Process for Chromosomal Engineering Using a Novel Dna Repair System

• US 20090227029A1
• Filed: 05/10/2006
• Published: 09/10/2009
• Est. Priority Date: 05/10/2006
• Status: Active Grant

• Current Assignee: Deinove
• Original Assignee: Deinove
• Inventors: Radman, Miroslav, Zahradka, Ksenija
• Granted Patent: US 9,193,974 B2
• Time in Patent Office: Days
• US Class Current: 435/441
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Abstract

This invention relates to chromosomal engineering via DNA repair process. The process of the invention comprises the steps of: 1) submitting at least one source of biological activity, e.g. Deinococcus radiodurans, to radiation, desiccation and/or chemical treatment liable to damage the DNA, so as to substantially shatter its chromosomes into short fragments; 2) annealing complementary single strand tails extended by the synthesis templated on partially overlapping DNA fragments of said shattered chromosomes; 4) converting the resulting long linear DNA intermediates into intact circular chromosomes, by means of a RecA dependent homologous recombination; whereas at least one foreign source of genetic material, e.g. DNA, can be introduced during steps 2 and/or 3; and 4) optionally separating and collecting the recombined chromosomes thus obtained.

Claims

1-31. -31. (canceled)

32. A process for chromosomal engineering, which comprises the steps of:

1). submitting at least one source of biological activity, e.g. Deinococcus radiodurans, to radiation, desiccation and/or chemical treatment liable to damage the DNA, so as to substantially shatter its chromosomes into short fragments;

2). annealing complementary single strand tails extended by the synthesis templated on partially overlapping DNA fragments of said shattered chromosomes;

3). converting the resulting long linear DNA intermediates into intact circular chromosomes, by means of a RecA dependent homologous recombination;

whereas at least one foreign source of genetic activity, e.g. DNA, is optionally introduced during steps

     2) and/or

     3); and

4). optionally separating and collecting the recombined chromosomes thus obtained.

33. The process of claim 32, wherein the at least one source of biological activity is selected from the group consisting of

(a). a bacterium resistant to ionizing radiation and/or desiccation and/or chemical treatment, and having a repair pathway, after radiation and/or desiccation, involving both extended synthesis dependent strand annealing (ESDSA) and homologous recombination processes;

(b). any living organism with a similar DNA repair pathway; and

(c). cell-free extracts of a bacterium according to (a) or of a living organism according to

(b). above.

34. The process of claim 32, which is carried out in the bacterium Deinococcus radiodurans, or any organism with similar DNA repair pathway, or in cell-free extracts of bacterium Deinococcus radiodurans, or any organism or organism extract with similar DNA repair pathway.

35. The process of claim 32, wherein the at least one source of biological activity is exposed to at least one gamma irradiation of about 0.7 megarad (7 kGy) or to desiccation.

36. The process of claim 32, wherein step 2) comprises an extended synthesis-dependent strand annealing.

37. The process of claim 32, wherein said foreign source of genetic activity comprises at least one DNA selected from the group consisting of intact or fragmented DNA from any biological species or DNA created by artificial synthesis.

38. The process of claim 32, wherein the foreign source of genetic activity comprises entire operons encoding biosynthetic pathways, e.g. for biodegradation of environmental pollutants, for bioconversion of energy, or for synthesis of antibiotics, therapeutic proteins or other useful compounds.

39. The process of claim 34, wherein integration of the foreign DNA fragments into the genome of D. radiodurans is promoted by the synthetic extensions of 3′

ends of foreign DNA.

40. The process of claim 34, wherein, to promote successful integration in the bacterial chromosome, the foreign DNA is extended by synthesis or end-addition by DNA ligase with a chosen bacterial sequence, preferably corresponding to one of its IS sequences, to offer multiple choices of harmless integration.

41. The process of claim 32, wherein all of the DNA fragments from any chosen source of genetic material or mixture of materials is extended by the same sequence such that—

in the same cell—

these DNA fragments can be directly end-annealed, in any possible combination, before being integrated into the chromosome.

42. The process of claim 32, which comprises the use of at least one means selected from the group consisting of natural transformation capacity, bacterial sex, such as conjugation or transduction capabilities, and artificial means, such as electroporation or chemical modification of the cellular membrane, for introducing foreign DNA into the living cells of at least one target source of biological activity undergoing DNA repair.

43. The process of claim 42, which further comprises triggering the DNA repair process by ionizing or ultraviolet irradiation, or by chemicals that damage DNA, such as mitomycin C, before or after the introduction of foreign DNA into the said source of biological activity.

44. An artificial DNA obtained by the process of claim 32.

45. A method for producing (i) useful small molecules or therapeutic proteins, (ii) enzymatic activities for biodegradation or bioremediation of the environment, (iii) energy generating metabolism or (iv) other useful compounds, which method uses the artificial DNA of claim 44.

46. A method for producing large repertoires of inter-species genomic mosaics encoding a wide variety of metabolic pathways, using the artificial DNA of claim 44.