Whole cell engineering by mutagenizing a substantial portion of a starting genome, combining mutations, and optionally repeating

Abstract

An invention comprising cellular transformation, directed evolution, and screening methods for creating novel transgenic organisms having desirable properties. Thus in one aspect, this invention relates to a method of generating a transgenic organism, such as a microbe or a plant, having a plurality of traits that are differentially activatable. Also, a method of retooling genes and gene pathways by the introduction of regulatory sequences, such as promoters, that are operable in an intended host, thus conferring operability to a novel gene pathway when it is introduced into an intended host. For example a novel man-made gene pathway, generated based on microbially-derived progenitor templates, that is operable in a plant cell. Furthermore, a method of generating novel host organisms having increased expression of desirable traits, recombinant genes, and gene products.

What is claimed is:  
     
         1 . A method of producing an improved organism having a desirable trait comprising: a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations, and c) detecting the presence of said improved organism.  
     
     
         2 . The method of  claim 1 , wherein the set of substantial genetic mutations in step b) is comprised of a knocking out of at least 15 different genes.  
     
     
         3 . The method of  claim 1 , wherein the set of substantial genetic mutations in step b) is comprised of a knocking out of at least 50 different genes.  
     
     
         4 . The method of  claim 1 , wherein the set of substantial genetic mutations in step b) is comprised of a knocking out of at least 100 different genes.  
     
     
         5 . The method of  claim 1 , wherein the set of substantial genetic mutations in step b) is comprised of an introduction of at least 15 different genes.  
     
     
         6 . The method of  claim 1 , wherein the set of substantial genetic mutations in step b) is comprised of an introduction of at least 50 different genes.  
     
     
         7 . The method of  claim 1 , wherein the set of substantial genetic mutations in step b) is comprised of an introduction of at least 100 different genes.  
     
     
         8 . The method of  claim 1 , wherein the set of substantial genetic mutations in step b) is comprised of an alteration in the expression of at least 15 different genes.  
     
     
         9 . The method of  claim 1 , wherein the set of substantial genetic mutations in step b) is comprised of an alteration in the expression of at least 50 different genes.  
     
     
         10 . The method of  claim 1 , wherein the set of substantial genetic mutations in step b) is comprised of an alteration in the expression of at least 100 different genes.  
     
     
         11 . A method of producing an improved organism having a desirable trait comprising: a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms each having at least one genetic mutation, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations c) detecting the manifestation of at least two genetic mutations, d) introducing at least two detected genetic mutations into one organism, and e) optionally repeating any of steps a), b), c), and d).  
     
     
         12 . The method of  claim 11 , wherein step d) is comprised of a knocking out of at least 15 different genes in one organism.  
     
     
         13 . The method of  claim 11 , wherein step d) is comprised of a knocking out of at least 50 different genes in one organism.  
     
     
         14 . The method of  claim 11 , wherein step d) is comprised of a knocking out of at least 100 different genes in one organism.  
     
     
         15 . The method of  claim 11 , wherein step d) is comprised of an introduction of at least 15 different genes into one organism.  
     
     
         16 . The method of  claim 11 , wherein step d) is comprised of an introduction of at least 50 different genes into one organism.  
     
     
         17 . The method of  claim 11 , wherein step d) is comprised of an introduction of at least 100 different genes into one organism.  
     
     
         18 . The method of  claim 11 , wherein step d) is comprised of an alteration in the expression of at least 15 different genes in one organism.  
     
     
         19 . The method of  claim 11 , wherein step d) is comprised of an alteration in the expression of at least 50 different genes in one organism.  
     
     
         20 . The method of  claim 11 , wherein step d) is comprised of an alteration in the expression of at least 100 different genes in one organism.  
     
     
         21 . A method for identifying a gene that alters a trait of an organism, comprising: a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations, and c) detecting the presence an organism having said altered trait, and d) determining the nucleotide sequence of a gene that has been mutagenized in the organism having the altered trait.  
     
     
         22 . A method for producing an organism with an improved trait, comprising: a) functionally knocking out an enogenous gene in a substantially clonal population of organisms; b) transferring a library of altered genes into the substantially clonal population of organisms, wherein each altered gene differs from the endogenous gene at only one codon; c) detecting a mutagenized organism having an improved trait; and d)determining the nucleotide sequence of an gene that has been transferred into the detected organism.