Gene encoding glycosyltransferase and its uses

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

1999-06-22

2001-06-05

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S075000, C435S119000, C435S127000, C435S252300, C435S252350, C435S320100, C435S325000, C536S023200

Reissue Patent

active

RE037206

ABSTRACT:

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 (12,13-dihydro-1,11-dihydroxy-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione) among microbial metabolites, and disclosed it in a previous patent application (Japanese Laid-Open Patent No. 20277/'91) [see The 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.
Seq ID. No. 2
ATGGGGGCAC GAGTGCTGGT GGCGACCACA
  30

CCGGGGGACG GGCACGTCAA CCCGATGGTG
  60

CCGGTCGCCC AGGAGATGGT CAGCCGTGGA
  90

CACGAGGTGC GGTGGTACAC GGGAAAGGCG
 120

TTCCGCTCCA CAGTGGAACG AACAGGAGCC
 150

CGCCACGAGC CGATGCGCGA CGCCCACGAC
 180

TTCGGCGGCA TGCCGAGGGA GGAGGCGTTT
 210

CCCCAGCACG CGGGGCTGAC CGGGATCACG
 240

GGGATGATCG CGGGGTTCAG GGACATCTTC
 270

ATCGAACCCG CGGCCGACCA GATGACAGAC
 300

CTGCTGGCAC TGCTGGAGGA CTTCCCGGCC
 330

GACGTGCTCG TGACCGACGA GACCTTCTTC
 360

GGCGCGGGTT TCGTCAGTGA GCGCACGGGA
 390

ATCCCCGTCG CCTGGATCGC CACGTCGATC
 420

TACGTCTTCA GCAGCCGCGA CACGGCACCG
 450

CTCGGGCTGG GCCTGCCGCC CAGCAGCTCC
 480

AGGCTGGGCA GGCTGCGCAA CACCGTGCTC
 510

AAACAGCTGA CAGACCGTGT CGTCATGCGA
 540

GATCTCCGGC GGCACGCGGA CGTGGTGCGC
 570

GACCGCGTCG GCCTGCCCCG CATCCGCAAG
 600

GGGGCGTTCG AGAACATCAT GCGCACGCCC
 630

GACCTCTACC TGCTGGGCAC CGTGCCGTCC
 660

TTCGAGTACC CGCGAGGCGA CATGCCACCC
 690

GAGGTGAGGT TCGTCGGGCC GTTCGTGAGC
 720

CCTGCTCCGC CGGACTTCAC CCCACCGGCG
 750

TGGTGGGGCG AGCTCGACTC CGGCCGGCCC
 780

GTCGTCCACG TCACCCAGGG CACCGTCGCC
 810

AACGACGCGG AACGCdTGCT GCTCCCCGCC
 840

ATCCGAGCGC TGGCAGCCGA AGACGTGCTC
 870

GTGGTCGCGA CCACCGGTGC CCCTCTGGAA
 900

CTGGAGCCGA TGCCGGCCAA CGTGCGGGTG
 930

GAACGGTTCA TCCCGCATCA CGCATTGCTT
 960

CCCCACGTGG ACGCCATGGT GACCAACGGG
 990

GGATACGGCG GCGTCAACAC GGCGCTCGCA
1020

CACGGCGTGC CGCTGGTCGT CGCGCGCACC
1050

GAGGAGAAGC AC9AGGTCGC GGCCAGAGTG
1080

AGCTGGTCAG GTGCGGGTGT TCACCTGAAG
1110

AAGCGCAGGC TGTCCGAACG GGACATCAGA
1140

CGGGCCGTGC GCGCCGTTCT CGACGAGCCG
1170

CGCTTCCGGG TCCACGCGGC GCGGCTGCGG
1200

GACGAGTACG CGGCGCGCGA CGCGGTCGTG
1230

GACGCGTCGA CCTGA
1245
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.
Seq. ID. No. 1
Met Gly Ala Arg Val Leu Val Ala Thr Thr Pro Gly
1               5                   10

Asp Gly His Val Asn Pro Met Val Pro Val A1a Gln
        15                  20

Glu Met Val Ser Arg Gly His Glu Val Arg Trp Tyr
25                  30                  35
50
Thr Gly Lys Ala Phe Arg Ser Thr Val Glu Arg Thr
            40                  45

Gly Ala Arg His Glu Pro Met Arg Asp Ala His Asp
    50                  55                  60

Phe Gly Gly Met Pro Arg Glu Glu Ala Phe Pro Gln
                65                  65

His Ala Gly Leu Thr Gly Ile Thr Gly Met Ile Ala
        75                  80

Gly Phe Arg Asp Ile Phe Ile Glu Pro Ala Ala Asp
85                  90                  95

Gln Met Thr Asp Leu Leu Ala Leu Leu Glu Asp Phe
            100             105

Pro Ala Asp Val Leu Val Thr Asp Glu Thr Phe Phe
    110                 1l5                 120

Gly Ala Gly Phe Val Ser Glu Arg Thr Gly Ile Pro
                125                 130

Val Ala Trp Ile Ala Thr Ser Ile Tyr Val Phe Ser
        135         &

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