Nucleic acids and proteins and methods for making and using them

Abstract

The invention provides polypeptides, including enzymes, structural proteins and binding proteins, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. Polypeptides, including enzymes and antibodies, and nucleic acids of the invention can be used in industrial, experimental, food and feed processing, nutritional and pharmaceutical applications, e.g., for food and feed supplements, colorants, neutraceuticals, cosmetic and pharmaceutical needs.

1 . An isolated or recombinant nucleic acid comprising
 (a) a nucleic acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63% 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NOs: from SEQ ID NO:1 through SEQ ID NO:26,897, over a region of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more residues,   wherein the nucleic acid encodes at least one polypeptide having an enzymatic activity, or encodes a polypeptide or peptide capable of generating an antibody that binds specifically to a polypeptide having a sequence comprising any of the even numbered SEQ ID NOs: in the sequence listing, including from SEQ ID NO:2 through SEQ ID NO:26,898;   (b) a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NOs: from SEQ ID NO:1 through SEQ ID NO:26,897,   wherein the nucleic acid encodes a polypeptide having an activity as set forth in Table 3, or encodes a polypeptide or peptide capable of generating an antibody that binds specifically to a polypeptide having a sequence comprising any of the even numbered SEQ ID NOs: in the sequence listing, including from SEQ ID NO:2 through SEQ ID NO:26,898,   and the stringent conditions include a wash step comprising a wash in 0.2×SSC at a temperature of about 65° C. for about 15 minutes,   and optionally the nucleic acid is at least about 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more residues in length or the full length of the gene or transcript; and   (c) a nucleic acid sequence complementary to (a) or (b).   
     
     
         2 . The isolated or recombinant nucleic acid of  claim 1 , wherein the sequence identity is at least about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63% 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NOs: from SEQ ID NO:1 through SEQ ID NO:26,897. 
     
     
         3 . (canceled) 
     
     
         4 . The isolated or recombinant nucleic acid of  claim 1 , wherein the nucleic acid sequence comprises a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NOs: from SEQ ID NO:1 through SEQ ID NO:26,897. 
     
     
         5 . The isolated or recombinant nucleic acid of  claim 1 , wherein the nucleic acid sequence encodes a polypeptide having a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NOs: from SEQ ID NO:1 through SEQ ID NO:26,897. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The isolated or recombinant nucleic acid of  claim 1 , wherein at least one enzymatic activity comprises aldolase, alpha-galactosidase, amidase, secondary amidase, amylase, catalase, dehalogenase, endoglucanase, epoxide hydrolase, esterase, hydrolase, glucosidase, glycosidase, intein, isomerase, laccase, lipase, monooxygenase, nitroreductase, nitrilase, P450 enzyme, pectate lyase, phosphatase, phospholipase, phytase, polymerase or xylanase activity. 
     
     
         9 . The isolated or recombinant nucleic acid of  claim 1 , wherein the protein is a structural or binding protein. 
     
     
         10 - 13 . (canceled) 
     
     
         14 . The isolated or recombinant nucleic acid of  claim 1 , wherein the enzyme or protein is thermostable or thermotolerant. 
     
     
         15 . The isolated or recombinant nucleic acid of  claim 14 , wherein the polypeptide retains enzyme activity under conditions comprising a temperature range of between about 37° C. to 95° C., or between about 55° C. to 85° C., or between about 70° C. to 75° C., or between about 70° C. to 95° C., or between about 90° C. to 95° C., or, the polypeptide retains enzyme activity after exposure to a temperature in the range from greater than 37° C. to about 95° C., from greater than 55° C. to about 85° C., or between about 70° C. to 75° C., or from greater than 90° C. to about 95° C. 
     
     
         16 . An isolated or recombinant nucleic acid encoding a polypeptide having an enzyme activity or encoding a protein, wherein the nucleic acid comprises a sequence that hybridizes under stringent conditions to a nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NOs: from SEQ ID NO:1 through SEQ ID NO:26,897, and the nucleic acid encodes a polypeptide having enzyme activity or encodes a protein. 
     
     
         17 . The isolated or recombinant nucleic acid of  claim 16 , wherein the nucleic acid is at least about 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more residues in length or the full length of the gene or transcript. 
     
     
         18 - 31 . (canceled) 
     
     
         32 . An expression cassette comprising a nucleic acid comprising a sequence as set forth in  claim 1 . 
     
     
         33 . A vector comprising a nucleic acid comprising a sequence as set forth in  claim 1 . 
     
     
         34 . A cloning vehicle comprising a nucleic acid comprising a sequence as set forth in  claim 1 , wherein the cloning vehicle comprises a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome. 
     
     
         35 . The cloning vehicle of  claim 34 , wherein the viral vector comprises an adenovirus vector, a retroviral vector or an adeno-associated viral vector. 
     
     
         36 . A bacterial artificial chromosome (BAC), a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC) or a mammalian artificial chromosome (MAC) comprising a sequence as set forth in  claim 1 . 
     
     
         37 . A transformed cell comprising a nucleic acid comprising a sequence as set forth in  claim 1 . 
     
     
         38 . A transformed cell comprising an expression cassette as set forth in  claim 32 . 
     
     
         39 . The transformed cell of  claim 38 , wherein the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell. 
     
     
         40 - 41 . (canceled) 
     
     
         42 . A transgenic plant comprising a sequence as set forth in  claim 1 . 
     
     
         43 . The transgenic plant of  claim 42 , wherein the plant is a corn plant, a sorghum plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant, a grass, or a tobacco plant. 
     
     
         44 . A transgenic seed comprising a sequence as set forth in  claim 1 . 
     
     
         45 . The transgenic seed of  claim 44 , wherein the seed is a corn seed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a rice, a barley, a peanut or a tobacco plant seed. 
     
     
         46 - 51 . (canceled) 
     
     
         52 . An isolated or recombinant polypeptide (i) having at least 50%; 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and all polypeptides disclosed in the SEQ ID listing, which include all even numbered SEQ ID NOs: from SEQ ID NO:2 through SEQ ID NO:26,898, over a region of at least about 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues, wherein optionally the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection, or, (ii) encoded by a nucleic acid having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NOs: from SEQ ID NO:1 through SEQ ID NO:26,897, over a region of at least about 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues, or encoded by a nucleic acid capable of hybridizing under stringent conditions to a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NOs: from SEQ ID NO:1 through SEQ ID NO:26,897. 
     
     
         53 . The isolated or recombinant polypeptide of  claim 52 , wherein the sequence identity is over a region of at least about at least about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or is 100% sequence identity. 
     
     
         54 . (canceled) 
     
     
         55 . The isolated or recombinant polypeptide of  claim 52 , wherein the polypeptide has a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and all polypeptides disclosed in the SEQ ID listing, which include all even numbered SEQ ID NOs: from SEQ ID NO:2 through SEQ ID NO:26,898. 
     
     
         56 . The isolated or recombinant polypeptide of  claim 52 , wherein the polypeptide has an enzymatic activity or encodes a protein. 
     
     
         57 . The isolated or recombinant polypeptide of  claim 56 , wherein the enzymatic activity comprises aldolase, alpha-galactosidase, amidase, secondary amidase, amylase, catalase, dehalogenase, endoglucanase, epoxide hydrolase, esterase, hydrolase, glucosidase, glycosidase, intein, isomerase, laccase, lipase, monooxygenase, nitroreductase, nitrilase, P450 enzyme, pectate lyase, phosphatase, phospholipase, phytase, polymerase or xylanase activity. 
     
     
         58 - 60 . (canceled) 
     
     
         61 . The isolated or recombinant polypeptide of  claim 56 , wherein the enzyme or the protein is thermostable or thermotolerant. 
     
     
         62 . The isolated or recombinant polypeptide of  claim 61 , wherein the polypeptide retains enzymatic activity under conditions comprising a temperature range of between about 1° C. to 5° C., between about 5° C. to 15° C., between about 15° C. to 25° C., between about 25° C. to 37° C., between about 37° C. to 95° C., between about 55° C. to 85° C., between about 70° C. to 95° C., between about 70° C. to 75° C., or between about 90° C. to 95° C., or, the polypeptide retains enzymatic activity after exposure to a temperature in the range from between about 1° C. to 5° C., between about 5° C. to 15° C., between about 15° C. to 25° C., between about 25° C. to 37° C., between about 37° C. to 95° C., between about 55° C. to 85° C., between about 70° C. to 75° C., or between about 90° C. to 95° C., or more. 
     
     
         63 . An isolated or recombinant polypeptide comprising a polypeptide as set forth in  claim 52  and lacking a signal sequence or a prepro sequence. 
     
     
         64 . An isolated or recombinant polypeptide comprising a polypeptide as set forth in  claim 52  and having a heterologous signal sequence or a heterologous prepro sequence. 
     
     
         65 . The isolated or recombinant polypeptide of  claim 56 ,
 wherein the enzymatic activity comprises a specific activity at about 37° C. in the range from about 100 to 1000 units per milligram of protein, from about 500 to 750 units per milligram of protein, from about 500 to 1200 units per milligram of protein, or from about 750 to 1000 units per milligram of protein.   
     
     
         66 . The isolated or recombinant polypeptide of  claim 61 , wherein thermotolerance comprises retention of at least half of the specific activity of the enzyme at 37° C. after being heated to an elevated temperature. 
     
     
         67 . The isolated or recombinant polypeptide of  claim 61 , wherein thermotolerance comprises retention of specific activity at 37° C. in the range from about 500 to 1200 units per milligram of protein after being heated to an elevated temperature. 
     
     
         68 . The isolated or recombinant polypeptide of  claim 52 , wherein the polypeptide comprises at least one glycosylation site. 
     
     
         69 . The isolated or recombinant polypeptide of  claim 68 , wherein the glycosylation is an N-linked glycosylation. 
     
     
         70 . The isolated or recombinant polypeptide of  claim 68 , wherein the polypeptide is glycosylated after being expressed in a  P. pastoris  or a  S. pombe.    
     
     
         71 . The isolated or recombinant polypeptide of  claim 56 , wherein the polypeptide retains an enzymatic activity under conditions comprising about pH 6.5, pH 6.0, pH 5.5, 5.0, pH 4.5 or 4.0. 
     
     
         72 . The isolated or recombinant polypeptide of  claim 56 , wherein the polypeptide retains an enzymatic activity under conditions comprising about pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10 or pH 10.5. 
     
     
         73 . A protein preparation comprising a polypeptide as set forth in  claim 52 , wherein the protein preparation comprises a liquid, a solid or a gel. 
     
     
         74 - 87 . (canceled) 
     
     
         88 . A method of producing a recombinant polypeptide comprising: (a) providing a nucleic acid operably linked to a promoter, wherein the nucleic acid comprises a sequence as set forth in  claim 1 ; and (b) expressing the nucleic acid of (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide. 
     
     
         89 . The method of  claim 88 , further comprising transforming a host cell with the nucleic acid of (a) followed by expressing the nucleic acid of (a), thereby producing a recombinant polypeptide. 
     
     
         90 - 139 . (canceled) 
     
     
         140 . A method for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising:
 (a) making a modified cell by modifying the genetic composition of a cell, wherein the genetic composition is modified by addition to the cell of a nucleic acid comprising a sequence as set forth in  claim 1 ;   (b) culturing the modified cell to generate a plurality of modified cells;   (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of (b) in real time; and,   (d) analyzing the data of (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifying an engineered phenotype in the cell using real-time metabolic flux analysis.   
     
     
         141 . The method of  claim 140 , wherein the genetic composition of the cell is modified by a method comprising deletion of a sequence or modification of a sequence in the cell, or, knocking out the expression of a gene. 
     
     
         142 . The method of  claim 141 , further comprising selecting a cell comprising a newly engineered phenotype. 
     
     
         143 . The method of  claim 142 , further comprising culturing the selected cell, thereby generating a new cell strain comprising a newly engineered phenotype. 
     
     
         144 - 148 . (canceled) 
     
     
         149 . A method of increasing thermotolerance or thermostability of an enzyme polypeptide, the method comprising glycosylating an enzyme, wherein the polypeptide comprises at least thirty contiguous amino acids of a polypeptide as set forth in  claim 52 , or a polypeptide encoded by a nucleic acid as set forth in  claim 1 , thereby increasing thermotolerance or thermostability of the enzyme. 
     
     
         150 . A method for overexpressing a recombinant enzyme in a cell comprising expressing a vector comprising a nucleic acid sequence as set forth in  claim 1 , wherein overexpression is effected by use of a high activity promoter, a dicistronic vector or by gene amplification of the vector. 
     
     
         151 . A method of making a transgenic plant comprising:
 (a) introducing a heterologous nucleic acid sequence into the cell, wherein the heterologous nucleic sequence comprises a sequence as set forth in  claim 1 , thereby producing a transformed plant cell;   (b) producing a transgenic plant from the transformed cell.   
     
     
         152 . The method as set forth in  claim 151 , wherein the (a) further comprises introducing the heterologous nucleic acid sequence by electroporation or microinjection of plant cell protoplasts. 
     
     
         153 . The method as set forth in  claim 152 , wherein the (a) comprises introducing the heterologous nucleic acid sequence directly to plant tissue by DNA particle bombardment or by using an  Agrobacterium tumefaciens  host. 
     
     
         154 . A method of expressing a heterologous nucleic acid sequence in a plant cell comprising:
 (a) transforming the plant cell with a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic sequence comprises a sequence as set forth in  claim 1 ;   (b) growing the plant under conditions wherein the heterologous nucleic acids sequence is expressed in the plant cell.   
     
     
         155 - 172 . (canceled) 
     
     
         173 . An isolated or recombinant nucleic acid comprising a sequence encoding a polypeptide having an enzyme activity and a signal sequence, wherein the nucleic acid comprises a sequence as set forth in  claim 1 . 
     
     
         174 . The isolated or recombinant nucleic acid of  claim 173 , wherein the signal sequence is derived from another enzyme. 
     
     
         175 . An isolated or recombinant nucleic acid comprising a sequence encoding a polypeptide having an enzyme activity or encoding a protein, wherein the sequence does not contain a signal sequence and the nucleic acid comprises a sequence as set forth in  claim 1 . 
     
     
         176 - 188 . (canceled)