Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-03-12
2002-12-17
Achutamurthy, Ponnathapu (Department: 1053)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S183000, C435S320100, C435S252300, C435S252350, C536S023100, C536S023200
Reexamination Certificate
active
06495348
ABSTRACT:
BACKGROUND OF THE INVENTION
Streptomyces are filamentous Gram-positive soil bacteria with a nucleotide base composition greater than 70 mole % G+C (Stackebrandt and Woese, 1981). They produce a wide array of biologically active compounds including over two thirds of the commercially important natural product metabolites (Alderson et al., 1993; Bevax, 1998). Genetic information accumulated over the past 15 years has demonstrated that genes encoding enzymes for natural product assembly are clustered on the Streptomyces genome (Martin, 1992). In addition, one or more pathway-specific transcriptional regulatory genes, and at least one resistance gene are typically found within the antibiotic biosynthetic gene cluster (Chater, 1992). Heterologous hybridization with gene probes based on highly conserved biosynthetic enzyme amino acid sequences has been useful to clone antibiotic biosynthetic genes (Hopwood, 1997; Seno and Baltz, 1989; Turgay and Marahiel, 1994).
The mitomycins are a group of natural products that contain a variety of functional groups, including aminobenzoquinone and aziridine ring systems. One representative of the family, mitomycin C (MC), was the first recognized bioreductive alkylating agent. In particular, since its discovery and demonstration of anticancer activity in the 1960s, many aspects of the chemistry and biology of MC have been investigated. This has provided detailed information on its unprecedented molecular mechanism, unique biological and pharmacological properties, drug resistance, and bioactive analogues (Hata et al., 1956; Verweij, 1997). MC is regarded as the prototype natural product alkylating agent whose activity is dependent on the reductive activation (either chemically, such as low pH, or enzymatically, such as DT-diaphorase, NADH cytochrome c reductase) (Boxer, 1997; Cummings et al., 1998). Activated MC crosslinks double-stranded DNA, which in turn induces diverse biological effects including selective inhibition of DNA synthesis, mutagenesis, induction of DNA repair (SOS response), sister chromatid exchange, signal transduction, and induction of apoptosis (Tomasz and Palem, 1997). Tumor hypoxia and the increased expression of bioreductive enzymes in malignant cells create a selective environment for drug activation and make MC an attractive agent for anti-tumor therapy (Spanswick et al., 1998). MC has become one of the most effective antitumor drugs against non-small cell lung carcinoma and other soft tumors, as well as a clinically important component of combination cancer chemotherapy and radiotherapy of solid tumors (Henderson, 1993).
In addition to its biological and pharmacological importance, MC is prominent because its molecular mechanism represents a model for structurally related antitumor antibiotics such as porfiromycin (Pan and Iracki, 1988), mitiromycin (Wakaki et al., 1958), FR66979 (Paz and Hopkins, 1997), FR900482 (Williams et al., 1997), FK973 (Hirai et al., 1994), and FK317 (Naoe et al., 1998), as well as structurally unrelated bioreductive agents such as EO9 (Smitskampwilms et al., 1996), and tirapazamine (Evans et al., 1998). Numerous MC derivatives have been synthesized and tested for enhanced activities, including the recently identified selective protein tyrosine kinase inhibitor, 1a-docosahexaenoyl MC (Kasai and Arai, 1995; Shikano et al., 1998).
Streptomyces lavendulae
produces MC. The molecule has an unusual structure comprised of aziridine, pyrrolizidine, pyrrolo-(1,2a)-indole, and amino-methylbenzoquinone rings to give the mitosane nucleus (Webb et al., 1962). The mitosane core of MC was shown to be derived from the junction of an amino-methylbenzoquinone (mC
7
N unit) and hexosamine (C
6
N unit) (Hornemann, 1981). The C
6
N unit consists of carbons 1, 2, 3, 9, 9a, 10, with the aziridine nitrogen derived intact from D-glucosamine (Hornemann et al., 1974).
The mC
7
N unit in MC and the ansamycins is derived from 3-amino-5-hydroxybenzoic acid (AHBA) (Becker et al., 1983; Kibby and Richards, 1981). AHBA was first shown to be incorporated into the ansamycin antibiotic actamycin (Kibby et al., 1980). Subsequently, it was confirmed as an efficient precursor for rifamycin (Becker et al., 1983; Kibby and Rickards, 1981; Ghilsalba and Neuesch, 1981), geldanamycin (Potgieter, 1983), ansamitocin (Hatano et al., 1982), ansatrienin (Wu et al., 1987), streptovaricin (Staley and Rinehart, 1991) and naphthomycin A (Lee et al., 1994). Anderson et al. (1980) demonstrated that [carboxy-
13
C] AHBA could be efficiently and specifically incorporated into the C-6 methyl group of porfiromycin, which contains the same mitosane core as MC. Incorporation experiments with radiolabeled precursors have demonstrated that the mitosane core of MC was derived from the junction of AHBA and D-glucosamine (Anderson et al., 1980; Hornemann, 1981).
Meanwhile the O— and N— (but not C—) methyl groups were shown to be derived from L-methionine, while the C-10 carbamoyl group came from L-arginine or L-citrulline (Bezanson and Vining, 1971; Hornemann and Eggert, 1975; Hornemann et al., 1974). [
14
C]-labeled precursor feeding studies with D-glucose, pyruvate and D-erythrose indicated that de novo biosynthesis of AHBA resulted directly from the shikimate pathway. However, no incorporation into the mC
7
N unit of either MC (Hornemann, 1981) or the ansamycin antibiotics (Chiao et al., 1998) was found from labeling studies with shikimic acid, the shikimate precursor 3-dehydroquinic acid, or the shikimate derived amino acids. These results led to the hypothesis of a modified shikimate pathway, in which a 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase-like enzyme catalyzes the conversion to 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid-7-phosphate (amino-DAHP), to give the ammoniated shikimate pathway (Kim et al., 1992). Floss (1997) provided strong support for this new variant of the shikimate pathway by showing that aminoDAHP, 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), and 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS) could be efficiently converted into AHBA by a cell-free extract of
Amycolatopsis mediterranei
(rifamycin producer), in contrast to the normal shikimate pathway intermediate DAHP which was not converted (Kim et al., 1992; Kim et al., 1996). Recently, the AHBA synthase (rifK) gene from
A. mediterranei
has been cloned, sequenced and functionally characterized (Kim et al., 1998).
Little is known regarding the details of the convergent assembly of MC from AHBA and D-glucosamine in
S. lavendulae
, i.e., whether its de novo biosynthesis is related to the primary metabolic shikimate pathway, an important route in microorganisms and plants for aromatic amino acid biosynthesis (Floss, 1997). In addition, it is unclear how
S. lavendulae
resists the activity of MC since the preferred MC alkylation sites in DNA are guanine and cytosine, and MC-induced cell death can result from a single crosslink per genome (Tomasz, 1995).
Thus, there is a continuing need for the identification and isolation of antibiotic biosynthetic genes, including genes which confer resistance to antibiotics or result in enhanced production of antibiotics.
SUMMARY OF THE INVENTION
The present invention provides an isolated and purified nucleic acid molecule, e.g., DNA, comprising a gene cluster for mitomycin, a variant or a fragment thereof (the mit/mmc gene cluster). As described hereinbelow, the
S. lavendulae
mitomycin gene cluster includes the mitomycin biosynthetic gene cluster comprising 47 mitomycin biosynthetic genes spanning 55 kb of contiguous DNA. The biosynthetic portion of the gene cluster includes genes that encode polypeptides involved in the generation of biosynthetic precursors, mitosane ring system assembly and functionalization (e.g., methylation, hydroxylation, aminotransfer, carbamoylation, and carbonyl reduction), a mitomycin resistance gene which is different than mrd and the unlinked mcr, as well as several regulatory genes. Gene disruption was employed to further characterize some of the genes. F
He Min
Mao Yingqing
Sheldon Paul
Sherman David H.
Varoglu Mustafa
Achutamurthy Ponnathapu
Kerr Kathleen
Regents of the University of Minnesota
Schwegman Lundberg Woessner & Kluth P.A.
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