Methods and compositions for the production of mannanase in plants

Abstract

Compositions and methods for expressing a nucleotide sequence encoding a mannanase polypeptide in a plant of interest and for the degradation of mannan in plants and plant parts are provided. Compositions having mannanase activity are provided. Compositions include isolated and recombinant polypeptide sequences having mannanase activity, recombinant and synthetic nucleic 5 acid molecules encoding the mannanase polypeptides, constructs or expression cassettes comprising at least one nucleic acid molecule encoding a mannanase polypeptide, and plants, plant cells, plant parts, and seed transformed with constructs comprising the nucleic acid molecules. Animal feed made from the transformed plants, plant parts, and seed are also encompassed.

Description

[0001] METHODS AND COMPOSITIONS FOR THE PRODUCTION OF MANNANASE IN PLANTS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 580,580, filed September 5, 2023, which is herein incorporated by reference in its entirety.

[0004] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is herein incorporated by reference in its entirety. Said XML copy, created on September 4, 2024, is named “Ml 17885_1040WO_seq_listing.xml” and is 13 kilobytes in size.

[0006] FIELD OF THE INVENTION

[0007] The invention is drawn to the expression of mannanase in plant cells and the use of transformed plants and seed in animal feed.

[0008] BACKGROUND OF THE INVENTION

[0009] P-Mannans (beta-galactomannans) are antinutritive fibers found in common feed ingredients, including soybean meal, and other leguminous feeds. P-Mannans have a molecular pattern like some pathogens, which triggers a Feed-Induced Immune Response (FUR). This unnecessary innate immune system stimulation consumes energy, and other valuable nutrients. This results in an increase in proliferation of macrophages and monocytes, and increased cytokine production, leading to an increased severity of disease symptoms and a decrease in the efficiency of nutrient use. P-mannan is present in most poultry diets currently used around the world. However, as noted, P-mannan has been found to be deleterious to animal performance, compromising weight gain and feed conversion.

[0010] Numerous studies have suggested that the addition of suitable crude enzyme preparations to diets for both animals, including pigs and poultry, can lead to improvements in feed conversion efficiency and live weight gain. P-mannanase supplementation in diet especially in high fiber diets or low energy diets has been reported to benefit the animals in many ways. Enzyme supplementation of poultry rations has been widely reviewed and well documented to improve efficiency of converting feedstuffs into broiler tissue.

[0011] To date the enzyme must be prepared by fermentation and isolation for incorporation into animal diets. Thus, a method to supply the enzyme directly into the animal feed would be advantageous. SUMMARY OF THE INVENTION

[0012] Compositions and methods for expressing a nucleotide sequence encoding a mannanase polypeptide in a plant of interest and for the degradation of mannan in plants and plant parts are provided. Compositions having mannanase activity are provided. Compositions include isolated and recombinant polypeptide sequences having mannanase activity, recombinant and synthetic nucleic acid molecules encoding the mannanase polypeptides, constructs or expression cassettes comprising at least one nucleic acid molecule encoding a mannanase polypeptide, and plants, plant cells, plant parts, and seed transformed with constructs comprising the nucleic acid molecules. Animal feed made from the transformed plants, plant parts, and seed are also encompassed.

[0013] The expressed mannanase polypeptide may be targeted to organelles, vacuoles or compartmental spaces in a plant or plant part. The mannanase enzyme is available to degrade the mannan in the transformed plant, plant part, or seed. Further, once the plant, seed, or plant part is harvested and ready for preparation as animal feed, upon grinding or processing of the plant or plant part, the mannanase enzyme breaks down P-mannans, allowing more energy to be available for growth and performance of the animal.

[0014] Transformed plants, plant cells, and seeds, as well as methods for making such plants, plant cells, and seeds are additionally provided. Such transformed plants, plant cells, and seeds are transformed with an expression construct encoding a mannanase polypeptide and may be operably linked to a targeting peptide. In this manner, the polypeptide of interest is directed to the cell wall, ER, or vacuoles of the plant cells. Animal feed comprising the transformed plants, plant parts, and / or seed are also encompassed.

[0015] The following embodiments are encompassed by the invention:

[0016] 1. A method for degrading mannan in a plant or plant part, said method comprising transforming said plant with an expression cassette comprising a nucleotide sequence encoding a mannanase polypeptide.

[0017] 2. The method of embodiment 1, wherein said mannanase is heat stable.

[0018] 3. The method of embodiment lor 2, wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a seed-preferred promoter.

[0019] 4. The method of embodiment 3, wherein said seed-preferred promoter is selected from the group consisting of Ciml (cytokinin-induced message), maize 15 kDa zein, maize 19 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, endl, end2, milps (myo-inositol- 1 -phosphate synthase, Gamma-zein, Globulin (Glb-1), bean P-phaseolin, napin, P- conglycinin, soybean lectin, and cruciferin.

[0020] 5. The method of embodiment 4, wherein said seed-preferred promoter is a globulin-1 promoter.

[0021] 6. The method of any one of embodiments 1-5, wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a nucleotide sequence which encodes a peptide that targets the mannanase polypeptide to a subcellular compartment.

[0022] 7. The method of embodiment 6, wherein said subcellular compartment is selected from the group consisting of a vacuole, embryo, apoplast, and endoplasmic reticulum.

[0023] 8. The method of any one of embodiments 1-7, wherein said plant is transformed with a second nucleotide sequence encoding an enzyme that degrades mannans.

[0024] 9. The method of embodiment 8, wherein said second enzyme is selected from the group consisting of P-mannosidase, acetyl mannan esterase, P-glucosidase, and a-galactosidase.

[0025] 10. The method of any one of embodiments 1-9, wherein said nucleotide sequence encoding a mannanase polypeptide is codon optimized for expression in said plant.

[0026] 11. The method of any one of embodiments 1-10, wherein said nucleotide sequence encoding a mannanase polypeptide comprises a sequence having 90% sequence identity to the sequence set forth in SEQ ID NO. 1.

[0027] 12. The method of any one of embodiments 1-11, wherein said mannanase polypeptide comprises the peptide sequence Lys-Asp-Glu-Leu (KDEL) at the C-terminus.

[0028] 13. The method of any one of embodiments 1-12, wherein said mannanase polypeptide is operably linked to the barley alpha amylase signal sequence (BAASS).

[0029] 14. A plant transformed with an expression cassette comprising a nucleotide sequence encoding a mannanase polypeptide.

[0030] 15. The transformed plant of embodiment 14, wherein said mannanase is heat stable.

[0031] 16. The plant of embodiment 14 or 15, wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a seed-preferred promoter.

[0032] 17. The plant of embodiment 16, wherein said seed-preferred promoter is selected from the group consisting of Ciml (cytokinin-induced message), maize 15 kDa zein, maize 19 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, endl, end2, milps (myo-inositol- 1 -phosphate synthase, Gamma-zein, Globulin (Glb-1), bean P-phaseolin, napin, P- conglycinin, soybean lectin, and cruciferin.

[0033] 18. The plant of embodiment 17, wherein said seed-preferred promoter is a globulin-1 promoter. 19. The plant of any one of embodiments 14-18, wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a nucleotide sequence which encodes a peptide that targets the mannanase polypeptide to a subcellular compartment.

[0034] 20. The plant of embodiment 19, wherein said subcellular compartment is selected from the group consisting of a vacuole, embryo, apoplast, and endoplasmic reticulum.

[0035] 21. The plant of any one of embodiments 14-20, wherein said nucleotide sequence encoding a mannanase polypeptide is codon optimized for expression in said plant.

[0036] 22. The plant of any one of embodiments 14-21, wherein said nucleotide sequence encoding a mannanase polypeptide comprises a sequence having 90% sequence identity to the sequence set forth in SEQ ID NO. 1.

[0037] 23. The plant of any one of embodiments 14-22, wherein said mannanase polypeptide comprises the peptide sequence Lys-Asp-Glu-Leu (KDEL) at the C-terminus.

[0038] 24. The plant of any one of embodiments 14-23, wherein said mannanase polypeptide is operably linked to the barley alpha amylase signal sequence (BAASS).

[0039] 25. Seed of any one of the plants of embodiments 14-24.

[0040] 26. Animal feed that comprises any of the plants of embodiments 14-24.

[0041] 27. Animal feed that comprises a seed of embodiment 25.

[0042] 28. An expression cassette comprising a nucleotide sequence encoding a mannanase polypeptide.

[0043] 29. The expression cassette of embodiment 28, wherein said mannanase is heat stable.

[0044] 30. The expression cassette of embodiment 28 or 29, wherein said nucleotide sequence has been codon optimized for expression in a plant of interest.

[0045] 31. The expression cassette of any one of embodiments 28-30, wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a seed-preferred promoter.

[0046] 32. The expression cassette of embodiment 31, wherein said seed-preferred promoter is selected from the group consisting of Ciml (cytokinin-induced message), maize 15 kDa zein, maize 19 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, endl, end2, milps (myo-inositol- 1 -phosphate synthase, Gamma-zein, Globulin (Glb-1), bean P- phaseolin, napin, P-conglycinin, soybean lectin, and cruciferin.

[0047] 33. The expression cassette of embodiment 32, wherein said seed-preferred promoter is a globulin- 1 promoter.

[0048] 34. The expression cassette of any one of embodiments 28-33, wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a nucleotide sequence which encodes a peptide that targets the mannanase polypeptide to a subcellular compartment. 35. The expression cassete of embodiment 34, wherein said subcellular compartment is selected from the group consisting of a vacuole, embryo, apoplast, and endoplasmic reticulum.

[0049] 36. The expression cassette of any one of embodiments 28-35, wherein said nucleotide sequence encoding a mannanase polypeptide comprises a sequence having 90% sequence identity to the sequence set forth in SEQ ID NO. 1.

[0050] 37. The expression cassette of any one of embodiments 28-36, wherein said mannanase polypeptide comprises the peptide sequence Lys-Asp-Glu-Leu (KDEL) at the C-terminus.

[0051] 38. The method of any one of embodiments 28-37, wherein said mannanase polypeptide is operably linked to the barley alpha amylase signal sequence (BAASS).

[0052] BRIEF DESCRIPTION OF THE FIGURES

[0053] Figure 1 shows approximate expression levels (pg / g) of mannanase in representative T1 generation maize seeds as measured in a blue dye assay.

[0054] Figure 2 shows approximate expression levels (pg / g) of mannanase in representative T2 generation maize seeds as measured in a blue dye assay.

[0055] Figure 3 shows approximate expression levels (pg / g) of mannanase in representative T3 generation maize seeds as measured in a blue dye assay.

[0056] Figure 4 shows approximate expression levels (pg / g) of mannanase in representative T4 generation maize seeds as measured in a blue dye assay.

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] Overview

[0059] The present invention provides compositions and methods for expressing mannanase in plant cells, plants, plant parts, and plant seeds. In some embodiments, the mannanase is heat stable or thermostable. The expressed mannanase may be directed to plant compartmental or subcellular spaces. Compositions of the invention include the transformed plants, plant parts, plant seeds expressing the mannanase, enzyme, animal feed made from the transformed plants or plant material, as well as expression cassetes for expressing the mannanase nucleotide sequence. The mannanase nucleotide sequence may be operably linked to promoters and targeting sequences to express the polypeptide in tissues of interest and direct storage of the polypeptide into cellular compartments such as vacuoles, organelles, endoplasmic reticulum, apoplast, and the like. Additional mannan degrading polypeptides may also be expressed in the plant cell or added to the animal feed to aid in the biodegradation of mannan. The combination of enzymes works together to break down the main and the side chains of mannan. In some instances, the combination of enzymes exhibits synergy.

[0060] Currently, the mannanase used to degrade the mannan in animal feed is produced by fermentation. The present method expresses the mannanase in the plant of interest where it is ready to degrade the mannan without the labor and cost of fermentation and isolation of mannanase. fl-mannanase

[0061] P-mannanase is a nutrient-sparing enzyme that breaks down P-mannans. P-Mannans (betagalactomannans) are antinutritive fibers found in common feed ingredients. Hemicellulose is the second most abundant polymer found in nature. It is usually present together with lignin and cellulose in the plant cell walls. It is estimated that hemicellulose is a third of the total components in plants, hetero- 1,4-P-D-mannans and hetero- 1,4-P-D-xylans are the two most significant types of hemicelluloses. In plant fruits and seeds the hemicellulose is mainly present in the form of mannan. The present invention expresses the mannanase enzyme in the plant, plant part, or seed of interest where the enzyme is readily available to degrade mannan. These mannanase enzymes can function under conditions of a wide range of pH and temperature.

[0062] Any of the mannan-degrading enzymes can be expressed in the plant including the mannandegrading enzymes classified in the different glycosyl hydrolase families (e.g., GH 1, GH 2, GH 3, GH 5, GH 26, GH 27, GH 113, etc.) catalyzing the production of oligosaccharides and monosaccharides. These enzymes can also be used to degrade plant mannan and to enhance plant metabolism, such as ripening and maturation by metabolizing mannan present in the cell wall.

[0063] Any mannanase polypeptide or enzyme or variant thereof may be used in the practice of the invention. In some embodiments, the enzyme is modified or mutated for enhanced expression, better stability, inactivation of glycosylation sites, and the like. See, for example, Li et al. (2014) J. Ind. Microbiol. Biotechnol. 41 :693-700, who modified the Aspergillus usamii P-mannanase to improve its substrate affinity by in silico design and site-directed mutagenesis; Li et al. (2017) J. Biosci. Bioeng. 123:294-299, who engineered a carbohydrate-binding module into an Aspergillus usamii P-mannanase to perfect its enzymatic properties; Li et al. (2017) Biotechnol. Biofuels 10:1— 12 who used directed evolution of a P-mannanase from Rhizomucor miehei to improve catalytic activity in acidic and thermophilic conditions.; Li et al. (2013) J. Biotechnol. 163:401-407 who used rational design for trypsin-resistant improvement of Armillariella tabescens P-mannanase based on molecular structure evaluation. In another embodiment, suitable substitution of amino acids can facilitate the access of substrate to the substrate binding groove and maintain it therein, thus improving the substrate affinity and catalytic efficiency of the enzyme. Likewise, the substrate affinity may be improved by rational modification or site directed mutagenesis, which can be predicted by in silico design, including molecular docking simulations and calculations of binding free energies.

[0064] In some embodiments, the mannanase polypeptide is the mannanase or a modified mannanase from Paenibacillus lentus (Accession No. WP 125083321.1), from Paenibacillus antarcticus (Accession No. 068648152.1), Paenibacillus sp. J45TS6 (Accession No. WP 213531700.1), Paenibacillus gallinarum (Accession No. WP 225229231.1 and MBD7968537.1) and the like. The mannanase polypeptide may also be from Bacillus lentus, Bacillus subtilis, Bacillus pumilus, Bacillus nealsonii, Bacillus clausii, Aspergillus niger, Aspergillus, kawachii, Talar omyces leycettanus, Trichoderma reesei, Trichoderma longibrachiatum, Thermotoga sp., Gloeophyllum trabeum, lactobacillus casei, Klebsiella pneumonia, Klebsiella oxytoca, Talaromyces trachyspermus, Lichtheimia ramose, etc. See, for example, Dawood, A. and Ma K., (2020) “Applications of Microbial B-Mannanases,” Sec. Industrial Biotechnology, found on the world wide web at doi: 10.3389 / fbioe.2020598630.

[0065] In some embodiments the mannanase enzyme is heat stable or thermostable. Thermostable enzymes are known in the art including the mannanase emzymes in GenBank Accession Nos. QIZ03395.1; AGW24296.1; ANH58352.1; 3MAN_A; 2MAN_A; 3WFL_A; 1BQC_A; AHH86071.1; G2Q4H7.1; AWK57895.1; AAF70202.1; CAA06924.1; P49425.3; UKS36951.1; 1PMJ X; 1PMH X; QBF51374.1; QBF51373.1; QBF51372.1; ASK09426.1; BAP19029.1; AHF8 1566.1 ; and C AA62442.1. All of which are herein incorporated by reference.

[0066] In some embodiments, the mannanase enzymes of the invention may be modified for heat stability. See, for example, CN103525790 “Optimized high-temperature resistant mannanase MAN5gy, and preparation method;” Cheng et al. (2016) Biomed Res lnt 2016:6380147 published online at doi: 10.1155 / 2016 / 6380147; Politz et al. (2000) Appl Microbiol Biotechnol 53(6):715-21 published online at doi: 10.1007 / s002530000351; dos Santos et al. (2012) J Struct Bio 177(2):469- 476; Liang et al. (2021) Front Microbiol 12 published online at doi.org / 10.3389 / fmicb.2021.722347; Kumagai et al. (2016) FEBS Letters published online at doi. org / 10.1002 / 1873-3468.12322; and, Tan et al. (2023) Front Microbiol 14:1119232, Thermostability modification of mannanase from Aspergillus niger via flexibility modification engineering published online at DOI: 10.3389 / fmib.2023.1119232; all of which are herein incorporated by reference. In one embodiment, the sequence from Paenibacillus lentus, set forth in SEQ ID NO: 1 can be modified by multiple sequence alignment with known thermostable mannanase enzymes followed by consensus mutation to generate a thermostable mannanase. In some embodiments, the mannanase may be used in combination with at least one additional enzyme selected from P-mannosidase, acetyl mannan esterase, P-glucosidase, a- galactosidase, and the like. Additional enzymes may be expressed in the plant or plant part or alternatively may be provided in the animal diet. In some instances, the enzyme combinations will provide synergistic results.

[0067] "Codon optimization" may be used to optimize sequences for increased expression in a particular plant of interest. Codon optimization may be used to modify a nucleic acid sequence for enhanced expression in the cells of the plant of interest, e.g., maize or soybean, by replacing at least one, more than one, or a significant number, of codons of the native sequence with codons that are more frequently or most frequently used in the genes of that plant. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1 -11. Various plants exhibit a particular bias for certain codons of a particular amino acid. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray et al., supra. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498. All of these references are herein incorporated by reference.

[0068] As discussed, the polynucleotides, polypeptides, or variants thereof may be utilized in the practice of the invention. By "variants" substantially similar sequences are intended. For nucleotide sequences of the nucleic acid molecules of the invention, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the mannanase polypeptides of the invention. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site- directed mutagenesis but which still encode a mannanase polypeptide of the invention. Generally, variants of a particular nucleotide sequence of the invention will have at least about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.

[0069] By "variant protein or polypeptide" a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and / or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein is intended. Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity. Biologically active variants of a mannanase polypeptide of the invention will have at least about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the mannanase polypeptide.

[0070] Methods for the determination of sequence identity between sequences are known in the art. See, for example, W02012 / 030759, herein incorporated by reference. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988). The ALIGN program is based on the algorithm of Myers and Miller (1988) CABIOS 4: 11- 17. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences.

[0071] Expression Cassettes

[0072] Polynucleotides encoding the mannanase proteins provided herein can be provided in expression cassettes for expression in a plant of interest. The cassette will include 5' and 3' regulatory sequences operably linked to a polynucleotide encoding a mannanase polypeptide that allows for expression of an active polynucleotide. The cassette may additionally contain at least one additional gene or genetic element to be cotransformed into the organism. Where additional genes or elements are included, the components may be operably linked. The term "operably linked" as used herein refers to a functional linkage between a genetic element and the polynucleotide expressing the polypeptide of interest.

[0073] Alternatively, the additional gene(s) or element(s) can be provided on multiple expression cassettes. The expression cassettes of the invention are provided with a plurality of restriction sites and / or recombination sites for insertion of the polynucleotides to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain a selectable marker gene.

[0074] The expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a mannanase polynucleotide of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in a plant. Targeting or signal sequences may also be included in the expression cassette and operably linked to the mannanase polynucleotide. The regulatory regions (i.e., promoters, transcriptional regulatory regions, targeting peptides, and translational termination regions) may be endogenous or heterologous to the plant cell or to each other. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

[0075] Convenient termination regions are available from the Ti-plasmid of A. lumefciciens. such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91 : 151- 158; Ballas et al. (1989) Nucleic Acids Res . 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639. Another terminator includes the pin / / terminator from the protease inhibitor II gene from potato (An et al. (1989) Plant Cell 1, 115-122. See also, Guerineau et al. (1991)Afo / . Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev . 5: 141-149; Mogen et al. (1990) Plant Cell 2 :1261-1212; Munroe et al. (1990) Gene 91 : 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res . 15:9627-9639.

[0076] Additional regulatory signals may be used and include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Patent Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) (hereinafter "Sambrook 11"); Davis et al, eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N. Y., and the references cited therein.

[0077] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) roc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991 ) Nature 353: 90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991 ) Virology 81 : 382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.

[0078] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0079] The expression construct expressing the mannanase polypeptide or enzyme will comprise other components. As indicated, in some embodiments, the mannanase may be targeted to plant compartments or subcellular spaces. Thus, the expression cassette or nucleotide sequence encoding the mannanase enzyme will be operably linked to a targeting or signal sequence. That is, in those instances where it is desirable to have the expressed polypeptide directed to a particular organelle, particularly the plastid, amyloplast, or to the endoplasmic reticulum, or secreted at the cell's surface or extracellularly to the apoplast, the expression cassette can further comprise a coding sequence for a transit peptide. Such transit peptides are well known in the art and include, but are not limited to, the transit peptide for the acyl carrier protein, the small subunit of RUBISCO, plant EPSP synthase, Zea mays Brittle-1 chloroplast transit peptide (Nelson et al. (1998) Plant Physiol 117(4): 1235-1252; Sullivan et al. Plant Cell 3(12): 1337-48; Sullivan et al. (1995) Planta 196(3):477-84; Sullivan et al. (1992) J. Biol. Chem. 267(26): 18999-9004) and the like.

[0080] One skilled in the art will readily appreciate the many options available in expressing a product to a particular organelle or plant subcellular compartment. Use of transit peptides is well known (e.g., see U.S. Patents Nos. 5,717,084; 5,728,925). A protein may be targeted to the endoplasmic reticulum of the plant cell. This may be accomplished by use of a localization sequence, such as KDEL. This sequence (Lys-Asp-Glu-Leu) contains the binding site for a receptor in the endoplasmic reticulum. See, Munro et al. (1987) “A C-terminal signal prevents secretion of luminal ER proteins.” Cell. 48:899-907. There are a wide variety of endoplasmic reticulum retention signal sequences available to one skilled in the art and the KDEL sequence is one example. Another example is HDEL (His-Asp-Glu-Leu). See, for example, Kumar et al. which discuses methods of producing a variety of endoplasmic reticulum proteins. Kumar et al. (2017) “prediction of endoplasmic reticulum resident proteins using fragmented amino acid composition and support vector machine” Peer J. doi: 10.7717 / peerj.3561. A barley alpha amylase signal sequence (BAASS) may be utilized for cell wall targeting.

[0081] Signal peptides may be used to transport a protein to the plant vacuole. See, for example, W02012 / 030759, herein incorporated by reference. Several vacuolar sorting signals / vacuolar sorting determinants have been described in plants and they can be divided into three major groups: ctVSS [C-terminal VSS (vacuolar sorting signals)], ssVSS (sequence-specific VSS) and physical structure VSS. The sequence specific sorting signals (ssVSD) require a conserved sequence of Asn- Pro-Ile-Arg (NPIR) or similar, which do not tolerate significant changes. Usually, this type of signal is located at the N-terminal of the protein directed to the lytic vacuole. The second main group comprises the signals present at the C-terminal region of the protein (ctVSD) and normally designates proteins destined to the protein storage vacuole. These signals do not have any homologous sequence or size set, having in common that they are rich in hydrophobic amino acids and need to be exposed at the C-terminus to operate. Finally, the third group corresponds to signals dependent on the tertiary (physical) structure of proteins (psVSD) and is most common in storage proteins. These sorting signals are present in the protein sequence, and may correspond to more than one polypeptide that function as whole when the protein acquires the native conformation. See, Neuhaus J.-M., Rogers J. (1998) Sorting of proteins to vacuoles in plant cells. Plant Mol. Biol. 38: 127-144; Zouhar J., Rojo E. (2009) Plant vacuoles: Where did they come from and where are they heading? Curr. Opin. Plant Biol. 12:677-684; and, Jolliffe N.A., Craddock C.P., Frigerio L. (2005) Pathways for protein transport to seed storage vacuoles. Biochem. Soc. Trans. 33: 1016- 1018.

[0082] As noted, signal sequences to target to the plant vacuole are well known. For example, Raikhel (U.S. Patent No. 5,360,726) shows a vacuole signal sequence as does Warren et al. (U.S. Patent No. 5,889,174). Vacuolar targeting signals may be present either at the amino-terminal portion, (Holwerda et al. (1992) The Plant Cell, 4:307-318, Nakamura et al. (1993) Plant Physiol., 101 : 1-5), carboxy -terminal portion, or in the internal sequence of the targeted protein. (Tague et al. (1992) The Plant Cell, 4:307-318, Saalbach et al. (1991) The Plant Cell, 3:695-708). Additionally, amino-terminal sequences in conjunction with carboxy-terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. (1990) Plant Molec. Biol. 14:357-368).

[0083] A number of promoters may be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissuepreferred, or other promoters for expression in the plant. Of interest are tissue specific promoters. Tissue specific promoters which may be used in the expression cassette include seed-preferred promoters, embryo-preferred promoters, and the like. "Seed-preferred" promoters include "seedspecific" promoters (those promoters active during seed development such as promoters of seed storage proteins). See Thompson et al. (1989) BioEssays 10: 108. Seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19B 1 (maize 19 kDa zein); milps (myo-inositol- 1 -phosphate synthase) (see WO 00 / 11177 and U.S. Patent No. 6,225,529). Another seed preferred promoter includes the phaseolin promoter (Bustos et al. (1989) The Plant Cell Vol. 1, 839-853). Gamma-zein is an endosperm-specific promoter. Globulin-1 (Glb-1) is a representative embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean P-phaseolin, napin, P-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, an Ltpl (See, for example, US Patent No. 7,550,579), an Ltp2 (Opsahl-Sorteberg, H-G. et al., (2004) Gene 341 :49-58 and US Patent 5,525,716), and oleosin genes. See also WO 00 / 12733, where seed-preferred promoters from endl and end2 genes are disclosed.

[0084] Seed-preferred promoters also include those promoters that direct gene expression predominantly to specific tissues within the seed such as, for example, the endosperm-preferred promoter of y-zein, the cryptic promoter from tobacco (Fobert et al. (1994) “T-DNA tagging of a seed coat-specific cryptic promoter in tobacco” Plant J. 4: 567-577), the P-gene promoter from corn (Chopra et al. (1996) “Alleles of the maize P gene with distinct tissue specificities encode Myb-homologous proteins with C-terminal replacements” Plant Cell 7: 1149-1158, Erratum in Plant Cell (1997) 1 : 109), the globulin-1 promoter from com (Belanger and Kriz (1991) “Molecular basis for Allelic Polymorphism of the maize Globulin-1 gene” Genetics 129: 863-972 and GenBank accession No. L22344), promoters that direct expression to the seed coat or hull of com kernels, for example the pericarp-specific glutamine synthetase promoter (Muhitch et al., (2002) “Isolation of a Promoter Sequence From the Glutamine Synthetase Gene Capable of Conferring Tissue-Specific Gene Expression in Transgenic Maize” Plant Science 163:865-872 and GenBank accession number AF359511) and to the embryo (germ) such as that disclosed at US Patent No. 7,169,967. When referring to a seed or an embryo preferred promoter is meant that it expresses an operably linked sequence to a higher degree in seed or embryo tissue that in other plant tissue. It may express during seed or embryo development, along with expression at other stages, may express strongly during seed or embryo development and to a much lesser degree at other times.

[0085] The globulin-1 promoter has been characterized and previously used in transgenic maize seeds. Storage promoters besides globulin-1 that are also active in cereal seed tissues may be used. The principal tissue types in maize seeds are the embryo, the endosperm including a surrounding aleurone cell layer and the maternally derived pericarp. Of these, the endosperm and to a lesser extent the embryo, comprise most of the volume of the seed. Thus, endosperm and embryo promoters are particularly important for modifying seed characteristics and content. The relatively high soluble protein content of the embryo makes it a particularly good target for foreign protein expression. The proximal 1.1 kb of the maize 27 kD g-zein promoter and the proximal 1.4 kb of the maize globulin- 1 promoter (GenBank accession L22344) are prominent examples of seed preferred promoters that have been used to express transgenes in endosperm and embryo tissues, respectively, of monocots. Cereal seeds consist principally of two tissue types, the embryonic plant or embryo and the surrounding endosperm. The endosperm is comprised almost entirely of nutritional reserves, primarily of complex carbohydrate and aqueous insoluble protein, but the embryo also contains considerable stores, mainly of oils and aqueous soluble proteins. Globulin-1 is one of the most abundant proteins in maize embryo tissue. It is largely limited to this tissue and becomes particularly concentrated in the scutellum late in embryo development. Given the high concentration of this protein observed in embryo tissues the maize, the globulin-1 promoter was previously identified as being a good candidate to direct high levels of transgene expression in the embryo. An approximately 1.4 kb extent of this promoter has been cloned (Genbank accession L22344) and used to drive high levels of transgene expression specifically in maize seed. Longer versions up to 3 kb of the globulin-1 promoter may be used.

[0086] For embryo specific promoters, see for example Streatfield et al. (2010) Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production, GM Crops, 1 :3, 162-172, DOI: 10.4161 / gmcr. l.3.12816. Other seed preferred promoters include those disclosed in US Patent No. 6,528,704; 6,403,862; 8,049,000; 9,227,794; 9,383,543; 9,703,754; 9,267,147; W02000 / 011177; and W02000 / 040710.

[0087] Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313: 810- 812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171 ); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991 ) Theor. Appt. Genet. 81 : 581 -588); MAS (Velten et al. (1984) EMBO J. 3:2723- 2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611. Transformed Plants and Animal Feed

[0088] Compositions of the invention include transformed organisms, such as a plant, including whole plants, as well as plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). "Transgenic plants" or "transformed plants" or "stably transformed" plants or cells or tissues refers to plants that have incorporated or integrated a polynucleotide encoding at least one mannanase polypeptide of the invention. It is recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments may also be incorporated into the plant cell.

[0089] Methods for plant transformation are known in the art. See, for example, US2018 / 0066277; US2020 / 0399324; US2022 / 0282273; WO2014 / 018853; WO2015 / 023846; US2021 / 0108223; W02020 / 056070; and CA3093297; all of which are herein incorporated in their entirety. Agrobacterium-and biolistic-mediated transformation remain the two predominantly employed approaches. However, transformation may be performed by infection, transfection, microinjection, electroporation, microprojection, biolistics or particle bombardment, electroporation, silica / carbon fibers, ultrasound mediated, PEG mediated, calcium phosphate co-precipitation, polycation DMSO technique, DEAE dextran procedure, Agrobacterium and viral mediated (Caulimoriviruses, Geminiviruses, RNA plant viruses), liposome mediated and the like.

[0090] Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Methods for transformation are known in the art and include those set forth in US Patent Nos: 8,575,425; 7,692,068; 8,802,934; 7,541,517; each of which is herein incorporated by reference. See, also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849- 858; Jones et al. (2005) Plant Methods 1 :5; Rivera etal. (2012) Physics of Life Reviews 9:308- 345; Bartlett et al. (2008) Plant Methods 4: 1-12; Bates, G.W. (1999) Methods in Molecular Biology 111 :359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575- 606; Christou, P. (1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica 85: 13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107: 1041-1047; Jones et al. (2005) Plant Methods 1 :5.

[0091] Transformation may result in stable or transient incorporation of the nucleic acid into the cell. "Stable transformation" is intended to mean that the nucleotide construct introduced into a host cell integrates into the genome of the host cell and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the host cell and does not integrate into the genome of the host cell.

[0092] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81- 84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

[0093] In specific embodiments, the sequences provide herein can be targeted to specific sites within the genome of the plant cell. Such methods include, but are not limited to, meganucleases designed against the plant genomic sequence of interest (D'Halluin et al. 2013 Plant Biotechnol J); CRISPR-Cas9, TALENs, and other technologies for precise editing of genomes (Feng et al. (2013) Cell Research 23: 1229-1232, Podevin et al. (2013) Trends Biotechnology, online publication, Wei et al. (2013) J Gen Genomics, Zhang et al (2013) WO 2013 / 026740); Cre-lox site-specific recombination (Dale et al. ( 1995) 77a / / / 77:649-659; Lyznik et al. (2007) Transgenic Plant J 1 : 1- 9; FLP-FRT recombination (Li et al. (2009) Plant Physiol 151 : 1087-1095); Bxbl-mediated integration (Yau et al. (2011) Plant J CC. 147-166); zinc-finger mediated integration (Wright et al. (2005) / awt 744:693-705); Cai et al. (2009) Plant Mol Biol 69:699-709); and homologous recombination (Lieberman-Lazarovich and Levy (2011) Methods Mol Biol 701 : 51-65); Puchta ( !) Plant Mol Biol . 173-182).

[0094] While any plant can be transformed, monocots and dicots, and used according to the methods of the invention, of special interest are feed grains, including corn, sorghum, barley, and oats as well as legumes, including soybean, beans, peas, alfalfa, peanuts, and the like.

[0095] Once transformed, plants can be selected, propagated, crossed, and used to obtain a plurality of plants that contain the mannanase enzyme of the invention. The plant, plant part, or plant seed expressing the recombinant mannanase polypeptide can be used or processed for animal feed. Accordingly, the invention encompasses the animal feed with the recombinant polypeptide of the invention. Such animal feed includes but is not limited to feedstuffs, including com meal soybean meal (SBM), palm kernel meal, copra meal, and sesame meal. The following examples are offered by way of illustration and not by way of limitation.

[0096] EXAMPLES

[0097] Construct Preparation

[0098] Six constructs for expression of mannanase in maize were prepared as described in the list below. The sequence of the mannanase protein from Peanibacillus Lentus strain (GenBank WP125083321.1x) modified for improved heat stability was utilized. The sequence of amino acids 33-331 was optimized using the algorithm developed by GenScript for maize codon usage. Previously characterized signal sequences for subcellular targeting to the cell wall / apoplast, endoplasmic reticulum (ER), and vacuole were added. An additional amino acid (glycine) was added to the predicted N-terminus of the protein to increase N-end stability once the signal sequence is cleaved. Sequences with inactivation of two potential N-linked glycosylation sites were designed with replacement of asparagine (N) with glutamine (Q) at positions 33 and 153. The nucleotide sequence of the coding region was outsourced for commercial gene synthesis by GenScript. In all constructs the mannanase coding region (aa 33-331) was synthesized for transfer into the maize transformation vector pSBl 1 using an Ncol site overlapping the initiating ATG and a PacI restriction site in the terminator region to add the coding region downstream of the promoter. All constructs incorporated the mannanase coding region under control of a synthetic promoter derived from the maize globulin-1 gene, pr44, which targets expression to the embryo. This promoter is an engineered version of the previously described pr25 promoter that contains two extra copies of the 5' region of pr25 (C. A. Hayden et al., Production of highly concentrated, heat-stable hepatitis B surface antigen in maize. Plant Biotechnology Journal 10, 979-984 (2012)). Each transcription unit also incorporated the terminator from potato proteinase inhibitor II. Subcellular localization and terminator sequences were as previously described (C. A. Hayden et al., Production of highly concentrated, heat-stable hepatitis B surface antigen in maize. Plant Biotechnology Journal 10, 979-984 (2012)). All constructs also include an expression cassette with a maize codon usage optimized phosphinothricin-acetyl- transf erase gene under control of a cauliflower mosaic virus 35S promoter for herbicide selection using bialaphos.

[0099] Mannanase CW (cell wall) and Mannanase ER (endoplasmic reticulum) differ only by the addition of the 4 amino acid KDEL targeting sequence at the end of the coding region (bp 975-986 / aa 325-328). Mannanase CW and Mannanase Vac (vacuole) differ only by the replacement of the CW signal sequence with the Vac signal sequence at the beginning of the coding region (bp 1-74 / aa 1-24 for CW signal vs. bp 1-131 / aa 1-43 for vac signal). Mannanase CW and Mannanase CW delglyc differ only by the replacement of two asparagine amino acids with glutamine (bp!74-176 / aa 58 and bp 534-536 / aa 178). Mannanase ER and Mannanase ER delglyc differ only by the replacement of two asparagine amino acids with glutamine (bpl74-176 / aa 58 and bp 534-536 / aa 178). Mannanase Vac and Mannanase Vac delglyc differ only by the replacement of two asparagine amino acids with glutamine (bp 231-233 / aa 77 and bp 591-593 / aa 197).

[0100] Constructs:

[0101] MAA (9307) pr44-BAASS:manannase (Mannanase CW)

[0102] MAB (9308) pr44-BAASS:mannanase (KDEL(Mannanase ER)

[0103] MAC (9309) pr44-vac: mannanase (Mannanase Vac)

[0104] MAD (9310) pr44-BAASS: mannanase w / inactivation of glycosylation sites (Mannanase CW delglyc)

[0105] MAE (9311) pr44-BAASS mannanase :KDEL w / inactivation of glycosylation sites (Mannanase ER delglyc)

[0106] MAF (9312) pr44-vac: mannanase w / inactivation of glycosylation sites (Mannanase Vac delglyc)

[0107] Plant transformation

[0108] Maize transformation was carried out as previously described with modifications (Y. Ishida et al.. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745-750 (1996)). In brief, the constructs are transferred into the LBA4404-pSB-JT Agrobacterium strain containing the vector pSBl by a triparental mating procedure (T. Komari, Y. Hiei, Y. Saito, N. Murai, T. Kumashiro, Vectors carrying two separate T- DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant Journal 10: 165-174 (1996)). The cointegrate DNA was then electroporated into Agrobacterium tumefaciens strain EHA101 (E. E. Hood, G. L. Helmer, R. T. Fraley, M. D. Chilton, The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacterio! 168: 1291-1301 (1986)). Hill maize embryos 1.5 to 3 mm in length are mixed with A. tumefaciens EHA101 with the appropriate vector for transformation (Y. Ishida etal., High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745-750 (1996)). Plants from events selected on bialaphos are grown to maturity in the greenhouse and pollinated with Hill to produce T1 generation seed. A total of 53 independent transformation events and 139 plants were obtained for the six different constructs.

[0109] Table 1 : Number of events and plants

[0110] Western blot analysis T1 seed

[0111] Proteins were extracted from ground maize seed with 1 X PBS + 1% SDS, loaded onto a 4-12% bistris gel (ThermoFisher Scientific), and transferred to PDVF membrane by iBlot (Thermofisher Scientific). Antibody binding demonstrated the presence of the enzyme. Extracts from individual seeds also showed significant enzymatic activity. (Data not shown.)

[0112] Screening of T1 and later generation seed with biochemical assay

[0113] Extract Preparation

[0114] For T1 generation analysis, individual seeds (100-200 mg) were pulverized in a custom machine in custom metal vials and resuspended in IX PBS +0.05% Tween (pH7.4). Samples were disrupted by addition of a ball bearing and mixing with a Genogrinder For later generation maize material (25-50 pooled seeds) were ground in a standard coffee grinder and resuspended in IX PBS +0.05% Tween (pH7.4) at lOOmg / mL.

[0115] Mannanase Blue Dye Protocol

[0116] The enzyme substrate Azo-Carob Galactomannan (ACGM), from Megazyme.com, Product Code: S-ACGLM was used. It was RBB (Remazol brilliant blue) labeled galactomannan which is the substrate of pD-mannanase. To prepare the substrate solution 0.35 g of ACGM was dissolved in 100 ml of 50 mM HEPES, pH 7.5 by adding dry powder slowly to a rapidly-stirred buffer. The solution was heated with stirring to boiling, then autoclaved for 20 minutes at 121 °C and cooled to room temperature. The reagent blank in the assay should give an absorbance at 590 nm of < 0.15 when read against 70% ethanol. If higher, the substrate was discarded.

[0117] To begin the assay 0.4 mL of ACGM substrate was dispensed into one 1.5 mL Eppendorf tube per sample and placed in a 40°C water bath for at least 10 minutes. This method is not intended to be as analytical as the reducing sugar method that defines 1 unit of activity is the amount of enzyme required to release 1 micromole of mannose reducing sugar equivalents per minute under defined assay conditions. The reaction was started by addition of 20 pL of sample or control enzyme. Each sample was incubated for exactly 10 minutes. Samples should be tested in duplicate for best results. The reaction was stopped by adding 0.9 mL of absolute ethanol (reagent alcohol) and mixing thoroughly. The tubes were then allowed to sit at room temperature for at least 10 minutes. The tubes were centrifuged at 12,000 RPM for 5 minutes. The absorbance at 590 nm was read using a reagent blank treated exactly as the test samples above using 20 pL of water or buffer instead of enzyme to zero the spectrophotometer. The A590 observed for the standards can be plotted against the activity in MU / Liter in the diluted standards and determine the slope. Seeds showing activity were observed. Expression in seed was estimated to be in the range of 100-500 mg / kg.

[0118] The test-tube scale Blue Dye assay was used to screen the activity of representative T1 generation plants crossed to maize line Hill in the greenhouse as well as representative plants from subsequent generations grown in the greenhouse or field over the course of a program of traditional hand pollination back-crossing into elite maize germplasm for better characteristics for commercial use.

[0119] Figures 1-4 show representative results over several generations.

[0120] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0121] All publications and patent applications mentioned in the specification are indicative of the level 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.

[0122] 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.

[0123] SEQUENCES

[0124] Paenibacillus lentus mannanase (SEQ ID NO.1): ASGFYVSGTILCDSTGNPFKIRGINHAHSWFKNDSATAMEAIAATGANTVRIVLSNGQQYA KDDANTVSNLLSLANQHKLIAILEVHDATGSDSVSALDHAVDYWIEMKNVLVGKEDRVLI NIANEWYGTWDSNGWADGYKSAIPKLRNAGINHTLIVDAAGWGQYPQSIVDKGNEVFNS DPLRNTIFSIHMYEYAGGNADMVRANIDQVLNKGLAVIIGEFGHYHTGGDVDETAIMSYT QQKGVGWLAWSWKGNGAEWLYLDLSYDWAGNHLTEWGETIVNGANGLKATSTRAPIF GN

[0125] Barley alpha amylase cell wall / apoplast targeting signal sequence (SEQ ID NO: 2): MANKHLSLSLFLVLLGLSASLASG

[0126] ER targeting signal sequence (SEQ ID NO: 3): KDEL

[0127] Vacuole targeting signal sequence (SEQ ID NO: 4):

[0128] MAHAR VLLLALAVLATAAVAVASSSSFADSNPIRPVTDRAAST

[0129] Synthetic promoter derived from the maize globulin-1 gene, pr44 (SEQ ID NO: 5):

[0130] GGCGCGCCGGTATGAATTTGGAAACAAATTCAGTACTTTTAAAAAAATTTGTTGTAGG GAGCAAATAATACATAAAATAATTTATGCATTATTTTATTTTTTATTTGTAATAATATG CTTGAAACGATAATTCAGTATGCATGTTGTGCCAGTGTACTACACGGGCGGGGGGAGG GGATTGAGTGGGCCAGCGCGGTGCGTAGGGTAGATGGGCTGAAATTGATAACTCAAGT CCGACTAGGTTCTCTTTTTATTTCCCTTCCTTTTCTATTTTCCTTTCTTTTAATTTTCATGC TTTCAAACTAAATTCAAATTCGAGTTTTGAATTTCAGCTTCTAAATTGTACACTAAAAT TATATGATAAGGTAACCCCTACTATTACTTTTAATTTTTTTATTCTACCCCATATTGTTT ACTTAGGGGAGAATAATTGACTTAATCACATTCTTCCTAGGTTTCAATTCTCAATCTTT CAAATCCACATTTTTAGATTTCTATTTTGAATTTAAATACCAGTTTGGATTTAGAGTTCA ATTTCAAAATACACAACCAAAATACCAGCATGAATGCAAATATATTTTATGTTTATGTA TTTACTTTTCTTTTATACTTTGCTCAAAATAGTTATTTTCATGTATGAAACTCAATAAGC AAGGAACTCACGTTATTATATAACCTAATAGGAATAATTTAGGTAACATAATTTATCAT CCTCTTGATTTAAAAGAGATATGCCTCCAGAATAAGACACATACTAAAAATAACTCTA ATATTGAATAACTAAAGTCGTACAAATCTCTACTATTATTCCTATAAAATAATAAAGA

[0131] ACTAGCTACAACTTCTTTAAGGCATTATTCAGGGTTTACAGCTTGAGAGGCATGAACCC ATCCTGTATACTCCTGGACTTGGAAGACAAAATGTCAACCAAAGTGAAAGGTTTTCTT ATGGTTGCTGCTAAGAGATAGATTGAACACTAGATCTCTCCTAAGACGTCAGGGCATG CGTTTAGACTCCTACACATGCGAAAACTGCATCTTACAGTTGGAAGAAACTATATCTC ACCACTTCCTGCGGTGTAACTTTGCCCAAAGATGTTGGCTCACTGTTGGAATCACTCCG CCCCGAACTTTGGATCTAACGCTTGCAGTGCTACATATTAGAGCAAGACTAACAATGC CGTGGAGAATGGAAGGTATTATAACCATGTCATGGTGCATATGGAAATGTCGAAATAA CTGGATATTCGAAAACATACCGCCAACGGTGGCGGCCTGCAAGGAAATGTTCAAGACT GAAATGAACTACATCTGCTACCAAGTTAAGCTCGAGACAGGAGCTAAAAGTAGAAAC TGGATACAACACTTTGTAACATAGTGACACTCCCCTTTTCCTTTCTTTTACCTTAGAACT ATACATACAATCCACATTCAATAAAAATTTGTAGGTACGCCATACACACTACCGGAAT CCGGCTCTTTGCCGAGTGTGAGGCGCTTTGTCGAGTGCTTTTTGTCCAGCACTCGGCAA AAAAGTCTTTGCCATGTGCCGCACTCGGCAAAGTCCTGCTCTCGGTAACGACCGCGTTT ACCGAGAGCAGGACTCTCGACACAGAAATACACTCGACAAAGAAATCTTTGCCGAGA

[0132] GCCAAACACTCGGCGAACGGCAGCGCTCGGCAAAGGGTCGTCAGCCGCCGTCTAAAG CTGACGGTCGTTATCTTTGTCGAGTGCCCCCTCGTCCGACACTCAGTAGAGCACGCGCC GGTATGAATTTGGAAACAAATTCAGTACTTTTAAAAAAATTTGTTGTAGGGAGCAAAT AATACATAAAATAATTTATGCATTATTTTATTTTTTATTTGTAATAATATGCTTGAAACG ATAATTCAGTATGCATGTTGTGCCAGTGTACTACACGGGCGGGGGGAGGGGATTGAGT GGGCCAGCGCGGTGCGTAGGGTAGATGGGCTGAAATTGATAACTCAAGTCCGACTAG GTTCTCTTTTTATTTCCCTTCCTTTTCTATTTTCCTTTCTTTTAATTTTCATGCTTTCAAAC TAAATTCAAATTCGAGTTTTGAATTTCAGCTTCTAAATTGTACACTAAAATTATATGAT AAGGTAACCCCTACTATTACTTTTAATTTTTTTATTCTACCCCATATTGTTTACTTAGGG GAGAATAATTGACTTAATCACATTCTTCCTAGGTTTCAATTCTCAATCTTTCAAATCCA CATTTTTAGATTTCTATTTTGAATTTAAATACCAGTTTGGATTTAGAGTTCAATTTCAAA ATACACAACCAAAATACCAGCATGAATGCAAATATATTTTATGTTTATGTATTTACTTT TCTTTTATACTTTGCTCAAAATAGTTATTTTCATGTATGAAACTCAATAAGCAAGGAAC TCACGTTATTATATAACCTAATAGGAATAATTTAGGTAACATAATTTATCATCCTCTTG ATTTAAAAGAGATATGCCTCCAGAATAAGACACATACTAAAAATAACTCTAATATTGA ATAACTAAAGTCGTACAAATCTCTACTATTATTCCTATAAAATAATAAAGAACTAGCT ACAACTTCTTTAAGGCATTATTCAGGGTTTACAGCTTGAGAGGCATGAACCCATCCTGT ATACTCCTGGACTTGGAAGACAAAATGTCAACCAAAGTGAAAGGTTTTCTTATGGTTG CTGCTAAGAGATAGATTGAACACTAGATCTCTCCTAAGACGTCAGGGCATGCGTTTAG ACTCCTACACATGCGAAAACTGCATCTTACAGTTGGAAGAAACTATATCTCACCACTTC CTGCGGTGTAACTTTGCCCAAAGATGTTGGCTCACTGTTGGAATCACTCCGCCCCGAAC TTTGGATCTAACGCTTGCAGTGCTACATATTAGAGCAAGACTAACAATGCCGTGGAGA ATGGAAGGTATTATAACCATGTCATGGTGCATATGGAAATGTCGAAATAACTGGATAT TCGAAAACATACCGCCAACGGTGGCGGCCTGCAAGGAAATGTTCAAGACTGAAATGA ACTACATCTGCTACCAAGTTAAGCTCGAGACAGGAGCTAAAAGTAGAAACTGGATACA ACACTTTGTAACATAGTGACACTCCCCTTTTCCTTTCTTTTACCTTAGAACTATACATAC AATCCACATTCAATAAAAATTTGTAGGTACGCCATACACACTACCGGAATCCGGCTCT TTGCCGAGTGTGAGGCGCTTTGTCGAGTGCTTTTTGTCCAGCACTCGGCAAAAAAGTCT TTGCCATGTGCCGCACTCGGCAAAGTCCTGCTCTCGGTAACGACCGCGTTTACCGAGA GCAGGACTCTCGACACAGAAATACACTCGACAAAGAAATCTTTGCCGAGAGCCAAAC ACTCGGCGAACGGCAGCGCTCGGCAAAGGGTCGTCAGCCGCCGTCTAAAGCTGACGGT CGTTATCTTTGTCGAGTGCCCCCTCGTCCGACACTCAGTAGAGCACGCGCCGGTATGAA TTTGGAAACAAATTCAGTACTTTTAAAAAAATTTGTTGTAGGGAGCAAATAATACATA AAATAATTTATGCATTATTTTATTTTTTATTTGTAATAATATGCTTGAAACGATAATTCA GTATGCATGTTGTGCCAGTGTACTACACGGGCGGGGGGAGGGGATTGAGTGGGCCAGC GCGGTGCGTAGGGTAGATGGGCTGAAATTGATAACTCAAGTCCGACTAGGTTCTCTTT TTATTTCCCTTCCTTTTCTATTTTCCTTTCTTTTAATTTTCATGCTTTCAAACTAAATTCA AATTCGAGTTTTGAATTTCAGCTTCTAAATTGTACACTAAAATTATATGATAAGGTAAC CCCTACTATTACTTTTAATTTTTTTATTCTACCCCATATTGTTTACTTAGGGGAGAATAA TTGACTTAATCACATTCTTCCTAGGTTTCAATTCTCAATCTTTCAAATCCACATTTTTAG ATTTCTATTTTGAATTTAAATACCAGTTTGGATTTAGAGTTCAATTTCAAAATACACAA CCAAAATACCAGCATGAATGCAAATATATTTTATGTTTATGTATTTACTTTTCTTTTATA CTTTGCTCAAAATAGTTATTTTCATGTATGAAACTCAATAAGCAAGGAACTCACGTTAT TATATAACCTAATAGGAATAATTTAGGTAACATAATTTATCATCCTCTTGATTTAAAAG AGATATGCCTCCAGAATAAGACACATACTAAAAATAACTCTAATATTGAATAACTAAA GTCGTACAAATCTCTACTATTATTCCTATAAAATAATAAAGAACTAGCTACAACTTCTT TAAGGCATTATTCAGGGTTTACAGCTTGAGAGGCATGAACCCATCCTGTATACTCCTGG ACTTGGAAGACAAAATGTCAACCAAAGTGAAAGGTTTTCTTATGGTTGCTGCTAAGAG ATAGATTGAACACTAGATCTCTCCTAAGACGTCAGGGCATGCGTTTAGACTCCTACAC ATGCGAAAACTGCATCTTACAGTTGGAAGAAACTATATCTCACCACTTCCTGCGGTGT AACTTTGCCCAAAGATGTTGGCTCACTGTTGGAATCACTCCGCCCCGAACTTTGGATCT AACGCTTGCAGTGCTACATATTAGAGCAAGACTAACAATGCCGTGGAGAATGGAAGGT ATTATAACCATGTCATGGTGCATATGGAAATGTCGAAATAACTGGATATTCGAAAACA

[0133] TACCGCCAACGGTGGCGGCCTGCAAGGAAATGTTCAAGACTGAAATGAACTACATCTG CTACCAAGTTAAGCTCGAGACAGGAGCTAAAAGTAGAAACTGGATACAACACTTTGTA ACATAGTGACACTCCCCTTTTCCTTTCTTTTACCTTAGAACTATACATACAATCCACATT CAATAAAAATTTGTAGGTACGCCATACACACTACCGGAATCCGGCTCTTTGCCGAGTG TGAGGCGCTTTGTCGAGTGCTTTTTGTCCAGCACTCGGCAAAAAAGTCTTTGCCATGTG

[0134] CCGCACTCGGCAAAGTCCTGCTCTCGGTAACGACCGCGTTTACCGAGAGCAGGACTCT

[0135] CGACACAGAAATACACTCGACAAAGAAATCTTTGCCGAGAGCCAAACACTCGGCGAA

[0136] CGGCAGCGCTCGGCAAAGGGTCGTCAGCCGCCGTCTAAAGCTGACGGTCGTTATCTTT

[0137] GTCGAGTGCCCCCTCGTCCGACACTCAGTAGAGCAAGCTTGCCGAGTGCCATCCTTGG

[0138] ACACTCGATAAAGTATATTTTATTTTTTTTTATTTTGCCAACCAAACTTTTTGTGGTATG

[0139] TTCCTACACTATGTAGATCTACATGTACCATTTTGGCACAATTACAAAAATGTTTTCTA

[0140] TAACTATTAGATTTAGTTCGTTTATTTGAATTTCTTCGGAAAATTCACATATGAACTGC

[0141] AAGTCACTCGAAACATGAAAAACCGTGCATGCAAAATAAATGATATGCATGTTATCTA

[0142] GCACAAGTTACGACCGAATTCAGAAGCAGACCAGAATCTTCAAGCACCATGCTCACTA

[0143] AACATGACCGTGAACTTGTTATCCAGTTGTTTAAAAATTGTATAAAACACAAATAAAG

[0144] TCAGAAATTAATGAAACTTGTCCACATGTCATGATATCATATATAGAGGTTGTGATAA

[0145] AAATTTGATAATGTTTCGGTAAAGTTGTGACGTACTATGTGTAGAAACCTAAGTGACCT

[0146] ACACATAAAATCATAGAGTTTCAATGTAGTTCACTCGACAAAGACTTTGTCAAGTGTC

[0147] CGATAAAAAGTATTCAGCAAAGAAGCCGTTGTCGATTTACTGTTCGTCGAGATCTCTTT

[0148] GCCGAGTGTCACACTAGGCAAAGTCTTTACGGAGTGTTTTTCAGGCTTTGACACTCGGC

[0149] AAAGCGCTCGATTCCAGTAGTGACAGTAATTTGCATCAAAAATAGCCGAGAGATTTAA

[0150] AATGAGTCAACTAATAGACCAACTAATTATTAGCTATTAGTCGTTAGCTTCTTTAATCT

[0151] AAGCTAAAACCAACTAATAGCTTATTTGTTGAATTACAATTAGCTCAACGGAATTCTCT

[0152] GTTTTTTGTATAAAAAAAAGGGAAAGTGGGGCTGATTTAGAGGAAAGTGTGGGCTGGCT

[0153] GTCGTCCAGATACAATGAACGTACCTAGTAGGAACTCTTTTACACGCTCGGTCGCTCGC

[0154] CGCGGATCGGAGTCCCAGGAACACGACACCACTGTGGAACACGACAAAGTCTGCTCA

[0155] GAGGCGGCCACACCCTGGCGTGCACCGAGCCGGAGCCCGGATAAGCACGGTAAGGAG

[0156] AGTACGGCGGGACGTGGCGACCCGTGTGTCTGCTGCCACGCAGCCTTCCTCCACGTAG

[0157] CCGCGCGGCCGCGCCACGTACCAGGGCCCGGCGCTGGTATAAATGCGCGCCACCTCCG

[0158] CTTTAGTTCTGCATACAGCCAACCCAACACACACCCGAGCATATCACAGTGACAGACA

[0159] CTACACCATG

[0160] Terminator from potato proteinase inhibitor II (SEQ ID NO: 6):

[0161] CTAGACTTGTCCATCTTCTGGATTGGCCAACTTAATTAATGTATGAAATAAAAGGATGC

[0162] ACACATAGTGACATGCTAATCACTATAATGTGGGCATCAAAGTTGTGTGTTATGTGTA

[0163] ATTACTAGTTATCTGAATAAAAGAGAAAGAGATCATCCATATTTCTTATCCTAAATGA

[0164] ATGTCACGTGTCTTTATAATTCTTTGATGAACCAGATGCATTTCATTAACCAAATCCAT

[0165] ATACATATAAATATTAATCATATATAATTAATATCAATTGGGTTAGCAAAACAAATCT

[0166] AGTCTAGGTGTGTTTTGCGAAT

Claims

WHAT IS CLAIMED IS:

1. A method for degrading mannan in a plant or plant part, said method comprising transforming said plant with an expression cassette comprising a nucleotide sequence encoding a mannanase polypeptide, wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a seed-preferred promoter.

2. The method of claim 1, wherein said seed-preferred promoter is a globulin- 1 promoter.

3. The method of claim 1, wherein said nucleotide sequence encoding a mannanase polypeptide is codon optimized for expression in said plant.

4. The method of claim 3, wherein said mannanase polypeptide comprises a sequence having 95% sequence identity to the sequence set forth in SEQ ID NO. 1.

5. The method of claim 2, wherein said promoter comprises the sequence set forth in SEQ ID NO: 5.

6. A plant transformed with an expression cassette comprising a nucleotide sequence encoding a mannanase polypeptide wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a seed-preferred promoter.

7. The plant of claim 6, wherein said seed-preferred promoter is a globulin- 1 promoter.

8. The plant of claim 6, wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a nucleotide sequence which encodes a peptide that targets the mannanase polypeptide to a subcellular compartment.

9. The plant of claim 8, wherein said subcellular compartment is a vacuole.

10. Seed of any one of the plants of claims 6-9.

11. Animal feed that comprises any of the plants of claims 6-9.

12. Animal feed that comprises a seed of claim 10.

13. An expression cassette comprising a nucleotide sequence encoding a mannanase polypeptide, wherein said nucleotide sequence has been codon optimized for expression in a plant of interest.

14. The expression cassette of claim 13, wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a seed-preferred promoter.

15. The expression cassette of claim 14, wherein said seed-preferred promoter is a globulin- 1 promoter.

16. The expression cassette of claim 13, wherein said nucleotide sequence encoding a mannanase polypeptide is operably linked to a nucleotide sequence which encodes a peptide that targets the mannanase polypeptide to a subcellular compartment.

17. The expression cassette of claim 16, wherein said subcellular compartment is a vacuole.

18. The expression cassette of claim 13, wherein said mannanase polypeptide comprises a sequence with 95% sequence identity to the sequence set forth in SEQ ID NO. 1.

19. The expression cassette of claim 15, wherein said promoter comprises the sequence set forth in SEQ ID NO. 5.

Images