METHOD FOR ISOLATING MUTANT CELLS

Abstract: A method for isolating a mutant cell that secretes a desired compound at a level greater than the starting cell from which the mutant cell is derived. The method includes providing a starting cell and a feeder cell. The starting cell has at least three characteristics. First, the starting cell has a requirement for a metabolite for survival or division of the starting cell in a solid growth medium. Second, the starting cell mutates to lack that requirement at a frequency of less than 10<-10> per cell division. Third, the starting cell has a rate of mutation of at least 10<-6> per nucleotide base per cell division. The feeder cell has at least two properties. First, the feeder cell requires the desired compound for survival or division of the feeder cell in a growth medium. Second, the feeder cell secretes the metabolite required by the starting cell into the solid growth medium. The method further includes contacting a plurality of the feeder cells and the starting cells together in a solid growth medium to allow the feeder cells to produce the desired compound and the starter cells to produce the metabolite. Both the cells will grow and divide to produce a plurality of colonies. Those colonies which are largest include one or more mutant cells.

Description: METHOD FOR ISOLATING MUTANT CELLS Background of the invention This invention relates to methods for isolation of a mutant cell containing one or more mutations which enhance production of a desired compound from the cell compared to a cell without such mutations. It is common to isolate mutant cells which overproduce a specific metabolite by selecting cells which are resistant to analogs of the metabolite. For example, Yamada et al. describe isolation of mutants which overproduce biotin by selection for cells resistant to a biotin analog. 47 Agricultural Biological Chemistry. 1011, 1983. Other examples include methionine overproducing mutants (Yamada et al.. Agricultural Biological Chemistry 46:47, 1982; isolated by selecting cells resistant to ethionine) ; valine overproducing mutants (Hibberd 62, Iowa State Journal of Research. 479, 1988; selecting valine resistance); pyrimidine nucleotide biosynthetic enzyme overproducers (Kempe et al., 9 Cell 541, 1976; selecting resistance to N-phosphonacetyl-1- aspartate) ; and amino acid overproducers in Cvanobacteria (Hall et al., 143 Journal Bacteriology. 981, 1980; selecting antimetabolite analog resistance) . In Hall et al., id., wild type Bacillus subtilis cells were used as a test lawn for screening obvious regulatory mutants from among collections of analog resistant strains. Auxotrophic strains of B. subtilis were convenient indicator strains for identification of mutants in Cvanobacteria through observation of syntrophic growth responses. Green, Second Chemical Congress of the North American Continent, San Francisco, August 22, 1980, Abstract No. 16 describes production of mutant Corn cells resistant to lysine- and threonine-induced growth inhibition. Grull et al. 137 Journal of Bacteriology. 480, 1979, describe isolation of a ino acid overproducing mutants of E. coli by mutagenesis and penicillin enrichment. Vincenzotto and Matagne, 90 Archive Internationales de Chemi, B88, 1982, describe isolation of mutants of an alga by mutagenesis and screening on agar medium containing various dyes. Santhaguru and Avissar, 21 Israel Journal of Medical Sciences. 185, 1985, describe use of levulinate, a competitive inhibitor of the heme biosynthetic pathway, for isolation of heme overproducing Rhizobium mutants. Summary of the Invention This invention concerns a method for symbiotic amplification of desired mutant cells by using two genetically different populations of cells which can cross-feed each other. The mutant cells are produced from starter cells grown on solid growth medium in the presence of feeder cells. Both the starter and feeder cells are dependent upon each other for continued survival or division of the cells in the growth medium. The more rapid the growth of the feeder cell the more rapid the growth of the surrounding starter cells, and vice versa. When mutations occur in the starting cell which increase production and secretion of a compound required for growth of the feeder cell the faster the surrounding feeder cells will grow. The faster such feeder cells produce a compound required for growth of the starter cells or mutant cell the faster the starting cell will grow. Thus, mutations which cause overproduction of the compound required by the feeder cells will result in formation of large colonies on the solid growth medium. The starter cell generally includes a gene, or a mutation in an existing gene, which causes rapid mutation of the DNA within the starting cell, for example, a mutator gene. Such mutator genes substantially increase the chance of production of a desired mutant cell. Thus, the invention features a method for isolating a mutant cell that secretes a desired compound at a level greater than the starter cell from which the mutant cell is derived. The method includes providing a starter cell and a feeder cell. The starter cell has at least three characteristics. First, the starting cell has a requirement for a metabolite for survival or division of the starting cell in a solid growth medium. Second, the starting cell mutates to lack that requirement at a frequency of less than 10~ 10 per cell division. Third, the starting cell has a rate of mutation of at least 10 per nucleotide base per cell division. The feeder cell has at least two properties. First, the feeder cell requires the desired compound for survival of division of the feeder cell in a growth medium. Second, the feeder cell secretes one or more metabolites which allow growth of the starter cell in the solid growth medium. The method further includes contacting a plurality of the feeder cells and the starter cells together in a solid growth medium to allow the feeder cells to produce the desired compound and the starter cells to produce the metabolite. Both the cells will grow and divide to produce a colony. Those colonies which are largest include one or more mutant cells. By "mutant cell" is meant any cell which contains one or more mutations which affect the secretion of the desired compound, compared to the starting cell from which the mutant cell is derived. By "derived" is merely meant that the mutant cell results from growth and cell division of the starting cell and includes one or more mutations compared to the starting cell. By "metabolite" is meant to include any compound which will allow growth of a starter cell and which is produced during metabolism by another cell. Such metabolite must be excreted into the medium surrounding the cell which produces it either by the cell's secretory mechanism, or other excretory mechanisms. Examples of such metabolites include vitamins, amino acids, nucleic acid components, fatty acids and lipids. Starting cells which require such metabolites generally have one or more mutations within their DNA which limit or prevent the ability of that starting cell to produce that metabolite. Preferably, the starting cell includes deletions at one or more regions of DNA responsible for production of that metabolite. Such deletions prevent the starting cell from mutating to lack the requirement for the metabolite. If the DNA is not deleted, that cell must contain two, or preferably more, mutations which prevent production of the metabolite, and do not mutate to produce a cell with an ability to produce a metabolite (i.e., revert) at a frequency of greater than 10~ 10 per cell division. Since the mutation rate at any particular nucleotide is between 10 "3 and 10~ 10 , it is preferable that starting cells contain at least 3 mutations within any gene encoding enzymes for the synthesis of the metabolite. Mutator genes are well known to those of ordinary skill in the art. Generally, they are mutations in a DNA polymerase which causes that polymerase to incorporate nucleotide bases incorrectly. Such incorrect incorporation results in a mutation. Generally, such mutator genes can increase the mutation rate of a cell by between 1000- and 100,000-fold. The mutation rate of any particular cell can be readily measured by one of ordinary skill in the art, for example, by providing a plasmid comprising the lacZ gene of E. coli and measuring the rate of mutation of nucleotide bases within the lacZ gene. It is preferred that the rate of mutation be at least 10 "6 per nucleotide base, preferably at least 10 "4 per nucleotide base,, in order to allow rapid mutation of DNA of the starting cells. Because of the presence of such mutator genes within the starting cell, and as discussed above, it is preferable that the genes encoding for the metabolite deleted so that the chance of reversion is practically zero. The desired compound can be any compound which affects the survival and division of the feeder cell. Generally, such desired compounds are chosen from cellular building blocks, e.g., lipids, fatty acids, vitamins, amino acids, nucleic acid components, and growth factors such as fibroblast growth factor. Interleukin 2, or any other equivalent compound. Generally, the feeder cell is able to survive or divide only minimally in the absence of the desired compound. For example, a bacterial feeder cell may have a rate of cell division of less than one cell division in three hours at 37°C in growth medium lacking the desired compound but otherwise having all required compounds. This compares to a rate of cell division of 1 in 30 minutes when the feeder cell is provided with the desired compound. The rate of cell division of plant and animal cells, or of cells grown under less optimum conditions, may be much lower. It is important in the invention only that the feeder cell have a greatly reduced rate of cell division in the absence of the desired compound compared to its rate of cell division in the presence of the desired compound. Thus, in the presence of the desired compound its rate of cell division is enhanced (e.g., at least three-fold and most preferably at least ten-fold) ; production of the metabolite required by the starting cell is also enhanced in the presence of the desired compound. Similarly, the starter cell requirement for the metabolite produced by the feeder cell need not be absolute. The absence of such metabolite need only significantly reduce the rate of cell survival or division (i.e., by at least 3-fold, preferably 10-fold). More importantly the starter cell must produce only a limited amount of the desired compound in the absence of the metabolite. Thus, both the starter cell and the feeder cell will grow, survive, and divide only poorly in the absence of the other of the starter or feeder cell or in the absence of the metabolite or desired compound. That is, both the starting cell and the feeder cell are dependent upon each other for growth and have a symbiotic growth relationship. By a "colony" is meant a group of cells including both starting and feeder cells which manifest themselves on the solid growth medium in a manner detectable by one of ordinary skill in the art. Generally, such colonies will be visible to a naked eye, having a size of greater than 0.5mm. In preferred embodiments, the starting cell and the feeder cell may belong to the same or a different species or genus; the starting or feeder cells may be grown on the surface of a solid medium or within a solid medium; and the starting cell or the feeder cell may include mutations which provide a selectable phenotype, for example, resistance to an antibiotic or inability of that cell to grow on a carbon source on which the other cell may grow. The selectable phenotypes allow later separation of the starter and feeder cells from one another, and allow isolation of a starting cell which has mutated to form a mutant cell. In other preferred embodiments, the starting cell and the feeder cell may include DNA which has been inserted into those cells by recombinant DNA technology, for example, the starting cell may include heterologous genes (i.e., genes from another organism) for the production of the desired compound. In a most preferred embodiment, the method further includes steps of isolating those colonies which include mutant cells (generally the largest colonies growing on a solid medium) and again contacting those cells with further feeder cells to allow further colonies to form. This process may be.repeated as many times as desired until a desired mutant cell which copiously excretes the desired compound is isolated. The invention provides a method for simple selection of desired mutant cells. There is no need to provide agents which cause mutations of the starting cells, nor a need for tedious screening of mutated cells to determine whether they contain a desired phenotype. The method automatically selects mutant cells which have the property of secreting desired compounds. These mutant cells can be readily isolated after selection in the method of this invention, and used in standard procedures for production of the desired compound. The invention allows isolation and selection of desired mutant cells without use of a large number of growth dishes. A large number of starter cells can be introduced onto one plate with feeder cells and growth of only one mutant cell-containing colony from such starter cells readily detected. The method can be used for concurrent selection of several mutant cells at once. For example when three or more cells are used, each of which depends on one of the other cells for growth, mutants in each of these cells may be selected. Specifically, if three cells, A, B, and C, are used and A requires a factor from C, B requires a factor from A, and C requires a factor from B, mutant cells of A and B, or A and C, or B and C can be simultaneously selected. One such cell, e.g.. A, may even be chosen so that it is able to supply B and C with their requirements. It is also possible to use this method to isolate symbiotic mixtures of two cells both of which are mutated but still depend on one another for growth. Other features and advantages of this invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiment The figure is a diagrammatic representation of symbiotic amplification by cross-feeder. By way of example only, the figure shows a symbiotic amplification method using two strains of Escherichia coli. This example is not limiting to the invention since, as described above, any desired combination of cell from any species or genus may be used in the invention. Referring to the figure. Cell A is a starting cell from which mutants which secrete a desired compound "X" may be isolated. Cell A includes in its chromosome a mutator gene, and a gene which genetically blocks the ability of Cell A to grow on glucose. Cell A thus requires a metabolite for growth, i.e., a carbon source other than glucose, e.g., acetate or succinate. These compounds must be provided by the feeder cell, Cell B. The mutator gene, for example, mutD, increases the frequency of spontaneous mutation about 1,000-fold. Also included in Cell A is a gene conferring resistance to streptomycin (rpsL) ; this resistance allows ready purification of Cell A, and its descendants from Cell B (which is sensitive to streptomycin) by growth in a medium containing streptomycin. Cell B, the feeder cell, is blocked for the synthesis of the desired compound X. For example, compound X may be a vitamin such as biotin. The starter Cell A secretes this vitamin during growth on acetate and succinate. The feeder cell is deleted for genes required for utilization of lactose (Δlac) which allows selection against growth of Cell B, even without the use of antibiotic streptomycin. The cell population is simply grown on a medium employing lactose as a carbon source for cell growth. Starter Cell A can grow on this medium; feeder Cell B cannot grow on this medium. Such medium may also contain streptomycin to ensure growth of starter Cell A. In order to determine whether any particular pair of Cell A and Cell B is suitable for use in the symbiotic amplification method, the cells are simply inoculated onto a solid medium lacking the desired compound but including the metabolite which allows growth of Cell A; for example, the medium lacks biotin but includes acetate. The starter cells are able to grow on this medium to form a patch of cells, or a streak, on the plate. The feeder Cell B is unable to grow on this plate unless the starter cells secrete some biotin. Growth of the Cell B adjacent the streak of Cell A demonstrates that the cells may act symbiotically with respect to biotin secretion. There follows an example by which selection for a desired mutant cell can be made. In this example, a mixture of Cell A (10 2 -10 3 cells) and Cell B (about 10 7 to 10 9 cells) are plated onto agar containing glucose. Cell A cannot grow on glucose as carbon and energy source but can secrete a small amount of biotin. The secreted biotin will stimulate the growth of a neighboring Cell B. Cell B will grow on glucose and will in turn excrete small amounts of acetate. Since Cell A requires acetate, any Cell A adjacent the secreting Cell B will be able to grow more rapidly than those not adjacent secreting Cell B. Growing Cell A will most effectively feed biotin to its immediate neighbors; that cell also stands to benefit the most when those neighbors start to grow and release acetate. A growth cycle is thus set up. Under favorable conditions a community of Cell A and Cell B will grow as a visible colony where each Cell A was originally seeded. This colony formation is known as symbiotic amplification by cross-feeding (SABCF) . During the formation of the colony. Cell A will multiply to a sizable population (a full grown bacterial colony on agar typically contains 10 9 cells) . During the course of this multiplication, a mutant of Cell A that secretes more biotin might arise. Its descendents will feed Cell B more effectively. The more rapid growth of Cell B around such a mutant cell will in turn help the mutant Cell A to grow more rapidly. If the mutational event occurs sufficiently early, the colony will be larger in size than colonies without a sizable community of mutant Cell A. Colonies appearing larger than normal are physically isolated and grown in a lactose medium containing streptomycin to eliminate B cells. The new population of mutant Cell A is then used to start a second round of selection by the above procedure. Colonies started by mutant Cell A will be relatively large. During the course of colony formation, a mutant of the mutant Cell A will arise from the growing population. Thus, a cascade of selection can occur and make the colony even larger and the method more effective. At the end of the growth phase the largest colonies will again be purified and the procedure repeated. In selecting the symbiotic cells for use in this method, the above-described cross-feeding experiment is useful. In addition, it is important to ensure that the physical diffusion of metabolites and the desired compound in the growth medium, for example, agar, is proportionate to the rate of consumption of that metabolite by surrounding cells requiring such compounds. The relative rates of these two processes can be changed by alteration of incubation temperature, which has little effect on physical diffusion of a metabolite, but has a significant effect on the growth rate of a cell and thus the consumption of the metabolite by that cell. An optimal temperature can be determined by standard procedure. In the example described above, a certain number of cells was used as feeder cells (10 7 -10 9 ) and another number of cells was used for starter cells (10 2 -10 3 ) . It is important that the optimal cell density and ratio of starter cells to feeder cells be established. This can be done by standard procedure. For example, by altering the ratio of those cells until the desired results are obtained. The above colonies may be grown within or on a surface of the solid growth medium, e.g., agar. In addition, the percentage of agar to liquid within the medium can be altered to reduce the rate of spread of growing cells. Again, optimal conditions for any pair of cells can be readily determined by standard procedure. When first starting the cross-feeder selection procedure described above it may be necessary to provide a small amount of the required metabolite and/or desired compound in order to allow some initial growth of the cells. Such initial growth should be limited to a few cell divisions, sufficient to allow a symbiotic relationship to start. Using the SABCF method, it is possible to engineer new biosynthetic pathways by recombinant DNA technology, for example, by cloning a gene into a plasmid or chromosome and transforming an appropriate cell with that plasmid. The SAPCF method can be used to improve such engineered pathways. Since different species of microbial cells, and even plant or animal cells, can be used in this method the method can be used to create mutant cells for production of almost any desired compound. Such cells can be genetically altered to allow selection of mutants that secrete a compound which is not a normal cellular component of that cell. Thus, for example, cells can be engineered to produce a growth factor, such as fibroblast growth factor (FGF) and overproducing mutants selected by growing those starter cells which secrete FGF in the presence of feeder cells which require FGF for growth or cell division. Such feeder cells may include a natural receptor for the FGF, or may naturally require FGF for cell growth. The starter cell may have a requirement for acetate, as described above, or any other analogous requirement well known to those of ordinary skill in the art. Other embodiments are described within the following claims.

Claims: Claims 1. A method for isolating a mutant cell that secretes a desired compound at a level greater than a starting cell from which said mutant cell is derived, comprising the steps of: providing the starter cell, said starting cell having: (a) a requirement for a first metabolite for survival or division of the starting cell in a solid growth medium, (b) mutating to lack said requirement at a frequency of less 10 "10 per cell division, and (c) having a rate of mutation of at least 10 "6 per nucleotide base per cell division; providing a feeder cell requiring the desired compound for survival or division of the feeder cell in said solid growth medium, said feeder cell secreting said first metabolite into said solid growth medium; and contacting a plurality of said feeder cells and said starting cells together in said solid growth medium to allow said feeder cells to produce said desired compound and said starting cells to produce said metabolite, and to allow said cells to grow and divide to produce a plurality of colonies; wherein those colonies which are largest include one or more mutant cells. 2. The method of claim 1 wherein said starting cell has a selectable phenotype allowing its ready isolation from said feeder cell. 3. The method of claim 1 wherein said feeder cell has a selectable phenotype allowing its ready isolation from said starting cell. 4. The method of claim 1 wherein said cells in a said colony are contacted with said feeder cells in said solid growth medium to allow said feeder cells to produce said desired compound and said cells from said colony to produce said metabolite, and to allow said cells to grow and divide to produce a plurality of colonies. 5. The method of claim 1 wherein said starting cell and said feeder cell are chosen from cells belonging to different species. 6. The method of claim 1 wherein said starting cell and said feeder cell are chosen from cells belonging to different genus. 7. The method of claim 1 wherein said cells are grown on the surface of said solid growth medium. 8. The method of claim 1 wherein said desired compound is selected from a cellular building block and a growth factor. 9. The method of claim 1 wherein said desired compound is a vitamin. 10. The method of claim 9 wherein said vitamin is biotin.

Inventors: LIN EDMUND C C, LIN, EDMUND, C., C.
Applicants: LIN EDMUND C C, LIN, EDMUND, C., C.
Application year: 1991
Main classification: C12N15
IPC codes: C07K14, C07K16, C12N1, C12N5, C12N9, C12N15, C12P13, C12P17, C12R1