Synthetic genes

• US 20050227316A1
• Filed: 04/07/2004
• Published: 10/13/2005
• Est. Priority Date: 04/07/2004
• Status: Abandoned Application

• Current Assignee: Kosan Biosciences, Inc. (Bristol-Myers Squibb Company)
• Original Assignee: Kosan Biosciences, Inc. (Bristol-Myers Squibb Company)
• Inventors: Patel, Kedar G., Reid, Ralph C., Santi, Daniel, Kodumal, Sarah J.
• Application Number:
  US10/820,975
• Publication Number:
  US 20050227316A1
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

The invention provides strategies, methods, vectors, reagents, and systems for production of synthetic genes, production of libraries of such genes, and manipulation and characterization of the genes and corresponding encoded polypeptides. In one aspect, the synthetic genes can encode polyketide synthase polypeptides and facilitate production of therapeutically or commercially important polyketide compounds.

Claims

1. A method for ligation of a plurality of DNA segments to obtain a ligation product that comprises sequences from each of said DNA segments in a predetermined order, said method comprising:

a) providing at least three DNA molecules, each comprising a DNA segment, wherein each segment is adjacent to one or two other segments in the ligation product, wherein each segment comprises a first region having sequence identity with a first adjacent DNA segment and a second region having sequence identity with a second adjacent DNA segment, if present;

b) cleaving each DNA molecule to produce a DNA segment with one or two ligatable ends, each ligatable end comprising at least a portion of the region having sequence identity with an adjacent DNA segment;

wherein at least one segment comprises two ligatable ends after cleavage,

c) simultaneously ligating each segment to the adjacent segment or segments; and

d) selecting a ligation product comprising sequences from each of said DNA segments in a predetermined order.

2. A method for ligation of a plurality of DNA segments to obtain a ligation product that comprises sequences from each of said DNA segments in a predetermined order, said method comprising:

a) providing a Type 1 DNA molecule, a Type 2 DNA molecule and at least one Type 3 DNA molecule, each comprising a DNA segment that is adjacent to one or two other segments in the final ligation product, wherein

i) said Type 1 DNA molecule comprises a first DNA segment, a first selectable marker, a first counter-selectable marker, a first cleavage site, and a second cleavage site, wherein cleavage of said second cleavage site produces a single-strand overhang in said first DNA segment which is ligatable to a single-strand overhang of an adjacent segment;

ii) said Type 2 DNA molecule comprises a second DNA segment, a second selectable marker, a second counter-selectable marker, a third cleavage site, and a fourth cleavage site, wherein cleavage of said fourth cleavage site produces a single-strand overhang in said second DNA segment which is ligatable to a single-strand overhang of an adjacent segment;

iii) each said Type 3 DNA molecule comprises a DNA segment, a third counter-selectable marker, a 5-prime cleavage site and a 3-prime cleavage site, wherein said 5-prime cleavage site, upon cleavage, produce a single-strand overhang in the segment that is ligatable to a single-strand overhangs of an adjacent segment, and said 3-prime cleavage site, upon cleavage, produce a single-strand overhang in the segment that is ligatable to a single-strand overhangs of a different adjacent segment;

wherein said first and second selectable markers are different;

wherein said first, second and third counter-selectable markers are independently selected and are the same or different;

wherein said first and third cleavage sites the same or are compatible;

wherein said second and fourth cleavage sites are independently selected and are the same or are different; and

,

wherein each 5-prime and 3-prime cleavage site is independently selected in each Type 3 DNA molecule and are the same or are different;

b) cleaving each DNA molecule at the first, second, third, and fourth cleavage sites, at the 5-prime cleavage site(s) and at the 3-prime cleavage site(s); and

c) ligating the resulting fragments to each other thereby producing a ligation product that comprises sequences from each of said DNA segments in a predetermined order.

3. The method of claim 2 further comprising the steps

d) transforming cells with ligation products produced in step (c);

and

e) selecting transformants that express said first and second selectable markers and do not express said first, second, or third counter-selectable marker.

4. The method of claim 3 further comprising the step:

f) isolating the ligation product comprising sequences from each of said DNA segments in a predetermined order from the transformants or their progeny.

5. The method of claim 2 wherein said first and second selectable markers are genes conferring drug resistance.

6. The method of claim 2 wherein said first, second and third counter-selectable markers are selected from the group consisting of ccdB (anti-DNA gyrase protein), sacB (sucrose sensitivity), araB (ribulose sensitivity), tetAR (tetracycline resistance/fusaric acid hypersensitivity),

7. The method of claim 2 wherein

a) said first and third cleavage sites are the same;

b) said second and fourth cleavage sites are the same;

c) the 5-prime cleavage site of at least one Type 3 DNA molecule is the same as the 3-prime cleavage site of the same Type 3 DNA molecule; and

/or

d) the 5-prime cleavage site of at least one Type 3 DNA molecule is the same as the 5-prime cleavage site of a different Type 3 DNA molecule.

8. The method of claim 2 wherein

a) said first and third cleavage sites are sites cleaved by a Type IIS restriction enzyme;

b) said second and fourth cleavage sites are sites cleaved by a Type IIS restriction enzyme; and

/or

c) said 5-prime and 3-prime cleavage sites of at least one Type 3 DNA molecule are sites cleaved by a Type IIS restriction enzyme.

9. The method of claim 2 wherein the first, second, third, fourth, 5-prime and 3-prime cleavage sites are sites cleaved by a Type IIS restriction enzyme.

10. The method of claim 2 wherein the first, second, third, fourth, 5-prime and 3-prime cleavage sites are sites cleaved by the same Type IIS restriction enzyme.

11. The method of claim 2 wherein the DNA segments of the Type 1, Type 2 and Type 3 DNA molecules comprise sequences encoding a polypeptide segment of a polyketide synthase.

12. The method of claim 2 wherein the DNA segments of the Type 1, Type 2 and Type 3 DNA molecules comprise sequences encoding a polyketide synthase domain.

13. The method of claim 2 wherein the DNA molecules cleaved in step (b) are cleaved in the same container.

14. A composition comprising:

i) a Type 1 DNA molecule, said DNA molecule comprising a first DNA segment, a first selectable marker, a first counter-selectable marker, a first cleavage site, and a second cleavage site;

wherein cleavage of said second cleavage site produces a single-strand overhang in said first segment which is ligatable to a single-strand overhang of an adjacent segment;

ii) a Type 2 DNA molecule, said DNA molecule comprising a second DNA segment, a second selectable marker, a second counter-selectable marker, a third cleavage site, and a fourth cleavage site wherein cleavage of said fourth cleavage site produces a single-strand overhang in said first segment which is ligatable to a single-strand overhang of an adjacent segment;

iii) at least one Type 3 DNA molecule, said DNA molecule comprising a DNA segment, a third counter-selectable marker, a 5-prime cleavage site and a 3-prime cleavage site, wherein said 5-prime and 3-prime cleavage sites, upon cleavage, produce single-strand overhangs in the segment that are ligatable to a single-strand overhangs of each of two adjacent segments;

wherein said first and second selectable markers are different;

wherein said first, second and third counter-selectable markers are independently selected and are the same or different;

wherein said first and third cleavage sites the same or are compatible;

wherein said second and fourth cleavage sites are independently selected and are the same or are different; and

,

wherein each 5-prime and 3-prime cleavage site is independently selected.

15. The composition of claim 14 comprising at least two Type 3 DNA molecules.

16. The composition of claim 14 comprising an endonuclease that cleaves at the first, second, third, or fourth cleavage sites or at one or more 5-prime or 3-prime cleavage sites.

17. The composition of claim 16 wherein the endonuclease cleaves at the first, second, third, and fourth cleavage sites and at one or more 5-prime or 3-prime cleavage sites.

18. The composition of claim 16 that contains at least two Type 3 DNA molecules comprising 5-prime or 3-prime cleavage sites and wherein the endonuclease cleaves at the third and fourth cleavage sites and at the 5-prime and 3-prime cleavage sites of said Type 3 DNA molecules.

19. A composition comprising the products resulting from cleavage of the Type 1, Type 2 and Type 3 DNAs of claim 14 at the first, second, third, fourth, 5-prime and 3-prime cleavage sites.

20. The composition of claim 19 additionally containing DNA ligase.

22. The vector of claim 20 wherein the SIS comprises —

N₁—

R₂—

N₂ —

where N₁ and N₂ are recognition sites for nicking enzymes, and may be the same or different, and R₂ is a recognition site for an endonuclease.

23. The vector of claim 20 wherein SM and USM are genes conferring drug resistance.

21. A cloning vector comprising, in the order shown,

a) SIS-CSM-R-USM or SIS-USM-R-CSM;

or

b) SIS-CSM-SM

where SIS is a synthon insertion site, CSM is a counter-selectable marker;

SM and USM are selectable markers, and R is an endonuclease cleavage site.

24. A vector comprising, in the order shown:

a) R₁-Sy-2S₁-CSM-R₂-USM

b) 2S₂-Sy-R₃-USM-R₄-CSM;

or

c) 2S₃-Sy-2S₄-CSM-SM

where 2S₁, 2S₂, 2S₃ and 2S₄ are recognition sites for Type IIS restriction enzymes, which may be the same or different,

Sy is a synthon coding region

R₁ and R₃ are endonuclease cleavage sites

R₂ and R₄ are endonuclease cleavage sites that, upon cleavage, result in compatible ends

CSM is a counter-selectable marker; and

,

USM and SM are selectable markers.

25. A composition comprising the vector of claim 24 and an endonuclease that cleaves at one or more of R₁, R₂, R₃, R₄, 2S₁, 2S₂, 2S₃, or 2S4.

26. A composition comprising each of the

a) a vector of the formula R₁-Sy-2S₁-CSM-R₂-USM

b) a vector of the formula 2S₂-Sy-R₃-USM-R₄-CSM;

and

c) a vector of the formula 2S₃-Sy-2S₄-CSM-SM

wherein 2S₁, 2S₂, 2S₃ and 2S4 are recognition sites for Type IIS restriction enzymes, which may be the same or different,

each Sy is a different synthon coding region,

R₁ and R₃ are endonuclease cleavage sites and are the same or are different,

R₂ and R₄ are endonuclease cleavage sites that, upon cleavage, result in compatible ends,

2S₁, 2S₂, 2S₃, and 2S₄ are endonuclease cleavage sites and are the same or are different,

CSM is a counter-selectable marker; and

,

USM and SM are selectable markers.

27. The composition of claim 26 that comprises 2, 3, 4, 5, or 6 vectors of the formula 2S₃ -Sy-2S₄-CSM-SM, each with a different Sy.

28. The composition of claim 26 further comprising an endonuclease that cleaves at one or more of R₁, R₂, R₃, R₄, 2S₁, 2S₂, 2S₃, and 2S₄.

29. A synthetic gene encoding a domain of a polyketide synthase that corresponds to a domain of the polyketide synthase encoded by a naturally occurring gene, wherein the domain-encoding sequence of the synthetic gene is different from the domain-encoding sequence of the naturally occurring gene, wherein

a) said domain-encoding sequence of said synthetic gene is less than about 80% identical to said domain-encoding sequence of said naturally occurring gene, and/or

b) said domain-encoding sequence of said synthetic gene comprises at least one unique restriction site that is not present or is not unique in the domain-encoding sequence of said naturally occurring gene, and/or

c) said domain-encoding sequence of said synthetic gene is free from at least one restriction site that is present in the domain-encoding sequence of said naturally occurring gene, and/or

d) the codon usage distribution in said domain-encoding sequence is substantially different from that of the naturally occurring gene.

30. A method for synthesis of a gene encoding a polypeptide segment comprising

a) designing a synthetic gene encoding the polypeptide segment;

b) designing component oligonucleotides for synthesis of the gene;

c) synthesizing the gene by generating synthons from said component oligonucleotides and stitching two or more synthons together.