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WO1991019799A1 - Procede d'isolement des cellules mutantes - Google Patents

Procede d'isolement des cellules mutantes Download PDF

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Publication number
WO1991019799A1
WO1991019799A1 PCT/US1991/004468 US9104468W WO9119799A1 WO 1991019799 A1 WO1991019799 A1 WO 1991019799A1 US 9104468 W US9104468 W US 9104468W WO 9119799 A1 WO9119799 A1 WO 9119799A1
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Prior art keywords
cell
cells
feeder
starting
desired compound
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PCT/US1991/004468
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English (en)
Inventor
Edmund C. C. Lin
Original Assignee
Lin Edmund C C
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Publication date
Application filed by Lin Edmund C C filed Critical Lin Edmund C C
Priority to EP91913873A priority Critical patent/EP0651795A1/fr
Publication of WO1991019799A1 publication Critical patent/WO1991019799A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C12N9/6475—Interleukin 1-beta convertase-like enzymes (3.4.22.10; 3.4.22.36; 3.4.22.63)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/185—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system
    • C12P17/186—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system containing a 2-oxo-thieno[3,4-d]imidazol nucleus, e.g. Biotin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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.
  • mutant cells which overproduce a specific metabolite by selecting cells which are resistant to analogs of the metabolite.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • mutator genes substantially increase the chance of production of a desired mutant cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the feeder cell is able to survive or divide only minimally in the absence of the desired compound.
  • 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.
  • 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).
  • the starter cell must produce only a limited amount of the desired compound in the absence of the metabolite.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a metabolite for growth i.e., a carbon source other than glucose, e.g., acetate or succinate.
  • the mutator gene for example, mutD, increases the frequency of spontaneous mutation about 1,000-fold.
  • Cell A 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.
  • rpsL streptomycin
  • Cell B the feeder cell
  • 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 ( ⁇ lac) 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.
  • 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.
  • 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) .
  • SABCF cross-feeding
  • mutant Cell A 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.
  • the above-described cross-feeding experiment is useful.
  • 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.
  • feeder cells 10 7 -10 9
  • starter cells 10 2 -10 3
  • 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.
  • 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.
  • 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.
  • 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.
  • FGF fibroblast growth factor
  • 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.

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Abstract

Procédé d'isolement d'une cellule mutante qui sécrète un composé voulu dans une quantité supérieure à celle de la cellule de départ à partir de laquelle est dérivée la cellule mutante. Le procédé consiste à prendre une cellule de départ et une cellule mourricière, où la cellule de départ présente au moins trois caractéristiques: elle a besoin d'un métabolite pour survivre ou se diviser dans un milieu de croissance solide; elle subit une mutation de manière à ne plus avoir besoin de ce métabolite, ladite mutation se produisant à une fréquence inférieure à 10-10 par division cellulaire; et elle présente un taux de mutation d'au moins 10-6 par base nucléotidique et par division cellulaire. La cellule nourricière présente au moins deux caractéristiques: elle a besoin du composé voulu pour survivre et se diviser dans un milieu de croissance; et elle libère par sécrétion dans le milieu de croissance solide le métabolite dont a besoin la cellule de départ. Le procédé consiste également à mettre une pluralité de cellules nourricières et de cellules de départ en contact les unes avec les autres dans un milieu de croissance solide afin de permettre aux cellules nourricières de produire le composé voulu et aux cellules de départ de produire le métabolite. Les deux cellules pourront croître et se diviser pour produire une pluralité de colonies, dont les plus grandes comprendront une ou plusieurs cellules mutantes.
PCT/US1991/004468 1990-06-21 1991-06-20 Procede d'isolement des cellules mutantes WO1991019799A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP91913873A EP0651795A1 (fr) 1990-06-21 1991-06-20 Procede d'isolement des cellules mutantes

Applications Claiming Priority (2)

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US54189590A 1990-06-21 1990-06-21
US541,895 1990-06-21

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2687167A1 (fr) * 1992-02-06 1993-08-13 Forschungszentrum Juelich Gmbh Procede de preparation de souches bacteriennes produisant un amino-acide, et leur utilisation.
WO1996006164A1 (fr) * 1994-08-23 1996-02-29 President And Fellows Of Harvard College Procede d'isolement de cellules mutantes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Agric. Biol. Chem., Vol. 47, No. 5, issued 1983, YAMADA et al., "Biotin Overproduction by Biotin Analog-resistant Mutants of Bacillus sphaericus", pages 1011-1016, see entire document. *
ELKIND et al., "The Radiobiology of Cultured Mammalian Cells", published 1967 by Gordon and Breach (NY), pages 551-552, see pages 551-552. *
Journal of Bacteriology, Vol. 112, No. 3, issued December 1972, PAI, "Mutant of Escherichia coli with Derepressed Levels of the Biotin Biosynthetic Enzymers", pages 1281-1287, see pages 1282-1283, Fig. 2 and Table 1-3. *
Methods in Enzymology, Volume LVIII, issued 1979, THOMPSON, "Mutant Isolation", pages 308-322, see entire document. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5646030A (en) * 1990-06-21 1997-07-08 President And Fellows Of Harvard College Method for isolating mutant cells
FR2687167A1 (fr) * 1992-02-06 1993-08-13 Forschungszentrum Juelich Gmbh Procede de preparation de souches bacteriennes produisant un amino-acide, et leur utilisation.
WO1996006164A1 (fr) * 1994-08-23 1996-02-29 President And Fellows Of Harvard College Procede d'isolement de cellules mutantes

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CA2085893A1 (fr) 1991-12-22
EP0651795A4 (fr) 1993-05-11
EP0651795A1 (fr) 1995-05-10
JPH06501149A (ja) 1994-02-10

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