Rebeccamycin (FIG. 1, A) is a natural product of Saccharothrix aerocolonigenes ATCC39243, a Gram-positive bacterium of the actinomycetes group (Bush et al. J. Antibiot. 40: 668-678, 1987). Actinomycetes are natural soil inhabitants with great industrial and biotechnological interest, especially the Streptomyces genus, because they are the source of many known bioactive compounds. Many of these compounds have pharmaceutical application due to their antitumor, antibacterial, antifungal, antiparasitic, or immunosupressor activity. Rebeccamycin shows antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus, Micrococcus luteus and Streptococcus faecalis (Bush et al. J. Antibiot. 40: 668-678, 1987). However, its major significance resides in its antitumor activity, demonstrated in vivo against several tumors implanted in mice, and in vitro against several tumor cell lines (Bush et al. J. Antibiot. 40: 668-678, 1987). There are currently two rebeccamycin derivatives in clinical trials for their future use as antineoplasic agents (NB-506, NSC655649).
Because of its chemical structure, rebeccamycin belongs to the indolocarbazole family of natural products. Since their discovery in 1977, more than 60 indolocarbazole natural products have been described, which can be classified in three groups containing either an indolo[2,3-a]pyrrolo[3,4-c]carbazole core (e.g. rebeccamycin), an indolo[2,3-a]carbazole core (e.g. tjipanazoles), or a bis-indolylmaleimide core (e.g. arcyriarubin). Due to their novel structures and the wide variety of activities displayed (antimicrobial, antifungal, immunosupressor, antitumor, etc.), this group of alkaloids has attracted great interest. In particular, indolopyrrolocarbazoles constitute a new class of antitumor agents, which can be further classified in two subgroups according to their mechanism of action. One subgroup consists of protein kinase inhibitors (especially protein kinase C inhibitors), and includes staurosporine (FIG. 1, B) and analogs. The second subgroup consists of DNA-damaging agents acting on topoisomerase I or II, but not on protein kinases, and includes rebeccamycin (FIG. 1, A) and analogs. Several indolocarbazoles have already entered clinical trials in the USA, Japan and Europe, including protein kinase inhibitors (UCN-01, CGP41251, CEP-751) and DNA-damaging agents (NB-506, NSC655649) (Akinaga et al. Anti-Cancer Drug Design 15: 43-52, 2000).
Nowadays there is a great need for new antitumor agents, with improved activity, lower undesirable secondary effects, and greater selectivity, as compared to drugs currently in use. Traditionally, pharmaceutical companies have developed new drugs by using two major approaches: (1) screening for new natural products, and (2) chemical synthesis and/or modification of specific compounds. These methods are still useful, but they usually need very important inputs of resources (time, money, energy), because analysis of thousands of products is generally required to find a new promising compound. Development of the genetic engineering of microorganisms has set the stage for generation of new bioactive compounds through manipulation of genes involved in biosynthesis of antitumor agents, mainly from actinomycetes. These techniques can also be used to improve present production levels of known natural drugs, as wild type strains usually yield low concentrations of the desired metabolite.
The chemical structures of most indolocarbazole natural products consist of two components: the indolocarbazole aglycon, and one or more sugar moieties. The indolocarbazole aglycon is biosynthesized from two tryptophan molecules, at least in the case of indolopyrrolocarbazoles. The sugar moiety present in rebeccamycin is a 4-O-methyl-β-D-glucose (FIG. 1, A). In the case of staurosporine, the sugar is an L-rhamnose derivative (FIG. 1, B). Recently, some genes involved in biosynthesis of the sugar moieties for the two mentioned indolocarbazoles have been reported:
(1) A chromosomal region of Streptomyces longisporoflavus DSM10189 involved in biosynthesis of the staurosporine sugar. This DNA region was able to complement a mutation impairing biosynthesis of the sugar moiety. There are not any reported evidences for the involvement of the mentioned DNA region in biosynthesis of the indolocarbazole aglycon (U.S. Pat. No. 6,210,935).
(2) A gene, called ngt, that codes for the rebeccamycin N-glucosyltransferase of Saccharothrix aerocolonigenes ATCC39243, responsible for sugar transfer to the indolocarbazole aglycon (Ohuchi et al. J. Antibiot. 53: 393-403, 2000). There are not any reported evidences for the involvement of the identified DNA region in the biosynthesis of the indolocarbazole aglycon. The DNA sequence of the ngt gene has previously been used for bioconversion of indolocarbazole aglycons to D-glucosilated derivatives. The procedure consisted of adding a particular indolocarbazole aglycon (either chemically synthesized or isolated from a producer strain) to the culture broth of a Streptomyces lividans strain harboring a plasmid containing the ngt gene, and isolating the glucosylated product from the culture (Ohuchi et al. J. Antibiot. 53: 393-403, 2000).
With the mentioned exception of the ngt gene (Ohuchi et al. J. Antibiot. 53: 393-403, 2000), there are not any previously reported descriptions of the nucleotide sequence which the present invention refers to. Moreover, there are not any previously reported descriptions of nucleotide sequences involved in biosynthesis of an indolocarbazole aglycon.
It was also known in the prior art (EP 0769555 A1) a gene encoding glycosyltransferase activity derived from Saccharothrix aerocolonigenes ATCC39243 strain, recombinant vectors having that gene, host cells transformed with such a vector, a process for preparing glycosyltransferase by culturing such a transformed host cell and a process for preparing glycosylated indolopyrrolocarbazole derivatives by culturing such a transformed host cell and using indolopyrrolocarbazole derivatives as starting compounds.