Gene encoding glycosyltransferase and its uses

Details Gene encoding glycosyltransferase and its uses Gene encoding glycosyltransferase and its uses

1996-12-12

1999-01-19

Grimes, Eric

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

435 75, 435119, 435127, 4352523, 4353201, 435325, 536 232, C12N 910, C12N 1554, C12N 1563, C12P 2102

Patent

active

058612939

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

This invention is useful in the field of medicine. More particularly, in the preparation of indolopyrrolocarbazole-derived antitumor substances, this invention is useful as a method for introducing a glycosyl group smoothly and efficiently.


BACKGROUND ART

As a result of search for antitumor substances, the present inventors found a novel antitumor substance BE-13793C 6H)-dione) among microbial metabolites, and disclosed it in a previous Journal of Antibiotics, Vol. 44, 20 pp. 723-728 (1991)!. Thereafter, they created a compound having more excellent antitumor activity by a chemical modification of BE-13793C, and disclosed it in another previous patent application (International Publication No. WO91/18003 and European Patent Laid-Open No. 0545195A1).
A problem to be solved by the present invention is to find a method which, in the preparation of indolopyrrolocarbazole derivatives having excellent antitumor activity as disclosed in the previous patent applications, can introduce a glycosyl group into such indolopyrrolocarbazole-derived antitumor substances smoothly and efficiently.


DISCLOSURE OF THE INVENTION

The present inventors have made intensive investigations with a view to solving the above-described problem, and have succeeded in cloning glycosyltransferase-encoding DNA derived from a microorganism having glycosylation capacity and expressing this gene in host cells transformed with a vector having the gene integrated thereinto. Thus, the present invention has been completed.
Accordingly, the present invention relates to genes encoding glycosyltransferase activity (including glycosyltransferase activity-encoding genes substantially biologically equivalent thereto), glycosyltransferase, recombinant vectors having a gene encoding glycosyltransferase activity (or a glycosyltransferase activity-encoding gene substantially biologically equivalent thereto), host cells transformed with such a vector, a process for preparing glycosyltransferase by culturing such a transformed host cell, and a process for preparing a glycosylated indolopyrrolocarbazole derivative by culturing such a transformed host cell.
As used herein, the term "gene encoding glycosyltransferase activity" means glycosyltransferase activity-encoding genes derived from microorganisms belonging to the order Actinomycetales or glycosyltransferase activity-encoding genes substantially biologically equivalent thereto. Among them, the glycosyltransferase activity-encoding genes derived from microorganisms belonging to the genus Saccharothrix or mutants thereof are preferred. A more preferred example thereof is that derived from Saccharothrix aerocolonigenes or a mutant thereof.
The glycosyltransferase gene derived from Saccharothrix aerocolonigenes ATCC39243 strain is a glycosyltransferase gene having the base sequence represented by the following Seq. I.D. No. 2. ##STR1##
The amino acid sequence of the transferase encoded by the glycosyltransferase gene represented by Seq. I.D. No. 2 is an amino acid sequence represented by the following Seq. I.D. No. 1. ##STR2##
The term "glycosyltransferase activity" means the ability to glycosylate, for example, an indolopyrrolocarbazole derivative of the general formula ##STR3## wherein R, X.sup.1 and X.sup.2 represent hydrogen atoms or substituent groups, and thereby convert it to a compound of the general formula ##STR4## wherein R, X.sup.1 and X.sup.2 have the same meanings as described above.
Specific examples of the aforesaid indolopyrrolocarbazole derivative include indolopyrrolocarbazole derivative in which each of X.sup.1 and X.sup.2 is a hydrogen atom, a halogen atom, an amino group, a mono(lower alkyl)amino group, a di(lower alkyl)amino group, a hydroxyl group, a lower alkoxy group, an aralkoxy group, a carboxyl group, a lower alkoxycarbonyl group, a lower alkanoyloxy group or a lower alkyl group, and R is a hydrogen atom, an amino group, a formylamino group, a lower alkanoylamino group, a mono(lower alkyl)amino group, a di(lower alkyl)amino group, a hydroxyl group, a lower al

REFERENCES:
patent: 4524145 (1985-06-01), Matson
patent: 5589365 (1996-12-01), Kojiri et al.
patent: 5643760 (1997-07-01), Kojiri et al.
Fujiwara et al., Biochem. Biophys. Res. Comm. 187:1432-1438, 1992.
C. Herdandez, et al. "Characterization of a Streptomyces antibioticus genes cluster . . . ", Gene, vol. 134, No. 1, 1993, pp. 139-140.
M.J. Zmijewski, Jr., et al. "Biosynthesis of vanomycin: identification of TDP-glucose . . . ", FEMS Microbiology Letters, vol. 59, No. 1-2, 1989, pp. 129-134.
Katsuhisa Kojiri, et al., A New Antitumor Substance, BE-13793C, Produced By A Streptomycete, The Journal of Antibiotics, vol. 44, No. 7, Jul. 1991.

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