# US 5,344,775 A · Synthesis of taxanes in culture using pseudocalluscells

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- Number: US 5,344,775 A
- Document ID: US5344775A
- Status: Granted
- Kind: A
- Application number: 07/794,711
- Filed: 1991-11-15
- Granted: 1994-09-06
- Earliest claimed: 1991-11-15
- Assignees: Escagenetics Corporation
- Inventors: Richard J. Smith
- Web page: https://patentgrep.com/patent/US5344775A

## CPC

- C12N 5/0025: {Culture media for plant cell or plant tissue culture}
- C12P 17/02: Oxygen as only ring hetero atoms
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

A method of synthesis of taxanes from pseudocallus of Taxus species, particularly T. baccata , and methods for pseudocallus production are described. Pseudocallus of the invention can be distinguished from callus tissue and cell suspensions by (1) its unique morphology which is characterized by an amorphous aggregation of cells lacking both differentiated tissues and clearly defined meristematic zones; by (2) very poor intercellular adhesion resulting in marked tissue friability; and by (3) relatively high initial rate of mass doubling. Production of taxanes is achieved by culturing the pseudocallus on support culture medium.

## Claims

### Claim 1

A method for obtaining pseudocallus cell aggregations from explants of a selected species of Taxus comprising the steps of:
(a) providing fragments of callus tissue induced by culture from Taxus explants containing meristematic tissue physically supported on a support culture medium containing as an auxin, alph-Napthaleneacetic acid (NAA), and as a cytokinin, 6-benzylamino purine (BAP)
(b) culturing said callus tissue fragments in an aqueous cell-suspension growth medium containing said auxin and said cytokinin to produce a suspension of a plurality of clusters of 1-10 cells having limited intercellular adhesion;
(c) plating cells removed from said cell suspension onto a support culture medium containing said auxin and said cytokinin, said medium capable of physically supporting growth of the cells on a surface thereof; and
(d) growing said plated cells on said support culture medium of step (c) to form pseudocallus cells, said pseudocallus cells being a loose, amorphous aggregation of cells lacking differentiated vascular or organ tissues and lacking clearly defined meristematic zones, said cell aggregation having poor intercellular adhesion, extreme friability, and falling apart into numerous individual cells and small cell clusters when mechanically disturbed, said pseudocallus cells showing an initial rate of mass doubling on fresh cell growth medium that is greater than the mass doubling of callus tissue of step (a), and exhibiting the property of producing higher levels of taxanes than that produced by callus tissue of step (a).

### Claim 2 (depends on 1)

The method of claim 1, wherein said growing step (d) occurs in the absence of light and in the absence of a taxanes production-inhibiting concentration of casein hydrolysate.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the selected species of Taxus is T. baccata `resembling Hessei`.

### Claim 4 (depends on 1)

The method of claim 1 wherein said growing step (d) is maintained until a concentration of taxanes in said pseudocallus cells aggregation is at least about 25 .mu.g taxanes/g of said pseudocallus cells.

### Claim 5 (depends on 1)

The method according to claim 1 wherein, said support for the culture medium is a solid or semi-solid and is further selected from the group consisting of a gelling agent Gelrite.RTM., agar, filter paper, cell mesh, and a micro-porous polypropylene membrane raft, and combinations thereof.

### Claim 6 (depends on 1)

Pseudocallus cells produced by the method of claim 1 from explants of a species of Taxus on a support culture medium containing said auxin and said cytokinin.

### Claim 7 (depends on 6)

The pseudocallus cells of claim 6, wherein the species of Taxus is T. baccata.

### Claim 8 (depends on 7)

The pseudocallus cells of claim 7, wherein the species is T. baccata VAR. `resembling Hessei`.

### Claim 9 (depends on 6)

The pseudocallus cells of claim 6 containing at least 25 .mu.g taxanes/g of the pseudocallus cells.

### Claim 10 (depends on 9)

The pseudocallus cells of claim 9, which contain at least 50 .mu.g taxanes/g of the pseudocallus cells.

### Claim 11 (depends on 6)

The method according to claim 6 wherein, said support for the culture medium is a solid or semi-solid and is further selected from the group consisting of a gelling agent, Gelrite.RTM., agar, filter paper, cell mesh, and a micro-porous polypropylene membrane raft, and combination thereof.

### Claim 12

A method of de novo production of taxanes in pseudocallus cell culture from Taxus-derived cells comprising the steps of:
(a) selecting callus cells grown from an explant containing meristematic tissue derived from a Taxus plant by culturing said explant physically supported on a support culture medium in the presence of both NAA, as an auxin, and BAP, as a cytokinin;
(b) culturing the selected callus cells from step (a) in an aqueous cell suspension growth medium containing both said auxin and said cytokinin until a suspension of cells in said suspension growth medium is formed, said cell suspension having clusters of from 1-10 cell, limited intercellular adhesion, and a cell mass doubling rate on the order of from about 7 to about 21 days;
(c) transferring the cells of said cell suspension onto a support culture medium capable of physically supporting said transferred cells on a surface thereof, said transfer of said cells occurring before active cell division halts in said culturing step (b);
(d) growing said cells transferred in step (c) on said support culture medium of step (c) in the presence of both said auxin and said cytokinin, to form pseudocallus cells, said pseudocallus cells formed being a loose, amorphous aggregation of cells lacking differentiated vascular or organ tissues and lacking clearly defined meristematic zones, said cell aggregation having poor intercellular adhesion, extreme friability, and falling apart into numerous individual cells and small cell clusters when mechanically disturbed, said pseudocallus cells further having a characteristic of an initial rate of mass doubling on the support culture medium greater than the mass doubling of said callus cells in step (a);
(e) maintaining growth of said pseudocallus cell aggregation in the presence of both said auxin and said cytokinin on said support culture medium to produce pseudocallus cells capable of producing high levels of taxanes;
(f) harvesting the pseudocallus cells; and
(g) recovering the taxanes from the harvested pseudocallus cells.

### Claim 13 (depends on 12)

The method or claim 12 wherein the explants are obtained from T. baccata.

### Claim 14 (depends on 12)

The method of claim 12 wherein said growth in steps (d) and (e) occurs in the absence of a taxane production-inhibitory concentration or casein hydrolysate.

### Claim 15 (depends on 13)

The method of claim 13, wherein the explants are obtained from T. baccata `resembling Hessei`.

### Claim 16 (depends on 12)

The method according to claim 12 wherein at least one of the taxanes is taxol.

### Claim 17 (depends on 14)

The method of claim 14, wherein said growth in steps (d) and (e) includes removing CO.sub.2 and ethylene from said pseudocallus cells and maintaining a concentration of CO.sub.2 in gases in contact with said pseudocallus cells below that normally present in the atmosphere.

### Claim 18 (depends on 12)

The method of claim 12, wherein said culturing is carried out until a concentration of taxanes in the pseudocallus cells of at least about 25 .mu.g taxanes/g of the pseudocallus cells, is reached.

### Claim 19 (depends on 12)

The method according to claim 12 wherein, said support for the culture medium is a solid or semi-solid and is further selected from the group consisting of a gelling agent, Gelrite.RTM., agar, filter paper, cell mesh, and a micro-porous polypropylene membrane raft, and combinations thereof.