Evolution of whole cells and organisms by recursive sequence recombination
The invention provides methods employing iterative cycles of recombination and selection/screening for evolution of whole cells and organisms toward acquisition of desired properties. Examples of such properties include enhanced recombinogenicity, genome copy number, and capacity for expression and/or secretion of proteins and secondary metabolites.
1 . A method of evolving a cell to acquire a desired function, comprising:
(i.) introducing a library of DNA fragments into a plurality of cells, whereby at least one of the fragments undergoes recombination with a segment in the genome or an episome of the cells to produce modified cells and, optionally, recombining a plurality of nucleic acids from the modified cells to produce additionally modified cells;
(ii.) screening the modified cells or the additionally modified cells for modified cells that have evolved toward acquisition of the desired function;
(iii.) recombining DNA from the modified cells that have evolved toward the desired function with a further library of DNA fragments, at least one of which undergoes recombination with a segment in the genome or the episome of the modified cells to produce further modified cells, or recombining DNA between the modified cells that have evolved toward the desired function to produce further modified cells;
(iv.) screening the further modified cells for further modified cells that have further evolved toward acquisition of the desired function;
repeating (iii.) and (iv.) as required until the further modified cells have acquired the desired function.
2 . The method of claim 1 , comprising recombining a plurality of nucleic acids from the modified cells to produce additionally modified cells, which additionally modified cells comprise more diverse DNA than the modified cells, wherein the additionally modified cells are screened to identify cells that have evoloved towards acquisition of the desired function.
3 . The method of claim 1 , wherein the library is a library of locked in prophage.
4 . The method of claim 1 , wherein the step of recombining DNA between the modified cells is performed by protoplast fusing the modified cells and allowing fused cells to recombine.
5 . The method of claim 4 , wherein DNA from the modified cells is reiteratively recombined by protoplast fusion prior to selection or screening.
6 . The method of claim 1 , wherein the step of recombining DNA between the modified cells is performed by protoplast fusing the modified cells and allowing fused cells to recombine, the method further comprising enriching the resulting fused cell population for fused cells comprising more than two cell genomes.
7 . The method of claim 6 , further comprising reiteratively recombining the modified cells by protoplast fusion, allowing the resulting protoplasts to form into spores and then into mycelia, and fusing the resulting mycelia into protoplasts, wherein said reiterative recombining is performed before or after enriching the resulting fused cell population for fused cells comprising more than two cell genomes.
8 . The method of claim 1 , wherein the library of DNA fragments is a substantially complete genomic library from at least one heterologous cell type.
9 . The method of claim 1 , wherein the library of fragments comprises natural variants of a gene from different individuals.
10 . The method of claim 1 , further comprising subdividing the modified cells into first and second pools, isolating the further library of DNA fragments from the second pool and introducing the further library of DNA fragments into the first pool.
11 . The method of claim 1 , wherein the library of DNA fragments are components of viruses and the introducing occurs by infection of the cells with the viruses.
12 . The method of claim 1 , wherein the library of DNA fragments is cloned into a suicide vector incapable of permanent episomal existence in the cells.
13 . The method of claim 12 , wherein the suicide vector further comprises a selective marker.
14 . The method of claim 1 , further comprising coating the library or further library of DNA fragments with recA protein to stimulate recombination with the segment of the genome.
15 . The method of claim 1 , further comprising denaturing the library of fragments to produce single-stranded DNA, reannealling the single-stranded DNA to produce duplexes some of which contain mismatches at points of variation in the fragments, and selecting duplexes containing mismatches by affinity chromatography to immobilized MutS.
16 . The method of claim 1 , further comprising fragmenting the library of fragments to produce subfragments before denaturation, and reassembling duplexes of subfragments containing mismatches into reassembled fragments.
17 . The method of claim 16 , wherein the average diversity between reassembled fragments is at least five times greater than the average diversity between fragments.
18 . The method of claim 17 , wherein the desired function is secretion of a protein, and the plurality of cells further comprises a construct encoding the protein.
19 . The method of claim 18 , wherein the protein is inactive unless secreted, and the modified or further modified cells having evolved toward acquisition of the desired function are screened by propagating the cells and recovering surviving cells.
20 . The method of claim 19 , wherein the protein is β-lactamase or alkaline phosphatase, and the modified or further modified cells having evolved toward acquisition of the desired function are screened by monitoring metabolism of a chromogenic substrate of the alkaline phosphatase, or by monitoring resistance to a β-lactamase antibiotic.
21 . The method of claim 18 , wherein the protein is an antibody and the plurality of cells is E. coli.
22 . The method of claim 21 , wherein the construct further encodes a marker which is expressed with the protein as a fusion protein, and the screening comprises propagating the modified or further modified cells and identifying cells secreting the fusion protein by FACS sorting.
23 . The method of claim 22 , wherein the marker protein is linked to a phospholipid anchoring domain that anchors the marker protein to the cell surface after secretion from the cell.
24 . The method of claim 22 , wherein the cells are contained in agar drops which confine secreted protein in proximity with the cell secreting the protein.
25 . The method of claim 18 , wherein at least one fragment in the library encodes a signal sequence, and the at least one fragment is incorporated into a construct operably linked to a sequence encoding a protein to be secreted from the cells.
26 . The method of claim 15 , wherein at least one fragment in the library encodes a signal processing enzyme and the cells contain a construct encoding a protein to be secreted operably linked to a signal sequence.
27 . The method of claim 15 , wherein at least one fragment in the library encodes a gene selected from the group consisting of SecA, SecB, SecE, SecD and SecF genes.
28 . The method of claim 1 , wherein the desired function is enhanced recombination.
29 . The method of claim 1 , wherein the library of fragments comprises a cluster of genes collectively conferring recombination capacity.
30 . The method of claim 1 , wherein the at least one gene is selected from the group consisting of recA, recBCD, recBC, recE, recF, recG, recO, recQ, recR, recT, ruvA, ruvB, ruvC, sbcB, ssb, topA, gyrA and B, lig, polA, uvrD, E, recL, mutU, and helD.
31 . The method of claim 30 , wherein the plurality of cells further comprises a gene encoding a marker whose expression is prevented by a mutation removable by recombination, and the modified or further modified cells are screened by their expression of the marker resulting from removal of the mutation by recombination.
32 . The method of claim 30 , wherein in the screening steps, the modified or further modified cells are exposed to a mutagen and modified or further modified cells having evolved toward acquisition of the desired function are selected by their survival of the exposure, survival being conferred by the cells' enhanced recombinational capacity to remove damage induced by the mutagen.
33 . The method of claim 30 , wherein the mutagen is radiation.
34 . The method of claim 33 , wherein enhanced recombination is conferred by increased genomic copy number of the modified or further modified cells.
35 . The method of claim 25 , wherein at least one gene is selected from a replication or cell septation gene.
36 . The method of claim 35 , wherein the modified or further modified cells having evolved toward acquisition of the desired function are selected by their capacity for syncytium formation or cell fusion.
37 . The method of claim 1 , wherein the plurality of cells are plant cells and the desired property is improved resistance to a chemical or microbe, and in the screening the steps, the modified or further modified cells are exposed to the chemical or microbe and modified or further modified cells having evolved toward the acquisition of the desired function are selected by their capacity to survive the exposure.
38 . The method of claim 1 , wherein the plurality of cells are microspores.
39 . The method of claim 1 , wherein the further modified cells are microspores.
40 . The method of claim 1 , wherein the further modified cells are microspores, and wherein the microspores are used to pollenate a population of plants.
41 . The method of claim 37 , wherein the microorganism is a virus, bacterium, or fungus.
42 . The method of claim 37 , wherein the chemical is a viricide, fungicide, insecticide, bactericide or herbicide.
43 . The method of claim 42 , wherein the chemical is BT-toxin.
44 . The method of claim 42 , wherein the chemical is glyphosate or atrazine.
45 . The method of claim 42 , further comprising propagating a plant cell having acquired the desired function to produce a transgenic plant.
46 . The method of claim 42 , wherein the plurality of cells are embryonic cells of an animal, and the method further comprises propagating the transformed cells to transgenic animals.
47 . The method of claim 46 , wherein the modified cells are screened as components of the transgenic animals.
48 . The method of claim 46 , further comprising obtaining embryonic cells from the transgenic animals having modified cells evolved toward acquisition of the property and transforming the cells with the further library.
49 . The method of claim 46 , further comprising isolating DNA from transgenic animals that have evolved toward acquisition of the property and introducing the DNA into fresh embryonic cells.
50 . The method of claim 46 , wherein the animal is a fish.
51 . The method of claim 46 , wherein at least one of the fragments encodes a growth hormone and the desired property is increased size of the animal.
52 - 125 . (canceled)
126 . A method of evolving a cell toward acquisition of a desired property, the method comprising:
(a) introducing a DNA fragment library cloned into an artificial chromosome into a population of cells;
(b) culturing the cells under conditions whereby sexual recombination occurs between the cells, whereby DNA fragments cloned into the artificial chromosome homologously recombine with corresponding segments of endogenous chromosomes of the populations of cells, and endogenous chromosomes recombine with each other; and
(c) screening or selecting for cells that have evolved toward acquisition of the desired property.
127 . The method of claim 126 , wherein the cells are yeast cells and the artificial chromosome is a YAC.
128 . The method claim 126 , further comprising:
(a) culturing the cells surviving the screening or selecting step under conditions whereby sexual recombination occurs between cells, whereby further recombination occurs between endogenous chromosomes;
(b) screening or selecting for further cells that have evolved toward acquisition of the desired property;
(c) repeating steps (d) and (e) as needed until the desired property has been acquired.
129 . A method of evolving a DNA segment for acquisition of a desired property, the method comprising:
(a) providing a library of variants of the segment, each variant cloned into separate copies of an artificial chromosome;
(b) introducing the copies of the artificial chromosome into a population of cells;
(c) culturing the cells under conditions whereby sexual recombination occurs between cells and homologous recombination occurs between copies of the artificial chromosome bearing the variants; and,
(d) screening or selecting for variants that have evolved toward acquisition of the desired property.
130 - 179 . (canceled)