been isolated and can be transformed into other hosts to allow cells to emit light through bioluminescence. Improving the efficiency of the Lux system is performed by shuffling the whole operon. The improved mutants are identified by eye or by established sensitive instrumental procedures.
  • Fig. 4 provides a schematic of the Vibro Lux system.
  • the following table provides the response in photon yield of Ml 7 cells to different fatty acids for the Vibro Lux system.
  • shuffled genes are expressed in oil bearing tissues using promoters that give preferential expression of the genes in those tissues (e.g. using a promoter that gives high expression in maturing
  • the enzymes to be shuffled for this example can include, for example, acyl-ACP carboxylase, Keto acyl-ACP synthases, keto acyl-ACP reductases, hydroxyacly-ACP dehydrases, and enoyl-ACP reductases.
  • the endoplasmic reticulum localized enzymes of the Kennedy pathway synthesize triglycerides from glycerol-3-phosphate and acyl-CoAs.
  • shuffling the genes encoding enzymes in this pathway is a method to increase oil yield.
  • the production of specific or unusual fatty acids can be limited by the ability of the Kennedy pathway
  • Kennedy pathway enzymes to utilize the specific fatty acids.
  • shuffling and/or other diversity generation methods as described herein, can be used to increase the pathway flux and increase the yield of the specific fatty acids.
  • the shuffled genes are expressed in oil bearing tissues using promoters that give preferential expression of the genes in those tissues (e.g. using a promoter that gives high expression in maturing embryos to drive expression of genes in oilseeds that produce oil in their embryos.
  • the enzymes to be shuffled for this example include, e.g., glycerol-3 -phosphate acyclytransferase, lysophosphatidyl choline acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase and the like.
  • Acyl-ACP desaturases can introduce double bonds at different positions on 18 carbon fatty acids such as stearic acid, and can also introduce double bonds on other chain length fatty acids such as palmitic acid when the substrate fatty acids are esterified to ACP.
  • acyl-ACP desaturases available as parents.
  • Acyl-ACP desaturases that introduce double bonds at different positions on aycl chains or that use fatty acids of different chain lengths as substrates can be evolved using the techniques herein to provide evolved desaturases that can produce novel unsaturated fatty acids.
  • a family of fatty acid desaturases related to the desaturases that form linoleic and linolenic acids in plants can form novel fatty acids such as hydroxy fatty acids, acetylenic fatty acids, epoxy fatty acids, and fatty acids with conjugated cis and trans double bonds.
  • Suitable parents can be identified using Arabidopsis FAD2 as a query to identify related sequences from GenbankTM (e.g., using BLAST or other suitable search/alignment algorithms).
  • GenbankTM e.g., using BLAST or other suitable search/alignment algorithms.
  • the family can be evolved using the methods described herein to develop enzymes capable of forming novel fatty acids with the above structures at different places on fatty acid molecules, and on fatty acids of different chain lengths. Providing this class of enzymes can also result in the production of novel functional groups not known to exist on fatty acids in nature.
  • FA fatty acid
  • Most of the enzymes involved in fatty acid (FA) synthesis are imported into chloroplasts or ER.
  • Most major systems that transport proteins across a membrane share the following features: an N-terminal transient signal sequence on the transported protein, a targeting system on the cis side of the membrane, a hetero-oligomeric transmembrane channel that is gated both across and within the plane of the membrane, a peripherally attached protein translocation motor that is powered by the hydrolysis of ATP, and a protein folding system on the trans side of the membrane.
  • Genetic engineering of FA synthesis commonly utilizes expression of genes from different species or even different kingdoms. In many cases, transit peptides of these enzymes do not efficiently guide the recombinant proteins into plastids of the new hosts, e.g.
  • a transit peptide from Soybean may not work efficiently in Canola.
  • some desired target enzymes e.g. bacterial or some fungal ones
  • Some transit peptides have been used successfully for recombinant expression, however, their effectiveness can not be generally applied.
  • large amounts of unprocessed (i.e., due to failure to import) proteins are detected, and the results have been speculated as a cause for poor phenotypes. It is desirable for transit peptides to be engineered to be highly specific and efficient.
  • Chloroplastic transit peptide sequences which can vary in length from 20 to 120 amino acids, contain no obvious blocks of conserved amino acid sequence or secondary structure.
  • the N-proximal portion lacks both positively charged residues as well as glycine and proline.
  • the central domain lacks acidic residues and is rich in hydroxylated amino acids such as serine and threonine.
  • the C-terminal domain has a loosely conserved consensus sequence Ile/Val-x-Ala/Cys-Ala close to the cleavage site. Plastid, but not mitochondrial precursor proteins, are phosphorylated at the serine or threonine within the transit peptide by a cytosolic protein kinase.
  • a group of transit peptides similar to that of the small subunit of ribulose-biphosphate carboxylase is shuffled and cloned into the N- terminal domain of a reporter protein.
  • the chimeric gene is cloned into an expression vector for expression in either E. coli or cynobacteria.
  • the cynobacteria expression library is transformed into Synechocystis. Import is monitored by the expression of the reporter.
  • the high performers are subjected to a second round import study in Synechosystis or by in vitro import experiments using isolated chloroplasts with E.coli produced chimeras.
  • Cells of Pseudomonas putida change the ratio of cis and trans monounsaturated fatty acids in response to growth temperature or membrane active compounds such as phenol or alcohol. These detoxification (or anti-stress) responses are attributed to a cis-trans isomerase.
  • Different strains of P. putida have been isolated from various highly stressful environments. The specificity of the enzyme is relatively na ⁇ ow.
  • Strain E-3 converses double bonds at positions 9, 10 or 11 but not 6 or 7 of cis-monounsaturated fatty acids with chain lengths of 14, 15, 16, and 17. However, 18:1 with double bonds at positions 9 or 11 are not substrates.
  • the isomerase from strain P8 catalyzes the conversion at position 9 of 18:1. Furthermore, some trans-fatty acids are of commercial interest. Therefore a library of such cis-trans isomerase is made to allow selection activities in a number of applications.
  • the resulting library is used for expression in E.coli or Pseudomonas.
  • a one-hybrid assay is based on an interaction between a target-specific
  • DNA-binding domain and a target-independent activation domain enables the rapid identification of novel DNA-binding proteins and access of their genes.
  • the two-hybrid system enables the detection of protein-protein interaction and subsequent isolation of their genes.
  • acyl-ACP In plant plastids, where lipid synthesis occurs, high levels of acyl-ACP are found. Certain genes encoding enzymes utilizing other acyl- molecules (e.g. acyl- CoA), therefore, are incomparable for catalysis in plastids. For example, it is desirable for PHB polymerase to utilize PHB-ACP other than its natural substrate PHB-CoA to achieve high level production of the biopolymer in plants.
  • PHB polymerase it is desirable for PHB polymerase to utilize PHB-ACP other than its natural substrate PHB-CoA to achieve high level production of the biopolymer in plants.
  • the substrate specificities of enzymes are modified from utilizing CoA or other linker molecules to acyl-carrier protein (ACP).
  • ACP acyl-carrier protein
  • These enzymes include, but not are limited to, desaturase, isomerase, thioesterase, and PHA polymerase. It is also desirable to modify regulatory elements, transcription factors and signal transduction elements for improved specificity and cross-species recognition. Examples of one and two hybrid shuffling protocols relevant to the present invention are found in Figures 3A and B. As shown in Figure 3A, a two-hybrid system can be used for screening.
  • KAS proteins which are known to form heterodimers, resulting in varied substrate specificities can be evolved.
  • PHA polymerase can be modified to use PHA- ACP instead of PHA-CoA.
  • Other enzymes PPS, Desaturases, TE, ACCase, etc.
  • PES Desaturases, TE, ACCase, etc.
  • genes for a target protein X
  • a random collection of cDNA Y
  • Both plasmids are co-transformed into yeast.
  • Hybrid proteins are expressed in the same cell, ⁇ -galactosidase activity is screened for to confirm interacting proteins.
  • a one-hybrid system is used to acquire modified genes of interest.
  • shuffled transcription factors are screened on a known target element ("E") connected to a known reporter system.
  • E target element
  • the Napin promoter which is a strong seed-specific promoter can be used (for some targets, the Napin promoter is activated too late or too early, depending on the target).
  • a transcription factor controlling an earlier promoter can be modified to bind the napin element.
  • the napin transcription factor can be modified to bind a weaker promoter.
  • Genomes are regulated at the level of transcription, primarily through the action of transcription factors that bind DNA in a sequence-specific fashion.
  • Zinc finger proteins a DNA-binding domain that localizes the protein to a specific site within the genome and through accessory effector domains that act to activate or repress transcription at or near that site.
  • Zinc finger proteins a DNA-binding domain that localizes the protein to a specific site within the genome and through accessory effector domains that act to activate or repress transcription at or near that site.
  • Cys2-His2 class of nucleic acid-binding proteins have unique structural features that allow recognition of specific DNA sequences, and can be engineered as fusion proteins with either an activator domain or a repressor domain (Beerli RR et al. 1998. Proc. Natul. Acad. Sci. USA. 95:14628).
  • the artificial transcriptional regulators can repress or activate gene expression in a specific manner. Results also indicated that gene activation or repression was achieved by targeting within the gene transcript, suggesting that information obtained from expressed sequence tags (ESTs) is sufficient for the construction of gene switches. These switches are useful, e.g., for controlling lipid synthetic enzymes.
  • Polydactyl zinc finger proteins are constructed from modular building blocks (Beerli et al. 1998, id.). These building blocks are substrates for DNA shuffling.
  • Libraries are made by shuffling individual blocks or shuffling blocks in combination, generating greater diversity of zinc finger proteins than simple genomic PCR based assembly methods.
  • the shuffled zinc finger DNAs are cloned into vectors with or without sequences encoding either activators or repressors. Identification of zinc fingers recognizing a specific DNA sequence.
  • a specific DNA recognition sequence can be, e.g., in the 5' untranslated region or 5' translated region of a known gene such as a lipid synthetic enzyme gene.
  • This specific sequence is cloned as a fusion with a reporter gene under the control of an appropriate promoter.
  • the hosts containing this vector are transformed with a library of shuffled zinc finger DNAs. The expression of the reporter gene is monitored. A particular zinc finger protein is identified by altered reporter gene expression due to the binding of the specific sequence by zinc finger.
  • a specific DNA sequence can be cloned without fusing with a reporter gene, the presence or absence of the transcripts of this DNA may be detected by hybridization.
  • Identification of zinc fingers associated with a particular phenotype Library of zinc fingers are transformed into prokaryotes (E . coli, etc.), or eukaryotes (fungi, plants, animals, etc.) and selected for desirable phenotypes. The production of the zinc fingers is controlled by appropriate promoters of desired timing and specificity. The zinc fingers may be fused with either an activator or a repressor.
  • ⁇ LISA assays with immobilized biotinylated hairpin oligonucleotides containing specific sequences.
  • a high-throughput system is used for this process.
  • Identification of zinc fingers can also be performed using a one-hybrid system as above.
  • This example provides methods to evolve methyltransferases, particularly cyclopropane fatty acid synthase related enzymes, to form branched chain fatty acids.
  • Branched chain fatty acids have the physical characteristics of unsaturated fatty acids, yet they have the oxidative stability of saturated fatty acids. Thus, they have desirable properties as industrial oils, and they may have some food oil applications.
  • shuffled genes e.g., derived from bacterial cyclopropane fatty acid synthases, can be used to transform oilseed crops to produce oils with branched chain fatty acids.
  • novel enzymes that can form cyclopropyl fatty acids, methoxy fatty acids or keto fatty acids can also be made by this approach.
  • a group of related genes from Mycobacterium also form methoxy fatty acids, methylene branched fatty acids, methyl branched fatty acids, and keto fatty acids.
  • Some forms of the enzymes act on fatty acids esterified to ACP or to fatty acids esterified to glycerol in phospholipids. These enzymes all act by the addition of methyl groups to double bonds of unsaturated fatty acids.
  • the parent sequences are therefore, easy to obtain and use in the shuffling procedures of the invention.
  • Parent sequences are identified, e.g., by using the E. coli CFA synthase as a query against Genbank or other protein or nucleotide databases.
  • a number of CFA synthase related parents are isolated, and used for DNA shuffling or the other diversity generation procedures noted herein.
  • the shuffled library is cloned into an ⁇ . coli expression vector.
  • the library is transformed, e.g., into an E. coli mutant deficient in the synthesis of unsaturated fatty acids (fabB).
  • This strain requires supplementation of unsaturated fatty acids in the growth medium, and thus can be fed oleic acid.
  • Oleic acid containing phospholipids would are suitable substrates for evolved methyltransferases, and provide a suitable screening system to predict the phenotype one observes in transgenic plant oils.
  • the shuffled library is screened, e.g., using gas chromatography to detect branched chain fatty acids and other unusual fatty acids such as keto or methoxy fatty acids.
  • Enzymes with desired methyltransferase activities identified through screening in E. coli are then tested for their ability to modify plant oils by expression in transgenic Arabidopsis plants.
  • Parents useful as substrates in shuffling or other diversity generation reactions are selected, e.g., from the following list of CFA synthase related genes (accession numbers are indicated): splP30010ICFA ECOLI CYCLOPROPANE-FATTY-ACYL-PHOSPHOLIPID SY... 804 0.0 gb
  • Kits will optionally additionally comprise instructions for performing methods or assays, packaging materials, one or more containers which contain assay, device or system components, or the like.
  • kits embodying the methods and apparatus herein optionally comprise one or more of the following: (1) a shuffled component as described herein; (2) instructions for practicing the methods described herein, and/or for operating the selection procedure herein; (3) one or more lipid assay component; (4) a container for holding lipids, nucleic acids, plants, cells, or the like and, (5) packaging materials.
  • the present invention provides for the use of any component 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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Abstract

L'invention concerne des procédés de modulation de la production de lipides dans des cellules et dans des organismes entiers. Des gènes uniques, des opérons, des cycles biosynthétiques de lipides et des génomes entiers peuvent être recombinés afin de produire des cellules et des organismes qui présentent une activité synthétique ou métabolique de lipides voulue. L'invention concerne également des banques d'acides nucléiques synthétiques de lipides recombinés et d'organismes recombinés.
PCT/US2000/009285 1999-04-10 2000-04-06 Production de lipides modifies WO2000061740A1 (fr)

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