Synthetic ligation reassembly in directed evolution

Abstract

Harvesting the full richness of biodiversity is instantly recognized by Diversa Corporation as a powerful means to access both novel molecules having direct commercial utility as well as molecular templates that could be retooled to acquire commercial utility. A directed evolution process for rapid and facilitated production from a progenitor polynucleotide template, of a library of mutagenized progeny polynucleotides wherein each of the 20 naturally encoded amino acids is encoded at each original codon position. This method, termed site-saturation mutagenesis, or simply saturation mutagenesis, is preferably based on the use of the degenerate N,N,G/T sequence. Also, a method of non-stochastically producing a library of chimeric nucleic acid molecules having an overall assembly order that is chosen by design. Accordingly, a set of progenitor templates, such as genes (e.g. a family of esterase genes) or genes pathways (e.g. encoding antibiotics) can be shuffled to generate a sizable library of distinct progeny polynucleotide molecules (e.g. 10 100 ) and correspondingly encoded polypeptides. Screening of these polynucleotide libraries enables the identification of a desirable molecular species that has a desirable property, such as a specific enzymatic activity serviceable for a commercial application, or a novel antibiotic. 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.

What is claimed is: 
     
       1. A method of producing a progeny library comprised of chimerized but pre-determined polynucleotide sequences each of which is comprised of a pre-determined number of building block sequences that are assembled in non-random order, the method comprising:
 (a) generating a plurality of pre-determined nucleic acid building block sequences comprised of sequences delineated by demarcation points selected from aligned progenitor nucleic acid sequences; and   (b) non-stochastically assembling said nucleic acid building block sequences to produce said chimerized but pre-determined polynucleotide sequences, such that a designed overall assembly order is achieved for each of said chimerized but pre-determined polynucleotide sequence.   
     
     
       2. The method of  claim 1  where the progenitor nucleic acid sequences comprise sequences derived from an uncultivated organism or an environmental sample. 
     
     
       3. The method of  claim 1  where the progenitor nucleic acid sequences are comprised of genomic nucleic acid sequences. 
     
     
       4. The method of  claim 1 , where the progeny library is comprised of at least 10 10  different pre-determined progeny molecular sequences. 
     
     
       5. The method of  claim 1 , where the progeny library is comprised of at least 10 15  different pre-determined progeny molecular sequences. 
     
     
       6. The method of any of  claims 1 - 5 , where the nucleic acid building block sequences are obtained from polynucleotide sequences that encode enzymes or fragments thereof. 
     
     
       7. The method of any of  claims 1 - 5 , where the nucleic acid building block sequences are assembled to produce polynucleotide encoding biochemical pathways from one or more operons or gene clusters of portions thereof. 
     
     
       8. The method of any of  claims 1 - 5 , where the nucleic acid building block sequences are obtained from polynucleotide encoding polyketides or fragments thereof. 
     
     
       9. The method of any of  claims 1 - 5 , where the nucleic acid building block sequences are obtained from polynucleotide encoding antibodies or antibody fragments or other peptides or polypeptides. 
     
     
       10. The method of any of  claims 1 - 5 , where the step of (b) non-stochastically assembling said nucleic acid building blocks is performed to generate a display library comprised of polypeptides or antibodies or peptidomimetic antibodies or antibody variable region sequences suitable for affinity interaction screening. 
     
     
       11. The method of any of  claims 1 - 5 , further comprising the step of
 (c) screening said progeny library to identify an evolved molecular property.   
     
     
       12. The method of  claim 1 , where step of (c) is comprised of expression screening to identify an evolved molecular property. 
     
     
       13. A method of producing a progeny library comprised of chimerized but pre-determined polynucleotide sequences each of which is comprised of a pre-determined number of building block sequences that are assembled in non-random order, the method comprising: (a) generating a plurality of pre-determined nucleic acid building block sequences comprised of sequences delineated by demarcation points selected to create nucleic acid building blocks of a pre-determined size from aligned progenitor nucleic acid sequences, wherein each of said plurality of pre-determined nucleic acid building blocks is a double stranded building block with two nucleotide overhangs generated by a method comprising the steps of (i) generating overlapping blunt-ended amplification products; (ii) melting the blunt-ended amplification products to produce single stranded nucleic acids; (iii) annealing the single stranded nucleic acids to produce a population of double stranded nucleic acids; and (iv) selecting for double stranded nucleic acids with two nucleotide overhangs; wherein selecting for double stranded nucleic acids with two nucleotide overhangs comprises degrading other nucleic acids in the population with a 3′ acting nuclease; and (b) non-stochastically assembling said nucleic acid building block sequences to produce said chimerized but pre-determined polynucleotide sequences, such that a designed overall assembly order is achieved for each of said chimerized but pre-determined polynucleotide sequence. 
     
     
       14. The method of claim 13 where the progenitor nucleic acid sequences comprise sequences derived from an uncultivated organism or an environmental sample. 
     
     
       15. The method of claim 13 where the progenitor nucleic acid sequences are comprised of genomic nucleic acid sequences. 
     
     
       16. The method of claim 13, where the progeny library is comprised of at least 10 10  different pre-determined progeny molecular sequences. 
     
     
       17. The method of claim 13, where the progeny library is comprised of at least 10 15  different pre-determined progeny molecular sequences. 
     
     
       18. The method of any of claims 13 to 17, where the nucleic acid building block sequences are obtained from polynucleotide sequences that encode enzymes or fragments thereof. 
     
     
       19. The method of any of claims 13 to 17, where the nucleic acid building block sequences are assembled to produce polynucleotide encoding biochemical pathways from one or more operons or gene clusters of portions thereof. 
     
     
       20. The method of any of claims 13 to 17, where the nucleic acid building block sequences are obtained from polynucleotide encoding polyketides or fragments thereof. 
     
     
       21. The method of any of claims 13 to 17, where the nucleic acid building block sequences are obtained from polynucleotide encoding antibodies or antibody fragments or other peptides or polypeptides. 
     
     
       22. The method of any of claims 13 to 17, where the step of (b) non-stochastically assembling said nucleic acid building blocks is performed to generate a display library comprised of polypeptides or antibodies or peptidomimetic antibodies or antibody variable region sequences suitable for affinity interaction screening. 
     
     
       23. The method of any of claims 13 to 17, further comprising the step of (c) screening said progeny library to identify an evolved molecular property. 
     
     
       24. The method of claim 13, where step of (c) is comprised of expression screening to identify an evolved molecular property. 
     
     
       25. The method of claim 13, wherein the 3′ acting nuclease is exonuclease III.