Methods and compositions for creating altered and improved cells and organisms

Abstract

The present invention relates to compositions involving randomized in-frame fusion polynucleotides and their introduction into a host organism to identify desirable phenotypic changes. The present invention further relates to methods of generating these randomized in-frame fusion polynucleotides by introducing randomized in-frame fusion polynucleotides into an organism, selecting for organisms with new or altered phenotypes, re-isolating the randomized in-frame fusion polynucleotides from the selected organisms, re-assembling the constituent polynucleotides of the re-isolated randomized in-frame fusion polynucleotides into new collections of randomized in-frame fusion polynucleotides, and repeating the selection for organisms with new or altered phenotypes.

1 . A method of producing an organism with a new or altered phenotype, comprising:
 (a) introducing a composition comprising a plurality of random in-frame fusion polynucleotides having sequences different from each other into an organism to produce one or more transformed organisms, each random in-frame fusion polynucleotide comprising at least two full-length open reading frames, one designated as a 5′ full-length open reading frame and one designated as a 3′ full-length open reading frame, the 5′ full-length open reading frame being joined, either directly, or indirectly via at least one intervening open reading frame, to the 3′ full-length open reading frame to form a composite open reading frame encoding a fusion polypeptide;   (b) isolating at least two transformed organism exhibiting a different phenotype as compared to a control organism cultivated under the same conditions;   (c) isolating at least the 5′ full-length open reading frame and the 3′ open-reading frame from at least two fusion polynucleotides present in the organisms of step (b);   (d) randomly combining the isolated full-length open reading frames of step (c) to form at least two random in-frame polynucleotides, each comprising a composite open reading frame encoding a fusion polypeptide;   (e) introducing the polynucleotide of step (d) into an organism to produce one or more transformed or and   (f) isolating at least one transformed organism from the organism(s) in step (e) exhibiting a different phenotype as compared to a control organism cultivated under the same conditions,   wherein steps (c) through (f) are optionally repeated one or more times.   
     
     
         2 . The method of  claim 1  wherein, each full-length 5′ open reading frame is selected from a collection comprising a plurality of full-length open reading frames having sequences different from each other and each full-length 3′ open reading frame is selected from a second collection comprising a plurality of full-length open reading frames having sequences different from each other. 
     
     
         3 . The method according to  claim 1 , wherein the at least two full-length open reading frames are nonhomologous. 
     
     
         4 .- 10 . (canceled) 
     
     
         11 . A method of producing an organism with a new or altered phenotype, comprising:
 (a) introducing a composition comprising a plurality of random in-frame fusion polynucleotides having sequences different from each other into an organism to produce one or more transformed organisms, each random in-frame fusion polynucleotide comprising at least two open reading frames isolated from the genome of the same species of organism, one designated as a 5′ open reading frame and one designated as a 3′ open reading frame, the 5′ open reading frame being joined either directly, or indirectly via at least one intervening open reading frame, to the 3′ open reading frame to form a composite open reading frame encoding a fusion polypeptide;   (b) isolating at least two transformed organisms exhibiting a different phenotype as compared to a control organism cultivated under the same conditions;   (c) isolating at least the 5′ open reading frame and the 3′ frame from each of the at least two fusion polynucleotides present in the organisms of step (b);   (d) randomly combining the isolated open reading frames of step (c) to form at least two random in-frame polynucleotides, each comprising a composite open reading frame encoding a fusion polypeptide;   (e) introducing the polynucleotide(s) of step (d) into an organism to produce one or more transformed organisms; and   (f) isolating at least one transformed organism from the organism(s) in step (e) exhibiting a different phenotype as compared to a control organism cultivated under the same conditions,   wherein steps (c) through (f) are optionally repeated one or more times, wherein the organism is fungus, an alga, a plant or an animal.   
     
     
         12 . The method of  claim 11 , wherein each 5′ open reading frame is selected from a collection comprising a plurality of open reading frames having sequences different from each other and each 3′ open reading frame is selected from a second collection comprising a plurality of open reading frames having sequences different from each other. 
     
     
         13 . The method according to  claim 11 , wherein the at least two open reading frames are nonhomologous. 
     
     
         14 . The method according to  claim 11 , wherein the at least two open reading frames are joined via a linker sequence. 
     
     
         15 . The method according to  claim 14 , wherein the linker sequence 1 to 1,000 codons in length. 
     
     
         16 . The method according to  claim 11 , wherein the random in-frame fusion polynucleotide further comprises an expression vector sequence. 
     
     
         17 . The method according to  claim 11 , wherein the random in-frame fusion polynucleotide further comprises at least one regulatory sequence. 
     
     
         18 . The method according to  claim 17 , wherein the regulatory sequence is a promoter or a terminator. 
     
     
         19 . The method according to  claim 11 , wherein the organism is a a fungus. 
     
     
         21 . The method according to  claim 11 , wherein the composite open reading frame encoding the fusion polypeptide of step (d) is different from the fusion polypeptide present in the organisms of step (b). 
     
     
         22 . The method according to  claim 11 , wherein the at least two open reading frames isolated from the genome of the same organism are full-length. 
     
     
         23 . The method according to  claim 19 , wherein the organism is an alga. 
     
     
         24 . A method of producing an organism with a new or altered phenotype, comprising:
 (a) introducing a composition comprising a plurality of random in-frame fusion polynucleotides having sequences different from each other into an organism to produce one or more transformed organisms, each random in-frame fusion polynucleotide comprising at least two open reading frames isolated from the genome of at least two different species of organism, one designated as a 5′ open reading frame and one designated as a 3′ open reading frame, the 5′ open reading frame being joined either directly, or indirectly via at least one intervening open reading frame, to the 3′ open reading frame to form a composite open reading frame encoding a fusion polypeptide, wherein the species are, bacteria, archaea, protozoa, yeast, yeast, cyanobacteria, fungus, alga, or plant;   (b) isolating at least two transformed organisms exhibiting a different phenotype as compared to a control organism cultivated under the same conditions;   (c) isolating at least the 5′ open reading frame and the 3′ frame from each of at least two fusion polynucleotides present in the organisms of step (b);   (d) randomly combining the isolated open reading frames of step (c) to form at least two random in-frame polynucleotides, each comprising a composite open reading frame encoding a fusion polypeptide;   (e) introducing the polynucleotide(s) of step (d) into an organism to produce one or more transformed organisms; and   (f) isolating at least one transformed organism from the organism(s) in step (e) exhibiting a different phenotype as compared to a control organism cultivated under the same conditions,   wherein steps (c) through (f) are optionally repeated one or more times, wherein the organism is a fungus, an alga, a plant or an animal.   
     
     
         25 . The method of  claim 24 , wherein the at least two different species of step (a) are species of fungus, alga, or plant. 
     
     
         26 . The method of  claim 25 , wherein the at least two different species of step (a) are both fungus. 
     
     
         27 . The method of  claim 25 , wherein the at least two different species of step (a) are both alga. 
     
     
         28 . The method of  claim 25 , wherein the at least two different species of step (a) are both plant.