# Patent

## Title
Modification of multidomain enzyme for expression in plants

## Abstract

Compositions and methods for expressing a multidomain enzyme in a plant are provided. The compositions include plants, seeds, plant tissues, and plant parts expressing a modified multidomain enzyme enzyme. The modified multidomain enzyme has a heterologous linker region that is not cleaved when the modified multidomain enzyme is expressed in a plant. In various embodiments, the linker region comprises the sequence set forth in SEQ ID NO:18, 19, or 20. Further provided are methods for producing a modified multidomain enzyme enzyme comprising cultivating plants expressing the modified multidomain enzyme. Downstream uses of transgenic plant material of the invention include agronomical and industrial uses, for example, human food, animal feed, pharmaceuticals, biofuel, industrial alcohol, fermentation feedstocks, and the like.

## Background
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named “72196WO Sequence Listing.txt”, created on Jan. 13, 2010, and having a size of 95 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
This invention relates to plant molecular biology, particularly to methods and compositions for increasing expression and/or activity of a protein in a plant.
BACKGROUND OF THE INVENTION
A number of heterologous expression systems have been devised over the last decade for the production of clinically and agronomically useful recombinant proteins. A significant challenge in most systems is to optimize the yield and quality of the recombinant protein product. Significant progress has been achieved over the last 15 years in the optimization of transgene transcription and translation in plants (Potenza et al., 2004, In Vitro Cell. Dev. Biol.-Plant, 40, 1-22; Streatfield, 2007, Plant Biotechnol. J. 5, 2-15) and the elucidation and modulation of the complex protein post-translational modifications characteristic of the plant cell machinery (Gomord and Faye, 2004, Curr. Opin. Plant Biol. 7, 171-181; Faye et al., 2005, Vaccine 23, 1770-1778). Despite these advances, ensuring satisfactory yield and quality of recombinant proteins often remains a difficult task.
One factor strongly influencing recombinant protein quality and yield is the relative inherent stability of polypeptide chains expressed in a heterologous environment (Faye et al., 2005).
Proteolytic enzymes, or proteases, contribute to the overall control of metabolic and transduction pathways by directing the activation or hydrolysis of proteins implicated in key regulatory processes, or by contributing to the elimination of misfolded proteins and the selective recycling of amino acids from short-lived proteins (Vierstra, 2003, Trends Plant Sci. 8, 135-142; Schaller, 2004, Planta, 220, 183-197). In plants, these enzymes also initiate the general recycling of proteins in senescing organs and the mobilization of amino acid constituents of seed or tuber storage proteins during germination (Müntz, 2007, J. Exp. Bot. 58, 2391-2407).
Proteases may affect the integrity of recombinant proteins in different ways, both in planta during protein expression and ex planta during extraction and subsequent downstream processing (Michaud et al., 1998, Methods Biotechnol. 3, 177-188; Rivard et al., 2006, Plant Biotechnol. J. 4, 359-368). Depending on the number of “susceptible” cleavage sites accessible to endogenous proteases for peptide bond hydrolysis, the protein may undergo complete hydrolysis directly impacting on its final yield or partial trimming, altering the activity or homogeneity of the final protein product. Although interesting yields may be obtained in terms of net protein levels, the final product may show altered integrity, structural heterogeneity and/or deficient biological activity, potentially altering its value for commercialization (Faye et al., 2005).
SUMMARY OF THE INVENTION
Compositions and methods thr expressing modified multidomain enzymes in a plant are provided. The compositions comprise plants, seeds, plant tissues, and plant parts expressing a modified multidomain enzyme, wherein the multidomain enzyme is composed of at least a first domain, at least a first linker sequence, and at least a second domain. The modified multidomain comprises a heterologous linker region that is not cleaved when the modified multidomain enzyme is expressed in a plant. Further provided are methods for producing a modified multidomain enzyme comprising cultivating plants expressing the modified multidomain enzyme.
Downstream uses of transgenic plants or plant material comprising the expression constructs of the invention include agronomical, pharmaceutical, and industrial uses, for example, human food, animal feed, biofuel, industrial alcohol, fermentation feedstocks, and the like.


DETAILED DESCRIPTION OF THE INVENTION
Overview
The present invention is directed to the use of plants for transgenic expression of multidomain enzymes. Higher plants are particularly useful for heterologous protein production since plants are amenable to large-scale production, they do not require aseptic conditions like bacterial recombinant protein production systems, and the levels of transgene-encoded proteins in plants may exceed 1% of the total protein content. To date, several proteins of commercial interest have been expressed successfully in plants, including a variety of antibodies, vaccine antigens, protein allergens, enzymes and enzyme inhibitors, coagulation factors, cytokines and hormones. However, expressing high levels of stable and functional proteins remains the bottleneck of many scientific and biotechnological endeavors including producing proteins for agricultural and therapeutic purposes
Thus, provided herein are methods and compositions for improving expression, stability, and/or activity of a multidomain enzyme in a plant cell. The methods comprise introducing into the plant cell a nucleic acid construct comprising a modified multidomain enzyme, wherein a native linker sequence in said multidomain enzyme has been replaced with a heterologous linker sequence that is not cleaved by a plant protease. By “heterologous” linker sequence is intended as linker sequence that is not native (i.e., does not naturally occur in the wild-type sequence) to the enzyme being modified. The linker sequence can be derived from a different species or organism, or may be a synthetic linker sequence (i.e., not existing in nature in any organism) or may be the native linker sequence modified. Linker regions that are not cleaved by plant proteases is not intended to be limited to the production of a single polypeptide by a host plant but is intended to refer to a preference for producing full length polypeptide as compared to the range of polypeptides produced when the linker region of the multidomain enzyme is the native sequence.
The modified multidomain enzyme is composed of at least one first domain, at least one heterologous linker, and at least one second domain. The first domain and the second domain are non-heterologous sequences. By “non-heterologous” it is intended that the first domain and the second domain are derived from the same native multidomain enzyme and may contain minor modifications which result in a domain polypeptide sequence which is greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, greater than 96% identical, greater than 97% identical, greater than 98% identical, or greater than 99% identical to the native polypeptide sequence.
In various embodiments, the nucleic acid constructs encoding the modified multidomain enzymes described herein result in an increased expression, stability and/or activity of the enzyme in the plant cell when compared to a control nucleic acid construct. An increase in expression, stability, or activity refers to an increase in a measurable amount of an enzymatically-active enzyme. The stability of an enzyme may also relate to its conformational stability, which is reflected in the enzyme's three-dimensional structure, or its chemical stability, which refers to the chemical composition of the enzyme's constituent amino acids.
It is recognized that polypeptides synthesized in heterologous systems can be produced in a range of sizes. A percentage of the produced polypeptide can be the full length enzyme which is defined as the polypeptide resulting from the translation of the coding sequence in its entirety; however, smaller polypeptides or larger polypeptides can also be produced. Smaller polypeptides may be the result of processing of the polypeptide by proteolytic processing of the polypeptide while larger polypeptides may be the result of the addition of carbohydrates to the polypeptide. The instant application describes a method for producing multidomain proteins in a plant host wherein the amount of full length polypeptide produced is greater when compared to the amount of full length polypeptide produced when the multidomain enzyme contains the native linker. Replacement of the native linker with heterologous linkers that are resistant to cleavage by plant proteases will lead to a greater amount of full length multidomain enzyme produced by the plant host.
While not bound by any particular theory or mechanism, the increase may result from an increase in translation or a decrease in degradation of the enzyme, and/or an increase in the catalytic activity of the enzyme. In another embodiment, the increase relates to an increase in the expression, stability, and/or activity of a full-length multidomain enzyme. For the purposes of the present invention, a full-length multidomain enzyme refers to a multidomain enzyme comprising at least a functional binding domain, a linker, and a functional catalytic domain. A modified protein having a “functional binding domain” is a protein in which the binding properties are substantially similar to, or improved relative to, the binding, properties of the native protein in its native environment. Likewise, a modified protein having a “functional catalytic domain” is a protein in which the catalytic properties are substantially similar to, or improved relative to, the catalytic properties of the native protein in its native environment. By “substantially similar” is intended at least about 80% or more of the binding properties or catalytic properties of the native protein. One of skill in the art will recognize that deletion of one or a few amino acids of any particular multidomain protein may have no significant effect on the stability or activity of the protein.
In one embodiment, the increase in expression, amount of full length polypeptide, stability, and/or activity is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 10-fold, at least about 20-fold, or greater when compared to a control. By “control” nucleic acid construct is intended a nucleic acid construct comprising a nucleotide sequence encoding a multidomain enzyme having a native linker sequence; or a linker sequence known to be cleaved by a plant protease. Unless otherwise specified, the control construct comprises a nucleic acid encoding a multidomain enzyme with a native linker sequence. A “native linker sequence” refers to the linker sequence present in the multidomain sequence in the organism from which the multidomain sequence was derived (i.e. the naturally-occurring linker sequence).
Thus, the methods of the invention find particular use in the integration of current practices for the cultivation of crop plants for the purpose of obtaining a commercially desired plant material with increased expression, stability and/or activity eta multidomain enzyme, and the use of the crop plant residues as a source of biomass for the production of fermentable sugars, or for agricultural, pharmaceutical, and/or human consumption.
By a “crop plant” is intended any plant that is cultivated for the purpose of producing plant material that is sought after by man for either oral consumption, or for utilization in an industrial, pharmaceutical, or commercial process. The invention may be applied to any of a variety of plants, including, but not limited to maize, wheat, rice, barley, soybean, cotton, sorghum, oats, tobacco, Miscanthus grass, Switch grass, trees, beans in general, rape/canola, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, cotton, coffee, sweet potato, flax, peanut, clover; vegetables such as lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli. Brussels sprouts, peppers, and pineapple; tree fruits such as citrus, apples, pears, peaches, apricots, walnuts, avocado, banana, and coconut; and flowers such as orchids, carnations and roses.
As used herein, the term “plant part” or “plant tissue” includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, withers, and the like.
In one embodiment, the plant is an indeterminate plat. These varieties grow vegetatively for indefinite periods in temperate regions. These varieties can be engineered to accumulate the polypeptide of interest in the vacuoles and can be grown until the first frost. At that time, the plant could be allowed to dessicate, harvested dry, and used for food, livestock feed, or in biomass conversion or other commercially-useful processes.
As used herein, “biomass” or “feedstock” refers to useful biological material including a product of interest, which material is to be collected and is intended for further processing to isolate or concentrate the product of interest. The biomass or feedstock may comprise the fruit or parts of it or seeds, leaves, or stems or roots where these are the parts of the plant that are of particular interest for the industrial purpose. “Biomass”, as it, refers to plant material, includes any structure or structures of a plant that contain or represent the product of interest.
The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
“Isolated” means altered “by the hand of man” from its natural state; i.e., that, if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a naturally occurring polynucleotide or a polypeptide naturally present in a living animal in its natural state is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein. For example, with respect to polynucleotides, the term isolated means that it is separated from the chromosome and cell in which it naturally occurs. A sequence is also isolated if separated from the chromosome and cell in which it naturally occurs in but inserted into a genetic context, chromosome, or cell in which it does not naturally occur.
Multidomain Enzymes
The methods of the present invention encompass modified multidomain enzymes. A “multidomain enzyme” or a “multidomain protein” refers to any protein containing two or more domains. The domains may be on single polypeptide; they may also be on different polypeptides. Domains are generally regarded as compact, semi-independent units (Richardson (1981) Advan. Protein Chem. 34:167-339) that could fold autonomously Wetlaufer (1973) Proc. Natl Acad. Sci. 70:697-701). Exemplary domains include an immunoglobulin superfamily constant domain such as a CH2 or CH3 domain, a receptor binding domain, a ligand binding domain, an enzymatic or catalytic domain, a fibronectin domain, a dockerin domain, and the like.
In various embodiments, the multidomain enzymes encompassed herein comprise at least a first domain, at least a first linker, and at least a second domain. In some embodiments, the multidomain enzyme comprises at least a first binding domain, at least a first linker, and at least a first catalytic domain. A binding domain is a noncatalytic domain involved in substrate binding or specific protein interactions. Upon binding, proteins may undergo a conformational change. Thus, these binding domains are essential for the function of many proteins. The term “catalytic domain” is defined herein as a structural portion or region of the amino acid sequence of the multidomain enzyme that possesses the catalytic activity of the enzyme. A “linker” is defined as the region connecting two domains. A connection between domains may play an important structural role in positioning domains with respect to one another, or the connection may merely tether two domains within a certain distance of one another. The linker region may also comprise sites for proteolytic cleavage. The linker region of multidomain enzymes is typically three dimensionally a linear region which is a flexible hinge connecting two domains together.
Numerous proteins of higher organisms have a multidomain architecture consisting of strings of mobile modules (Doolittle (1995) Annu Rev Biochem. 64:287-114). Many of the modules identified so far have defined binding and/or catalytic functions (i.e., binding domains or catalytic domains), but some may just act as simple spacer elements required only to arrange binding surfaces in space (i.e., linker regions).
A variety of software applications for protein structure prediction, including domain recognition and linker sequence predictions, are described in Lobley (2009) Bioinformatics Advance Access Online, May 7, 2009; Bryson (2005) Nucl. Acids Res. 33 (Web server issue):W36-38; Jones (1999) J. Mol. Biol. 292: 195-202; McGruffin and Jones (2003) Bioinformatics 19:8740881; Jones (1999) J. Mol. Biol 287: 797-815; Jones (2007) Bioinformatics 23: 538-544; Jones et al (1994) Biochem. 33: 3038-3049; Ebina et al. (2009) Biopolymers 92(1):1-8, and several programs are available on the internet, for example, at tuat.ac.jp/ ̃domserv/cgi-bin/DLP-SVM.cgi; at predictprotein.org/about.html; and at bioinf.cs.ucl.ac.uk/psipred/index.html#more.
Linkers
Provided herein are methods and compositions for improving expression, stability, and/or activity of a multidomain enzyme in a plant cell. The methods comprise introducing into the plant cell a nucleic acid construct comprising a modified multidomain enzyme, wherein a native linker sequence in the modified multidomain enzyme has been replaced with a heterologous linker sequence that is not cleaved by a plant protease. The heterologous linker sequence may be resistant to cleavage by a plant protease due to the replacement of protease sensitive sites with protease insensitive sites or by altering the structural conformation of the multidomain enzyme such that protease-sensitive sites are inaccessible to the plant proteases. A “protease sensitive” site is an amino acid residue or sequence that is recognized by and cleaved by a particular plain protease. As discussed supra, enzymes susceptible to protease cleavage may undergo complete hydrolysis, directly impacting its final yield, or may undergo partial trimming thereby altering the activity or homogeneity of the final protein product. Thus, replacement of native linker sequences with heterologous linker sequences in a multidomain protein may result in improvement in the integrity, structural heterogeneity and/or biological activity of the enzyme.
The linker sequences front different enzymes rarely share any apparent sequence homology but their amino acid composition is typically rich in proline and hydroxyl amino acids (Gilkes et al, (1991) Microbiol. Rev. S5, 303-315; and Claeyssens and Tomme (1989) in Tkichalenna reesei Cellulases: Biochemistry, Genetics, Physiology and Application (Kubicek, C. P., Eveleigh, D. E., Esterbauer, H., Steiner, W., and Kubicek-Pranz, E. M., eds) pp. 1-11, Proceedings, Tricell (1989) Royal Society of Chemistry)). In general, a linker may be between about 5 to 60 amino acid residues, between about 15 to 50 amino acid residues, and between about 25 to 45 amino acid residues. See, for example. Srisodsuk et al., 1993, J. Biol. Chem. 268(28): 20756-20761 (herein incorporated by reference in its entirety) for a discussion of the linker peptide of T. reesei CBHI.
In one embodiment of the present invention, the native linker sequence is replaced by a linker sequence derived from a fungal organism or from a bacterium. While not bound by any particular theory or mechanism, linker sequences derived from bacterial or fungal organisms may be less susceptible to cleavage by plant enzymes. By “derived from” is intended that the heterologous linker sequence is identified in a protein expressed by the organism and utilized as a linker sequence in the modified multidomain enzyme encompassed herein. The native linker sequence in the modified multidomain enzyme may be replaced with a linker sequence that is identical to the linker sequence identified in the fungal or bacterial protein, or may be modified further to improve the functionality of the linker sequence in a plant (including, but not limited to, using plant-preferred codons to improve expression of the modified enzyme in the plant and/or replacing one or more plant protease-sensitive sites with plant protease-insensitive sites).
In another embodiment, methods for improving the expression, stability, and/or activity of the multidomain enzyme comprise replacement of one or more cleavage-sensitive residues within the linker region with one or more residues comprising a glycosylation site sequence, or by adding one or more glycosylation site sequences. The role of glycosylation in many multidomain enzymes includes providing sufficient spatial separation between the catalytic core and binding domains, and protecting the linker peptide against proteolysis (Srisodsuk et al., 1993. J. Biol. Chem., 268, 20756-20761; Clarke, 1997, Biodegradation of cellulose. In Enzymology and biotechnology. Technomic Publishing, Pennsylvania, p. 55). Thus, while not being bound by any particular theory or mechanism, of the linker region of multidomain enzymes to increase glycosylation may prevent proteolytic degradation of the modified multidomain enzyme by plant enzymes.
In one embodiment, the heterologous linker region comprises one or more N-linked glycosylation sites. An “N-linked glycosylation” site comprises an amino acid residue or sequence that is susceptible to N-linked glycosylation. In various embodiments, the heterologous linker comprises one or more N-linked glycosylation consensus sequences, including one or more Asn-X-Ser/Thr/Cys sequences where X is any amino acid except proline.
Alternatively, or in addition, the heterologous linker region comprises one or more O-finked glycosylation sites. An “O-linked glycosylation site” comprises an amino acid residue or sequence that is susceptible to O-linked glycosylation. To date, a consensus primary amino acid sequence for O-glycosylation has not been identified, however, different structural motifs have been proposed (see, for example. Young, et al., 1979, Biochemistry. 18(20):4444-4448, Muller et al., 1997, J Biol. Chem. 272(40):24780-24793; Yoshida et al., 1997, J Biol Chem, 272(27):16884-16888; Gooley et al. (1991) Biochem Biophys Res Commun. 178(3):1194-1201; and Christlet Veluraja (2001) Biophys. J. 80(2): 952-960, each of which is herein incorporated by reference in its entirety). Thus, in various embodiments, the heterologous linker region comprises one or more O-linked glycosylation structural motifs, including but not limited to one or more of Thr-Ala-Pro-Pro, Thr-Val-X-Pro, Ser/Thr-Pro-X-Pro, and Thr-Ser-Ala-Pro.
Alternatively, the heterologous linker sequence may be derived from the linker sequence of a glycosylated protein, including a plant glycoprotein. The sequences of a number of glycosylated proteins have been published in recent years. SWISSPROT, PIR, PROSITE, PDB, EMBL, HSSP, LISTA, and MIM databases contain glycosylated protein entries. Many O-linked glycosylated proteins are listed in the O-GLYCBASE database (Gupta et al, (1999) Nucleic Acids Research 27:370-372).
In yet another embodiment, the native linker region of a multidomain enzyme may be replaced with all or part of the transmembrane domain of a transmembrane protein. Certain membrane proteins are “transmembrane proteins” and have an extracellular domain, which interacts with the external cellular environment, an intracellular domain, which interacts with the internal cellular environment, and a transmembrane domain which traverses the cellular lipid bilayer, “Transmembrane domain,” which comprises the “transmembrane regions,” refers to the domain of transmembrane proteins that lies within the plasma membrane, and may also include the corresponding cytoplasmic (intracellular) and extracellular loops. Thus all or substantially all of the transmembrane region of a transmembrane protein can be used as a linker sequence in a multidomain enzyme. The TMPDB database of transmembrane proteins is described in (Ikeda et al. (2003) Nucleic Acids Res. 31, 406-409) The Protein Data Bank of Transmembrane Proteins (PDBTM) is described in Tusnády et al, (2004) Bioinformatics 20(17):2964-72 and Tusnády et al, (2005) Nucleic Acids Res. 33(Database issue):D275-8.
The expression, stability, and/or activity of a multidomain enzyme may also be improved by removal of protease cleavage sites within the linker sequences. A variety of plant proteases and their target cleavage site sequences are known in the art.
Modified multidomain enzymes may be generated by replacement of native linker regions with linker regions derived from other proteins, or may be generated by mutagenesis approaches. In one embodiment, site-directed or random mutagenesis is used to modify one or more sues within a linker sequence to generate a linker that is less sensitive to protease cleavage. In another embodiment, directed evolution approaches are used to improve the linker regions. In the past several years, directed evolution has emerged as an alternative approach to rational design, enabling the improvement of structural and functional properties, such as stability and performance under different conditions, or changes in their reaction and substrate specificity (Tao and Cornish (2002) Curr Opin Chem Biol 6:858-864). Rather than designing a limited number of site-directed mutants, directed evolution implements an iterative Darwinian optimization process, whereby the fittest variants are selected from an ensemble of random mutations. Improved variants are identified by screening or selection for the properties of interest and then encoding genes are then used as parent genes for the following round of evolution (Roodveldt et al, (2005) Current Opinion in Structural Biology 15(1):50-56).
Screening or selecting for improved variants can be done in two ways: screening or selecting for the protein's own function or screening or selecting for the activity of a reporter protein. Screening or selecting for the protein's own function will vary according to the activity of the multidomain protein being evaluated. Methods for screening or selecting for the activity of a reporter protein are known in the art. See, for example, U.S. Patent Publication 20090092982, which describes a method which couples the folding status and/or stability of the protein (or variant of a protein) to a screenable (e.g., selectable) phenotype imparted by a separate entity (e.g., antibiotic resistance). This screenable phenotype is used to assess stability.
Cellulase
In various embodiments of the present invention, the modified multidomain enzyme is a cellulose-degrading, enzyme. Plants are an abundant source of cellulosic substrate, therefore, expression of cellulose-degrading enzymes within the cellulosic feedstock will minimize or eliminate the need for exogenous addition of enzyme. Thus, provided herein are nucleotide sequences encoding a modified cellulose enzyme. For the purposes of the present invention, a “cellulase” is an enzyme that is capable of catalyzing the hydrolysis of a 1-4-beta-D-glycosidic linkage, and is composed of at least one catalytic domain and at least one other domain selected from the group consisting of a catalytic domain and cellulose binding domain. The structure of many cellulases is described in Gilkes et al. (1991) Microbiological Reviews 55(4303-315, which is herein incorporated by reference in its entirety. Cellulose degradation processes using transgenic biomass produced according to the present invention can be carried out more inexpensively, easily, and more environmentally safe than can conventional methods.
The modified cellulase enzymes encompassed herein have a linker sequence that results in less cleavage when the modified cellulase is expressed in plants. In some embodiments, less than about 90% of the modified enzyme is cleaved when expressed in plants, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or none of the modified enzyme is cleaved when expressed in plants. The heterologous linker sequence may result in less cleavage due to the replacement or protection of protease-sensitive cleavage sites as discussed supra. Thus, the modified cellulase encompassed herein has improved expression, stability, and/or activity relative to a control cellulase.
In various embodiments, replacement of one or more native linker sequences with an improved linker sequence results in an increase in the expression, stability, and/or activity of the full-length cellulase enzyme. This full-length enzyme comprises at least one binding domain, at least one heterologous linker, and at least one catalytic domain. A particular advantage of this full-length protein is the retention of the binding domain, particularly the cellulose-binding domain. While not bound by any particular theory or mechanism, the presence of the cellulose-binding domain in the modified cellulase may result in an improvement in hydrolysis of insoluble cellulosic substrates, such as crystalline cellulose.
In some embodiments, the native linker sequence corresponding to amino acid residues 471 through 499 of SEQ ID NO:2 is replaced with a heterologous linker sequence. Alternatively, a native linker sequence in a cellulose homologous to SEQ. ID NO:2 is replaced with a heterologous linker sequence. It will be understood that the native linker region of homologous cellulose sequences may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids shorter or longer than the linker region defined by amino acid residues 471 through 499 of SEQ ID NO:2.
In various embodiments of the present invention, the nucleotide sequence encodes a cellulase comprising the linker sequence set forth in SEQ ID NO:18, 19, or 20. Based on this information, as well as detailed information in the art regarding the structural features of cellulose enzymes, additional heterologous linker sequences can be designed and tested for expression in plant cells. Methods for monitoring expression, processing (including cleavage), and activity of cellulase enzymes are known in the art.
In one embodiment, the modified multidomain enzyme is a cellobiohydrolase enzyme or an endoglucanase enzyme. Cellobiohydrolases and endoglucanases are structurally similar and are frequently composed of multiple domains. At least one of the domains is a catalytic core domain which may be associated with additional catalytic domains or at least one cellulose-binding domain (CBD). The two domains are connected by relatively long, glycosylated linker peptides of 6-59 amino acids.
The term “cellobiohydrolase” (CBH) refers to a group of cellulase enzymes classified as EC 3.2.1.91. These enzymes are also known as exoglucanases or exo-cellobiohydrolases. CBH enzymes have been isolated from a variety of sources, microbial sources such as bacteria, yeast, and fungi, each of which is encompassed herein. In various embodiments, the CBH enzyme is a modified cellobiohydrolase I (CBHI) enzyme. CBHI plays a key role in the decomposition of crystalline cellulose (Claeyssens et al. (1990) Biochem J 270(1):251-256; and Wood et al. (1989) Biochem J 260(1):37-44). In general, a CBHI type enzyme preferentially hydrolyzes cellobiose from the reducing end of cellulose and a cellobiohydrolase II (CBH2) type enzyme preferentially hydrolyzes the non-reducing end of cellulose.
Endoglucanases (1,4-p-D-glucan glucanohydrolase; EC 3.2.1.4) are ubiquitous enzymes that hydrolyze 1,4-β linkages adjacent to unsubstituted glucose residues (Henrissat et al. (1989) Gene 81:83-95), are produced by a broad range of organisms, including fungi, bacteria, plants, and insects.
Glucoamylase
In various embodiments of the present invention, the modified multidomain enzyme is a starch-degrading enzyme. Starch-degrading enzymes are widely distributed throughout many species of animals, plants and microorganisms. These enzymes have been classified as alpha-amylases and glucoamylases belonging to glycoside hydrolase families 13, 14 or 15. In various embodiments, the present invention encompasses a modified glucoamylase enzyme. Glucoamylases (alpha-1,4-glucan glucohydrolases E.C.3.2.1.3) are starch hydrolyzing exo-acting carbohydrases. Glucoamylases catalyze the removal of successive glucose units from the non-reducing ends of starch or related oligo and polysaccharide molecules and can hydrolyze both linear and branched glucosidic linkages of starch (amylase and amylopectin). Commercially glucoamylases are very important enzymes that have been used in a wide variety of applications requiring the hydrolysis of starch. Glucoamylases can be provided through the heterologous expression of glucoamylase in at least one variety in the harvested plant material of the invention.
Similar to other polysaccharide-degrading enzymes die majority of glucoamylases have a modular structure consisting of a catalytic domain, a starch-binding domain, and a highly O-glycosylated linker that connects the two domains (Bourne and Henrissat (2001) Curr. Opin. Struct. Biol, 11(5): 593-600; Sauer et al. (2000) Biochim. Biophys, Acta. 1543(2): 275-293), A description of the catalytic site, mechanism of action, substrate recognition, the linker region, and multidomain architecture of this class of enzymes can be found in Sauer et al. (2000) Biochim. Biophys. Acta. 1543(2): 275-293, which is herein incorporated by reference in its entirety.
The modified glucoamylase enzymes encompassed herein have a linker sequence that results in less cleavage when the modified glucoamylase is expressed in plants. In some embodiments, less than about 90% of the modified enzyme is cleaved when expressed in plants, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or none of the modified enzyme is cleaved when expressed in plants. The heterologous linker sequence may result in less cleavage due to the replacement or protection of protease-sensitive cleavage sites as discussed supra. In various embodiments of the present invention, the modified glucoamylase comprises the linker sequence set forth in SEQ ID NO: 18, 19, or 20. Thus, the modified glucoamylase encompassed herein has improved expression, stability, and/or activity relative to a control glucoamylase.
Plant Expression Cassettes
The compositions of the invention also comprise nucleic acid sequences for transformation and expression of a multidomain enzyme in a plant cell of interest. The nucleic acid sequences may be present in DNA constructs or expression cassettes, “Expression cassette” as used herein means a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest (i.e., a nucleotide sequence encoding a polypeptide of interest) which is operatively linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette May be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. Additionally, the promoter can also be specific to a particular tissue or organ or stage of development.
The present invention encompasses the transformation of plants with expression cassettes capable of directing expression of a multidomain enzyme in a plant cell. The expression cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter) and a polynucleotide encoding the modified multidomain enzyme. The expression cassette may optionally comprise a transcriptional and translational termination region (i.e. termination region) functional in plants.
In addition, the construct may further comprise additional regulatory elements to facilitate transcription, translation, or transport of the modified multidomain enzyme. The regulatory sequences of the expression construct are operably linked to the polynucleotide encoding the modified multidomain enzyme. By “operably linked” is intended a functional linkage between a regulatory element and a second sequence wherein the regulatory element initiates and/or mediates transcription, translation, or translocation of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleotide sequences being linked are contiguous. The regulatory elements include promoters, enhances, and signal sequences useful for targeting cytoplasmically-synthesized proteins to the endomembrane system of the plant cell.
The expressed multidomain enzymes may also be targeted to certain organelles such as vacuoles to alleviate toxicity problems. For vacuole-targeted expression of multidomain enzymes, plants are transformed with vectors that include a vacuolar targeting sequence such as that from a tobacco chitinase gene. In this case, the expressed multidomain enzyme will be stored in the vacuoles where they will not be able to degrade cellulose and harm the plant in one embodiment of the present invention, the vacuole sorting signal sequence is derived from the barley polyamino oxidase 2 (BPAO2) signal sequence. BPAO2 has an N-terminal signal peptide for entry into the secretory pathway. The presence of a C-terminal extension of this signal peptide results in vacuolar localization of BPAO in a plant cell (see Cervelli et al. (2004) The Plant Journal 40:410-418). In another embodiment, useful vacuole sorting, signals are described in U.S. application Ser. No. 12/359,421, which is herein incorporated by reference in its entirety.
In various embodiments of the present invention, modified multidomain enzyme coding sequences are fused to promoters active in plants and transformed into the nuclear genome or the plastid genome. Chloroplast expression has the advantage that the multidomain enzyme is less damaging to the plastid as it contains little or no cellulose.
In other embodiments, the construct comprises, in the 5′ to 3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a polynucleotide encoding an endoplastic reticulum signal sequence, and a polynucleotide encoding the modified multidomain enzyme. Exemplar signal sequences include the SEKDEL (SEQ ID NO:23) endoplasmic reticulum targeting sequence, the gamma zein 27 kD signal sequence, and the Glycine max glycinin GY1 signal sequence. Others useful in the methods of the invention will be apparent to one of skill in the art.
Any promoter capable of driving expression in the plant of interest may be used in the practice of the invention. The promoter may be native or analogous or foreign or heterologous to the plant host. The terms “heterologous” and “exogenous” when used herein to refer to a nucleic acid sequence (e.g. a DNA or RNA sequence) or a gene, refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally mewing multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
A “homologous” nucleic acid (e.g. DNA) sequence is a nucleic acid (e.g. DNA or RNA) sequence naturally associated with a host cell into which it is introduced.
The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a sequence by appropriately selecting and positioning promoters and other regulatory regions relative to that sequence.
Some suitable promoters initiate transcription only, or predominantly, in certain cell types. Thus, as used herein a cell type- or tissue-preferential promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other cell types or tissues as well. Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in the following references: Jordano, et al., Plant Cell, 1:855-866 (1989); Bustos, et al., Plant Cell, 1:839-854 (1989); Green, et al., EMBO J. 7, 4035-4044 (1988); Meier, et al., Plant Cell, 3, 309-316 (1991); and Zhang, et al., Plant Physiology 110: 1069-1079 (1996).
Promoters active in photosynthetic tissue in order to drive transcription in green tissues such as leaves and stems are also of interest for the present invention. Most suitable are promoters that drive expression only or predominantly in such tissues. The promoter may confer expression constitutively throughout the plant, or differentially with respect to the green tissues, or differentially with respect to the developmental stage of the green tissue in which expression occurs, or in response to external stimuli.
Examples of such promoters include the ribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Larix laricina), the pine cab6 promoter (Yamamoto et al, (1994) Plant Cell Physiol, 35:773-778), the Cab-1 gene promoter from wheat (Fejes et al. (1990) Plain Mol. Biol. 15:921-932), the CAB-1 promoter from spinach (Lubberstedt et al. (1994) Plant Physiol, 104:997-1006), the cab1R promoter front rice (Luan et al. (1992) Plant Cell 4:971-981), the pyruvate orthophosphate dikinase (PPDK) promoter from corn t Matsuoka et al, (1993) Proc Natl Acad Sci USA 90:9586-9590), the tobacco Lhcb1*2 promoter (Cerdan et al. (1997) Plant Mol. Biol. 33:245-255), the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al. (1995) Planta 196:564-570), and thylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS. Other promoters that drive transcription in stems, leafs and green tissue are described in U.S. Patent Publication No. 2007/0006346, herein incorporated by reference in its entirety.
A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a green tissue-specific manner in transgenic plants.
In some other embodiments of the present invention, inducible promoters may be desired. Inducible promoters drive transcription in response to external stimuli such as chemical agents or environmental stimuli. For example, inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought. With a chemically inducible promoter, expression of the multidomain enzyme genes transformed into plants may be activated at an appropriate time by foliar application of a chemical inducer.
A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and correct mRNA polyadenylation. The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the DNA sequence of interest, the plant host or any combination thereof). Appropriate transcriptional terminators are those that are known w function in plants and include the CAMV 35S terminator, the mil terminator, the nopaline synthase terminator and the pea rbcs E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a gene's native transcription terminator may be used.
In some embodiments, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues.
Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.
Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adh1 gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200 (1987)). In the same experimental system, the intron from the maize bronze 1 gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)). Other leader sequences known in the art include but are not limited to: picomavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, O., Fuerst T. R., and Moss, B. PNAS USA 86:6126-6130 (1989)); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et. al., 198); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20 human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak, D. G., and Samow, P., Nature 353: 90-94 (1991); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L., Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV), (Gallic, D. R. et al., Molecular Biology of RNA, pages 237-256 (1989); and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al., Virology 81:382-385 (1991). See also, Della-Cioppa et at, Plant Physiology 84:965-968 (1987).
It will also be recognized that the nucleotide sequence encoding the modified multidomain enzyme may be optimized for increased expression in the transformed host cell. That is, the nucleotide sequences can be synthesized using host cell-preferred codons for improved expression, or may be synthesized using codons at a host-preferred codon usage frequency. Generally, the GC content of the gene will be increased. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. Sec, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et at (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
Plants
Plants useful in the present invention include plants that are transgenic for the modified multidomain enzyme. One of skill in the art will recognize that plants may express one or more additional polypeptide sequences associated with or contributing to one or more secondary trains) of interest. These polypeptides may be cytoplasmically-expressed, may be targeted to a subcellular organelle, or may be secreted by the plant cell. Secondary traits of interest include agronomic traits that primarily are of benefit to a seed company, a grower, or a grain processor, for example, herbicide resistance, virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, and fungal resistance. See, e.g., U.S. Pat. Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431. A secondary trait of interest may also be one that increases plant vigor or yield (including traits that allow a plant to grow at different temperatures, soil conditions and levels of sunlight and precipitation), or one that allows identification of a plant exhibiting a trait of interest (e.g., selectable marker gene, seed coat color, etc.). A plethora of genes useful for generating plants with desired secondary traits are available in the art.
The type of plant selected depends on a variety of factors, including for example, the downstream use of the harvested plant material, amenability of the plant species to transformation and the conditions wider which the plants will be grown, harvested, and/or processed. One of skill will further recognize that additional factors for selecting appropriate plant varieties for use in the present invention include high yield potential, good stalk strength, resistance to specific diseases, drought tolerance, rapid dry down and grain quality sufficient to allow storage and shipment to market with minimum loss.
It is further contemplated that the constructs of the invention may be introduced into plant varieties having improved properties suitable or optimal for a particular downstream use.
For example, naturally-occurring genetic variability in plants with altered starch metabolism is useful in the methods of the invention. Many such plants carry mutations in genes encoding isoforms of starch synthesis or starch degradation enzymes. For example, plants have been identified which are heterozygous or homozygous for one or more of the waxy (wx), amylose extender (ae); dull (du), horny (h), shrunken (sh), brittle (bt), floury (fl), opaque (o), or sugary (su) mutant alleles. See, for example, U.S. Pat. Nos. 4,428,972; 4,767,849; 4,774,328; 4,789,738; 4,789,557; 4,790,997; 4,792,458; 4,798,735; and 4,801,470, herein incorporated by reference. These plants can be used in their native form, or can be modified to exhibit one or more additional traits of interest.
For plants with increased nutritional quality, several varieties of corn are available, such as those with increased lysine (Crow's Hybrid Corn Company, Milford, Ill.), protein (BASF) and oil (Pfister Hybrid Corn Company, El Paso, Ill. under the trademark KERNOIL®) levels. Other suitable high oil corn includes the corn populations known as Illinois High Oil (IHO) and Alexander High Oil (Alexo); samples of which are available from the University of Illinois Maize Genetics Cooperative—Stock Center (Urbana, Ill.).
Sweet corn is also available in which there is a reduction in the amount of starch and an increase in the amount of glucose, sucrose and/or water soluble polysaccharides normally found in the immature corn kernel (Creech, R. and Alexander, D. E. In Maize Breeding and Genetics; D. B. Walden, Ed.; John Wiley and Sons: New York, 1978; pp. 249-264). In several plant species such as corn (Shannon & Garwood, 1984), pea (Bhattacharyya et al, 1990), potato (Hovenkamp-Hermelink et al., 1987), Arabidopsis (Caspar et al., 1985; Lin et al., 1988a; Lin et al., 1988b) and tobacco (Hanson et al., 1988), mutants with an altered carbohydrate composition have been found. Brown mid rib (Bmr) corn has been used as an alternative for improving digestibility for silage hybrids for decades. The improvement in ruminal intakes and digestibility is derived from reduced lignin content in Bmr mutated hybrids. Additional varieties, both naturally-occurring and transgenic, with desired traits that are useful for downstream processing as described herein are well known to those of skill in the art.
Plants useful in the present invention also include, but are not limited to, crops producing edible flowers such as cauliflower (Brassica oleracea), artichoke (Cynara scolvmus), and safflower (Carthamus, e.g. tinctorius); fruits such as apple (Malus, e.g. domesticus), banana (Musa, e.g. acuminata), berries (such as the currant; Ribes, e.g. rubrum), cherries (such as the sweet cherry, Prunus, e.g. avium), cucumber (Cucumis, e.g. sativus), grape (Vitis, e.g. vinifera), lemon (Citrus limon), melon (Cucumis melo), nuts (such as the walnut, Juglans, e.g. regia; peanut. Arachis hypoaeae), orange (Citrus, e.g. maxima), peach (Prunus, e.g. persica), pear (Pyra, e.g. communis), pepper (Solanum, e.g. capsicum) plum (Prunus, e.g. domestica), strawberry (Fragaria, e.g. moschata), tomato (Lycopersicon, e.g. esculentum); leafs, such as alfalfa (Medicago, e.g. saliva), sugar cane (Saccharum), cabbages (such as Brassica oleracea), endive (Cichoreum, e.g. endivia), leek (Allium, e.g. porrum), lettuce (Lactuca, e.g. sativa), spinach (Spinacia e.g. oleraceae), tobacco (Nicotiana, e.g. tabacum); roots, such as arrowroot (Maranta, e.g. arundinacea), beet (Beta, e.g. vulgaris), carrot (Daucus, e.g. carota), cassava (Manihot, e.g. esculenta), turnip (Brassica, e.g. rapa), radish (Raphanus, e.g. sativus) yam (Dioscorea, e.g. esculenta), sweet potato (Ipomoea batatas); seeds, such as bean (Phaseolus, e.g. vulgaris), pea (Pisum, e.g. sativum), soybean (Glycine, e.g. max), wheat (Triticum, e.g. aestivum), barley (Hordeum, e.g. vulgare), corn (Zea, e.g. mays), rice (Oryza, e.g. sativa); grasses, such as Miscanthus grass (Miscanthus, e.g., giganteus) and switchgrass (Panicum, e.g. virgatum); trees such as poplar (Populus, e.g. tremula), pine (Pinus); shrubs, such as cotton (e.g., Gossypium hirsutum); and tubers, such as kohlrabi (Brassica, e.g. oleraceae), potato (Solanum, e.g. tuberosum), and the like.
Plant Transformation
The expression constructs described herein can be introduced into the plant cell in a number of art-recognized ways. The term “introducing” in the context of a polynucleotide, for example, a nucleotide construct of interest, is intended to mean presenting to the plant the polynucleotide in such a manner that the polynucleotide gains access to the interior of a cell of the plant. Where more than one polynucleotide is to be introduced, these polynucleotides can be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and can be located on the same or different transformation vectors. Accordingly, these polynucleotides can be introduced into the host cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol. The methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a plant, only that the polynucleotide(s) gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotides into plants are known in the art including, but not limited to, transient transformation methods, stable transformation methods, and virus-mediated methods.
“Transient transformation” in the context of a polynucleotide is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant.
By “stably introducing” or “stably introduced” in the context of a polynucleotide introduced into a plant is intended the introduced polynucleotide is stably incorporated into the plant genome, and thus the plant is stably transformed with the polynucleotide.
“Stable transformation” or “stably transformed” is intended to mean that a polynucleotide, for example, a nucleotide construct described herein, introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations.
Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the npt11 gene, which confers resistance to kanamycin and related, antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet. 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), the EPSPS gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642), and the mannose-6-phosphate icon gene, which provides the ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).
Methods for regeneration of plants are also well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants, as well as a representative plastid transformation technique.
Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). For the construction of vectors useful in Agrobacterium transformation, see, for example, US Patent Application Publication No. 2006/0260011, herein incorporated by reference.
Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and Microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. For the construction of such vectors, see, for example, US Application No. 20060260011, herein incorporated by reference.
For expression of a nucleotide sequence of the present invention in plant plastids, plastid transformation vector pPH143 (WO 97/32011, example 36) is used. The nucleotide sequence is inserted into pPH143 thereby replacing the PROTOX coding sequence. This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance. Alternatively, the nucleotide sequence is inserted in pPH143 so that it replaces the aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors.
Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J. 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).
Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.
Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both of these techniques are suitable for use with this invention. Co-transformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).
Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 813-839 (1990)) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.
Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)). Furthermore, WO 93/21335 describes techniques for the transformation of rice via electroporation.
Patent Application EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnologyl 11:1553-1558 (1993)) and Weeks et al, (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 hours and are then bombarded. Twenty embryos per target plate is typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSOG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont BIOLISTICS®, helium device using a burst pressure of about 1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed hack into the dark to recover for about 24 hours (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as “GA7s” which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.
Transformation of monocotyledons using Agrobacterium has also been described. See, WO 94/00977 and U.S. Pat. No. 5,591,616, both of which are incorporated herein by reference. See also, Negrotto et al., Plant Cell Reports 19: 798-803 (2000), incorporated herein by reference.
For example, rice (Oryza sativa) can be used for generating transgenic plants. Various rice cultivars can be used (Hiei et al., 1994, Plant Journal 6:271-282; Dong et al., 1996, Molecular Breeding 2:267-276; Hiei et al., 1997, Plant Molecular Biology, 35:205-218). Also, the various media constituents described below may be either varied in quantity or substituted. Embryogenic responses are initiated and/or cultures are established from mature embryos by culturing on MS-CIM medium (MS basal salts, 4.3 g/liter; B5 vitamins (200×), 5 ml/liter; Sucrose, 30 g/liter; proline, 500 mg/liter; glutamine, 500 mg/liter; casein hydrolysate, 300 mg/liter; 2,4-D (1 mg/ml). 2 ml/liter; adjust pH to 5.8 with 1 N KOH; Phytagel, 3 g/liter). Either mature embryos at the initial stages of culture response or established culture lines are inoculated and co-cultivated with the Agrobacterium tumefaciens strain LBA4404 (Agrobacterium) containing the desired vector construction. Agrobacterium is cultured from glycerol stocks on solid YPC medium (100 mg/L spectinomycin and any other appropriate antibiotic) for about 2 days at 28° C. Agrobacterium is re-suspended in liquid MS-CIM medium. The Agrobacterium culture is diluted to an OD600 of 0.2-0.3 and acetosyringone is added to a final concentration of 200 uM. Acetosyringone is added before mixing the solution with the rice cultures to induce Agrobacterium for DNA transfer to the plant cells. For inoculation, the plant cultures are immersed in the bacterial suspension. The liquid bacterial suspension is removed and the inoculated cultures are placed on co-cultivation medium and incubated at 22° C. for two days. The cultures are then transferred to MS-CIM medium with Ticarcillin (400 mg/liter) to inhibit the growth of Agrobacterium. For constructs utilizing the PMI selectable marker gene (Reed et al., In Vitro Cell. Dev. Biol.-Plant 37:127-132), cultures are transferred to selection medium containing Mannose as a carbohydrate source (MS with 2% Mannose, 300 mg/liter Ticarcillin) after 7 days, and cultured for 3-4 weeks in the dark. Resistant colonies are then transferred to regeneration induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1 mg/liter zeatin. 200 mg/liter timentin 2% Mannose and 3% Sorbitol) and grown in the dark for 14 days. Proliferating colonies are then transferred to another round of regeneration induction media and moved to the light growth room. Regenerated shoots are transferred to GA7 containers with GA7-1 medium (MS with no hormones and 2% Sorbitol) for 2 weeks and then moved to the greenhouse when they are large enough and have adequate roots. Plants are transplanted to soil in the greenhouse (To generation) grown to maturity, and the T1 seed is harvested.
The plants obtained via transformation with a nucleic acid sequence of the present invention can be any of a wide variety of plant species, including those of monocots and dicots; however, the plants used in the method of the invention, are preferably selected from the list of agronomically important target crops set forth supra. The expression of a gene of the present invention in combination with other characteristics important for production and quality can be incorporated into plant lines through breeding. Breeding, approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wis. (1983); Mayo O., The Theory of Plant Breeding, Second Edition, Clarendon Press, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986).
For the transformation of plastids, seeds of Nicotiana tabacum c.v. “Xanthienc” are germinated seven per plate in a 1′′ circular array on T agar medium and bombarded 12-14 days after sowing with 1 um tungsten particles (M10, Biorad, Hercules, Calif.) coated with DNA from plasmids pPH143 and pPH145 essentially as described (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917). Bombarded seedlings are incubated on T medium for two days after which leaves are excised and placed abaxial side up in bright light (350-500 umol photons/m2/s) on plates of RMOP medium (Svab, Z. Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526-8530) containing 5001.10111 spectinomycin dihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearing underneath the bleached leaves three to eight weeks after bombardment are subcloned onto the same selective medium, allowed to form callus, and secondary shoots isolated and subcloned. Complete segregation of transformed plastid genome copies (homoplasmicity) in independent subclones is assessed by standard techniques of Southern blotting (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digested total cellular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5, 346349) is separated on 1% Tris-borate (TBE) agarose gels, transferred to nylon membranes (Amersham) and probed with sup.32P-labeled random primed DNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment from pC8 containing a portion of the rps 7/12plastid targeting sequence. Homoplasmic shoots are rooted aseptically on spectinomycin-containing MS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301-7305) and transferred to the greenhouse.
The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting.
Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding. Depending on the desired properties, different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding. Multi-line breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines that, for example, increase the effectiveness of conventional methods such as herbicide or pesticide treatment or allow one to dispense with said methods due to their modified genetic properties.
Use
Plant material harvested from the transgenic plants described herein is useful in downstream agronomical and industrial uses, such as human food, animal feed, biofuel, industrial alcohol, fermentation feedstock, and the like. Thus provided herein are methods for producing a modified multidomain enzyme comprising cultivating plants expressing the modified multidomain enzyme. Methods for producing ethanol comprising fermenting a modified multidomain enzyme-expressing plant are also encompassed, as well as methods for enhancing the digestibility of animal feed by adding a modified multidomain enzyme-expressing plant to the feed mix.
In one embodiment, this plant material can be used to formulate food or beverage for human consumption or animal feed, can be used to formulate diet with easily digestible starch and hence more extractable energy, or can be used to improve the nutritional quality of the food or feed (e.g., increased vitamin content, increased oil content, increased protein content, etc). The food, feed, or beverage can be flour, dough, bread, pasta, cookies, cake, thickener, beer, malted beverage, or a food additive. The food, feed, or beer product of can have reduced allergenicity and/or increased digestibility. Further, a dough product can have increased, strength and volume in comparison to a dough made from a non-transgenic seed or grain of the same species. The food, feed, or beverage can have hyperdigestible protein and/or hyperdigestible starch. The food, feed, or beverage can be hypoallergenic.
Oil extracted from the harvested plant material of the invention can be used as a raw material for chemical modification, a component of biodegradable material, a component of a blended food product, a component of an edible oil or cooking oil, lubricant, or a component thereof, biodiesel or a component thereof, a component of a snack food, a fermentation process raw material, or a component of cosmetics.
The harvested plant material of the invention can also be combined with other ingredients to produce a useful product. The specific ingredients included in a product will be determined according to the ultimate use of the product. Exemplary products include animal feed, raw material for chemical modification, biodegradable materials, blended food product, edible oil, cooking oil, lubricant, biodiesel process raw material, snack food, cosmetics, cleaning and detergent compositions (e.g., laundry detergents, dish washing detergents, and hard surface cleaning compositions), and fermentation process raw material. Products incorporating the harvested plant material described herein also include complete or partially complete swine, poultry, and cattle feeds, pet foods, and human food products such as extruded snack foods, breads, as a food binding agent, aquaculture feeds, fermentable mixtures, food supplements, sport drinks, nutritional food bars, multi-vitamin supplements, diet drinks, and cereal foods. Products incorporating the harvested plant material described herein include, e.g., cardboard, paper products, and industrial materials. These products may incorporate the raw harvested plant material, or may incorporate a processed or extracted form of the harvested plant material (e.g., oil, protein, starch, etc, extracted from the harvested plant material).
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989) and T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).
Example 1
Dicot Optimized Cellulase Genes
Dicot plant synthetic genes were designed using the backtranslation program in Vector NTI 9.0. Six protein sequences were backtranslated into dicot optimized coding sequences using the preferred codons for dicots. Additional sequence was added to the 5′ and 3′ end of each cellulase gene coding sequence for cloning and differential targeting to subcellular compartments. For construction of dicot transient expression vectors, an AscI, BamHI, and tobacco Kozak sequences were added at the 5′ end. An ER targeting sequence and SacI-NotI cloning sites were added at the 3′ end. Silent mutations were introduced to remove any restriction sites which interfered with cloning strategies. Synthetic genes were synthesized by GENEART (Germany).
Example 2
Construction of Plant Expression Vectors
Expression vectors capable of directing the expression of an optimized cellobiohydrolase gene (CBHI) with novel linkers in transgenic plants were designed. Table 1 outlines the sequences and vectors generated.
The constitutive CaMV 35S promoter was used to drive expression of the dicot optimized cellulase genes. Tobacco expressed cellulases were targeted to the endoplasmic reticulum (ER) via fusion to the tobacco PR1a signal sequence (SEQ ID NO:13) and the ER retention sequence (SEQ ID NO:14).
Tobacco expression vectors used the binary vector, pGR106, containing potato virus X (PVX) amplicon (Lu et al., 2003; EMBO J, 22:5690-5699). Vector component information is shown in Table 1.







TABLE 1







Description of sequences










SEQ ID NO:





of cellulase
Description of
Subcellular
Signal


insert
gene
targeting
sequence





1, 2
CBH1 with native
ER retention signal
PR1a ER targeting



linker (pSM439)

sequence


1, 2
CBH1 with native
None
None



linker




3, 4
CBH1 with GGG
ER retention signal
PR1a ER targeting



linker (pSM449)

sequence


3, 4
CBH1 with GGG
None
None



linker




5, 6
CBH1 with
ER retention signal
PR1a ER targeting



SGGGG linker

sequence



(pSM450)




5, 6
CHB1 with
None
None



SGGGG linker




7, 8
CBH1 with AP
ER retention signal
PR1a ER targeting



linker (pSM451)

sequence


7, 8
CBH1 with AP
None
None



linker




9, 10
CBH1 with VP
ER retention signal
PR1a ER targeting



linker (pSM452)

sequence


9, 10
CBH1 with VP
None
None



linker




11, 12
CBH1 with SP
ER retention signal
PR1a ER targeting



linker (pSM453)

sequence


11, 12
CHB1 with SP
None
None



linker (pSM453)









Description of C-Terminal Additions











SEQ ID NO:
Element







13
Tobacco PR1a ER targeting sequence


14
ER retention signal


15
Beta-conglycinin protein storage vacuole targeting sequence


16
Maize gamma zein 27 kD ER targeting sequence


17
Barley polyamine oxidase vacuole targeting sequence









The Glycine max glycinin GY1 promoter and signal sequence (GenBank Accession X15121) will be used to drive soybean seed specific expression of celluloses. Targeting of the cellulose in soybean will involve either the C-terminal addition of ER retention sequence (SEQ ID NO:14) or protein storage vacuole (PSV) sequence, (SEQ ID NO:15) from beta-conglycinin (Plant Phys 2004:134, 625-639).
A variety of promoters will be used to drive expression of monocot optimized cellulases with altered linker regions in transgenic plants. The maize PepC promoter (The Plant Journal 1994: 6(3), 311-319) will be used to drive leaf preferred expression of monocot optimized cellulose genes. The maize TrpA promoter (U.S. Pat. No. 6,018,104 and Plant Mol. Biol 27:1183-1188, 1995)) will be used to drive monocot stem specific expression of the monocot optimized cellulase. Each of the maize optimized cellulases will be cloned behind the rice glutei in promoter for expression in the endosperm of the maize seed.
The expression constructs will use a variety of targeting sequences to target the cellulases with altered linker regions to different organelles. For vacuole targeting, the cellulases with altered linkers will be operably linked to the gamma zein 27 kD signal sequence (SEQ ID NO:16) at the N terminus to targets through ER and fused to the vacuole sequence domain (VSD) from barley polyamine oxidase (SEQ ID NO:17) to direct the cellulose into the leaf vacuole (Plant Phys 2004: 134, 625-639). For retention of the cellulose with altered linker regions in the ER, the ER retention sequence (SEQ ID NO:14) will be used in place of the VSD.
All expression cassettes will be subcloned into a binary vector for transformation into tobacco, soybean, sugarcane, sugar beet and maize using recombinant DNA techniques that are known in the art.
Example 3
Transient Expression of Cellulases in Tobacco Leaves
Expression cassettes described in Example 2 above were cloned into PVX vector pGR106 (Lu et al., 2003). The PVX constructs were transferred into Agrobacterium tumefaciens strain GV3101 containing the helper plasmid pJIC SA_Rep (available on the Internet at jic.ac.uk/sainsbury-lab/dcb/Services/vigsprotocol.htm) using the freeze-thaw method (An et al., Binary vector. In: Gelvin S B, Schilproot R A (eds), Plant molecular biology manual. Kluwar Academic Publishers, Dordrecht. pp A3 1-19 (1988)).
Transgenic TEV-B tobacco plants (made in the tobacco cultivar Xanthi) containing a mutated P1/HC-Pro gene from TEV that suppresses post-transcriptional gene silencing (Mallory et al., Nat Biotechnol 20:622 (2002)) were used for transient expression of the cellulase genes described in Example 1. Preparation of Agrobacterium cultures and infiltration, of tobacco plants were carded out as described by Azhakanandam et al., Plant Mol. Biol, 63: 393-404 (2007). In brief, the genetically modified Agrobacteria were grown overnight in 50 ml of LB medium containing 100 μM acetosyringone and 10 μM MES (pH 5.6), and subsequently were pelleted by centrifugation at 4000 g for 10 min. The pellets were resuspended in the infection medium [Murashige and Skoog salts with vitamins, 2% sucrose, 500 μM MES (pH 5.6), 10 μM MgSO4, and 100 μM acetosyringone] to OD600=1.0 and subsequently held at 28° C. for 3 h. Infiltration of individual leaves was carried out on 4 weeks old recipient plants using a 5 ml syringe by pressing the tip of the syringe (without a needle) against the abaxial surface of the leaf. Infiltrated plants were maintained at 21-22° C. with a photoperiod of 16 h light and 8 h dark. Plant tissue was harvested after 5 days post infiltration for subsequent analysis.
Example 4
Enzyme Analysis of Tobacco Leaves Transiently Expressing Cellulases
Protein extracts were obtained from approximately 100-500 mg of leaf tissue collected from tobacco plants transiently expressing cellulases as described in Example 3. Leaf material from tobacco transiently expressing cellulases was placed into 24 deep well blocks containing small steel balls and pre-cooled on dry ice. Samples were ground to a fine powder rising a Genogrinder (SPEC/CertiPrep, Metuchen, N.J.). Samples were extracted in 500-1000 □l of Western Extraction Buffer (WEB=12.5 mM sodium borate, pH10; 2% BME; and 1% SDS at room temperature for approximately 30 minutes followed by centrifugation for 5 minutes at 13,000 rpm.
Alternatively, leaf or seed tissue will be collected from transgenic plants expressing cellulases with altered linkers. Flour samples will be prepared from seed by pooling approximately 10-20 seed and grinding to a fine powder using, a Kleco Grinder (Gracia Machine Company, Visalia, Ca.). Flour will be extracted as described in the paragraph above for tobacco transiently expressing cellulases.
SDS—polyacrylamide gel electrophoresis (SDS-PAGE) was performed by transferring 100 □l of WEB samples to an eppendorf tube and add 25 □l 4×BioRad LDS or modified BioRad loading buffer (4×BioRad LDS:BME at a ratio of 2:1). Samples were heated for 10 minutes at 70° C. then immediately place on ice for 5 minutes. Following incubation on ice, samples were centrifuged briefly. Sample extracts (5-10 □l) were run on BioRad 4-12% Bis/Tris protein gel (18 well) using MOPS buffer.
Immunoblot analysis was performed by transferring SDS-PAGE gels onto a nitrocellulose membrane using chilled Nupage transfer buffer (Invitrogen) for 30 minutes at 100 volts. Total protein transferred to the blot was visualized using Ponceau stain (Sigma). Following Ponceau stamina the membrane was incubated in blocking buffer for 30 minutes in TBST wash buffer (30 mM Tris-HCL, pH 7.5, 100 mM NaCl, and 0.05% Tween 20) with 3% dry milk, then washed three times for 5 minutes in TBST. Primary antibody was added at 1 ug/ml in TBST wash buffer with 3% milk, and the blot incubated 2 hours to overnight. Following overnight incubation, the blot was washed three times for 5 minutes each in TBST wash buffer. Secondary antibody (Rabbit-AP) was diluted 1:8000 (in TBST) and added to blot for at least 30 minutes. Following incubation in the secondary antibody, the blot was again washed three times for 5 minutes each. Visualization of immuno reactive bands was carried out by adding Moss BCIP/NBT—alkaline phosphatase substrate. Blots were rinsed thoroughly in water following incubation in the BCIP/NBT substrate and allowed to air dry.
Western blot analysis (see Table 2) was carried out using sample extracts from tobacco transient expression of cellulases using the constructs described in Example 2. Immuno-reactive bands detected on the western blot varied in apparent size and staining intensity. The predicted size, based on amino acid sequence, of each of the CBHI proteins with either the native linker (pSM439) or the heterologous linker (pSM449, pSM450, pSM451, pSM452, and pSM453) was 53.3, 52.3, 52.1, 53.1, 53.5, and 53.4 kD, respectively. Immuno-reactive bands were detected for constructs pSM439, pSM449, pSM452, and pSM453. Samples from leaves infiltrated with constructs pSM450 and pSM451, along with controls, showed no detectable protein in this experiment. The CBHI-native linker protein (pSM439) was detected as a band that migrates at about 55 kD The CBHI-Gly linker protein (pSM449) showed a band that migrated slightly below the CBHI-native linker protein. Interestingly, the CBHI-VP linker protein showed broad immuno-detectable band between 55-60 kD. Unexpectedly the CBHI-SP linker protein showed a broad immuno-detectable band between 55-64 kD. These results show that the strategy of using heterologous linkers is capable of improving the expression, accumulation and/or stability of the CBHI protein in plant tissue and that the type of linker appears to influence expression, accumulation and/or stability of the CBHI protein in tobacco.







TABLE 2







Western blot results for plant expression vectors


used for tobacco transient experiments.










Western
Construct


Linker Region
Results
number





STGGSSTTTASGTTTTKASSTSTSSTSTGTGV
+++
pSM439


(residues 468 through 499 of 




SEQ ID NO: 2)




 


GGGGGGGGGG GGGGGGGGGG GGGGGGGGGG
+
pSM449


(SEQ ID NO: 21)




 


SGGGGSGGGGSGGGGSGGGGSGGGGS
−
pSM450


(SEQ ID NO. 22)




 


APAPAPAPAPAPAPAPAPAPAPAPAPAPAP
−
pSM451


SEQ ID NO: 20)




 


VPVPVPVPVPVPVPVPAPVPVPVPVPVPVP
+++
pSM452


(SEQ ID NO: 18)




 


SPSPSPSPSPSPSPSPSPSPSPSPSPSPSP
++++
pSM453


(SEQ ID NO: 19)









Example 5
Enzyme Extraction and Activity Analysis of Transient Expressed CBHI
Approximately 100-500 mg of fresh leaf tissue from tobacco plants transiently expressing cellulases as described in Example 3, was extracted in 2 to 10 ml 100 mM Na Acetate buffer (pH 4.75), 0.02% NaN3, 0.02% Tween, and 1 Complete protease inhibitor cocktail tablet (Roche) per 50 ml of buffer. One of ordinary skill in the art would be able to identify other standard extraction buffers that would be appropriate for extracting protein from leaf. Samples were placed on benchtop rotators for 30-60 minutes then centrifuged at 3000 rpm for 10 minutes. For fresh leaf samples the amount of total protein extracted was measured by Pierce BCA protocol as outlined in product literature. Cellulase activity assays were carried out using methylumbelliferyl-lactoside (MUL). Additional substrates such as pNP-lactoside, carboxymethyl-cellulose, oat-b glucan, phosphoric acid treated cellulose (PASC). Avicel, or other commercially available substrates can used for measuring cellulase activity following previously published protocols (Methods in Enzymology, Vol 160).
CBHI activity results for transient expression of CBHI with native and heterologous linkers are shown in Table 3. All of the constructs, except pSM450, showed activity on the MUL substrate. Unexpectedly, the CBHI-VP and CBHI-SP proteins showed activity which was slightly lower than that observed for the CBHI-native linker protein. These results are significant, showing that it is possible to create proteolytically stable linkers without disrupting the functionality of the CBHI enzyme when expressed in tobacco.







TABLE 3







Summary of CBH1 activity in tobacco leaves transiently


expressing cellulases. Activity is reported as an average of


four individual leaves from four different plants. Samples were


extracted in buffer and CBH1 activity was assayed on


methylumbelliferyl-lactoside as the substrate.












Avg CBH1 Activity





(nmoles/min/mg




Sample
TSP)
StDev















unifiltrated leaf
0.09
0.08



culture medium only
0.15
0.17



PV X vector only
0.20
0.22



pSM439
1.55
0.46



pSM449
0.40
0.09



pSM450
0.21
0.22



pSM451
0.56
0.37



pSM452
0.97
0.25



pSM453
0.89
0.29










Example 6
Linker Modifications of Glucoamylase
Variants of the glucoamylase of SEQ ID NO: 24 will be generated in which the linker sequence has been replaced with the sequences outlined in Table 4. The glucoamylase variants will be generated by replacing bp 459-513 of SEQ ID: 24 with the sequence described in Table 4. The linker variants described in Table 4 are designed to alter the linker region of the native glucoamylase such that more full length protein accumulates in plant tissues. Glucoamylase variants made with the linkers of SEQ ID NO: 35-69 will generate glucoamylase variants that are less sensitive to degradation by plant proteases. The recognition site for some proteases are known; however, this recognition site may not be a specific string of amino adds but may be a pattern of specific types of amino acids assembled into a string. In particular the recognition sequence for the protease pepsin will be used to design variant linkers that have less similarity to the known recognition sequence for pepsin. The procine pepsin cleavage site is described by Powers in Adv. Exp. Med. Biol 95: 141-157 (1977).
As outlined in Table 4, the variant linker sequences are designed to avoid plant mediated cleavage of the linker region when the variant is expressed in plant cells. Several approaches will be taken to generate the linker variants. One approach is to eliminate protease cleavage sites of the native glucoamylase linker. Variants of the native glucoamylase linker sequence are outlined m Table 4 which have less similarity to the pepsin cleavage site. Additionally, variants have been designed which incorporate sites for plant based N or O linked glycosylation by increasing, the presence of the amino acids threonine and serine. Glycosylation may protect protease cleavage sites from protease attack by physically blocking access of the protease to the linker region. Another alternative outlined in Table 4 is the replacement of the native glucoamylase linker sequence with linkers from other enzymes which may be less sensitive to plant proteases, or replacement of the native glucoamylase linker sequence with synthetic linker sequences which promote plant based N or O linked glycosylation of the linker sequence.
The glucoamylase variants generated by replacing the native linker sequence (bp 459-513 of SEQ ID NO: 24 with the sequences outlined in Table 4) will be cloned into an expression vector which operably links the following components together; a promoter which is functional in plant cells such as the NOS promoter of SEQ ID NO: 70, subcellular targeting sequence such as the ER targeting sequence from the PR1a gene of SEQ ID NO: 13, dicot-optimized gene encoding the glucoamylase variant polypeptide, ER retention sequence of SEQ ID NO: 14. This expression construct will be designed to target the glucoamylase varient protein to the endoplasmic reticulum and to retain the glucoamylase variant protein in the endoplasmic reticulum.
As an alternative, the glucoamylase variants may be targeted to the vacuole in order to accumulate the variant protein for analysis. The expression construct for vacuolar targeting of the variant glucoamylase will consist of the following components operably linked together; a promoter which is functional in plant cells such as the NOS promoter of SEQ ID NO: 70, an ER targeting sequence such SEQ ID NO: 13, a dicot-optimized gene encoding the glucoamylase variant polypeptide, a vacuolar targeting sequence such as SEQ ID NO: 17.
The expression constructs described above will be generated by synthesis of the described expression cassette DNA by Gene Art. The synthesized expression cassette will be cloned into a binary vector also containing an origin of replication from BCTV, beet curly top virus. The BCTV containing binary vectors will be transferred into Agrobacterium tumefaciens strain LBA4404 containing a helper plasmid containing a BCTV replicase sequence using the freeze-thaw method (An et al., Binary vector, In: Gelvin S B, Schilproot R A (eds), Plant molecular biology manual. Kluwar Academic Publishers, Dordrecht, pp A3 1-19 (1988)).







TABLE 4 







Linker sequences to be used to create glucoamylase variants.









SEQ. ID




NO:
Variant linker sequence
properties












35
CSTGSATGTYSTPTGTSWPSTSTSGTAGT
Eliminate protease



TTTSATTTTSTSVSSTTTTTTSTTSC
cleavage site


 


36
CSTGSATGPYSTPTGTSWPSTSTSGTAGT
Eliminar protease



TTTSATTTTSTSVSSTTTTTTSTTSC
cleavage site


 


37
CSTGSATGPYSTPTGTSWPSTSTSGTAGT
Eliminate protease



TTTSATTTTSTSVSTTTTTTSTTSC
cleavage site


 


38
CSTGSATGPYSTPTGTSWPSTTSGTAGTT
Eliminate protease



TTSATTTTSTSVSGTTTTTTSTTSC
cleavage site


 


39
CSTGSATGTSTPTGTSWPSTVTSGTAGTT
Eliminate protease



TTSATTTTSTSVSGTTTTTTSTTSC
cleavage site


 


40
CSTGSATGPYSTPTGTSWPSTSTSGTAGT
Eliminate protease



TTTSATTTTSTTTTTTTSTTSC
cleavage site


 


41
CSTGSATGPYSTPTGTSWPSTSTSGTAGT
Eliminate protease



TTTSATTTTTTTTTTSTTSC
cleavage site


 


42
CSTGSATGPYSTPTGTSWPSTSTSGTAGT
Eliminate protease



TTTSATTTTSTSVSGTTTTTTSTTSC
cleavage site


 


43
CSTGSATGPYSTPTGTSWPSTVTSGTAGT
Eliminate protease



TTTSATTTTSTSVSGTTTTTTSTTSC
cleavage site


 


44
CSTGSATGPYSTPTGTSWPSTSTSGTAGT
Eliminate protease



TTTSATTTTSTSVSKTNTTTTSTTSC
cleavage site and add




a N-glycosylation site


 


45
CSTGSATGPYSTPTGTSWPPSQTPGTAGT
Eliminate protease



TTTSATTTTSTSVSSTTTTTTSTTSC
cleavage site; add 




linker from other enzyme


 


46
CSTGSATGPYSTPTGTSWPSTSTSGTAGT
Eliminate protease



TTTSATTTTSTTSSASTSTTSC
cleavage site; add 




linker from other enzyme


 


47
CSTGSATGPYSTPTGTSWPSTSTSGGVPTP
Eliminate protease



TGTTTTTTSTTSC
cleavage site; add 




linker from other enzyme


 


48
CSTGSATGPYATPTNTAWPTTTQPGTAG
Eliminate protease



TTTTSATTTTSTSVSSTTTTTTSTTSC
cleavage site; add 




linker from other enzyme


 


49
CSTGSATGPYATPTNTAWPTTTQPGTAG
Eliminate protease



TTTTSATTTTSTTTTTTTSTTSC
cleavage site; add 




linker from other enzyme


 


50
CSTGSATGTYSTPTGTSWPPSQTPKPGVP
Add linker from other



SGTPYTPLPC
enzyme


 


51
CSTGSATGTYSTPTGTSWPPSQTPSPGVPS
Add linker from other



GTPSTPLPC
enzyme


 


52
CSTGSATGTYSTPTGTSWPPSQTPSPGVPS
Add linker from other



GTPSTPSPC
enzyme


 


53
CSTGSATGTSSTPTGTSWPPSQTPSPGVPS
Add linker from other



GTPSTPSPC
enzyme


 


54
CSTGSATGTSSTPTGTSWPTKSPTTTTAT
Add linker from other



ATTTTAPSTSTTPPSSSEPATFPTGNC
enzyme and promote N




glycosylation


 


55
CSTGSATGTSSTPTGTSWPTSSPTTTTATA
Add linker from other



TTTTAPSTSTTPPSSSTPATFPTGNC
enzyme and promote N




glycosylation


 


56
CSTGSATGTSSTPTGTSWPTSSPTTTTATS 
Add linker from other



TTPPSSSTPATFPTGNC
enzyme and promote N




glycosylation


 


57
CSTGSATGTSSTPTGTSWPTSSPTTSTTPP
Add linker from other



SSSTPATFPTGNC
enzyme and promote N




glycosylation


 


58
CSATSATGPYATPTNTAWPSTVTSGTAGT
Eliminate protease



TTTATTTTSTSVSGTTTQPPERPAC
cleavage site; add 




linker from other enzyme


 


59
CSATSATGPYATPTNTAWTTTQPPERPAC
Add linker from other




enzyme


 


60
CSATSATGPYATPTNTAWGGGGSTTTQP
Add linker from other



PERPAC
enzyme plus use of




synthetic sequence


 


61
CSATSATGPYATPTNTAWGGGGSGGGGS
Add linker from other



TTTQPPERPAC
enzyme plus use of




synthetic sequence


 


62
CSATSATGPYATPTNTAWGTAGVPTPTG
Eliminate protease



PTPTTTTQPPERPAC
cleavage site; add 




linker from other enzyme


 


63
CSTGSATGTYSTPTGTSWPGGGGSGGGG
synthetic



SGGGGSC



 


64
CSTGSATGTYSTPTGTSWPGGGGSGGGG
synthetic



SGGGGSGGGGSC



 


65
CSTGSATGTYSTPTGTSWPGGGGSGGGG
synthetic



SGGGGSGGGGSGGGGSC



 


66
CSTGSATGTYSTPTGTSWPGGGGSGGGG
synthetic



SGGGGSGGGGSGGGGSSGGGGSC



 


67
CSTGSATGTYSTPTGTSWPGGGCSCGGG
Synthetic plus N



SGGGGSGGGGSATFPTGNC
glycosylation sites


 


68
CSTGSATGTYSTPTGTSWPGGGGSGGGG
Synthetic plus N



SGGGGSGGGGSGNSTISSC
glycosylation site


 


69
CSTGSATGTYSTPTGTSWPGGGGSGGGG
Synthetic plus N



SGGGGSGGGGSGNSTISSATFPTGNC
glycosylation sites









Glucoamylase variant enzymes will be produced by transient expression of the enzyme in the leaves of tobacco plants Transgenic TEV-B tobacco plants (made in the tobacco cultivar Xanthi) containing a mutated P1/HC-Pro gene from TIN that suppresses post-transcriptional gene silencing (Mallory et al., Nat Biotechnol 20:622 (2002)) will be used for transient expression of selected enzymes in tobacco leaves. Alternatively, leaves from non-transgenic tobacco plants will be used for transient expression of selected enzymes in tobacco leaves. Preparation of Agrobacterium cultures and infiltration of tobacco plants will be carried out as described by Azhakanandam et al., Plant Mol. Biol. 63: 393-404 (2007). In brief, the genetically modified agrobacteria will be grown overnight in 50 mL of LB medium containing 100 μM acetosyringone and 10 μM MES (pH 5.6), and subsequently will be pelleted by centrifugation at 4000×g for 10 min. The pellets will be resuspended in the infection medium [Murashige and Skoog salts with vitamins, 2% sucrose, 500 μM. MES (pH 5.6), 10 μM MgSO4, and 100 μM acetosyringone] to OD600=1.0 and subsequently held at 28 degrees C. for 3 hours. Infiltration of individual leaves will be carried out on TEV-B or non-transgenic tobacco plants (about 4 weeks old) using a 5 mL syringe by pressing the tip of the syringe (without a needle) against the abaxial surface of the leaf. Infiltrated plants will be maintained at 22-25 degrees C. with a photoperiod of 16 hours light and 8 hours dark. Plant tissue will be harvested after 5 days post infiltration for subsequent analysis.
Analysis of variant glucoamylase production by transient expression in tobacco plants will be performed by western blot of plant extracts essentially as described in Example 4. Leaves from tobacco plants transiently expressing the glucoamylase variants will be collected and total protein extracted from the leaves essentially as described in Example 4. The total protein from the tobacco leaves will be separated on a polyacrylamide gel and subsequently transferred to a nylon membrane essentially as described in Example 4. Antibodies which bind to glucoamylase of SEQ ID NO: 24 will be used to detect the glucoamylase variants using standard assays and techniques for western blot analysis. The size of the glucoamylase variants will be determined by comparing the mobility of the variant glucoamylase with a protein standard. Based upon the size of the glucoamylase variant, it will be determined if the linker variant was less sensitive to cleavage by plant proteases. The percentage of the produced glucoamylase variant that is less sensitive to protease cleavage will be determined by comparing the amount of protein in the varying molecular weight bands identified by western blot. Methods for determining the amount and ratio of different sized proteins are known and include imaging the western blot and determining the area of a specific band of protein.
Example 7
Linker Modifications of Cellobiohydrolase (SEQ ID NO: 25-34)
Dicot optimized plant synthetic genes will be designed using the backtranslation program in Vector NTI 9.0. Protein sequences will be backtranslated into dicot optimized coding sequences using the preferred codons for dicots. Additional sequence may be added to the 5′ and/or 3′ end of each cellulose gene coding sequence for cloning and differential targeting to subcellular compartments. Synthetic genes will be synthesized by GENEART (Germany).
Dicot optimized expression vectors capable of directing the expression of an optimized cellobiohydrolase gene (CBHI) with novel linkers in transgenic plants will be designed. Table 5 outlines the sequences and vectors that will be generated for transient expression in tobacco leaves. A constitutive promoter will be operably linked to the polynucleotide sequence encoding the cellobiohydrolase variants. An example of a constitutive promoter is the NOS promoter from Agrobacterium of SEQ ID NO: 70. The cellulose variant enzyme will be targeted to different subcellular compartments. The use of an endoplasmic reticulum (ER) targeting sequence such as SEQ ID NO: 14 will be used to promote passage of the cellobiohydrolase through the ER for appropriate processing. The use of an ER retention sequence such as SEQ ID NO: 23 will be used to retain the cellobiohydrolase in the ER. Alternatively, the use of a vacuolar targeting sequence such as SEQ ID NO: 15 will be used to target the cellobiohydrolase to the vacuole for accumulation. Alternatively, no targeting sequence will be used to accumulate the cellobiohydrolase in the apoplast of the plant cell.
Monocot optimized expression vectors capable of directing the expression of an optimized cellobiohydrolase with novel linkers in transgenic plants will be designed. The expression constructs designed for monocot expression will use a variety of targeting sequences to target the cellulases with altered linker regions to different organelles. For vacuole targeting, the cellulases with altered linkers will be operably linked to the gamma zein 27 kD signal sequence (SEQ ID NO: 16) at the N terminus to target through the ER, and fused to the vacuole sequence domain (VSD) from barley polyamine oxidase (SEQ ID NO:17) to direct the cellulose into the leaf vacuole (Plant Phys 2004: 134, 625-639). For retention of the cellulose with altered linker regions in the ER, the ER retention sequence (SEQ ID NO:14) will be used in place of the VSD. A variety of promoters will be used to drive expression of monocot optimized cellulases with altered linker regions in transgenic plants. The maize PepC promoter (The Plant Journal 1994: 6(3), 311-319) will be used to drive leaf preferred expression of monocot optimized cellulose genes. The maize TrpA promoter (U.S. Pat. No. 6,018,104 and Plant Mot Biol 27:1183-1188, 1995)) will be used to drive monocot stem specific expression of the monocot optimized cellulose. Each of the maize optimized cellulases will be cloned behind the rice glutelin promoter for expression in the endosperm of the maize seed.
All monocot optimized expression cassettes will be subcloned into a binary vector for transformation into tobacco, soybean, sugarcane, sugar beet and maize using recombinant DNA techniques that are known in the art.







TABLE 5







Expression constructs containing variants of CBH1 with modified linkers.












Subcelluar




Description of gene 
targeting
Signal sequence


Construct
(SEQ ID NO:)
(SEQ ID NO:)
(SEQ ID NO:)













CBH1
CBH1, linker modified to
Vacuole (15)
PR1a ER targeting sequence


linker 9
promote glycosylation (25)

(14)


CBH1
CBH1, linker modified to
ER retention
ER targeting sequence (13)


linker 8
promote glycosylation 26)
(23)



CBH1
CBH1, linker modified to
ER retention
ER targeting sequence (13)


linker 7
promote glycosylation (27)
(23)



CBH1
CBH1, linker modified to
ER retention
ER targeting sequence (13)


linker 6
promote glycosylation (28)
(23)



CBH1
CBH1, linker modified to
ER retention
ER targeting sequence (13)


linker 5
promote glycosylation (29)
(23)



CBH1
CBH1, linker modified to
ER retention
ER targeting sequence (13)


linker 4
promote glycosylation (30)
(23)



CBH1
CBH1, linker modified to
ER retention
ER targeting sequence (13)


linker 3
promote glycosylation (31)
(23)



CBH1
CBH1, linker modified to
ER retention
ER targeting sequence (13)


linker 2
promote glycosylation (32)
(23)



CBH1
CBH1, linker modified to
none
ER targeting sequence (14)


linker 10
promote glycosylation (33)




CBH1
CBH1, linker modified to
ER retention
ER targeting sequence (I3)


linker 1
promote glycosylation (34)
(23)



pSM439
CBH1 with native linker
Vacuole (17)
PR1a ER targeting sequence


CBH1
(pSM439) (1, 2)

(14)


pSM449
CBH1 with GGG linker
Vacuole (17)
PR1a ER targeting sequence


CBH1
(pSM439) (3, 4)

(14)


pSM450
CBH1 with SGGGG linker
Vacuole (17)
PR1a ER targeting sequence


CBH1
(pSM450) (5, 6)

(14)


pSM451
CBH1 with AP linker
Vacuole (17)
PR1a ER targeting sequence


CBH1
(pSM451) (7, 8)

(14)


pSM452
CBH1 with VP linker
Vacuole (17)
PR1a ER targeting sequence


CBH1
(pSM452) (9, 10)

(14)


pSM453
CBH1 with SP linker
Vacuole (17)
PR1a ER targeting sequence


CBH1
(pSM453) (11, 12)

(14)


pSM439
CBH1 with native linker
None
PR1a ER targeting sequence


CBH1
(pSM439) (1, 2)

(14)


pSM449
CBH1 with GGG linker
None
PR1a ER targeting sequence


CBH1
(pSM439) (3, 4)

(14)


pSM450
CBH1 with SGGGG linker
None
PR1a ER targeting sequence


CBH1
(pSM450) (5, 6)

(14)


pSM451
CBH1 with AP linker
None
PR1a ER targeting sequence


CBH1
(pSM451) (7, 8)

(14)


pSM452
CBH1 with VP linker
None
PR1a ER targeting sequence


CBH1
(pSM452) (9, 10)

(14)


pSM453
CBH1 with SP linker
None
PR1a ER targeting sequence


CBH1
(pSM453) (11, 12)

(14)









Analysis of plant expression of cellobiohydrolase linker variants will be performed by western blot analysis of variants expressed transiently in tobacco leaves. The leaves from tobacco plants transiently expressing the cellobiohydrolase constructs will be generated essentially as described in Example 6 for glucoamylase variants. Briefly, tobacco leaves will be infiltrated with agrobacterium containing an expression cassette containing the variant cellobiohydrolase constructs. Tobacco leaves will be collected after approximately 5 days and the protein in the leaves extracted essentially as described in Example 4. Western blot analysis will be formed essentially as described in Example 4 using antibodies that react with the cellobiohydrolase variant protein. Table 6 outlines Western blot analysis of cellobiohydrolase linker variants expressed transiently in tobacco leaves.







TABLE 6







Western blot analysis of Cellobiohydrolase linker variants.












Subcellular
Relative




targeting (SEQ
proportion full-



Construct (SEQ ID NO:)
ID NO:)
lenght protein *







CBH1 linker 8 (26)
ER retention (23)
+++



CBH1 linker 9 (25)
Vacuole (15)
−



CBH1 linker 10 (33)
None
+++



pSM439 CBH1 (1, 2)
Vacuole (17)
+



pSM449 CBH1 (3, 4)
Vacuole (17)
++++



pSM450 CBH1 (5, 6)
Vacuole (17)
++++



pSM451 CBH1 (7, 8)
Vacuole (17)
++++



pSM452 CBH1 (9, 10)
Vacuole (17)
++++



pSM453 CBH1 (11, 12)
Vacuole (17)
++++



pSM439 CBH1 (1, 2)
None
+



pSM449 CBH1 (3, 4)
None
+++



pSM450 CBH1 (5, 6)
None
+++



pSM451 CBH1 (7, 8)
None
+++



pSM452 CBH1 (9, 10)
None
+++



pSM453 CBH1 (11, 12)
None
++++







* Refers to proportion of full-length protein as detected by Western blot by anti CBH1 antibody probe, indicating that all (++++), some (++) or none (−) of the detectable protein had an apparent molecular weight similar to, or greater than, the predicted full length CBH1 protein.






The modified linker variants of CBHI were transiently expressed in tobacco leaves and the lysates of tobacco leaves collected as described above. The lysates were analyzed by Western blot using an antibody that recognizes CBHI and showed significant increase in proportion of full-length CBHI protein compared to similarly expressed CBHI with native linker sequence (pSM439). CBHI linker 8 and CBHI linker 10, displayed resistance to proteolytic cleavage in the ER and apoplast targeted constructs. Linker variants pSM449, pSM450, pSM451, pSM452 and pSM453 were directed to the vacuole and these linker variants resulted in protein with a molecular weight similar to, or greater than pSM439 directed to the vacuole. All of the protein produced is likely full length. Apoplast targeted CBHI linker variants of pSM449, pSM450, pSM451, pSM452 and pSM453, was predominantly similar to or greater than, the predicted molecular weight of full length CBHI, indicating a minimal amount of proteolytic cleavage. The data show that linker amino acid sequences may be engineered to reduce and/or eliminate proteolytic cleavage of CBHI.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

## Claims

1. A method for expressing a multidomain enzyme in a plant cell comprising introducing into said plant cell a nucleic acid construct comprising a nucleotide sequence encoding a modified multidomain enzyme, wherein said multidomain enzyme comprises at least one first domain, at least one first linker sequence, and at least one second domain, wherein said first domain and said second domain are non-heterologous sequence, wherein the native linker sequence in said modified multidomain enzyme has been replaced with a heterologous linker sequence, wherein said heterologous linker sequence is a linker sequence that is not cleaved by a plant protease, wherein said heterologous linker sequence is selected from the group consisting of SEQ ID NO:18, 19, and 20, and wherein the multidomain enzyme is targeted to a vacuole.

2. The method of claim 1, wherein said plant cell expresses said modified multidomain enzyme, wherein a full length multidomain enzyme is produced by said plant cell.

3. The method of claim 1, wherein said multidomain enzyme is selected from the group consisting of a cellulase enzyme and a glucoamylase enzyme.

4. The method of claim 1, wherein said plant cell is selected from the group consisting of a rice, wheat, corn, soybean, sugar beet, and sugar cane plant cell.

5. A plant cell comprising a nucleic acid construct comprising a nucleotide sequence encoding a modified multidomain enzyme, wherein said multidomain enzyme comprises at least one first domain, at least one first linker sequence, and at least one second domain, wherein the native linker sequence in said modified multidomain enzyme has been replaced with a heterologous linker sequence, wherein said first domain and said second domain are non-heterologous sequence, wherein the native linker sequence in said modified multidomain enzyme has been replaced with a heterologous linker sequence, wherein said heterologous linker sequence is a linker sequence that is not cleaved by a plant protease, wherein said heterologous linker sequence is selected from the group consisting of SEQ ID NO:18, 19, and 20, and wherein the multidomain enzyme is targeted to a vacuole.

6. The plant cell of claim 5, wherein said plant cell expresses said modified multidomain enzyme, wherein a full length multidomain enzyme is produced by said plant cell.

7. The plant cell of claim 5, wherein said multidomain enzyme is a cellulase enzyme.

8. The plant cell of claim 5, wherein said plant cell is selected from the group consisting of a rice, wheat, corn, soybean, sugar beet, and sugar cane plant cell.

9. A plant comprising the plant cell of claim 5.

10. A transgenic seed produced from said plant of claim 9.

11. A method of producing fermentable sugars from plant biomass, said method comprising:
(a) obtaining a plant comprising a nucleic acid construct comprising a nucleotide sequence encoding a modified multidomain enzyme, wherein said multidomain enzyme comprises at least one first domain, at least one first linker sequence, and at least one second domain, wherein the native linker sequence in said modified multidomain enzyme has been replaced with a heterologous linker sequence, wherein said first domain and said second domain are non-heterologous sequence, wherein said heterologous linker sequence is a linker sequence that is not cleaved by a plant protease, wherein said heterologous linker sequence is selected from the group consisting of SEQ ID NO:18, 19, and 20, wherein the multidomain enzyme is targeted to a vacuole, and wherein said multidomain enzyme is involved in the conversion of plant material to fermentable sugar;
(b) growing said plant under conditions in which the nucleic acid construct is expressed; and
(c) using said plant in a biomass conversion method.