Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
1998-03-20
2001-04-03
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S006120, C435S041000, C435S252300, C435S252350, C435S320100, C435S471000, C536S023200, C536S024320, C536S024330
Reexamination Certificate
active
06210935
ABSTRACT:
Staurosporin, an indole-carbazole alkaloid antibiotic, was first isolated from cultures of the microorganism
Streptomyces staurosporens
and described by Omura et al. (Omura et al., J. Antibiot. (1977), 30, 275-282). The biological properties of that secondary metabolite are of exceptional interest and include the following activities:
inhibitory activity against fungi and yeasts (Omura et al., J. Antibiot. (1977), 30, 275-282),
strong inhibition of Ca
2°
/phospholipid-dependent serine/threonine protein kinases (PKC) (Tamoki et al., Biochem. Biophys. Res. Comm. (1986), 135, 397-402),
antiproliferative activity (Tamoki et al, Biochem. Biophys. Res. Comm. (1986), 135, 397-402),
inhibition of platelet aggregation (Oka et al., Biol. Chem. (1986), 50, 2723-2727).
The isoenzyme family of the protein kinase Cs (PKC) plays an important part in signal transduction and cell regulation (Nishizuka, Nature (1988), 334, 661-665). The observation that phorbol esters, which have a tumour-stimulating property, stimulate PKC activity in cells (Nishizuka, Nature (1984), 308, 693-698) led to the conclusion that the inhibition of those enzymes by staurosporin and by similar staurosporin-like compounds could perhaps be used in the chemotherapy of tumours.
Later, staurosporins were isolated from other strains of Streptomyces, for example
Streptomyces longisporoflavus
(strain R-19, DSM 10189),
Streptomyces actuosus
(Morioka et al., Agric. Biol. Chem. (1985), 49, 1959-1963) and Streptomyces species, strain M-193 (Oka et al, Biol. Chem. (1986), 50, 2723-2727) and Streptomyces species, strain 383. Other alkaloids very similar to staurosporin, which contain the same chromophore as staurosporin and exhibit similar biological activity, have also been isolated. Examples are rebeccamycin (Nettleton et al., Tetrahedron Lett. (1985), 26, 4011-4014), UCN-01, UCN02 (Takahashi et al, J. Antibiot. (1987), 40, 1782-1783; Takahashi et al., J. Antibiot. (1989), 42, 571-576) and K-252 (Kase et al., J. Antibiot. (1986), 39,1059-1065), which have also been described as PKC inhibitors or anti-tumour compounds.
Staurosporin has the structure of formula (1)
and is an exceptionally strong inhibitor of protein kinase C, but the molecule lacks the selectivity required for pharmaceutical applications involving the very specific inhibition of individual protein kinases. For that reason, analogous compounds based on the fermentation product staurosporin have been prepared by chemical derivatisation at different centres (Ruegg & Burgess, Trends in Pharmacological Science (1989), 10, 218-220). An example thereof is the compound of formula (2) (Meyer et at, Int. J. Cancer (1989), 43, 851-856)
which has selectivity for protein kinase C inhibition and exhibits antiproliferative activity in vitro and anti-tumour properties in vivo.
Streptomyces are gram-positive filamentous bacteria that are found ubiquitously in soil. Streptomyces cultures grow in the form of branching mycelia which, when nutrients are limited, are capable of differentiating further to form aerial mycelia and, finally, to form spores. A special property of that group of microorganisms is their enormous potential for producing an extremely large variety of differently structured metabolites, known as secondary metabolites. Many of those compounds have antibacterial, antifungal, anti-tumour, immunomodulating or herbicidal properties and are therefore of great practical importance for pharmaceutical or agrochemical use.
Because of the practical importance of microbial secondary metabolites, there is a great deal of interest in understanding the genetic basis of their synthesis in order to create the means to influence them in a targeted manner. That is desirable especially because natural production strains, as in the case of the biosynthesis of staurosporin, generally yield only low concentrations of the secondary metabolites that are of interest. Those concentrations are not sufficient to satisfy the demand for the substance for wide-ranging activity tests and for preclinical and clinical trials, let alone for commercial production.
The genetic basis of secondary metabolite biosynthesis consists essentially in the genes that code for the individual biosynthesis enzymes and in the regulatory elements that control the expression of the biosynthesis genes. In all of the systems investigated hitherto, the secondary metabolite synthesis genes of Streptomyces have been found as clusters of adjacent genes. The size of such antibiotic gene clusters ranges from approximately 10 kilobases (kb) to approaching 100 kb. The clusters normally also contain specific regulator genes and genes for the resistance of the producing organism to its own antibiotic (Chater, Ciba Found. Symp. (1992), 171, 144-162).
In the invention described herein, success has now been achieved, by identifying and cloning genes of staurosporin biosynthesis, in providing the genetic basis for improving in a targeted manner the productivity of staurosporin-synthesising Streptomyces and, especially, of
S. longisporoflavus
or, using genetic methods, for synthesising staurosporin analogues, such as other indole-carbazole alkaloids. In a first step, a staurosporin biosynthesis gene of
S. longisporoflavus
was successfully identified by complementation of a mutant blocked in a biosynthesis step and cloned. Using DNA sequencing, the expected function of the protein derived from the cloned gene in the relevant biosynthesis step of staurosporin was confirmed. On the basis of the DNA sequence, there was found on a cloned 2.1 kb BgIII fragment a second gene that is involved in the synthesis of staurosporin and is likewise capable of complementing a mutant that is blocked in the synthesis of the sugar moiety of the staurosporin molecule. Finally, the cloned DNA fragment was used as a DNA probe for isolating the other staurosporin synthesis genes on large chromosomal DNA fragments of
S. longisporoflavus.
The gene cluster thus isolated and characterised forms the basis for the targeted optimisation of staurosporin production in
S. longisporoflavus
and other Streptomyces or Actinomyces. The following molecular genetic objectives and/or techniques are of primary importance therein:
overexpression of individual genes in production strains using plasmid vectors or by the incorporation of additional copies into the chromosome
study of the expression and transcriptional regulation of the gene cluster during fermentation in different production strains and optimisation thereof by means of physiological parameters and appropriate fermentation conditions
identification of regulator genes and of the DNA binding sites of the corresponding regulator proteins in the gene cluster. Characterisation of the effect of those regulatory elements on staurosporin production and influencing thereof by means of controlled mutations in those genes or in the DNA binding sites
duplication of the whole gene cluster or of parts thereof in production strains.
In addition to its use for improving fermentative staurosporin production in accordance with the above description, the gene cluster can likewise be used for the biosynthetic preparation of novel staurosporin analogues. The following possibilities may be mentioned:
inactivation of individual biosynthesis steps by means of gene disruption
use of genes of the cluster as DNA probe for isolating from nature Actinomyces or other microorganisms that produce metabolites similar to staurosporin
replacement of individual elements of the staurosporin gene cluster with those of other indole-carbazole alkaloid-producing Actinomyces, such as rebeccamycin, UCN-01, UCN-02 or K-252, and expression of novel, so-called hybrid metabolites.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an isolated DNA fragment comprising a DNA region that is involved directly or indirectly in the biosynthesis of indole-carbazole alkaloids, including the adjacent DNA regions to the right and left which, because of their function in connection with indole-carbazole alkaloid biosynthesis, qualify a
Bietenhader Jürg
Engel Nathalie
Pospiech Andreas
Schupp Thomas
Toupet Christiane
Achutamurthy Ponnathapu
Lee Michael U.
McCormack Myra H.
Moore William W.
Novartis AG
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