Patent Application
20120079627
The present specification makes reference to a Sequence Listing (submitted electronically as a .txt file named “SequenceListing.txt” on Jun. 1, 2010). The .txt file was generated on Jun. 1, 2010 and is 671 kb in size. The entire contents of the Sequence Listing are herein incorporated by reference.
Plant gene expression is highly regulated in a tissue-specific and developmental stage-specific manner. Plant gene expression is also regulated in response to many external factors, including biotic and abiotic stress. Nucleotide sequences upstream of gene coding sequences, commonly known as promoters, precisely regulate when and where any particular gene is expressed. Promoters also control the extent of foreign gene expression in transgenic plants and hence are crucial in determining the levels to which a desirable gene can be expressed.
Over the last three decades, plant biologists have isolated and characterized several plant promoters that can drive heterologous transgene expression. These well-characterized promoters include CaMV 35S promoter (Odell et al. (1985) Nature. 313:810-812), Opine promoters (U.S. Pat. No. 5,955,646), the rice actin promoter (McElroy et al. (1991) Mol Gen Genet. 231:150-160), the maize ubiquitin promoter (Christensen et al. (1992) Plant Mol Biol. 18:675-89.), the maize ADH1 promoter (U.S. Pat. No. 5,001,060) and the Rubisco promoter (Outchkourov et al. (2003) Planta 216:1003-1012).
Many of the dicot promoters do not perform satisfactorily in monocots such as maize and other cereal crops or grasses. In general, dicot promoters do not require intron sequences downstream of the transcription initiation site to enhance gene expression in transgenic dicot plants, whereas the first intron downstream of monocot promoters often enhances gene expression in transgenic monocot plants (McElroy et al. (1991) Mol Gen Genet. 231:150-160 and Christensen et al. (1992) Plant Mol Biol. 18:675-89).
Functional assays have demonstrated that differences in required promoter elements of dicot and monocot promoters may be one of the reasons why dicot promoters do not necessarily work well in monocots and vice versa.
The present invention encompasses the recognition that while transgenic dicot plants containing multiple transgenes (stacked traits) are desirable, the ability to create such plants is limited by the availability of suitable promoters for each transgene. The present invention further encompasses the recognition that a collection of novel dicot promoters, with divergent DNA sequences and an optimal range of functional characteristics, would, among other things, facilitate creating of transgenic dicot plants.
In various aspects, provided are a collection of novel dicot gene regulatory elements (including promoters) from the poplar genome, as well as nucleic acids and vectors (including gene expression vectors) comprising such novel gene regulatory elements. In one aspect, transgenic plants expressing a heterologous gene under the control of novel dicot gene regulatory elements are provided. Novel gene regulatory elements of the invention may in some be embodiments be used in other plants, including other dicots, as well as monocots and multicotyledonous plants.
Throughout the specification, several terms are employed that are defined in the following paragraphs.
As used herein, the terms “about” and “approximately”, in reference to a number, is used herein to include numbers that fall within a range of 20%, 10%, 5%, or 1% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
As used herein, the phrase “binary vector” refers to cloning vectors that are capable of replicating in both E. coli and Agrobacterium tumefaciens. In a binary vector system, two different plasmids are employed for generating transgenic plants. In many embodiments, the first plasmid is a small vector known as disarmed Ti plasmid has an origin of replication (ori) that permits the maintenance of the plasmid in a wide range of bacteria including E. coli and Agrobacterium. In many embodiments, the small vector contains foreign DNA in place of T-DNA, the left and right T-DNA borders (or at least the right T-border), markers for selection and maintenance in both E. coli and A. tumefaciens, and a selectable marker for plants. In many embodiments, the second plasmid is known as helper Ti plasmid, harbored in A. tumefaciens, which lacks the entire T-DNA region but contains an intact vir region essential for transfer of the T-DNA from Agrobacterium to plant cells.
As used herein, the phrase “cell wall-modifying enzyme polypeptide” refers to a polypeptide that modifies at least one component (e.g., xylans, xylan side chains, glucuronoarabinoxylans, xyloglucans, mixed-linkage glucans, pectins, pectates, rhamnogalacturonans, rhamnogalacturonan side chains, lignin, cellulose, mannans, galactans, arabinans, oligosaccharides derived from cell wall polysaccharides, and combinations thereof) or interaction (e.g., covalent linkage, ionic bond interaction, hydrogen bond interaction, and combinations thereof) in plant cell wall. In some embodiments, cell wall-modifying enzyme polypeptides have at least 50%, 60%, 70%, 80% or more overall sequence identity with a polypeptide whose amino acid sequence is set forth in Table 1 of co-pending U.S. patent application Ser. No. 12/476,247 (filed on Jun. 1, 2009), the contents of which are herein incorporated by reference in their entirety. Alternatively or additionally, in some embodiments, cell wall-modifying enzyme polypeptide shows at least 90%, 95%, 96%, 97%, 98%, 99%, or greater identity with at least one sequence element found in a polypeptide whose amino acid sequence is set forth in Table 1 of co-pending U.S. patent application Ser. No. 12/476,247, which sequence element is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids long. In some embodiments, a provided cell wall-modifying enzyme polypeptide disrupts a linkage selected from the group consisting of hemicellulose-cellulose-lignin, hemicellulose-cellulose-pectin, hemicellulosediferululate-hemicellulose, hemicellulose-ferulate-lignin, mixed beta-D-glucan-cellulose, mixed-beta-D-glucan-hemicellulose, pectin-ferulate-lignin linkages, and combinations thereof.
As used herein, the term “construct”, when used in reference to a gene and/or nucleic acid, refers to a functional unit that allows expression of a gene of interest. Nucleic acid constructs typically comprise, in addition to the gene of interest (i.e., the heterologous gene whose expression is desired), a gene regulatory element capable of driving expression of the gene of interest (such as a promoter) and a terminator (also known as a stop signal), both of which are operably linked to the gene of interest. In some embodiments, constructs comprise additional sequences, e.g. marker genes that are also accompanied by a gene regulatory element (such as a promoter) and a terminator. In many embodiments, the sequences for each of the elements in the construct do not exist in this combination and arrangement in nature and/or are arranged and/or combined by the hand of man.
As used herein, the phrase “externally applied”, when used to describe enzyme polypeptides used in the processing of biomass, refers to enzyme polypeptides that are not produced by the organism whose biomass is being processed. “Externally applied” enzyme polypeptides as used herein does not include enzyme polypeptides that are expressed (whether endogenously or transgenically) by the organism (e.g., plant) from which the biomass is obtained.
As used herein, the term “extract”, when used as noun, refers to a preparation from a biological material (such as lignocellulosic biomass) in which a substantial portion of proteins are in solution. In some embodiments of the invention, the extract is a crude extract, e.g., an extract that is prepared by disrupting cells such that proteins are solubilized and optionally removing debris, but not performing further purification steps. In some embodiments of the invention, the extract is further purified in that certain substances, molecules, or combinations thereof are removed.
As used herein, the term “gene” refers to a discrete nucleic acid sequence responsible for a discrete cellular product and/or performing one or more intracellular or extracellular functions. More specifically, the term “gene” refers to a nucleic acid that includes a portion encoding a protein and optionally encompasses regulatory sequences, such as promoters, enhancers, terminators, and the like, which are involved in the regulation of expression of the protein encoded by the gene of interest. The gene and regulatory sequences may be derived from the same natural source, or may be heterologous to one another. The definition can also include nucleic acids that do not encode proteins but rather provide templates for transcription of functional RNA molecules such as tRNAs, rRNAs, etc. Alternatively, a gene may define a genomic location for a particular event/function, such as the binding of proteins and/or nucleic acids.
As used herein, the term “gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, proteins post-translationally modified, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP ribosylation, myristilation, and glycosylation.
The terms “genetically modified” and “transgenic” are used herein interchangeably. A transgenic or genetically modified organism is one that has a genetic background which is at least partially due to manipulation by the hand of man through the use of genetic engineering. For example, the term “transgenic cell”, as used herein, refers to a cell whose DNA contains an exogenous nucleic acid not originally present in the non-transgenic cell. A transgenic cell may be derived or regenerated from a transformed cell or derived from a transgenic cell. Exemplary transgenic cells in the context of the present invention include plant calli derived from a stably transformed plant cell and particular cells (such as leaf, root, stem, or reproductive cells) obtained from a transgenic plant. A “transgenic plant” is any plant in which one or more of the cells of the plant contain heterologous nucleic acid sequences introduced by way of human intervention. Transgenic plants typically express DNA sequences, which confer the plants with characters different from that of native, non-transgenic plants of the same strain. The progeny from such a plant or from crosses involving such a plant in the form of plants, seeds, tissue cultures and isolated tissue and cells, which carry at least part of the modification originally introduced by genetic engineering, are comprised by the definition.
As used herein, the term “genetic probe” refers to a nucleic acid molecule of known sequence, which has its origin in a defined region of the genome and can be a short DNA sequence (or oligonucleotide), a PCR product, or mRNA isolate. Genetic probes are gene-specific DNA sequences to which nucleic acids from a sample (e.g., RNA from a plant extract) are hybridized. Genetic probes specifically bind (or specifically hybridize) to nucleic acid of complementary or substantially complementary sequence through one or more types of chemical bonds, usually through hydrogen bond formation.
As used herein, the term “gene regulatory element” means an element, typically within a nucleic acid, that has the ability to regulate genes, whether it is a by promoting, enhancing, or attenuating expression. In some embodiments, the gene regulatory element is a promoter. In some embodiments, the gene regulatory element is an enhancer. In some embodiments, gene regulatory elements are located at or near the 5′ end of the first exon of a gene. In some embodiment, gene regulatory elements are located within the region of a gene involved in transcriptional and translational initiation.
As used herein the term “heterologous”, when used in reference to genes, refers to genes that are not normally associated with other genetic elements with which they are nevertheless associated (e.g., in a nucleic acid construct) in such an arrangement in nature and/or refers to genes that are associated with such other elements by the hand of man. “Heterologous gene products” refers to products of heterologous genes.
As used herein, the term “lignocellulolytic enzyme polypeptide” refers to a polypeptide that disrupts or degrades lignocellulose, which comprises cellulose, hemicellulose, and lignin. The term “lignocelluloytic enzyme polypeptide” encompasses, but is not limited to cellobiohydrolases, endoglucanases, β-D-glucosidases, xylanases, arabinofuranosidases, acetyl xylan esterases, glucuronidases, mannanases, galactanases, arabinases, lignin peroxidases, manganese-dependent peroxidases, hybrid peroxidases, laccases, ferulic acid esterases and related polypeptides. In some embodiments, disruption or degradation of lignocellulose by a lignocellulolytic enzyme polypeptide leads to the formation of substances including monosaccharides, disaccharides, polysaccharides, and phenols. In some embodiments, a lignocellulolytic enzyme polypeptide shares at least 50%, 60%, 70%, 80% or more overall sequence identity with a polypeptide whose amino acid sequence is set forth in Table 1. Alternatively or additionally, in some embodiments, a lignocellulolytic enzyme polypeptide shows at least 90%, 95%, 96%, 97%, 98%, 99%, or greater identity with at least one sequence element found in a polypeptide whose amino acid sequence is set forth in Table 1, which sequence element is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids long. It will be appreciated that the present invention describes use of lignocellulolytic enzyme polypeptides generally, but also of particular lignocellulolytic enzyme polypeptides (e.g., Acidothermus cellulolyticus E1 endo-1,4-β-glucanase polypeptide, Acidothermus cellulolyticus xylE polypeptide, Acidothermus cellulolyticus gux1 polypeptide, Acidothermus cellulolyticus aviIII polypeptide, and Talaromyces emersonii cbhE polypeptide).
As used herein, the term “mixed linkage glucans” refer to non-cellulosic glucans present in plants and often enriched in seed bran. β-D-glucan residues of mixed-linkage glucans are unbranched but contain both (1→3) and (1→4)-linkages. In some embodiments, enzymes that modify mixed-linkage glucans include laminarinase (E.C. 3.2.1.39), licheninase (E.C. 3.2.1.73/74). In some embodiments, some cellulases can hydrolyze certain (1→4)-linkages.
As used herein, the term “nucleic acid construct” refers to a polynucleotide or oligonucleotide comprising nucleic acid sequences not normally associated in nature. A nucleic acid construct of the present invention is prepared, isolated, or manipulated by the hand of man. The terms “nucleic acid”, “polynucleotide” and “oligonucleotide” are used herein interchangeably and refer to a deoxyribonucleotide (DNA) or ribonucleotide (RNA) polymer either in single- or double-stranded form. For the purposes of the present invention, these terms are not to be construed as limited with respect to the length of the polymer and should also be understood to encompass analogs of DNA or RNA polymers made from analogs of natural nucleotides and/or from nucleotides that are modified in the base, sugar and/or phosphate moieties.
As used herein, the term “operably linked” refers to a relationship between two nucleic acid sequences wherein the expression of one of the nucleic acid sequences is controlled by, regulated by or modulated by the other nucleic acid sequence. In some embodiments, a nucleic acid sequence that is operably linked to a second nucleic acid sequence is covalently linked, either directly or indirectly, to such second sequence, although any effective three-dimensional association is acceptable. A single nucleic acid sequence can be operably linked to multiple other sequences. For example, a single promoter can direct transcription of multiple RNA species.
As will be clear from the context, the term “plant”, as used herein, can refer to a whole plant, plant parts (e.g., cuttings, tubers, pollen), plant organs (e.g., leaves, stems, flowers, roots, fruits, branches, etc.), individual plant cells, groups of plant cells (e.g., cultured plant cells), protoplasts, plant extracts, seeds, and progeny thereof. The class of plants that may be used in the methods of the present invention is as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants, as well as certain lower plants such as algae. The term includes plants of a variety of a ploidy levels, including polyploid, diploid and haploid. In certain embodiments of the invention, plants are green field plants. In other embodiments, plants are grown specifically for “biomass energy”. For example, suitable plants include, but are not limited to, alfalfa, bamboo, barley, canola, corn, cotton, cottonwood (e.g. Populus deltoides), eucalyptus, miscanthus, poplar, pine (pinus sp.), potato, rape, rice, soy, sorghum, sugar beet, sugarcane, sunflower, sweetgum, switchgrass, tobacco, turf grass, wheat, and willow. Using transformation methods, genetically modified plants, plant cells, plant tissue, seeds, and the like can be obtained.
As used herein, “plant biomass” refers to biomass that includes a plurality of components found in plant, such as lignin, cellulose, hemicellulose, beta-glucans, homogalacturonans, and rhamnogalacturonans. Plant biomass may be obtained, for example, from a transgenic plant expressing at least one cell wall-modifying enzyme polypeptide as described herein. Plant biomass may be obtained from any part of a plant, including, but not limited to, leaves, stems, seeds, and combinations thereof.
As used herein, the term “polypeptide” generally has its art-recognized meaning of a polymer of at least three amino acids. However, the term is also used to refer to specific functional classes of polypeptides, such as, for example, lignocellulolytic enzyme polypeptides (including, for example, Acidothermus cellulolyticus E1 endo-1,4-β-glucanase polypeptide, Acidothermus cellulolyticus xylE polypeptide, Acidothermus cellulolyticus gux1 polypeptide, Acidothermus cellulolyticus aviIII polypeptide, and Talaromyces emersonii cbhE polypeptide). For each such class, the present specification provides specific examples of known sequences of such polypeptides. Those of ordinary skill in the art will appreciate, however, that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein (or in a reference or database specifically mentioned herein), but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Other regions of similarity and/or identity can be determined by those of ordinary skill in the art by analysis of the sequences of various polypeptides presented herein.
As used herein, the term “pretreatment” refers to a thermo-chemical process to remove lignin and hemicellulose bound to cellulose in plant biomass, thereby increasing accessibility of the cellulose to cellulases for hydrolysis. Common methods of pretreatment involve using dilute acid (such as, for example, sulfuric acid), ammonia fiber expansion (AFEX), steam explosion, lime, and combinations thereof.
As used herein, the terms “promoter” and “promoter element” refer to a polynucleotide that regulates expression of a selected polynucleotide sequence operably linked to the promoter, and which effects expression of the selected polynucleotide sequence in cells. The term “plant promoter”, as used herein, refers to a promoter that functions in a plant. In some embodiments of the invention, the promoter is a constitutive promoter, i.e., an unregulated promoter that allows continual expression of a gene associated with it. A constitutive promoter may in some embodiments allow expression of an associated gene throughout the life of the plant. Examples of constitutive plant promoters include, but are not limited to, rice act1 promoter, Cauliflower mosaic virus (CaMV) 35S promoter, and nopaline synthase promoter from Agrobacterium tumefaciens. In some embodiments, the promoter is a promoter from poplar. In some embodiments, the promoter comprises a polynucleotide having a sequence of at least one of SEQ ID NO: 1 to 158. In some embodiments of the invention, the promoter is a tissue-specific promoter that selectively functions in a part of a plant body, such as a flower. In some embodiments of the invention, the promoter is a developmentally specific promoter. In some embodiments of the invention, the promoter is an inducible promoter. In some embodiments of the invention, the promoter is a senescence promoter, i.e., a promoter that allows transcription to be initiated upon a certain event relating to the age of the organism.
As used herein, the term “protoplast” refers to an isolated plant cell without cell walls which has the potency for regeneration into cell culture or a whole plant.
As used herein, the term “regeneration” refers to the process of growing a plant from a plant cell (e.g., plant protoplast, plant callus or plant explant).
As used herein, the term “stably transformed”, when applied to a plant cell, callus or protoplast refers to a cell, callus or protoplast in which an inserted exogenous nucleic acid molecule is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. The stability is demonstrated by the ability of the transformed cells to establish cell lines or clones comprised of a population of daughter cells containing the exogenous nucleic acid molecule.
As used herein, the term “tempering” refers to a process to condition lignocellulosic biomass prior to pretreatment so as to favor improved yield from hydrolysis and/or allow use of less severe pretreatment conditions without sacrificing yield. In some embodiments, the lignocellulosic biomass transgenically expresses a lignocellulolytic enzyme polypeptide and tempering facilitates activation of the lignocellulolytic enzyme polypeptide. In some embodiments, tempering facilitates improved yield from subsequent hydrolysis as compared to yield obtained from processing without tempering. In some embodiments, tempering facilitates comparable or improved yield from subsequent hydrolysis using less severe pretreatment conditions than would be required without tempering. In some embodiments, tempering comprises a process selected from the group consisting of ensilement, grinding, pelleting, forming a warm water suspension and/or slurry, incubating at a specific temperature, incubating at a specific pH, and combinations thereof. In some embodiments, tempering comprises separating liquid from a slurry that contains soluble sugars and crude enzyme extracts and re-addition of the separated liquid back to the solid biomass after pretreatment. Specific conditions for tempering may depend on specific traits (such as, e.g., traits of the transgene) of the biomass.
As used herein, the term “tissue-preferred”, when used in reference to a gene regulatory element (such as a promoter) or an expression pattern, means characterized by expression preferences in certain tissues. For example, a tissue-preferred promoter can drive and/or facilitate expression that is high in certain tissues (eg. stem) but in low in others.
As used herein, the term “tissue-specific”, when used in reference to a gene regulatory element (such as a promoter) or an expression pattern, means characterized by expression only in certain tissues. For example, a tissue-specific promoter can drive and/or facilitate expression in some tissues but not others.
As used herein, the term “transformation” refers to a process by which an exogenous nucleic acid molecule (e.g., a vector or recombinant DNA molecule) is introduced into a recipient cell, callus or protoplast. The exogenous nucleic acid molecule may or may not be integrated into (i.e., covalently linked to) chromosomal DNA making up the genome of the host cell, callus or protoplast. For example, the exogenous polynucleotide may be maintained on an episomal element, such as a plasmid. Alternatively, the exogenous polynucleotide may become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. Methods for transformation include, but are not limited to, electroporation, magnetoporation, Ca2+ treatment, injection, particle bombardment, retroviral infection, and lipofection. In some circumstances, an exogenous nucleic acid is introduced in to a cell by mating with another cell. For example, in S. cerevisiae, cells mate with one another.
The term “transgene”, as used herein, refers to an exogenous gene which, when introduced into a host cell through the hand of man, for example, using a process such as transformation, electroporation, particle bombardment, and the like, is expressed by the host cell and integrated into the cell's DNA such that the trait or traits produced by the expression of the transgene is inherited by the progeny of the transformed cell. A transgene may be partly or entirely heterologous (i.e., foreign to the cell into which it is introduced). Alternatively, a transgene may be homologous to an endogenous gene of the cell into which it is introduced, but is designed to be inserted (or is inserted) into the cell's genome in such a way as to alter the genome of the cell (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can also be present in a cell in the form of an episome. A transgene can include one or more transcriptional regulatory sequences and other nucleic acids, such as introns. Alternatively or additionally, a transgene is one that is not naturally associated with the vector sequences with which it is associated according to the present invention.
In various embodiments, the present invention provides, among other things, novel nucleic acids and vectors comprising novel gene regulatory elements from poplar that can be used to express a gene of interest in a variety of cells, including both monocot and dicot plants. Monocot and dicot transgenic plants expressing heterologous genes under the control of a novel gene regulatory element are also provided. Gene regulatory elements of the present invention include those that, in their endogenous contexts, collectively regulate several classes of genes that are involved in plant cell structure and function, intermediary metabolism, tissue-specific and developmental stage-specific functions. Gene regulatory elements of the present invention collectively demonstrate a useful range of properties with regard to gene expression, including, but not limited to, promoter strength, tissue- and/or developmental stage-specificity, and responsiveness to stimuli.
Nucleic acids of the present invention generally comprise a characteristic sequence corresponding to a novel gene regulatory element from sorghum.
Nucleotide sequences of certain provided sorghum gene regulatory elements are listed as SEQ ID NOs: 1 to 158 and presented in Table 3. In some embodiments, nucleotide sequences of provided nucleic acids comprise a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to at least one of SEQ ID NO.: 1 to 158. In some embodiments, nucleotide sequences of provided nucleic acids comprise a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to at least one of SEQ ID NO: 90, 94, 103, 117, 131, 137, 145, and 158. (See, e.g., Examples 2, 3, 4, and 5). In some embodiments, the nucleotide sequences of provided nucleic acids comprise a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to at least one of SEQ ID NO: 90 and 103.
In many embodiments, provided nucleic acids comprise gene regulatory elements from poplar. In some such embodiments, the gene regulatory elements are promoters, that is, they can drive expression of a gene that is operably linked.
Nucleic acids of the invention may include, in addition to nucleotide sequences described above, sequences that can facilitate manipulations such as molecular cloning. For example, restriction enzyme recognition sites and/or recombinase recognition sites may be included in inventive nucleic acids.
Nucleic acids of the present invention included single stranded and double stranded nucleic acids. DNA, RNA, DNA:RNA heteroduplexes, RNA:RNA duplexes, and DNA-RNA hybrid molecules are contemplated and included. In some embodiments, nucleic acids of the present invention include unconventional nucleotides, chemically modified nucleotides, and/or labeled nucleotides (e.g., radiolabeled, fluorescently labeled, enzymatically labeled, etc.). For example, modifications, labels, and/or use of unconventional nucleotides may facilitate downstream manipulations and/or analyses.
Gene vectors of the present invention generally contain a nucleic acid construct that includes one or more expression cassettes for expression of a gene of interest (e.g., a heterologous gene) in a plant of interest. Nucleic acid constructs (also known as “gene constructs”) act as a functional unit that allows expression of a gene of interest. Nucleic acid constructs typically comprise, in addition to the gene of interest (e.g., a heterologous gene whose expression is desired), a gene regulatory element capable of driving expression of the gene of interest (such as a promoter) and a terminator (also known as a stop signal), both of which are operably linked to the gene of interest.
In many embodiments, the gene regulatory element regulates expression of the gene of interest (such as a heterologous gene).
In some embodiments, constructs comprise additional sequences, e.g. marker genes, which are also accompanied by a gene regulatory element (such as a promoter) and a terminator. In many embodiments, the sequences for each of the elements in the construct do not exist in this combination and arrangement in nature and/or are arranged and/or combined by the hand of man.
Expression cassettes generally include 5′ and 3′ regulatory sequences operably linked to a nucleotide sequence encoding a gene of interest.
Techniques used to isolate or clone a gene of interest are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. Cloning of a gene from such genomic DNA, can be effected, e.g., by using polymerase chain reaction (PCR) or antibody screening or expression libraries to detect cloned DNA fragments with shared structural features (Innis et al., “PCR: A Guide to Method and Application”, 1990, Academic Press: New York). Alternatively or additionally, other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used.
Expression cassettes generally include the following elements (presented in the 5′-3′ direction of transcription): a transcriptional and translational initiation region, a coding sequence for a gene of interest, and a transcriptional and translational termination region functional in the organism where it is desired to express the gene of interest (such as a plant).
Other sequences that can be present in a nucleic acid construct include sequences that enhance gene expression (such as, for example, intron sequences and leader sequences). Examples of introns that have been reported to enhance expression include, but are not limited to, introns of the Maize Adh1 gene and introns of the Maize bronze1 gene (J. Callis et. al., Genes Develop. 1987, 1: 1183-1200). Examples of non-translated leader sequences that are known to enhance expression include, but are not limited to, leader sequences from Tobacco Mosaic Virus (TMV, the “omegasequence”), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AlMV) (see, for example, D. R. Gallie et al., Nucl. Acids Res. 1987, 15: 8693-8711; J. M. Skuzeski et. al., Plant Mol. Biol. 1990, 15: 65-79).
Where appropriate, the gene(s) or polynucleotide sequence(s) encoding the enzyme(s) of interest may be modified to include codons that are optimized for expression in the transformed plant (Campbell and Gowri, Plant Physiol., 1990, 92: 1-11; Murray et al., Nucleic Acids Res., 1989, 17: 477-498; Wada et al., Nucl. Acids Res., 1990, 18: 2367, and U.S. Pat. Nos. 5,096,825; 5,380,831; 5,436,391; 5,625,136, 5,670,356 and 5,874,304). Codon optimized sequences are synthetic sequences, and preferably encode the identical polypeptide (or an enzymatically active fragment of a full length polypeptide which has substantially the same activity as the full length polypeptide) encoded by the non-codon optimized parent polynucleotide.
Transcriptional initiation regions (also known as gene promoters, which may be said to comprise ‘promoter elements’) in nucleic acid constructs of the present invention can be native or analogous (i.e., found in the native organism such as a plant) and/or foreign or heterologous (i.e., not found in the native plant) to the plant host. Promoters can comprise a naturally occurring sequence and/or a synthetic sequence.
A given nucleic acid construct may contain more than one promoter, for example, in embodiments wherein expression of more than one heterologous gene is desired. In some embodiments, the two or more promoters include promoters that are the same. In the some embodiments, the two or more promoters are different from one another. In some embodiments that involve at least two different promoters, one promoter drives expression of a heterologous gene in cells of one species (such as a species bacterium) while one other promoter drives expression of a heterologous gene in cells of another species (such as a plant species). In some embodiments, the two or more promoters include at least two promoters that drive expression in cells of the same species.
As mentioned previously, the present invention provides in certain embodiments gene regulatory elements from poplar, which include poplar promoters capable of driving gene expression in plants, including poplar and plants other than poplar (including both monocotyledonous and dicotyledonous plants). In many embodiments, provided gene regulatory elements comprise isolated nucleic acids as described above. Nucleotide sequences of certain provided poplar gene regulatory elements are listed as SEQ ID NOs: 1 to 158. In some embodiments, the nucleotide sequence of the gene regulatory element has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to at least one of SEQ ID NO.: 1 to 158. In some embodiments, the nucleotide sequence of the gene regulatory element has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to at least one of SEQ ID NO:90, 94, 103, 117, 131, 137, 145, and 158. (See, e.g., Examples 2, 3, 4, and 5). In some embodiments, the nucleotide sequence of the gene regulatory element has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to at least one of SEQ ID NO: 90 and 103.
Provided gene regulatory elements can be used alone, in combination with each other, and/or in combination with known promoters (such as known plant promoters) to drive and/or facilitate expression of a gene of interest (such as a heterologous gene). For example, in embodiments wherein two heterologous gene products are expressed in the same plant or other organism, expression of one heterologous gene product may be driven and/or facilitated by a gene regulatory element from poplar provided herein, while expression of the other heterologous gene product may be driven and/or facilitated by another second gene regulatory element from poplar provided herein. Alternatively or additionally, expression of one heterologous gene product may be driven and/or facilitated by a gene regulatory element from poplar provided herein, while expression of the other heterologous gene product may be driven and/or facilitated by a known promoter such as a known plant promoter. Any number of heterologous gene products may be expressed with the aid of and/or under the control of any combinations of gene regulatory elements or promoters.
Provided gene regulatory elements include several types of plant promoters, such as constitutive plant promoters, tissue-specific promoters, and developmental-stage specific plant promoters.
In certain embodiments, at least one promoter in the nucleic acid construct is a constitutive plant promoter, i.e., an unregulated promoter that allows continual expression of a gene associated with it. Examples of known plant promoters that can be used in addition to provided gene regulatory elements include, but are not limited to, the 35S cauliflower mosaic virus (CaMV) promoter, a promoter of nopaline synthase, and a promoter of octopine synthase. Examples of other constitutive promoters used in plants are the 19S promoter and promoters from genes encoding actin and ubiquitin. Promoters may be obtained from genomic DNA by using polymerase chain reaction (PCR), and then cloned into the construct.
Constitutive promoters may allow expression of an associated gene throughout the life of an organism such as a plant. In some embodiments, the heterologous gene product is produced throughout the life of the organism. In some embodiments, the heterologous gene product is active throughout the life of the organism. Alternatively or additionally, a constitutive promoter may allow expression of an associated gene in all or a majority of tissues in the organism. In some embodiments, the heterologous gene product is present in all tissues during the life of the organism.
In certain embodiments, at least one promoter in the nucleic acid construct is a tissue-specific plant promoter, i.e., a promoter that allows expression of a gene in a specific tissue or tissues associated with it.
In certain embodiments, at least one promoter in the nucleic acid construct is a tissue-preferred plant promoter, i.e., a promoter that allows preferential expression in one or some tissues (e.g., higher in one or some tissues than in others). For example, a tissue-preferred plant promoter may allow a high level of expression in stem but a low level of expression in leaves and seed.
The gene of interest can be any gene whose expression is desired. In a nucleic acid construct (particularly expression constructs), genes of interest are generally heterologous, i.e., they are not normally associated with the other elements in the construct in such an arrangement in nature and/or they are associated with such other elements by the hand of man. In some embodiments, heterologous gene products (which may be polypeptides and/or RNA molecules) are expressed in cells, tissues, and/or organisms in which they are not expressed in nature; and/or are expressed at levels different than they are expressed in nature.
A given nucleic acid construct may have one or more than one heterologous gene.
a. Enzyme Polypeptides
In some embodiments, the heterologous gene encodes an enzyme polypeptide. A wide variety of enzyme polypeptides may be expressed under the control of, or facilitated by, poplar gene regulatory elements provided by the present invention. A discussion of some classes of such enzyme polypeptides is presented below. The discussion below is not intended to be exhaustive; provided gene regulatory elements may be used to drive and/or facilitate expression of other enzyme polypeptides as well.
i. Lignocellulolytic Enzyme Polypeptides
In some embodiments, the heterologous gene is a lignocellulolytic enzyme polypeptide.
Plants generally comprise lignocellulosic biomass, a complex substrate in which crystalline cellulose is embedded within a matrix of hemicellulose and lignin. Lignocellulose represents approximately 90% of the dry weight of most plant material with cellulose making up between 30% to 50% of the dry weight of lignocellulose and hemicellulose making up between 20% and 50% of the dry weight of lignocellulose.
Disruption and degradation (e.g., hydrolysis) of lignocellulose by lignocellulolytic enzyme polypeptides leads to the formation of substances including monosaccharides, disaccharides, polysaccharides and phenols. In some embodiments, the lignocellulolytic enzyme polypeptide are characterized by and/or are employed under conditions and/or according to a protocol that achieves enhanced disruption and/or degradation of lignocellulose.
Lignocellulolytic enzyme polypeptides whose expression may be driven with gene regulatory elements of the invention include enzymes that are involved in the disruption and/or degradation of lignocellulose. Lignocellulolytic enzyme polypeptides include, but are not limited to, cellulases, hemicellulases and ligninases. Representative examples of lignocellulolytic enzyme polypeptides are presented in Table 1.
Acidothermus
cellulolyticus
Acidothermus
cellulolyticus
Acidothermus
cellulolyticus
Acidothermus
cellulolyticus
Talaromyces
emersonii
A—Cellulases
Cellulases are lignocellulolytic enzyme polypeptides involved in cellulose degradation. Cellulase enzyme polypeptides are classified on the basis of their mode of action. There are two basic kinds of cellulases: the endocellulases, which cleave the polymer chains internally; and the exocellulases, which cleave from the reducing and non-reducing ends of molecules generated by the action of endocellulases. Cellulases include cellobiohydrolases, endoglucanases, and β-D-glucosidases. Endoglucanases randomly attack the amorphous regions of cellulose substrate, yielding mainly higher oligomers. Cellulobiohydrolases are exocellulases which hydrolyze crystalline cellulose and release cellobiose (glucose dimer). Both types of enzymes hydrolyze β-1,4-glycosidic bonds. β-D glucosidases or cellulobiase converts oligosaccharides and cellubiose to glucose. Beta-glucan glucohydrolase hydrolyzes oligosaccharides to glucose.
According to the present invention, the heterologous gene may encode a cellulase enzyme polypeptide. Transgenic plants of the invention may be engineered to comprise one or more than one gene encoding a cellulase enzyme polypeptide. For example, plants may be engineered to comprise one or more genes encoding a cellulase of the cellubiohydrolase class, one or more genes encoding a cellulase of the endoglucanase class, and/or one or more genes encoding a cellulase of the 13-D glucosidase class.
Examples of endoglucanase genes that can be used in the present invention include those that can be obtained from Aspergillus aculeatus (U.S. Pat. No. 6,623,949; WO 94/14953), Aspergillus kawachii (U.S. Pat. No. 6,623,949), Aspergillus oryzae (Kitamoto et al., Appl. Microbiol. Biotechnol., 1996, 46: 538-544; U.S. Pat. No. 6,635,465), Aspergillus nidulans (Lockington et al., Fungal Genet. Biol., 2002, 37: 190-196), Cellulomonas fimi (Wong et al., Gene, 1986, 44: 315-324), Bacillus subtilis (MacKay et al., Nucleic Acids Res., 1986, 14: 9159-9170), Cellulomonas pachnodae (Cazemier et al., Appl. Microbiol. Biotechnol., 1999, 52: 232-239), Fusarium equiseti (Goedegebuur et al., Curr. Genet., 2002, 41: 89-98), Fusarium oxysporum (Hagen et al., Gene, 1994, 150: 163-167; Sheppard et al., Gene, 1994, 150: 163-167), Humicola insolens (U.S. Pat. No. 5,912,157; Davies et al., Biochem J., 2000, 348: 201-207), Hypocrea jecorina (Penttila et al., Gene, 1986, 45: 253-263), Humicola grisea (Goedegebuur et al., Curr. Genet., 2002, 41: 89-98), Micromonospora cellulolyticum (Lin et al., J. Ind. Microbiol., 1994, 13: 344-350), Myceliophthora thermophila (U.S. Pat. No. 5,912,157), Rhizopus oryzae (Moriya et al., J. Bacteriol., 2003, 185: 1749-1756), Trichoderma reesei (Saloheimo et al., Mol. Microbiol., 1994, 13: 219-228), and Trichoderma viride (Kwon et al., Biosci. Biotechnol. Biochem., 1999, 63: 1714-1720; Goedegebuur et al., Curr. Genet., 2002, 41: 89-98).
In certain embodiments, the heterologous gene encodes the endo-1,4-β-glucanase E1 gene (GenBank Accession No. U33212, See Table 1). This gene was isolated from the thermophilic bacterium Acidothermus cellulolyticus. Acidothermus cellulolyticus has been characterized with the ability to hydrolyze and degrade plant cellulose. The cellulase complex produced by A. cellulolyticus is known to contain several different thermostable cellulase enzymes with maximal activities at temperatures of 75° C. to 83° C. These cellulases are resistant to inhibition from cellobiose, an end product of the reactions catalyzed by endo- and exo-cellulases.
The E1 endo-1,4-β-glucanase is described in detail in U.S. Pat. No. 5,275,944. This endoglucanase demonstrates a temperature optimum of 83° C. and a specific activity of 40 μmol glucose release from carboxymethylcellulose/min/mg protein. This E1 endoglucanase was further identified as having an isoelectric pH of 6.7 and a molecular weight of 81,000 Daltons by SDS polyacrylamide gel electrophoresis. It is synthesized as a precursor with a signal peptide that directs it to the export pathway in bacteria. The mature enzyme polypeptide is 521 amino acids (aa) in length. The crystal structure of the catalytic domain of about 40 kD (358 aa) has been described (J. Sakon et al., Biochem., 1996, 35: 10648-10660). Its pro/thr/ser-rich linker is 60 aa, and the cellulose binding domain (CBD) is 104 aa. The properties of the cellulose binding domain that confer its function are not well-characterized. Plant expression of the E1 gene has been reported (see for example, M. T. Ziegler et al., Mol. Breeding, 2000, 6: 37-46; Z. Dai et al., Mol. Breeding, 2000, 6: 277-285; Z. Dai et al., Transg. Res., 2000, 9: 43-54; and T. Ziegelhoffer et al., Mol. Breeding, 2001, 8: 147-158).
Examples of cellobiohydrolase genes that can be used in the present invention can be obtained from Acidothermus cellulolyticus, Acremonium cellulolyticus (U.S. Pat. No. 6,127,160), Agaricus bisporus (Chow et al., Appl. Environ. Microbiol., 1994, 60: 2779-2785), Aspergillus aculeatus (Takada et al., J. Ferment. Bioeng., 1998, 85: 1-9), Aspergillus niger (Gielkens et al., Appl. Environ. Microbiol., 65: 1999, 4340-4345), Aspergillus oryzae (Kitamoto et al., Appl. Microbiol. Biotechnol., 1996, 46: 538-544), Athelia rolfsii (EMBL accession No. AB103461), Chaetomium thermophilum (EMBL accession Nos. AX657571 and CQ838150), Cullulomonas fimi (Meinke et al., Mol. Microbiol., 1994, 12: 413-422), Emericella nidulans (Lockington et al., Fungal Genet. Biol., 2002, 37: 190-196), Fusarium oxysporum (Hagen et al., Gene, 1994, 150: 163-167), Geotrichum sp. 128 (EMBL accession No. AB089343), Humicola grisea (de Oliviera and Radford, Nucleic Acids Res., 1990, 18: 668; Takashima et al., J. Biochem., 1998, 124: 717-725), Humicola nigrescens (EMBL accession No. AX657571), Hypocrea koningii (Teeri et al., Gene, 1987, 51: 43-52), Mycelioptera thermophila (EMBL accession No. AX657599), Neocallimastix patriciarum (Denman et al., Appl. Environ. Microbiol., 1996, 62: 1889-1896), Phanerochaete chrysosporium (Tempelaars et al., Appl. Environ. Microbiol., 1994, 60: 4387-4393), Thermobifida fusca (Zhang, Biochemistry, 1995, 34: 3386-3395), Trichoderma reesei (Terri et al., BioTechnology, 1983, 1: 696-699; Chen et al., BioTechnology, 1987, 5: 274-278), and Trichoderma viride (EMBL accession Nos. A4368686 and A4368688).
Examples of β-D-glucosidase genes that can be used in the present invention can be obtained from Aspergillus aculeatus (Kawaguchi et al., Gene, 1996, 173: 287-288), Aspergillus kawachi (Iwashita et al., Appl. Environ. Microbiol., 1999, 65: 5546-5553), Aspergillus oryzae (WO 2002/095014), Cellulomonas biazotea (Wong et al., Gene, 1998, 207: 79-86), Penicillium funiculosum (WO 200478919), Saccharomycopsis fibuligera (Machida et al., Appl. Environ. Microbiol., 1988, 54: 3147-3155), Schizosaccharomyces pombe (Wood et al., Nature, 2002, 415: 871-880), and Trichoderma reesei (Barnett et al., BioTechnology, 1991, 9: 562-567).
Other examples of cellulases that can be used in accordance with the present invention include family 48 glycoside hydrolases such as gux1 from Acidothermus cellulolyticus, avicelases such as aviIII from Acidothermus cellulolyticus, and cbhE from Talaromyces emersonii. (See Table 1.)
Transgene expression of cellulases in plants for the conversion of cellulose to glucose has been reported (see, for example, Y. Jin Cai et al., Appl. Environ. Microbiol., 1999, 65: 553-559; C. R. Sanchez et al., Revista de Microbiologica, 1999, 30: 310-314; R. Cohen et al., Appl. Environ., 2995, 71: 2412-2417; Z. Dai et al., Transg. Res., 2005, 14: 627-543).
B—Hemicellulases
Hemicellulases are lignocellulolytic enzyme polypeptides that are involved in hemicellulose degradation. Hemicellulases include xylanases, arabinofuranosidases, acetyl xylan esterases, ferulic acid esterases, xyloglucanases, β-glucanases, β-xylosidases, glucuronidases, mannanases, galactanases, and arabinases. Similar to cellulase enzyme polypeptides, hemicellulases are classified on the basis of their mode of action: the endo-acting hemicellulases attack internal bonds within the polysaccharide chain; the exo-acting hemicellulases act progressively from either the reducing or non-reducing end of polysaccharide chains.
According to the present invention, heterologous genes may encode a hemicellulase enzyme polypeptide. Transgenic plants of the invention may be engineered to comprise one or more than one gene encoding a hemicellulase enzyme polypeptide. For example, plants may be engineered to comprise one or more genes encoding a hemicellulase of the xylanase class, one or more genes encoding a hemicellulase of the arabinofuranosidase class, one or more genes encoding a hemicellulase of the acetyl xylan esterase class, one or more genes encoding a hemicellulase of the glucuronidase class, one or more genes encoding a hemicellulase of the mannanase class, one or more genes encoding a hemicellulase of the galactanase class, and/or one or more genes encoding a hemicellulase of the arabinase class.
Examples of endo-acting hemicellulases include endoarabinanase, endoarabinogalactanase, endoglucanase, endomannanase, endoxylanase, and feraxan endoxylanase. Examples of exo-acting hemicellulases include α-L-arabinosidase, β-L-arabinosidase, α-1,2-L-fucosidase, α-D-galactosidase, β-D-galactosidase, β-D-glucosidase, β-D-glucuronidase, β-D-mannosidase, β-D-xylosidase, exo-glucosidase, exo-mannobiohydrolase, exo-mannanase, exo-xylanase, xylan α-glucuronidase, and coniferin β-glucosidase.
Hemicellulase genes can be obtained from any suitable source, including fungal and bacterial organisms, such as Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium, Trichoderma, Humicola, Thermomyces, and Bacillus. Examples of hemicellulases that can be used in the present invention can be obtained from Acidothermus cellulolyticus, Acidobacterium capsulatum (Inagaki et al., Biosci. Biotechnol. Biochem., 1998, 62: 1061-1067), Agaricus bisporus (De Groot et al., J. Mol. Biol., 1998, 277: 273-284), Aspergillus aculeatus (U.S. Pat. No. 6,197,564; U.S. Pat. No. 5,693,518), Aspergillus kawachii (Ito et al., Biosci. Biotechnol. Biochem., 1992, 56: 906-912), Aspergillus niger (EMBL accession No. AF108944), Magnaporthe grisea (Wu et al., Mol. Plant. Microbe Interact., 1995, 8: 506-514), Penicillium chrysogenum (Haas et al., Gene, 1993, 126: 237-242), Talaromyces emersonii (WO 02/24926), and Trichoderma reesei (EMBL accession Nos. X69573, X69574, and AY281369).
In certain embodiments, the heterologous gene comprises the A. cellulolyticus endoxylanase xylE.
C—Ligninases
Ligninases are lignocellulolytic enzyme polypeptides that are involved in the degradation of lignin. Lignin-degrading enzyme polypeptides include, but are not limited to, lignin peroxidases, manganese-dependent peroxidases, hybrid peroxidases (which exhibit combined properties of lignin peroxidases and manganese-dependent peroxidases), and laccases. Hydrogen peroxide, required as co-substrate by the peroxidases, can be generated by glucose oxidase, aryl alcohol oxidase, and/or lignin peroxidase-activated glyoxal oxidase.
According to the present invention, heterologous genes may encode a ligninase enzyme polypeptide. Transgenic plants of the invention may be engineered to comprise one or more than one gene encoding a ligninase enzyme polypeptide. For example, plants may be engineered to comprise one or more genes encoding a ligninase of the lignin peroxidase class, one or more genes encoding a ligninase of the manganese-dependent peroxidase class, one or more genes encoding a ligninase of the hybrid peroxidase class, and/or one or more genes encoding a ligninase of the laccase class.
Lignin-degrading genes may be obtained from Acidothermus cellulolyticus, Bjerkandera adusta, Ceriporiopsis subvermispora (see WO 02/079400), Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.
Examples of genes encoding ligninases that can be used in the invention can be obtained from Bjerkandera adusta (WO 2001/098469), Ceriporiopsis subvermispora (Conesa et al., J. Biotechnol., 2002, 93: 143-158), Cantharellus cibariusi (Ng et al., Biochem. and Biophys. Res. Comm., 2004, 313: 37-41), Coprinus cinereus (WO 97/008325; Conesa et al., J. Biotechnol., 2002, 93: 143-158), Lentinula edodes (Nagai et al., Applied Microbiol. and Biotechnol., 2002, 60: 327-335, 2002), Melanocarpus albomyces (Kiiskinen et al., FEBS Letters, 2004, 576: 251-255, 2004), Myceliophthora thermophila (WO 95/006815), Phanerochaete chrysosporium (Conesa et al., J. Biotechnol., 2002, 93: 143-158; Martinez, Enz, Microb, Technol, 2002, 30: 425-444), Phlebia radiata (Conesa et al., J. Biotechnol., 2002, 93: 143-158), Pleurotus eryngii (Conesa et al., J. Biotechnol., 2002, 93: 143-158), Polyporus pinsitus (WO 96/000290), Rigidoporus lignosus (Garavaglia et al., J. of Mol. Biol., 2004, 342: 1519-1531), Rhizoctonia solani (WO 96/007988), Scytalidium thermophilum (WO 95/033837), Tricholoma giganteum (Wang et al., Biochem. Biophys. Res. Comm., 2004, 315: 450-454), and Trametes versicolor (Conesa et al., J. Biotechnol., 2002, 93: 143-158).
For example, transgenic plants of the invention may be engineered to comprise one or more lignin peroxidases. Genes encoding lignin peroxidases may be obtained from Phanerochaete chrysosporium or Phlebia radiata. Lignin-peroxidases are glycosylated heme proteins (MW 38 to 46 kDa) which are dependent on hydrogen peroxide for activity and catalyze the oxidative cleavage of lignin polymer. At least six (6) heme proteins (H1, H2, H6, H7, H8 and H10) with lignin peroxidase activity have been identified Phanerochaete chrysosporium in strain BKMF-1767. In certain embodiments, plants are engineered to comprise the white rot filamentous Phanerochaete chrysosporium ligninase (CGLS) (H. A. de Boer et al., Gene, 1988, 69(2): 369) (see the Examples section).
D—Other Lignocellulolytic Enzyme Polypeptides
In addition to cellulases, hemicellulases and ligninases, lignocellulolytic enzyme polypeptides that can be used in the practice of the present invention also include enzymes that degrade pectic substances or phenolic acids such as ferulic acid. Pectic substances are composed of homogalacturonan (or pectin), rhamno-galacturonan, and xylogalacturonan. Enzymes that degrade homogalacturonan include pectate lyase, pectin lyase, polygalacturonase, pectin acetyl esterase, and pectin methyl esterase. Enzymes that degrade rhamnogalacturonan include alpha-arabinofuranosidase, beta-galactosidase, galactanase, arabinanase, alpha-arabinofuranosidase, rhamnogalacturonase, rhamnogalacturonan lyase, and rhamnogalacturonan acetyl esterase. Enzymes that degrade xylogalacturonan include xylogalacturonosidase, xylogalacturonase, and rhamnogalacturonan lyase.
Phenolic acids include ferulic acid, which functions in the plant cell wall to cross-link cell wall components together. For example, ferulic acid may cross-link lignin to hemicellulose, cellulose to lignin, and/or hemicellulose polymers to each other. Ferulic acid esterases cleave ferulic acid, disrupting the cross linkages.
Other enzymes that may enhance or promote lignocellulose disruption and/or degradation may be expressed under the control of a gene regulatory element provided in the present disclosure and include, but are not limited to, amylases (e.g., alpha amylase and glucoamylase), esterases, lipases, phospholipases, phytases, proteases, and peroxidases.
E—Combinations of Lignocellulolytic Enzyme Polypeptides
According to the present invention, heterologous genes may encode a lignocellulolytic enzyme polypeptide, e.g., a cellulase enzyme polypeptide, a hemicellulase enzyme polypeptide, or a ligninase enzyme polypeptide. Transgenic plants of the invention may be engineered to comprise one or more than one gene encoding lignocellulolytic enzyme polypeptides, e.g., enzymes from different classes of cellulases, enzymes from different classes of hemicellulases, enzymes from different classes of ligninases, or any combinations thereof. For example, combinations of genes may be selected to provide efficient degradation of one component of lignocellulose (e.g., cellulose, hemicellulose, or lignin). Alternatively, combinations of genes may be selected to provide efficient degradation of the lignocellulosic material.
In certain embodiments, genes are optimized for the substrate (e.g., cellulose, hemicellulase, lignin or whole lignocellulosic material) in a particular plant (e.g., corn, tobacco, switchgrass). Tissue from one plant species is likely to be physically and/or chemically different from tissue from another plant species. Selection of genes or combinations of genes to achieve efficient degradation of a given plant tissue is within the skill of artisans in the art.
In some embodiments, combinations of genes are selected to provide for synergistic enzyme activity (i.e., genes are selected such that the interaction between distinguishable enzyme polypeptides or enzyme activities results in the total activity of the enzymes taken together being greater than the sum of the effects of the individual activities).
Efficient lignocellulolytic activity may be achieved by production of two or more enzyme polypeptides in a single transgenic plant. As mentioned above, plants may be transformed to express more than one enzyme polypeptide, for example, by employing the use of multiple gene constructs encoding each of the selected enzymes or a single construct comprising multiple nucleotide sequences encoding each of the selected enzymes. Alternatively, individual transgenic plants, each stably transformed to express a given enzyme, may be crossed by methods known in the art (e.g., pollination, hand detassling, cytoplasmic male sterility, and the like) to obtain a resulting plant that can produce all the enzymes of the individual starting plants.
Alternatively or additionally, efficient lignocellulolytic activity may be achieved by production of two or more lignocellulolytic enzyme polypeptides in separate plants. For example, three separate lines of plants (e.g., corn), one expressing one or more enzymes of the cellulase class, another expressing one or more enzymes of the hemicellulase class and the third one expressing one or more enzymes of the ligninase class, may be developed and grown simultaneously. The desired “blend” of enzymes produced may be achieved by simply changing the seed ratio, taking into account farm climate and soil type, which are expected to influence enzyme yields in plants.
Other advantages of this approach include, but are not limited to, increased plant health (which is known to be adversely affected as the number of introduced genes increases), simpler transformations procedures and great flexibility in incorporating the desired traits in commercial plant varieties for large-scale production.
G—Thermophilic and Thermostable Enzyme Polypeptides
It may be sometimes desirable to expressing thermophilic and/or thermostable enzyme polypeptides. Gene regulatory elements provided by the present invention may be used to drive and/or facilitate expression of genes encoding such polypeptides as well. For example, enzyme polypeptides whose optimal range of temperature for activity (thermophilic enzyme polypeptides) may be expressed in transgenic plants in accordance with the invention. Without wishing to be bound by any particular theory, the limited activity or absence of activity during growth of the plant (at moderate or low temperatures, at which the enzyme polypeptide is less active) may be beneficial to the health of the plant. Alternatively or additionally, and without wishing to be bound by any particular theory, such enzyme polypeptides may facilitate increased hydrolysis because of their high activity at high temperature conditions commonly used in the processing of cellulosic biomass.
In some embodiments, the present invention provides a transgenic plant, the genome of which is augmented with a recombinant polynucleotide encoding at least one lignocellulolytic enzyme polypeptide that exhibits low activity at a temperature below about 60° C., below about 50° C., below about 40° C., or below about 30° C. In some embodiments, the present invention provides a transgenic plant, the genome of which is augmented with a recombinant polynucleotide encoding at least one lignocellulolytic enzyme polypeptide that exhibits high activity at a temperature above about 50° C., above about 60° C., above about 70° C., above about 80° C., or above about 90° C.
In some embodiments, the present invention provides a transgenic plant, the genome of which is augmented with a recombinant polynucleotide encoding at least one lignocellulolytic enzyme polypeptide that is or is homologous to a lignocellulolytic enzyme polypeptide found in a thermophilic microorganism (e.g., bacterium, fungus, etc.). In some such embodiments, the thermophilic organism is a bacterium that is a member of a genus selected from the group consisting of Aeropyrum, Acidilobus, Acidothermus, Aciduliprofundum, Anaerocellum, Archaeoglobus, Aspergillus, Bacillus, Caldibacillus, Caldicellulosiruptor, Caldithrix, Cellulomonas, Chaetomium, Chloroflexus, Clostridium, Cyanidium, Deferribacter, Desulfotomaculum, Desulfurella, Desulfurococcus, Fervidobacterium, Geobacillus, Geothermobacterium, Humicola, Ignicoccus, Marinitoga, Methanocaldococcus, Methanococcus, Methanopyrus, Methanosarcina, Methanothermobacter, Nautilia, Pyrobaculum, Pyrococcus, Pyrodictium, Rhizomucor, Rhodothermus, Staphylothermus, Scylatidium, Spirochaeta, Sulfolobus, Talaromyces, Thermoascus, Thermobifida, Thermococcus, Thermodesulfobacterium, Thermodesulfovibrio, Thermomicrobium, Thermoplasma, Thermoproteus, Thermothrix, Thermotoga, Thermus, and Thiobacillus; in some such embodiments, the thermophilic microorganism is a bacterium that is a member of a species selected from the group consisting of Acidothermus cellulolyticus, Pyrococcus furiosus, and Talaromyces emersonii.
ii. Cell Wall-Modifying Enzyme Polypeptides
In some embodiments, the heterologous gene (whose expression is driven by a provided gene regulatory element) encodes a cell wall-modifying enzyme polypeptide described in U.S. patent application Ser. No. 12/476,247 (filed on Jun. 1, 2009), the contents of which are herein incorporated by reference in their entirety. In some embodiments, cell wall-modifying enzyme polypeptides are lignocelluloytic enzyme polypeptides.
Cell wall-modifying enzyme polypeptides useful in accordance with the present invention include those having at least 50%, 60%, 70%, 80% or more overall sequence identity with a polypeptide whose amino acid sequence is set forth in Table 1 of U.S. patent application Ser. No. 12/476,247. Alternatively or additionally, in some embodiments, cell wall-modifying enzyme polypeptide shows at least 90%, 95%, 96%, 97%, 98%, 99%, or greater identity with at least one sequence element found in a polypeptide whose amino acid sequence is set forth in Table 1 of U.S. patent application Ser. No. 12/476,247, which sequence element is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids long.
A variety of organisms produce cell wall-modifying enzyme polypeptides. Cell wall-modifying enzyme polypeptides may have, for example, archaeal, fungal, insect, animal, or plant origins.
In some embodiments, the cell wall-modifying enzyme polypeptide has cellulase activity. In some embodmients, the cell wall-modifying enzyme polypeptide has an activity selected from the group consisting of feruloyl esterase (also known as ferulic acid esterase), xylanase, alpha-L-arabinofuranosidase, endogalactanase, acetylxylan esterase, beta-xylosidase, xyloglucanase, glucuronoyl esterase, endo-1,5-alpha-L-arabinosidase, pectin methylesterase, endopolygalacturonase, exopolygalacturonase, pectin lyase, pectate lyase, rhamnogalacturonan lyase, pectin acetylesterase, alpha-L-rhamnosidase, mannanase, exoglucanase, glucan glycohydrolase, licheninase, laminarinase, beta-(1,3)-(1,4)-glucanase and beta-glucosidase activity. Such activities may be similar to that of other enzyme polypeptides, including those known in the art that are classified by an EC class and/or listed in enzyme databases (such as CaZY, www.cazy.org, which lists carbohydrate-active enzymes).
In some embodiments, the cell wall-modifying enzyme polypeptide modifies a plant cell wall component. In many such embodiments, the cell wall-modifying enzyme polypeptide modifies the plant cell wall component in such a way that the plant biomass is more amenable to processing steps (e.g., enzymatic digestion). For example, cell wall-modifying enzyme polypeptides may modify plant cell wall components in such a way as to allow increased digestability, increased hydrolysis, and/or increased sugar yields.
In some embodiments, modifying comprises cleavage and/or hydrolysis of the plant cell wall component. Examples of plant cell wall components that may be modified include, but are not limited to, xylans, xylan side chains, glucuronoarabinoxylans, xyloglucans, mixed-linkage glucans, pectins, pectates, rhamnogalacturonans, rhamnogalacturonan side chains, lignin, cellulose, mannans, galactans, arabinans, oligosaccharides derived from cell wall polysaccharides, and combinations thereof.
In some embodiments, the cell wall-modifying enzyme polypeptide disrupts an interaction in the plant biomass such as a covalent linkage, an ionic bonding interaction, a hydrogen bonding interaction, or a combination thereof. Examples of linkages that may be disrupted include, but are not limited to, hemicellulose-cellulose-lignin, hemicellulose-cellulose-pectin, hemicellulose-diferululate-hemicellulose, hemicellulose-ferulate-lignin, mixed beta-D-glucan-cellulose, mixed-beta-D-glucan-hemicellulose, pectin-ferulate-lignin linkages, and combinations thereof. In some embodiments, disrupting comprises hydrolyzing a linkage, such as a feruloyl ester linkage.
b. Heterologous Gene Products Conferring Resistance to Pests, Disease, and Environmental Stress
Heterologous genes may express products that confer benefits) to the transgenic plant such as herbicide resistance, insect resistance, disease resistance, resistance against parasites, and/or increased tolerance to environmental stress (e.g., drought).
Herbicide Resistance
A number of gene products are known in the art that can confer resistance to herbicides. For example, glyphosate (N-(phosphonomethyl) glycine) is a broad-spectrum systemic herbicide and the active ingredient of ROUNDUP™ formulations. Glyphosate acts by inhibiting 5-enolpyruvoyl-shikimate-3-phosphate synthetase (EPSPS) (encoded in some organisms by the aroA gene), starving the affected cells for aromatic amino acids. Some micro-organisms have a mutant form of EPSPS that is resistant to glyphosate inhibition, and this form of the enzyme can be used to impart glyphosate resistance.
As a further example, the herbicide bromoxynil (marketed as Buctril) is applied post-emergence to kill broadleaf weeds, and works by inhibiting photosynthesis in plants. Bromoxynil nitrilase (BXN), a gene from the bacterium Klebsiella pneumoniae, detoxifies bromoxynil in genetically engineered plants and therefore can confer resistance to herbicides.
The L-isomer of phosphinothricin (PPT, glufosinate ammonium) is the active ingredient of several commercial broad spectrum herbicide formulation. An analogue of L-glutamic acid, PPT, is a competitive inhibitor of glutamine synthetase, the only enzyme that can catalyze assimilation of ammonia into glutamic acid into plants. Inhibition of glutamine synthetase ultimately results in the accumulation of toxic ammonia levels, resulting in plant cell death. Phosphosphinothricin acetyltransferase, which is encoded by the bar gene from Streptomyces hygroscopicus, confers resistance to herbicides that contain PPT.
Dalapon is an herbicide used to control grasses in a wide variety of crops. Dalapon dehalogenase is capable of degrading high concentrations of the herbicide dalapon.
Additional non-limiting examples of genes that provide resistance to herbicides include, but are not limited to, mutant genes that confer resistance to imidazalinone or sulfonylurea, such as genes encoding mutant form of acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) (Lee at al., EMBO J., 1988, 7: 1241; Miki et al., Theor. Appl. Genet., 1990, 80: 449; and U.S. Pat. No. 5,773,702); and genes that confer resistance to phenoxy propionic acids and cyclohexones such as the ACCAse inhibitor-encoding genes (Marshall et al., Theor. Appl. Genet., 1992, 83: 435).
Resistance to Pests and/or Diseases
Genes that confer resistance to pests and/or disease include, but are not limited to, genes whose products confer resistance to infestation from an organism selected from the group consisting of insects, bacteria, fungi, and nematodes. Heterologous genes whose products confer resistance to viruses may also be expressed using gene regulatory elements of the present invention.
Gene products that can confer resistance to insects and/or insect disease include, but are not limited to, Bt (Bacillus thuringiensis) proteins (such as delta-endotoxin (U.S. Pat. No. 6,100,456)); vitamin-binding proteins such as avidin and avidin homologs (which can be used as larvicides against insect pests); insect-specific hormones or pheromones such as ecdysteroid and juvenile hormone, and variants thereof, mimetics based thereon, or an antagonists or agonists thereof; insect-specific peptides or neuropeptides which, upon expression, disrupts the physiology of the pest; insect-specific venom such as that produced by a wasp, snake, etc.; enzyme polypeptides responsible for the accumulation of monoterpenes, sesquiterpenes, asteroid, hydroxamic acid, phenylpropanoid derivative or other non-protein molecule with insecticidal activity; insect-specific antibodies or antitoxins (Tavladoraki et al., Nature, 1993, 366: 469); and TcdA protein (Liu et al., 2003 Nature Biotechnology 21: 1222-1228).
Gene products that can confer resistance to bacteria and/or bacterial diseases include, but are not limited to, nucleotide-binding-sequence LRR (also known as ‘NBS-leucine rich repeat’) proteins (Van Der Biezen and Jones, 1998 Trends in Biochemical Sciences 23: 454-456).
Gene products that can confer resistance to fungi and/or fungal diseases include, but are not limited to, Pi-ta (U.S. Pat. No. 6,743,969), Pathogenesis-related (PR) proteins, chitinases and β-1,3-glucanases, ribosome-inactivating proteins (RIPs), thionins, hydrophobic moment peptides (such as derivatives of Tachyplesin which inhibit fungal pathogens), and antifungal peptides such as LCI.
Gene products that can confer resistance to viruses and/or viral diseases include, but are not limited to, nucleotide-binding site-leucine-rich repeat (NBS-LRR proteins), virus-specific antibodies and antitoxins (Tavladoraki et al., Nature, 1993, 366: 469), viral invasive proteins or complex toxins derived therefrom (Beachy et al., Ann. Rev. Phytopathol., 1990, 28: 451), PR proteins, and Rx proteins (genetically engineered cross protection is conferred by expressing viral coat protein genes in the plant genome).
Gene products that can confer resistance to nematodes and/or nematode diseases include, but are not limited to, peroxidases, chitinases, lipoxygenases, proteinase inhibitors, Mi proteins, Gro, Gpa and Cre proteins.
Other gene products that can confer resistances to diseases or pests include, but are not limited to, lectins (Van Damme et al., Plant Mol. Biol., 1994, 24: 825); protease or amylase inhibitors, such as the rice cysteine proteinase inhibitor (Abe et al., J. Biol. Chem., 1987, 262: 16793) and the tobacco proteinase inhibitor I (Hubb et al., Plant Mol. Biol., 1993, 21: 985); enzyme polypeptides involved in the modification of a biologically active molecule (U.S. Pat. No. 5,539,095); peptides that stimulate signal transduction; membrane permeases (channel formers or channel blockers) (Jaynes et al., Plant Sci., 1993, 89: 43); and developmental-arrestive proteins produced by a plant, pathogen or parasite that prevents disease.
Resistance to Stress
Gene products that confer resistance to environmental stress include both biotic and abiotic stress proteins.
Biotic stress in plants can be caused by bacteria, fungi, viruses, insects and nematodes. Non-limiting examples of proteins that can provide biotic stress resistance/tolerance in plants include those that confer resistance to diseases and pests mentioned above, as well as DREB transcription factors (Agarwal et al., 2006 Plant Cell Reports 25: 1263-1274) and MAP Kinases (U.S. Pat. No. 7,345,219).
Abiotic stress in plants can be caused by a variety of factors, including, but not limited to, nutrient imbalances, light (high light, UV, darkness), water imbalances (deficit, desiccation, flooding), temperature imbalances (frost, cold, heat), oxidation stress, hypoxia, physical factors (such as wind and touch), salt, and heavy metals. Examples of gene products that can provide abiotic stress resistance/tolerance in plants include HSFs, LEAs, CORs, CBFs and ABFs (Vinocur and Altman, 2005 Current Opinion in Biotechnology 16:123-132).
Examples of genes whose products confer resistance to environmental stress include, but are not limited to, mtld and HVA1 (which confer resistance to environmental stress factors); and rd29A and rd19B (Arabidopsis thaliana genes that encode hydrophilic proteins induced in response to dehydration, low temperature, salt stress, and/or exposure to abscisic acid and enable the plant to tolerate the stress (Yamaguchi-Shinozaki et al., Plant Cell, 1994, 6: 251-264)). Other such genes contemplated can be found in U.S. Pat. Nos. 5,296,462 and 5,356,816.
c. Other Heterologous Gene Products
Gene regulatory elements provided by the present invention may also be used to drive and/or facilitate other heterologous gene products that confer advantages to the plants that express them.
For example, nutrient utilization polypeptides can be expressed in transgenic plants. Such polypeptides can maximize utilization of nutrients by plants and may lead to increased yields. Nutrients whose utilization maximization may be desired include, but are not limited to, nitrogen, phosphorous, potassium, iron, zinc etc.
It may be desirable to trnasgenically express anthranilate synthase, which catalyzes the conversion of chorismate into anthranilate. Anthranilate is the biosynthetic precursor of both tryptophan and numerous secondary metabolites, including inducible plant defense compounds.
It may be desirable to express mycotoxin reduction polypeptides in plants. Mycotoxins are toxic and carcinogenic chemicals produced by fungi in plants during growth or storage of grains and are major concern for growers. Bt proteins, when expressed in plants reduce mycotoxin content (Wu et al., 2004 Toxin Reviews 23: 397-424).
Male sterility polypeptides may also be expressed in transgenic plants using gene regulatory elements of the present invention. Male sterility in plants can be induced by expressing several types of polypeptides such as RNase/Barnase (Mariani et al., 1990 Nature 347: 737-741).
Heterologous gene products that affect grain composition or quality (e.g., by altering key components of grain, such as starch, protein, bran, etc.) may also be expressed. Desired changes in composition may include, for example, relative proportions of starch fractions such amylose and amylopectin; decreased amounts of undesirable components such as phytic acid; and/or improved amino acid content conferred, for example, by modified seed storage proteins that have been. For example, corn zeins modified to contain more lysine can be expressed.
Polypeptides having therapeutic value can also be expressed in plants using provided gene regulatory elements. Such polypeptides can be harvested from plants transgenically expressing them and then purified for downstream applications. Such polypeptides include, but are not limited to, antibodies, blood products, cytokines, growth factors, hormones, recombinant enzymes, and vaccines that would have a variety of applications in human and animal health. For example, lactoferrin and lysozyme has been produced in rice grains (Ventria Bioscience).
Heterologous gene products that may be expressed also include RNA molecules, for example, those that regulate a plant gene.
The transcriptional and translational termination region generally comprises a sequence that encodes a “terminator” (the “terminator sequence”). The transcriptional and translational termination region can be native with the transcription initiation region, can be native with the operably linked polynucleotide sequence of interest, and/or can be derived from another source. Convenient termination regions are available from the T1-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions (An et al., Plant Cell, 1989, 1: 115-122; Guerineau et al., Mol. Gen. Genet. 1991, 262: 141-144; Proudfoot, Cell, 1991, 64: 671-674; Sanfacon et al., Genes Dev. 1991, 5: 141-149; Mogen et al., Plant Cell, 1990, 2:1261-1272; Munroe et al., Gene, 1990, 91:151-158; Ballas et al., Nucleic Acids Res., 1989, 17: 7891-7903; and Joshi et al., Nucleic Acid Res., 1987, 15: 9627-9639).
In some embodiments, nucleic acid constructs include one or more marker genes. Marker genes are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow transformed cells to be distinguished from cells that do not have the marker. Such genes may encode, for example, a selectable and/or screenable marker. In some embodiments, nucleic acid constructs comprise a marker that allows selecting and/or screening in a transformed cell.
In some embodiments, the transformed cell is grown in culture medium under conditions that select for cells that either have (positive selection) or do not have (negative selection) the marker. In some embodiments, a combination of positive and negative selection is used.
In some so-called positive selection schemes, most cells in a population are unable to divide and because they lack the ability to use a nutrient (such as, for example, a carbon source) present in the selection medium. In these schemes, the selectable marker confers an ability to use the nutrent. Thus, cells that have the selectable marker gain an advantage over other cells in the population and therefore can be selected.
In some so-called negative screening/selection schemes, most cells in a population are unable to divide because of the effects of a toxic agent (such as, for example, an antibiotic present in the selection medium). In these schemes, the selectable marker confers an ability to overcome the toxicity (for example, by blocking uptake or by chemically modifying the toxic agent). Thus, cells that have the selectable marker gain an advantage over other cells in the population and therefore can be selected.
In some embodiments, the transformed cell undergoing selection is a prokaryotic cell, such as E. coli and Agrobacterium. In some embodiments, the transformed cell undergoing selection is a eukaryotic cell, such as a yeast (for example, S. cerevisiae), mammalian, insect, or plant cell.
In some embodiments, the characteristic phenotype allows the identification of cells, groups of cells, tissues, organs, plant parts or whole plants containing the construct.
Many examples of suitable marker genes are known in the art and can be used in screening and/or selection schemes. Reagents such as appropriate components of selection media are also known in the art. Examples of such marker genes include, but are not limited to, phosphomannose isomerase, phosphinothricin, neomycin phosphotransferase, hygromyci phosphotransferase, enolpyruvoyl-shikimate-3-phosphate synthetase, etc.
For example, phosphomannose isomerase (PMI) catalyses the interconversion of mannose 6-phosphate and fructose 6-phosphate in prokaryotic and eukaryotic cells. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. Accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. Phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. On media containing mannose, manA expression in transformed plant cells relieves the growth inhibiting effect of mannose-6-phosphate accumulation and permits utilization of mannose as a source of carbon and energy, allowing transformed cells to grow.
Reporter proteins (such as GUS (β-glucuronidase), green fluorescent protein and derivatives thereof, and luciferase). Reporter genes may allow easy visual detection of transformed cells by visual screening and may also be used as marker genes. Non-limiting examples of eporter proteins include GUS (a β-glucuronidase), green fluorescent protein and derivatives thereof, and luciferase.
In some embodiments, the marker confers benefit(s) to the transgenic plant such as herbicide resistance, insect resistance, disease resistance, and increased tolerance to environmental stress (e.g., drought). (See, for example, the section on genes of interest above for an expanded discussion of some of these genes.)
Alternatively or additionally, a marker gene can provide some other visibly reactive response (e.g., may cause a distinctive appearance such as color or growth pattern relative to plants or plant cells not expressing the selectable marker gene in the presence of some substance, either as applied directly to the plant or plant cells or as present in the plant or plant cell growth media). It is now well known in the art that transcriptional activators of anthocyanin biosynthesis, operably linked to a suitable promoter in a construct, have widespread utility as non-phytotoxic markers for plant cell transformation.
B. Tissue-Specific and/or Tissue-Preferred Expression
In certain embodiments, heterologous gene products) is/are targeted to specific tissues of the transgenic plant such that the heterologous gene products) is/are present in only some plant tissues during the life of the plant. For example, tissue specific expression may be performed to preferentially express polypeptides encoded by heterologous genes in leaves and stems rather than grain or seed (which can reduce concerns about human consumption of genetically modified organism (GMOs)). Tissue-specific expression has other benefits including targeted expression of enzyme polypeptide(s) to the appropriate substrate.
In certain embodiments, heterologous gene product(s) is/are preferentiallly expressed certain tissues of the transgenic plant such that the heterologous gene products) is/are present at higher levels in some plant tissues than in others during the life of the plant.
Tissue-specific and/or tissue-preferred expression may be functionally accomplished by using one or more tissue-specific and/or tissue-preferred gene regulatory elements, such as some of the poplar promoters disclosed herein. A number of known tissue-specific promoters may be used in combination with gene regulatory elements disclosed herein. For example, in embodiments wherein two heterologous gene products are expressed in the same plant or other organism, expression of one heterologous gene product may be driven by a gene regulatory element from poplar as disclosed herein, while expression of the other heterologous gene product may be driven by a gene regulatory element that is known, such as a known tissue-specific promoter. Several tissue-specific regulated genes and/or promoters have been reported in plants. Some reported tissue-specific genes include without limitation genes encoding seed storage proteins (such as napin, cruciferin, β-conglycinin, and phaseolin), genes encoding zein or oil body proteins (such as oleosin), genes involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase, and fatty acid desaturases (fad 2-1)), and other genes expressed during embryo development (such as Bce4 (Kridl et al., Seed Science Research, 1991, 1: 209)). Examples of tissue-specific promoters that have been described in the art include the lectin (Vodkin, Prog. Clin. Biol. Res., 1983, 138: 87; Lindstrom et al., Der. Genet., 1990, 11: 160), corn alcohol dehydrogenase 1 (Dennis et al., Nucleic Acids Res., 1984, 12: 983), corn light harvesting complex (Bansal et al., Proc. Natl. Acad. Sci. USA, 1992, 89: 3654), corn heat shock protein, pea small subunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase (van Tunen et al., EMBO J., 1988, 7:125), bean glycine rich protein 1 (Keller et al., Genes Dev., 1989, 3: 1639), truncated CaMV 35s (Odell et al., Nature, 1985, 313: 810), potato patatin (Wenzler et al., Plant Mol. Biol., 1989, 13: 347), root cell (Yamamoto et al., Nucleic Acids Res., 1990, 18: 7449), maize zein (Reina et al., Nucleic Acids Res., 1990, 18: 6425; Kriz et al., Mol. Gen. Genet., 1987, 207: 90; Wandelt et al., Nucleic Acids Res., 1989, 17 2354), PEPCase, R gene complex-associated promoters (Chandler et al., Plant Cell, 1989, 1: 1175), and chalcone synthase promoters (Franken et al., EMBO J., 1991, 10: 2605). Particularly useful for seed-specific expression is the pea vicilin promoter (Czako et al., Mol. Gen. Genet., 1992, 235: 33).
Tissue-specific and/or tissue-preferred expression may also be functionally accomplished by introducing a constitutively expressed gene in combination with an antisense gene that is expressed only in those tissues where the gene product is not desired, or where it is desired that the gene be expressed at lower levels. For example, a gene encoding an heterologous or homologous polypeptide may be expressed in all tissues under the control of a constitutive promoter such as constitutive poplar promoters disclosed herein and/or a known constitutive promoter such as the 35S promoter from Cauliflower Mosaic Virus. Expression of an antisense transcript of the gene in a particular tissue, using for example tissue-specific promoter or tissue-preferred promoter, would prevent accumulation of the enzyme polypeptide in that tissue. A tissue-specific and tissue-preferred poplar promoter disclosed herein and/or a known tissue-specific or tissue-preferred promoter may be used to drive expression of the antinsense transcript. For example, an antisense transcript of the gene for which tissue-specific or tissue-preferred expression is desired may be expressed in maize kernel using a zein promoter, thereby preventing accumulation of the gene product in seed. Hence the polypeptide encoded by the heterologous gene would be present in all tissues except the kernel.
In certain embodiments, heterologous gene product(s) is/are targeted to specific cellular compartments or organelles, such as, for example, the cytosol, the vacuole, the nucleus, the endoplasmic reticulum, the cell wall, the mitochondria, the apoplast, the peroxisomes, plastids, or combinations thereof. In some embodiments of the invention, the heterologous gene is expressed in one or more subcellular compartments or organelles, for example, the cell wall and/or endoplasmic reticulum, during the life of the plant.
In some embodiments, directing the product (e.g., a polypeptide and/or RNA molecule) of the heterologous gene to a specific cell compartment or organelle allows the product to be localized such that it will not come into contact with another molecule until desired. For example, if the product is an enzyme polypeptide, it may be possible to prevent the enzyme polypeptide from coming into contact with its substrate during plant growth. Thus, the enzyme polypeptide would not act until it is allowed to contact its substrate, e.g., following physical disruption of cell integrity by milling.
As another example, targeting expression of a cell wall-modifying and/or lignocellulolytic enzyme polypeptide to the cell wall (as in the apoplast) can help overcome the difficulty of mixing hydrophobic cellulose and hydrophilic enzymes that make it hard to achieve efficient hydrolysis with external enzymes.
In some embodiments, gene products are targeted to more than one subcellular compartments or organelles. Such targeting may allow one to increase the total amount of heterologous gene product in the plant. In some embodiments, targeting to one or more subcellular compartments or organelles is achieved using a gene regulatory element (such as a promoter) that drives expression specifically or preferentially in one or more subcellular compartments or organelles. Thus, for example, using an apoplast promoter with the E1 endo-1,4-β-glucanase gene and a chloroplast promoter with the E1 gene in a plant would increase total production of E1 compared to a single promoter/E1 construct in the plant.
Furthermore, in the case of expression of enzyme polypeptides that modify the cell wall (e.g., cell wall-modifying enzyme polypeptides and/or lignocellulolytic enzyme polypeptides)) one can minimize in vivo (pre-processing) deconstruction of the cell wall that occurs when multiple synergistic enzymes are present in a cell by using promoters targeted to different locations in the plant. For example, combining an endoglucanase with an apoplast promoter, a hemicellulase with a vacuole promoter, and an exoglucanase with a chloroplast promoter, sequesters each enzyme in a different part of the cell and achieves the advantages listed above. This method circumvents the limit on polypeptide or other heterologous gene product mass that can be expressed in a single organelle or location of the cell.
Localization of a nuclear-encoded protein (e.g., enzyme polypeptide) within the cell is known to be determined by the amino acid sequence of the protein. Protein localization can be altered, for example, by modifying the nucleotide sequence that encodes the protein in such a manner as to alter the protein's amino acid sequence. Polynucleotide sequences encoding polypeptides can be altered to redirect cellular localization of the encoded polypeptides by any suitable method (see, e.g., Dai et al., Trans. Res., 2005, 14: 627, the entire contents of which are herein incorporated by reference). In some embodiments of the invention, polypeptide localization is altered by fusing a sequence encoding a signal peptide to the sequence encoding the polypeptide. Signal peptides that may be used in accordance with the invention include without limitation a secretion signal from sea anemone equistatin (which allows localization to apoplasts) and secretion signals comprising the KDEL motif (which allows localization to endoplasmic reticulum).
Generally, any vector that can be used constructed to express a product (e.g., polypeptide or RNA molecule) of a gene after introduction of such a vector in a host cell is considered an “expression vector.” Expression vectors typically contain nucleic acid constructs such as expression cassettes described above inserted into a vector. Expression vectors can be designed for expressing a gene product in any of a variety of host cells, including both prokaryotic (e.g., bacteria such as E. coli and Agrobacterium) and eukaryotic (e.g. insect, yeast (such as S. cerevisiae), and mammalian cells) host cells.
Nucleic acid constructs according to the present invention may be cloned into any of a variety of vectors, such as binary vectors, viral vectors, phage, phagemids, cosmids, and plasmids. Vectors suitable for transforming plant cells include, but are not limited to, Ti plasmids from Agrobacterium tumefaciens (J. Darnell, H. F. Lodish and D. Baltimore, “Molecular Cell Biology”, 2nd Ed., 1990, Scientific American Books: New York); plasmid containing a β glucuronidase gene and a cauliflower mosaic virus (CaMV) promoter plus a leader sequence from alfalfa mosaic virus (J. C. Sanford et al., Plant Mol. Biol. 1993, 22: 751-765); and plasmids containing a bar gene cloned downstream from a CaMV 35S promoter and a tobacco mosaic virus (TMV) leader. Other plasmids may additionally contain introns, such as that derived from alcohol dehydrogenase (Adh1) and/or other DNA sequences. The size of the vector is not a limiting factor.
For constructs that are intended be used in Agrobacterium-mediated transformation, the plasmid may contain an origin of replication that allows it to replicate in Agrobacterium and a high copy number origin of replication functional in E. coli. This permits facile production and testing of transgenes in E. coli prior to transfer to Agrobacterium for subsequent introduction in plants. Resistance genes can be carried on the vector, one for selection in bacteria, for example, streptomycin, and another that will function in plants, for example, a gene encoding kanamycin resistance or herbicide resistance. Also present on the vector are restriction endonuclease sites for the addition of one or more transgenes and directional T-DNA border sequences which, when recognized by the transfer functions of Agrobacterium, delimit the DNA region that will be transferred to the plant.
Methods of preparation of nucleic acid constructs and expression vectors are well known in the art and can be found described in several textbooks such as, for example, J. Sambrook, E. F. Fritsch and T. Maniatis, “Molecular Cloning: A Laboratory Manual”, 1989, Cold Spring Harbor Laboratory: Cold Spring Harbor, and T. J. Silhavy, M. L. Berman, and L. W. Enquist, “Experiments with Gene Fusions”, 1984, Cold Spring Harbor Laboratory Cold Spring Harbor; F. M. Ausubel et al., “Current Protocols in Molecular Biology”, 1989, John Wiley & Sons: New York.
In one aspect, the present invention provides novel transgenic plants that express one or more polypeptides or RNA molecules under the control of a gene regulatory element provided by the present disclosure. The polypeptides or RNA molecules may be any polypeptide or RNA molecule for which expression in a plant is desired, including, but not limited to, those described herein.
In certain embodiments, provided are transgenic plants, the genomes of which are augmented with a recombinant polynucleotide comprising a gene regulatory element from poplaras described herein. In some embodiments, the nucleotide sequence of the gene regulatory element has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to at least one of SEQ ID NO: 1 to 158. In some embodiments, the nucleotide sequence of the gene regulatory element is one of SEQ ID NO: 1 to 158. In some embodiments, the nucleotide sequence of the gene regulatory element has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to at least one of SEQ ID NO:90, 94, 103, 117, 131, 137, 145, and 158. (See, e.g., Examples 2, 3, 4, and 5). In some embodiments, the gene regulatory element has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to at least one of SEQ ID NO: 90 and 103.
In some embodiments, the transgenic plant further comprises a heterologous gene operably linked to the gene regulatory element. In some such embodiments, the gene regulatory element regulates expression of the heterologous gene.
The heterologous gene may encode any polypeptide or RNA molecule for which expression in a plant is desired, including, but not limited to, those described herein. In some embodiments, the recombinant polynucleotide further comprises a gene terminator sequence that is operably linked to the heterologous gene.
Nucleic acid constructs, such as those described above, can be used to transform any plant. In some embodiments, plants are green field plants. In some embodiments, plants are grown specifically for “biomass energy” and/or phytoremediation.
In some embodiments, the plants are monocotyledonous plants. Examples of monocotyledonous plants that may be transformed in accordance with the practice of the present invention include, but are not limited to, bamboo, barley, maize (corn), millet, miscanthus, rice, rye, sorghum, sugarcane, switchgrass, turfgrass, and wheat. Alternatively or additionally, any grass species may be used.
In some embodiments, the plants are dicotyledonous plants. Examples of dicotyledonous plants that may be transformed in accordance with the practice of the present invention include, but are not limited to, alfalfa, Arabidopsis, aspen, birch, eucalyptus, flax, canola, cotton, cottonwood (e.g., Populus deltoides), hemlock, hemp, larch, oil seed rape, potato, poplar, sisal, spruce, soybean, sunflower, sweetgum, tobacco, tomato, and willow. Alternatively or additionally, any tree species may be used.
In some embodiments, the plants is a multicotyledonous plant. A non-limiting example of a multicotyledonous plant that may be transformed in accordance with the practice of the present invention is a pine tree (pinus sp.).
In some embodiments, the plant is a monocotyledonous or dicotyledonous plant of a genus selected from the group consisting of Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale, Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.
In some embodiments, the plant is a monocotyledonous or dictoyledonous plant of a species selected from the group consisting of Abelmoschus esculentus (okra), Abies spp. (fir), Acer spp. (maple), Agrostis spp. (bentgrass), Allium cepa (onion), Alstroemeria spp., Ananas comosus (pineapple), Andropogon gerardii (big bluestem), Andrographis paniculata, Artemisia annua, Arundo donax (giant reed), Atropa belladonna, bamboo, Berberis spp., Beta vulgaris (sugarbeet), Bixa orellana, Brassica juncea, Brassica napus (canola), Brassica oleracea (broccoli, cauliflower, brussel sprouts), Calendula officinalis, Camellia sinensis (tea), Camptotheca acuminate, Cannabis sativa, Capsicum annum (hot and sweet pepper), Carthamus tinctorius (safflower), Catharanthus roseus, Cephalotaxus spp., Chrysanthemum parthenium, Cinchona officinalis, Citrullus lanatus (watermelon), Coffea arabica (coffee), Colchicum autumnale, Coleus forskohlii, Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Cynodon dactylon (bermudagrass), Datura stomonium, Dianthus caryophyllus (carnation), Digitalis lanata, Digitalis purpurea, Dioscorea spp., Elaeis guineensis (palm), Ephedra sinica, Ephedra spp., Erianthus spp., Erythroxylum coca, Eucalyptus spp. (eucalyptus), Fragaria ananassa (strawberry), Festuca arundinacea (tall fescue), Galanthus wornorii, Glycine max (soybean), Gossypium hirsutum (cotton), Helianthus annuus (sunflower), Hevea spp. (rubber), Hordeum vulgare (barley), Hyoscyamus spp., Jatropha curcas (Jatropha), Lactuca sativa (lettuce), Linum usitatissimum (flax), Lolium spp. (ryegrass), Lupinus albus (lupin), Lycopersicon esculentum (tomato), Lycopodium serratum (Huperzia serrata), Lycopodium spp., Manihot esculenta (cassaya), Medicago sativa (alfalfa), Mentha spicata (mint), Menthapiperita (mint), Miscanthus giganteus (miscanthus), Miscanthus spp., Musa paradisiaca (banana), Nicotiana tabacum (tobacco), Oryza sativa (rice), Panicum spp., Panicum virgatum (switchgrass), Papaver somniferum (opium poppy), Papaver orientale, Parthenium argentatum (guayule), Pennisetum glaucum (pearl millet), Pennisetum purpureum (elephant grass), Petunia spp. (petunia), Phalaris arundinacea (reed canarygrass), Phleum pratense (timothy), Pinus spp. (pine), Poa pratensis (bluegrass), Poinsettia pulcherrima (poinsettia), Populus balsamifera (poplar), Populus spp., Populus tremuloides (aspen), Rosa spp. (rose), Saccharum sp. (energycane), Salix spp. (willow), Sanguinaria canadensis, Scopolia spp., Secale cereale (rye), Solanum melongena (eggplant), Solanum tuberosum (potato), Sorghum spp., Sorghum bicolor (sorghum, sudangrass), Spartina pectinata (prairie cord-grass), Spinacea oleracea (spinach), Rauwolfia serpentina, Rauwolfia spp., Ricinus communis (castor), Tanacetum parthenium, Taxus baccata, Taxus brevifolia, Theobroma cacao (cocoa), Triticosecale (triticum-wheat X rye), Triticum aestivum (wheat), Uniola paniculata (oats), Veratrum californica, Vinca rosea, Vitis vinifera (grape), and Zea mays (corn).
In some embodiments, the transgenic plant is fertile. In some embodiments, the transgenic plant is not fertile (i.e., sterile).
Using transformation methods, genetically modified plants, plant cells, plant tissue, seeds, and the like can be obtained.
Transformation according to the present invention may be performed by any suitable method. In certain embodiments, transformation comprises steps of introducing a nucleic acid construct, as described above, into a plant cell or protoplast to obtain a stably transformed plant cell or protoplast; and regenerating a whole plant from the stably transformed plant cell or protoplast.
Cell Transformation
Delivery or introduction of a nucleic acid construct into eukaryotic cells may be accomplished using any of a variety of methods. The choice of a particular method used for the transformation is not critical to the instant invention. Suitable techniques include, but are not limited to, non-biological methods, such as microinjection, microprojectile bombardment, electroporation, induced uptake, and aerosol beam injection, as well as biological methods such as direct DNA uptake, liposome-mediated transfection, polyethylene glycol-mediated transfection, and Agrobacterium-mediated transformation. Any combinations of the above methods that provide for efficient transformation of plant cells or protoplasts may also be used in the practice of the invention.
Methods of introduction of nucleic acid constructs into plant cells or protoplasts have been described. See, for example, “Methods for Plant Molecular Biology”, Weissbach and Weissbach (Eds.), 1989, Academic Press, Inc; “Plant Cell, Tissue and Organ Culture: Fundamental Methods”, 1995, Springer-Verlag: Berlin, Germany; and U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,240,855; 5,302,523; 5,322,783; 5,324,646; 5,384,253; 5,464,765; 5,538,877; 5,538,880; 5,550,318; 5,563,055; and 5,591,616).
In particular, electroporation has frequently been used to transform plant cells (see, for example, U.S. Pat. No. 5,384,253). This method is generally performed using friable tissues (such as a suspension culture of cells or embryogenic callus) or target recipient cells from immature embryos or other organized tissue that have been rendered more susceptible to transformation by electroporation by exposing them to pectin-degrading enzymes or by mechanically wounding them in a controlled manner Intact cells of maize (see, for example, K. D'Halluin et al., Plant cell, 1992, 4: 1495-1505; C. A. Rhodes et al., Methods Mol. Biol. 1995, 55: 121-131; and U.S. Pat. No. 5,384,253), wheat, tomato, soybean, and tobacco have been transformed by electroporation. As reviewed, for example, by G. W. Bates (Methods Mol. Biol. 1999, 111: 359-366), electroporation can also be used to transform protoplasts.
Another method of transformation is microprojectile bombardment (e.g., through use of a “gene gun”) (see, for example, U.S. Pat. Nos. 5,538,880; 5,550,318; and 5,610,042; and WO 94/09699). In this method, nucleic acids are delivered to living cells by coating or precipitating the nucleic acids onto a particle or microprojectile (for example tungsten, platinum or gold), and propelling the coated microprojectile into the living cell. Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any monocotyledonous or dicotyledonous plant species (see, for example, U.S. Pat. Nos. 5,036,006; 5,302,523; 5,322,783 and 5,563,055; WO 95/06128; A. Ritala et al., Plant Mol. Biol. 1994, 24: 317-325; L. A. Hengens et al., Plant Mol. Biol. 1993, 23: 643-669; L. A. Hengens et al., Plant Mol. Biol. 1993, 22: 1101-1127; C. M. Buising and R. M. Benbow, Mol. Gen. Genet. 1994, 243: 71-81; C. Singsit et al., Transgenic Res. 1997, 6: 169-176).
The use of Agrobacterium-mediated transformation of plant cells is well known in the art (see, for example, U.S. Pat. No. 5,563,055). This method has long been used in the transformation of dicotyledonous plants, including Arabidopsis and tobacco, and has recently also become applicable to monocotyledonous plants, such as rice, wheat, barley and maize (see, for example, U.S. Pat. No. 5,591,616). In plant strains where Agrobacterium-mediated transformation is efficient, it is often the method of choice because of the facile and defined nature of the gene transfer. In some embodiments, Agrobacterium-mediated transformation of plant cells is carried out in two phases. First, the steps of cloning and DNA modifications are performed in E. coli, and then the plasmid containing the gene construct of interest is transferred by heat shock treatment into Agrobacterium, and the resulting Agrobacterium strain is used to transform plant cells. In some embodiments, Agrobacterium infiltrates plant leaves. In some embodiments, the bacterial strain Agrobacterium tumefaciens is used to transform plant cells.
Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., I. Potrykus et al., Mol. Gen. Genet. 1985, 199: 169-177; M. E. Fromm et al., Nature, 1986, 31: 791-793; J. Callis et al., Genes Dev. 1987, 1: 1183-1200; S. Omirulleh et al., Plant Mol. Biol. 1993, 21: 415-428).
Alternative methods of plant cell transformation, that have been reviewed, for example, by M. Rakoczy-Trojanowska (Cell Mol. Biol. Lett. 2002, 7: 849-858; the contents of which are herein incorporated by reference in their entirety), can also be used in the practice of the present invention.
In some embodiments, successful delivery of the nucleic acid construct into the host plant cell or protoplast is preliminarily evaluated visually. Selection of stably transformed plant cells can be performed, for example, by introducing into the cell a nucleic acid construct comprising a marker gene which confers resistance to some normally inhibitory agent, such as an antibiotic or herbicide. Examples of antibiotics that may be used include aminoglycoside antibiotics (such as neomycin, kanamycin, and paromomycin) and the antibiotic hygromycin. Several aminoglycoside phosphotransferases confer resistance to aminoglycoside antibiotics, and include aminoglycoside phosphotransferase I (aph-I) enzyme and aminoglycoside (or neomycin) phosphotransferase II (APH-II or NPTII), which, though unrelated, both have ability to inactivate the antibiotic G418. The hygromycin phosphotransferase (denoted hpt, hph or aphIV) gene was originally derived from Escherichia coli. Hygromycin phosphotransferase (HPT) detoxifies the aminocyclitol antibiotic hygromycin B. As is known in the art, plants have been transformed with the hpt gene, and hygromycin B has proved very effective in the selection of a wide range of plants.
Examples of herbicides that may be used include phosphinothricin and glyphosate. Potentially transformed cells then are exposed to the selective agent. Cells where the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival will generally be present in the population of surviving cells.
Alternatively or additionally, host cells comprising a nucleic acid sequence of the invention and expressing a gene product encoding by inventive nucleic acids may be identified and selected by a variety of procedures, including, but not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques such as membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acids or proteins.
Plant cells are available from a wide range of sources including the American Type Culture Collection (Rockland, Md.), or from any of a number of seed companies including, for example, A. Atlee Burpee Seed Co. (Warminster, Pa.), Park Seed Co. (Greenwood, S.C.), Johnny Seed Co. (Albion, Me.), or Northrup King Seeds (Hartsville, S.C.). Descriptions and sources of useful host cells can be found in I. K. Vasil, “Cell Culture and Somatic Cell Genetics of Plants”, Vol. I, II and II; 1984, Laboratory Procedures and Their Applications Academic Press: New York; R. A. Dixon et al., “Plant Cell Culture—A Practical Approach”, 1985, IRL Press: Oxford University; and Green et al., “Plant Tissue and Cell Culture”, 1987, Academic Press: New York.
Plant cells or protoplasts stably transformed according to the present invention are provided herein.
Plant Regeneration
In plants, every cell is capable of regenerating into a mature plant and contributing to the germ line such that subsequent generations of the plant will contain the transgene of interest. Stably transformed cells may be grown into plants according to conventional ways (see, for example, McCormick et al., Plant Cell Reports, 1986, 5: 81-84). Plant regeneration from cultured protoplasts has been described, for example by Evans et al., “Handbook of Plant Cell Cultures”, Vol. 1, 1983, MacMilan Publishing Co: New York; and I. R. Vasil (Ed.), “Cell Culture and Somatic Cell Genetics of Plants”, Vol. I (1984) and Vol. II (1986), Acad. Press: Orlando.
Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a Petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently roots. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and plant hormones, such as auxin and cytokinins. Glutamic acid and proline may also be added to the medium. Efficient regeneration generally depends on the medium, on the genotype, and on the history of the culture.
Regeneration from transformed individual cells to obtain transgenic whole plants has been shown to be possible for a large number of plants. For example, regeneration has been demonstrated for dicots (such as apple; Malus pumila; blackberry, Rubus; Blackberry/raspberry hybrid, Rubus; red raspberry, Rubus; carrot; Daucus carota; cauliflower; Brassica oleracea; celery; Apium graveolens; cucumber; Cucumis sativus; eggplant; Solanum melongena; lettuce; Lactuca sativa; potato; Solanum tuberosum; rape; Brassica napus; soybean (wild); Glycine canescens; strawberry; Fragaria×ananassa; tomato; Lycopersicon esculentum; walnut; Juglans regia; melon; Cucumis melo; grape; Vitis vinifera; and mango; Mangifera indica) as well as for monocots (such as rice; Oryza sativa; rye, Secale cereale; and Maize).
Primary transgenic plants may then be grown using conventional methods. Various techniques for plant cultivation are well known in the art. Plants can be grown in soil, or alternatively can be grown hydroponically (see, for example, U.S. Pat. Nos. 5,364,451; 5,393,426; and 5,785,735). Primary transgenic plants may be either pollinated with the same transformed strain or with a different strain and the resulting hybrid having the desired phenotypic characteristics identified and selected. Two or more generations may be grown to ensure that the subject phenotypic characteristics is stably maintained and inherited and then seeds are harvested to ensure that the desired phenotype or other property has been achieved.
As is well known in the art, plants may be grown in different media such as soil, growth solution or water.
Selection of plants that have been transformed with the construct may be performed by any suitable method, for example, with northern blot, Southern blot, herbicide resistance screening, antibiotic resistance screening or any combinations of these or other methods. The Southern blot and northern blot techniques, which test for the presence (in a tissue such as a plant tissue) of a nucleic acid sequence of interest and of its corresponding RNA, respectively, are standard methods (see, for example, Sambrook & Russell, “Molecular Cloning”, 2001, Cold Spring Harbor Laboratory Press: Cold Spring Harbor).
Transgenic plants and plant parts disclosed herein may be used advantageously in a variety of applications. In many embodiments, transgenic plants of the present invention express polypeptides that confer desirable traits to the plant and/or plant biomass (e.g., resistance to herbicides, resistance to environmental stress, resistance to pests and diseases). In some embodiments, expression of such polypeptides results in downstream process innovations and/or improvements in a variety of applications including ethanol production, phytoremediation and hydrogen production.
In some embodiments, plants transformed according to the present invention provide a means of increasing ethanol yields, reducing pretreatment costs by reducing acid/heat pretreatment requirements for saccharification of biomass; and/or reducing other plant production and processing costs, such as by allowing multi-applications and isolation of commercially valuable by-products. For example, a gene regulatory element provided by the present disclosure may drive expression of one or more lignocellulolytic enzyme polypeptide(s) and/or cell wall modifying enzyme polypeptide(s) in a transgenic plant and such enzyme polypeptides may allow biomass from the transgenic plant to be processed to produce more easily and/or cost effectively.
Plant Culture
Farmers can grow different transgenic plants of the present invention (e.g., different variety of transgenic corn, each expressing a transgenic polypeptide or RNA) simultaneously, achieving the desired “blend” of gene products produced by changing the seed ratio.
Plant Harvest
Transgenic plants of the present invention can be harvested as known in the art. For example, current techniques may cut corn stover at the same time as the grain is harvested, but leave the stover lying in the field for later collection. However, dirt collected by the stover can interfere with ethanol production from lignocellulosic material. The present invention provides a method in which transgenic plants are cut, collected, stored, and transported so as to minimize soil contact. In addition to minimizing interference from dirt with ethanol production, this method can result in reduction in harvest and transportation costs.
Tempering
In some embodiments, provided transgenic plants undergo a tempering phase that conditions the biomass for pretreatment and hydrolysis. Tempering may facilitate reducing severity of pretreatment conditions to achieve a desired glucan conversion yield and/or improving hydrolysis and glucan conversion after treatment. For example, a typical yield from biomass that has been pretreated under standard pretreatment conditions (e.g., 1% sulfuric acid, 170° C., for 10 minutes) is at least 80% glucan conversion. When tempered as described herein, the same typical yield may be achieved under less severe pretreatment conditions and/or with reduced amounts of externally applied enzymes. Less severe pretreatment conditions may comprise, for example, reduced acid concentrations, lower incubation temperatures, and/or shorter pretreatment times.
In some embodiments, when tempered as described herein and using the same pretreatment conditions, typical yield may be increased above at least 80% glucan conversion.
Without wishing to be bound by any particular theory, tempering may facilitate such improvements by, for example, allowing activation of endoplant enzyme polypeptides after harvest, increasing susceptibility of lignin and hemicellulose to traditional pretreatment, and/or increasing accessibility of polysaccharides (e.g., cellulose).
A variety of techniques for tempering may be used. In some embodiments, tempering comprises increasing the temperature of the biomass to activate thermophilic enzymes. Increasing the temperature to activate thermophilic enzymes may be achieved, for example, by one or more of ensilement, grinding, pelleting, and warm water suspension/slurries. In some embodiments, tempering comprises disrupting cell walls. Cell wall disruption may be achieved, for example, by sonication and/or liquid extraction to release enzyme polypeptides from sequestered locations in the plant (which may allow further activation and/or extraction to be added back after pretreatment). In some embodiments, tempering comprises adding accessory enzyme polypeptides during an incubation period before pretreatment. Such accessory enzyme polypeptides may weaken cross linking and improve accessibilty of the biomass to embedded glucanases or xylanases. In some embodiments, tempering comprises incubating the biomass in a particular set of conditions (e.g., a particular temperature, particular pH, and/or particular moisture conditions). Such incubations may in some embodiments increase susceptibility to various glucanases and/or accessory enzyme polypeptides present in the plant tissues or added to the sample. For example, samples may be tempered as a liquid slurry (e.g., comprising about 10% to about 30% total solids) under conditions favorable to activate cell wall-modifying enzymes. In some embodiments, samples are tempered as a liquid slurry for about 1 to about 48 hours. In some embodiments, conditions favorable to activate cell wall-modifying enzymes comprise a pH of about 4 to about 7 and a temperature of about 25° C. to about 100° C. Alternatively or additionally, samples may be tempered as a lower moisture ensilement (e.g., about 40% to about 60% total solids) under anaerobic conditions. In some embodiments, samples are ensiled for about 21 days to several months.
In some embodiments, tempering is integrated with other processes such as one or more of harvest, storage, and transportation of biomass. For example, biomass can be ensiled under conditions that condition the biomass for subsequent pretreatment and hydrolysis; that is, storage and tempering are combined. In some embodiments, during ensilement of biomass, temperatures are increased in the ensiled material such that thermally active embedded enzymes are activated. Ensilement conditions may allow preservation of biomass while providing sufficient time for enzyme polypeptides to affect characteristics of the biomass (such as, for example, amenability to pretreatment and improvement of subsequent hydrolysis).
In some embodiments, the tempering phase precedes entirely the pretreatment phase. In some embodiments, the tempering phase overlaps with the pretreatment phase.
In some embodiments as described herein, transgenic plants express more than one cell wall-modifying enzyme polypeptide. In some such embodiments, it may be desirable to activate enzyme polypeptides sequentially. It may be desirable to do so, for example, if the efficiency of endoplant enzymes is a function of the sequence in which they are activated. For example, beta-glucosidases may be most efficient after endo- and exoglucanases have cleaved cellulose into dimers, and cellulases and hemicellulases may be more efficient when accessory enzymes have reduced cross-linkages between cellulose, hemicellulose, and lignin. Accordingly, in some embodiments, cellulases might be activated after ferulic acid esterases (FAEs) have had the opportunity to cleave ferulate-polysaccharide-lignin complexes, or after other accessory enzymes have had the opportunity to cleave cellulose-hemicellulose cross linkages.
Sequential activation could be attained, for example, by using enzymes with different peak temperature and/or pH optima. Increasing temperature continually or stepwise (e.g., during a tempering step), could thereby allow activation of enzyme polypeptides with lower temperature optima first. For example, a wound-induced promoter could be used to produce a non-thermostable enzyme polypeptide after harvesting that breaks lingin cross-links and leads to cell death, before increasing temperature during tempering to activate a thermostable cellulase in the biomass.
In some embodiments as described herein, cell wall-modifying enzyme polypeptides are specifically targeted to organelles and/or plant parts. In some embodiments, cell wall-modifying enzyme polypeptides are specifically targeted to seeds. Cell wall hydrolyzing enzymes in the grain could improve yields of fermentable sugars by targeting the cellulose and hemicelluolose in the grain bran and fiber, or could loosen or weaken the outer layers of the grain kernel, making it easier to mill. Starch in corn grain is often processed to produce ethanol, but significant quantities of cellulose and hemicellulose from the bran and fiber are not used. In some embodiments, incorporating a tempering step prior to starch hydrolysis (e.g., of transgenic corn grain), endogenous enzymes can act on the fiber and bran and increase the yield of fermentable sugars. In some embodiments, dry seed (e.g., dry wheat) is tempered by soaking in water at a slightly elevated temperature for several hours before further processing. Such a tempering step may decrease the energy required for milling and increase the quality and eventual yield. Endogenous enzymes in the grain may also provide additional benefits.
In some embodiments, tempering comprises externally applying an amount of at least one cell wall-modifying enzyme polypeptide. External application of cell wall-modifying enzyme polypeptides is discussed in more detail in the “Saccharification” section.
In some embodiments, the seed or grain of a transgenic plant is tempered.
Pretreatment
Conventional methods for processing plant biomass include physical, chemical, and/or biological pretreatments. For example, physical pretreatment techniques can include one or more of various types of milling, crushing, irradiation, steaming/steam explosion, and hydrothermolysis. Chemical pretreatment techniques can include acid, alkaline, organic solvent, ammonia, sulfur dioxide, carbon dioxide, and pH-controlled hydrothermolysis. Biological pretreatment techniques can involve applying lignin-solubilizing microorganisms (T.-A. Hsu, “Handbook on Bioethanol: Production and Utilization”, C. E. Wyman (Ed.), 1996, Taylor & Francis: Washington, D.C., 179-212; P. Ghosh and A. Singh, A., Adv. Appl. Microbiol., 1993, 39: 295-333; J. D. McMillan, in “Enzymatic Conversion of Biomass for Fuels Production”, M. Himmel et al., (Eds.), 1994, Chapter 15, ACS Symposium Series 566, American Chemical Society: B. Hahn-Hagerdal, Enz. Microb. Tech., 1996, 18: 312-331; and L. Vallander and K. E. L. Eriksson, Adv. Biochem. Eng./Biotechnol., 1990, 42: 63-95). The purpose of the pretreatment step is to break down the lignin and carbohydrate structure to make the cellulose fraction accessible to cellulolytic enzymes.
Simultaneous use of transgenic plants that express one or more enzyme polypeptides (e.g., lignocellulolytic enzyme polypeptides and/or cell wall-modifying enzyme polypeptides) according to the present invention may reduce or eliminate expensive grinding of the biomass and/or reduce or eliminate the need for heat and strong acid required to strip lignin and hemicellulose away from cellulose before hydrolyzing the cellulose.
In some embodiments, lignocellulosic biomass of plant parts obtained from inventive transgenic plants is more easily hydrolyzable than that of non-transgenic plants. Thus, the extent and/or severity of pretreatment required to achieve a particular level of hydrolysis is reduced. Therefore, the present invention in some embodiments provides improvements over existing pretreatment methods. Such improvements may include one or more of: reduction of biomass grinding, elimination of biomass grinding, reduction of the pretreatment temperature, elimination of heat in the pretreatment, reduction of the strength of acid in the pretreatment step, elimination of acid in the pretreatment step, and any combination thereof.
In some embodiments, lower temperatures of pretreatment may be used to achieve a desired level of hydrolysis. In some embodiments, pretreating is performed at temperatures below about 175° C., below about 145° C., or below about 115° C. For example, under some conditions, the yield of hydrolysis products from lignocellulosic biomass from transgenic plant parts pretreated at about 140° C. is comparable to the yield of hydrolysis products from non-transgenic plant parts pretreated at about 170° C. Under some conditions, the yield of hydrolysis products from lignocellulosic biomass from transgenic plant parts pretreated at about 170° C. is above about 60%, above about 70%, above about 80%, or above about 90% of theoretical yields. Under some conditions, the yield of hydrolysis products from lignocellulosic biomass from transgenic plant parts pretreated at about 140° C. is above about 60%, above about 70%, or above about 80% of theoretical yields. Under some conditions, the yield of hydrolysis products from lignocellulosic biomass from transgenic plant parts pretreated at about 110° C. is above about 40%, above about 50%, or above about 60% of theoretical yields. Such yields from transgenic plant parts can represent an increase of up to about 20% of yields from non-transgenic plant parts.
In some embodiments, such improvements are observed in inventive transgenic plants expressing an enzyme polypeptide (e.g., a cell wall-modifying enzyme polypeptide and/or lignocellulolytic enzyme polypeptide) at a level less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1% of total soluble protein. Without wishing to be bound by any particular theory, the inventors propose that low levels of enzyme expression may facilitate modifying the cell wall, possibly by nicking cellulose or hemicellulose strands. Such modification of the cell wall may make the biomass more susceptible to pretreatment. Thus, biomass from inventive transgenic plants expressing low levels of cell wall-modifying enzymes may require less pretreatment, and/or pretreatment in less severe conditions.
In certain embodiments, the pretreated material is used for saccharification without further manipulation. In other embodiments, it is desired to process the plant tissue so as to produce an extract comprising the cell wall-modifying enzyme polypeptide(s). In this case, the extraction is carried out in the presence of components known in the art to favor extraction of active enzymes from plant tissue and/or to enhance the degradation of cell-wall polysaccharides in the lignocellulosic biomass. Such components include, but are not limited to, salts, chelators, detergents, antioxidants, polyvinylpyrrolidone (PVP), and polyvinylpolypyrrolidone (PVPP). The remaining plant tissue may then be submitted to a pretreatment process.
Saccharification
In saccharification (or enzymatic hydrolysis), lignocellulose is converted into fermentable sugars (i.e., glucose monomers) by enzyme polypeptides present in the pretreated material. If desired, externally applied cellulolytic enzyme polypeptides (i.e., enzymes not produced by the transgenic plants being processed) may be added to this mixture. Extracts comprising transgenically expressed enzyme polypeptides obtained as described above can be added back to the lignocellulosic biomass before saccharification. Here again, externally applied cellulolytic enzyme polypeptides may be added to the saccharification reaction mixture.
In some embodiments, the amount of externally applied enzyme polypeptide that is required to achieve a particular level of hydrolysis of lignocellulosic biomass from inventive transgenic plants is reduced as compared to the amount required to achieve a similar level of hydrolysis of lignocellulosic biomass from non-transgenic plants. For example, in some embodiments, processing transgenic lignocellulosic biomass in the presence of as low as 15 mg externally applied cellulase per gram of biomass (15 mg/g) yields a similar level of hydrolysis as processing non-transgenic lignocellulosic biomass in the presence of 100 mg/g cellulase. This represents a reduction of almost 90% of cellulases needed for hydrolysis can be achieved when processing biomass from inventive transgenic plants. Such a reduction in externally applied cellulases used can represent significant cost savings.
In some embodiments, a mixture of enzyme polypeptides each having different enzyme activities (e.g., exoglucanase, endoglucanase, hemi-cellulase, beta-glucosidase, and combinations thereof), and/or an enzyme polypeptide having more than one enzyme activity (e.g., exoglucanase, endoglucanase, hemi-cellulase, beta-glucosidase, and combinations thereof) is added during a “treatment” step to promote saccharification. Without wishing to be bound by any particular theory, such combinations of enzyme activity, whether through the activity of an enzyme complex or other mixture of enzymes, may allow a greater degree of hydrolysis than can be achieved with a single enzyme activity alone. Commercially available enzyme complexes that can be employed in the practice of the invention include, but are not limited to, Accellerase™ 1000 (Genencor), which contains multiple enzyme activities, mainly exoglucanase, endoglucanase, hemi-cellulase, and beta-glucosidase.
Saccharification is generally performed in stirred-tank reactors or fermentors under controlled pH, temperature, and mixing conditions. A saccharification step may last up to 200 hours. Saccharification may be carried out at temperatures from about 30° C. to about 65° C., in particular around 50° C., and at a pH in the range of between about 4 and about 5, in particular, around pH 4.5. Saccharification can be performed on the whole pretreated material.
The present Applicants had previously shown that adding cellulases to plants transgenically expressing E1, an endoglucanse (EC 3.2.1.4) increases total glucose production compared to adding cellulases to non-transgenic plants, which suggests that simply using transgenic E1 plants with current external cellulase techniques can substantially increase yields of products (e.g., such as ethanol, methanol, butanol, and/or other alcohols). The experiment also indicates that adding cellulases to E1 plants increases total glucose production compared to adding cellulases to non-transgenic plants. This is an important result since it suggests that simply using transgenic E1 plants with current external cellulase techniques can substantially increase yields of products (e.g., of ethanol, methanol, butanol, and/or other alcohols) in the presence or absence of pretreatment processes.
Fermentation
In the fermentation step, sugars, released from the lignocellulose as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to one or more organic substances (e.g., ethanol, methanol, butanol, or other alcohols) by a fermenting microorganism, such as yeasts and/or bacteria. The fermentation can also be carried out simultaneously with the enzymatic hydrolysis in the same vessels, again under controlled pH, temperature and mixing conditions. When saccharification and fermentation are performed simultaneously in the same vessel, the process is generally termed simultaneous saccharification and fermentation or SSF.
Fermenting microorganisms and methods for their use inproduction (e.g., of ethanol, methanol, butanol, and/or other alcohols) are known in the art (Sheehan, “The Road to Bioethanol: A Strategic Perspective of the US Department of Energy's National Ethanol Program” In: “Glycosyl Hydrolases For Biomass Conversion”, ACS Symposium Series 769, 2001, American Chemical Society: Washington, D.C.). Existing ethanol production methods that utilize corn grain as the biomass typically involve the use of yeast, particularly strains of Saccharomyces cerevisiae. Such strains can be utilized in the methods of the invention. While such strains may be preferred for the production of alcohols (e.g., ethanol, methanol, and butanol) from glucose that is derived from the degradation of cellulose and/or starch, the methods of the present invention do not depend on the use of a particular microorganism, or of a strain thereof, or of any particular combination of said microorganisms and said strains.
Yeast or other microorganisms are typically added to the hydrolysate and the fermentation is allowed to proceed for 24-96 hours, such as 35-60 hours. The temperature of fermentation is typically between 26-40° C., such as 32° C., and at a pH between 3 and 6, such as about pH 4-5.
A fermentation stimulator may be used to further improve the fermentation process, in particular, the performance of the fermenting microorganism, such as, rate enhancement and yield (e.g., of ethanol, methanol, butanol, and/or other alcohols). Fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamin, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and vitamins A, B, C, D, and E (Alfenore et al., “Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process”, 2002, Springer-Verlag). Examples of minerals include minerals and mineral salts that can supply nutrients comprising phosphate, potassium, manganese, sulfur, calcium, iron, zinc, magnesium and copper.
Recovery
Following fermentation (or SSF), the mash is distilled to extract the alcohol (e.g., ethanol, methanol, butanol, and/or other alcohols). Alcohol with a purity greater than 96 vol. % can be obtained.
By-Products
The hydrolysis process of lignocellulosic raw material also releases by-products such as weak acids, furans, and phenolic compounds, which are inhibitory to the fermentation process. Removing such by-products may enhance fermentation.
In some embodiments, processing of provided transgenic plants comprise removing, from the hydrolysate, products of the enzymatic process that cannot be fermented. Such products comprise, but are not limited to, lignin, lignin breakdown products, phenols, and furans. In certain embodiments, products of the enzymatic process that cannot be fermented are separated and used subsequently. For example, products can be burned to provide heat required in some steps of the alcohol (e.g., ethanol, methanol, butanol) production such as saccharification, fermentation, and alcohol (e.g., ethanol, methanol, butanol) distillation, thereby reducing costs by reducing the need for current external energy sources such as natural gas. Alternatively or additionally, such by-products may have commercial value. For example, phenols can find applications as chemical intermediates for a wide variety of applications, ranging from plastics to pharmaceuticals and agricultural chemicals. Phenol condensed to with aldehydes (e.g., methanol) make resinous compounds, which are the basis of plastics which are used in electrical equipment and as bonding agents in manufacturing wood products such as plywood and medium density fiberboard (MDF).
Separation of by-products from the hydrolysate can be done using a variety of chemical and physical techniques that rely on the different chemical and physical properties of the by-products (e.g., lignin and phenols). Such techniques include, but are not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, distillation, or extraction.
Some of the hydrolysis by-products, such as phenols, or fermentation/processing products, such as methanol, can be used as ethanol denaturants. Currently about 5% gasoline is added immediately to distilled ethanol as a denaturant under the Bureau of Alcohol, Tobacco and Firearms regulations, to prevent unauthorized non-fuel use. This requires shipping gasoline to the ethanol production plant, then shipping the gas back with the ethanol to the refinery. The gas also impedes the use of ethanol-optimized engines that make use of ethanol's higher compression ratio and higher octane to improve performance. Using transgenic plant derived phenols and/or methanol as denaturants in lieu of gasoline can reduce costs and increase automotive engine design alternatives.
Reducing Lignin Content
Another way of reducing lignin and lignin breakdown products that are not fermentable in hydrolysate is to reduce lignin content in a transgenic plant of the present invention. Such methods have been developed and can be used to modify the inventive plants (see, for example, U.S. Pat. Nos. 6,441,272 and 6,969,784, U.S. Pat. Appln. No. 2003-0172395, US and PCT publication No. WO 00/71670).
Combined Starch Hydrolysis and Cellulolytic Material Hydrolysis
Transgenic plants and plant parts disclosed herein can be used in methods involving combined hydrolysis of starch and of cellulosic material for increased yields (e.g, of ethanol, methanol, butanol, and/or other alcohols). In addition to providing enhanced yields, these methods can be performed in existing starch-based alcohol processing facilities.
Starch is a glucose polymer that is easily hydrolyzed to individual glucose molecules for fermentation. Starch hydrolysis may be performed in the presence of an amylolytic microorganism or enzymes such as amylase enzymes. In certain embodiments of the invention, starch hydrolysis is performed in the presence of at least one amylase enzyme. Examples of suitable amylase enzymes include α-amylase (which randomly cleaves the α(1-4)glycosidic linkages of amylose to yield dextrin, maltose or glucose molecules) and glucoamylase (which cleaves the α(1-4) and α(1-6)glycosidic linkages of amylose and amylopectin to yield glucose).
Hydrolysis of starch and hydrolysis of cellulosic material from provided transgenic plants can be performed simultaneously (i.e., at the same time) under identical conditions (e.g., under conditions commonly used for starch hydrolysis). Alternatively, the hydrolytic reactions can be performed sequentially (e.g., hydrolysis of lignocellulose can be performed prior to hydrolysis of starch). When starch and cellulosic material are hydrolyzed simultaneously, the conditions are preferably selected to promote starch degradation and to activate cell wall-modifying enzyme polypeptide(s) for the degradation of lignocellulose. Factors that can be varied to optimize such conditions include physical processing of the plants or plant parts, and reaction conditions such as pH, temperature, viscosity, processing times, and addition of amylase enzymes for starch hydrolysis.
Provided transgenic plants (or plant parts) may be used alone or in a mixture with non-transgenic plants (or plant parts). Suitable plants include any plants that can be employed in starch-based alcohol production (e.g., corn, wheat, potato, cassaya, etc.). For example, the present inventive methods may be used to increase ethanol yields from corn grains.
The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the invention.
In the present example, gene regulatory elements were identified and isolated from the poplar genome. Promoters of poplar genes were identified by searching for gene sequences similar to that of genes having or suspected of having desirable expression patterns in other plants. Nucleic acids containing identified promoters were isolated by polymerase chain reaction (PCR)-based amplification. These gene regulatory elements may be useful, for example, in driving expression of genes in transgenic plants.
Two methods were used to identify gene regulatory elements of interest from the poplar genome. In one method, protein sequences of interest were identified in the poplar proteome (see the website whose address is “http://” followed immediately by “genome.jgi-psf.org/Poptr1—1/Poptr1—1.home.html”) using the BLASTp tool by searching for sequences similar to protein sequences of Arabidopsis or maize gene products having functions and/or expression patterns of interest. Protein sequences of gene products from Arabidopsis or maize that are known to be involved in cell structure and function, intermediary metabolism, or tissue-specific and developmental stage-specific functions were used as input in the BLASTp searches. Genomic DNA sequences that encode the putative homologous proteins were then identified.
In a second method, publicly available EST databases were analyzed for the abundance of particular transcripts in various tissues and corresponding full length genes were identified in the poplar genome.
Both approaches resulted in identification of poplar genomic DNA sequences of interest, and novel regulatory DNA sequences within and/or in the proximity of such genomic DNA sequences were discovered for several classes of genes. The 3 kb region upstream of the genes were identified using a poplar genome database and designated as regulatory DNA sequences.
These novel regulatory DNA sequences are listed as SEQ ID NO: 1 through SEQ ID NO: 158 of the accompanying Sequence Listing.
Using genomic information obtained as described above, oligonucleotides (see Table 2) were designed for use as PCR primers to amplify various poplar gene regulatory elements. Poplar promoter sequences were amplified with high-fidelity Phusion Taq Polymerase (New England Biolabs, MA) or PLANTAMP™ PCR system (Epicentre Biotechnologies WI) using genomic DNA isolated from young leaves of two month old hybrid poplar plants (Poplulus tremula×P. alba, clone 717) as template. Gradient PCR was performed using a dual block thermal cycler (Biorad, CA) for optimum amplification of promoter sequences. PCR-amplified fragments were gel-purified and cloned into intermediate pCR Blunt vectors using the Zero Blunt PCR cloning kit (Invitrogen, CA).
A variety of promoters were isolated and cloned in this manner, including the promoters for the L5L2 gene, (SEQ ID NO: 117) which encodes an L5-like ribosomal protein; an ubiquitin protein that is constitutively expressed in eukaryotic genes (Ubi2) (SEQ ID NO: 90); early response to dehydration 4 (ERD4) (SEQ ID NO: 137), a gene within a family that signals water deficiency and results in metabolic pathways to mitigate osmotic stress; methionine adenosyltransferase 2 (SAM2) (SEQ ID NO: 131), which synthesizes S-adenosylmethionine (SAM) from methionine and ATP; calmodulin protein (CalL2) (SEQ ID NO:103), which induces responses to change in Ca2+ concentrations in cells; and elongation factor protein (Elf1a) (SEQ ID NO: 94), which plays a role in translational elongation. A 2 kb PtPAL2 (SEQ ID NO: 158) promoter belonging to the class of phenyl alanine ammonia lyase gene family, which is involved in the biosynthesis of phenylpropanoids, was also isolated and cloned.
Construction of base expression vectors to generate reporter gene fusions pUC18 (Invitrogen, CA), a high-copy number cloning vector pUC18, was used for creating base expression vectors. First, a region comprising the coding sequences of the β-glucuronidase (GUS) gene with (GUSintron) or without (GUS) the catalase intron and the NOS terminator (NOS) was PCR-amplified from a plasmid. The catalase intron within the GUS gene is spliced out during the process of transcription in plant cells and eliminates background expression in bacteria. Restriction enzyme recognition sites BamHI-KpnI were engineered into PCR primers ES190 and ES191 (see Table 2). PCR amplified GUSintron-NOS and GUS-NOS fragments were digested with BamHI-KpnI enzymes and cloned into pUC18 vector to create the pUC18-GUSintron-NOS and pUC18-GUS-NOS vectors. Next, a multi cloning site (MCS) cassette comprising HindIII-AscI-PstI-SalI-PacI-NotI-XhoI-SpeI-HpaI-XbaI-BamHI restriction enzyme recognition sites was PCR amplified, digested with HindIII-BamHI enzymes, and cloned into pUC18-GUSintron-NOS and pUC18-GUS-NOS to create pUC18-MCS-GUSintron-NOS (
Cloning of Poplar Promoters into Expression Vectors
In general, poplar promoters isolated as described in Example 1 were classified into two categories depending upon the presence or absence of the first intron located within the promoter region. Poplar promoters (PtP) without the first intron were digested from the vectors into which they had been cloned in Example 1 with appropriate restriction enzyme(s) and then cloned into the pUC18-MCS-GUSintron-NOS vector. Poplar promoters with the first intron were cloned into the pUC18-MCS-GUS-NOS vector to create pUC18-PtP-GUSintron-NOS (
Particle Bombardment of P. tremula×P. alba Leaves
Preparation of Tungsten Particles
M10 Tungsten particles (Sylvania, Mass.) were used for microprojectile bombardment experiments. Stock solution was prepared by washing 50 mg of tungsten particles in 500 μl 95% ethanol followed by washing them in water 4-6 times. Finally the particles were suspended in 500 μl ddH2O, Stock solution was used for a maximum of 12 hours after preparation. Twenty-five μl of the resuspended tungsten particles were mixed with 5 μl of DNA (200 to 500 ng/μl) in a microcentrifuge tube and vortexed for a few seconds. The mixture was allowed to sit at room temperature (RT) for 1 minute. DNA was precipitated by adding 25 μl of 2.5 M CaCl2 and 10 μl of 100 mM Spermidine and leaving the mixture on ice for 4 minutes; precipitated DNA adhered to the tungsten particles. Fifty microliters of the supernatant was discarded and the remaining coated particles were kept on ice. Two microliters of the tungsten particle preparation was used per shot within 15 minutes. The mixture was discarded after 15 minutes and freshly coated particles were prepared as needed for subsequent rounds of particle bombardment.
Particle Bombardment
Young and fully developed leaves from 2 to 3 month old poplar plants were used in the experiments. The leaves were placed in a petri dish with wet filter paper and bombarded with coated particles at 60 psi pressure and 28 psi in HG vacuum. After particle bombardment, leaf tissue samples were kept in Petri plates under moist conditions for a 24 hr period.
In order to monitor the GUS expression pattern for each transformed plasmid, bombarded poplar leaves were incubated with 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) in a standard procedure for 24-48 hr as previously described (Jefferson et al. 1987, “GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants,” EMBO J. 6:3901-3907, the entire contents of which is herein incorporated by reference). Tissue samples were cleared using 70% ethanol repeatedly until most of the chlorophyll was removed. Samples were observed for GUS expression (seen as blue spots) and images were taken using a Leica stereo microscope (Leica, N.J.). Results are presented in
Based on the strength of GUS expression as evaluated histochemically, poplar promoters were classified into high expressers (PtUbi2 and PtCal L2 (SEQ ID NOs: 90 and 103 respectively); medium expressers (PtL5L2 (SEQ ID NO: 117)) and weak expressers (PtElf1a (SEQ ID NO:94)). A well-known dicot promoter, CMPS (Cestrum yellow leaf curling virus promoter; U.S. Pat. No. 7,166,770) was included as a control. These results suggest that poplar promoters PtUbi2 and PtCal L2 can drive high levels of heterologous gene expression in dicots.
In the present Example, poplar gene regulatory elements of the present invention demonstrated an ability to drive expression in tissue-preferred manner. Experiments described in this Example also demonstrate that inventive gene regulatory elements can drive expression in tissues other than leaves.
Stem sections were used for particle bombardments. Both transverse and longitudinal sections were used from 3 month old plants. Particle bombardment and GUS activity experiments were performed as described in Example 2.
Results are presented in
These results demonstrate that the suite of inventive regulatory elements provided by the present invention include regulatory elements that can direct diverse tissue-preferred expression patterns.
In the present Example, inventive regulatory elements are used to drive expression of structural genes that encode proteins or polypeptides. Genes of interest (GOI) may include, but are not limited to, genes that encode cell wall modifying enzymes and genes that confer agronomically important traits, as described in the claims.
Stable Poplar Transformation with Poplar Promoters Driving Genes of Interest (GOIs)
A plant transformation binary vector pED-MCS-GOI-NOS was created to allow cloning of different poplar regulatory elements (including promoters) to drive genes of interest (
Total plant protein extract was made from fresh leaf tissue as described below. Leaf tissue was frozen in liquid nitrogen and homogenized in a bead beater (MINI BEADBEATER™, Biospec products) with 3 to 4 zirconium beads (2.0 mm size, Biospec products). Leaf protein was extracted by suspending the homogenized tissue in protein extraction buffer (50 mM MES, pH 5.6, 2 mM DTT, 1 mM EDTA, 1× protease inhibitor cocktail (Sigma P9599), 0.1% (w/v) Triton X-100). Samples were centrifuged and supernatants were used to determine total protein concentration using a Bradford protein assay. Two microliters of the total protein sample was incubated for 10 minutes with 200 μL of Bradford reagent (Quick Start Bradford 1× Dye Reagent, Bio-Rad, CA). The dye in the reagent turns blue when bound to the protein, which is measured at the 590 nm absorbance. Protein concentrations were determined by comparing absorbance readings to a standard curve of known bovine serum albumin (BSA) concentrations.
Glucan glucohydrolase (GGH) enzyme activity was determined using methyl-umbelliferyl-cellobioside (MUC) as a substrate. Fifteen microliters of protein extract was incubated for 1 hr at 85° C. in 200 μL of buffer (0.05 M sodium acetate, pH 5.0 and 0.1 M calcium chloride) containing 10 mM MUC fluorescent substrate. GGH enzyme activity cleaves the MUC substrate and produces fluorescent MU product, which is then read in a plate reader (Tecan GENios, MTX lab systems Inc, VA) at 355 nm excitation and 450 nm emission wavelengths. GGH enzyme activity was measured as strength of fluorescent signal normalized to mass in grams of protein used for analysis. The promoter strengths of the CMPS and PtL5L2 promoters were compared by measuring the level of GGH activity in protein extracts prepared from 5 different transgenic events. Based on the level of GGH expression, we concluded that the PtL5L2 promoter had similar or greater activity than the CMPS promoter (
Trangenic poplar plants were transformed with constructs containing poplar promoters driving GUS gene expression. These promoters included PtDREP4, PtERD4, PtSAM2, PtCal2, PtL5L2 and PtUbi2. Transgenic poplar leaf tissues were harvested for GUS protein expression from one month old plants, while roots were sampled from transgenic seedlings grown in magenta boxes. Tissues were incubated with 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) in a standard procedure for 24-48 hr as previously described (Jefferson et al. 1987). Tissue samples were cleared using 70% ethanol repeatedly until most of the chlorophyll was removed. Samples were observed for GUS activity (as indicated by blue color), and images were taken using a Leica stereo microscope (Leica, N.J.). Results are presented in
The present Example demonstrates that regulatory elements of the present invention can also be used to drive gene expression in other dicot plants.
Promoters PtUbi2 (SEQ ID NO: 90), PtL5L2 (SEQ ID NO: 117), PtPAL2 (SEQ ID NO: 158) and 35S were cloned into expression vectors to drive expression of an E1 gene encoding an endoglucanase. PtERD4 (SEQ ID NO:137), PtSAM2 (SEQ ID NOL 131) and PtUbi2 (SEQ ID NO: 90) were cloned into binary vectors to drive GUS gene expression (
In Vitro Enzyme Activity in Nicotiana benthamiana Infiltrations
Nine-week old Nicotiana benthamiana plants were infiltrated with suspensions of Agrobacteria containing the construct of interest. Each suspension was adjusted to an optical density (OD600) of 0.1 to ensure that equivalent concentrations of bacteria were injected into each leaf sample. Young leaves were injected with 0.75-1 mL of bacteria suspended in infiltration media. The infiltration media was buffered to pH 5.5 and contains a source of sugar and phosphate for the bacteria as well as acetylsyringone, which aids the bacteria in infection of plant cells. Following infiltration, plants were grown for three days at 74° F. in a room with a 14:10 photoperiod, supplemented with 50 μmol m−1 s−2 florescent and incandescent light.
The infiltrated region of each leaf was cut out and ground in buffer. Total leaf proteins were extracted through an ammonium sulfate precipitation before washing and resuspension in buffer. Total protein concentration was obtained with a bicinchoninic acid (BCA) protein assay and compared to a standard curve of known bovine serum albumin (BSA) concentrations by a colormetric reaction (Fisher, Rockford, Ill.).
Enzyme Assays
Total protein extracts were used to measure enzyme activity on methyl-umbelliferyl-cellobioside (MUC) and carboxymethyl cellulose (CMC) substrates. For MUC assays, proteins were incubated with the florescent substrate in buffer for 30 minutes. Endoglucanases cleave the fluorescent substrate; thus, the plate reader will read a stronger fluorescent signal from samples with higher endoglucanase activity. As shown in
The polysaccharide CMC is also hydrolysable by endoglucanases. Plant protein extracts were prepared from leaves infiltrated with either PtUbi2:E1 or 35S:E1, then were incubated with the CMC for 1 day at 65° C. before being measured by a colorimetric dinitrosalicylic acid (DNS) assay to quantify glucose concentration. If a promoter drives high expression of E1, then a high glucose concentration is expected in the corresponding protein extract. After 24-hr incubation of endoclucanase-containing constructs on CMC substrates, glucose equivalents were ˜0.217 mg for the PtUbi2 promoter and ˜0.211 mg for the 35S promoter, consistent with the MUC data in
Similarly to constructs used in in vitro enzyme assays, constructs with promoters of interest driving GUS were infiltrated into N. benthamiana and stained for GUS activity as described in Example 4. The PtERDL4 (pABC262) and PtSAML2 (pABC263) promoters drove low constitutive expression throughout the infiltrated leaf area (
All literature and similar material cited in this application, including, patents, patent applications, articles, books, treatises, dissertations and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including defined terms, term usage, described techniques, or the like, this application controls.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.
Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the invention being indicated by the following claims.
The present application claims priority to and benefit of U.S. provisional patent application 61/182,467, filed on May 29, 2009, the contents of which are herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2010/036945 | 6/1/2010 | WO | 00 | 11/29/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61182467 | May 2009 | US |
