Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-07-29
2001-10-23
Achutamurthy, Ponnathapu (Department: 1852)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S069100, C435S183000, C435S252300, C435S320100, C536S023100
Reexamination Certificate
active
06306627
ABSTRACT:
The present invention relates to the isolation of genes which encode enzymes for the biosynthesis of &agr;-amylase inhibitors, so-called pseudo-oligosaccharides. The genes concerned are, in particular, genes from the Streptomycetes strain
Streptomyces glaucescens
GLA.O (DSM 40716). In addition, this present patent describes the use of these genes for producing acarbose and homologous substances with the aid of
Streptomyces glaucescens
GLA.O, the heterologous expression of these genes in other strains which produce pseudo-oligosaccharides (e.g. Actinoplanes sp SE50/100) for the purpose of increasing and stabilizing production, and also their heterologous expression in other microorganisms such as
E. coli, Bacillus subtilis
, Actinomycetales, such as Streptomyces, Actinoplanes, Ampullariella and Streptoporangium strains,
Streptomyces hygroscopicus var. limoneus
and
Streptomyces glaucescens
, and also biotechnologically relevant fungi (e.g.
Aspergillus niger
and
Penicillium chrysogenum
) and yeasts (e.g.
Saccharomyces cerevisiae
). The invention also relates to homologous genes in other microorganisms and to methods for isolating them.
Streptomyces glaucescens
GLA.O produces the two antibiotics hydroxystreptomycin (Hütter (1967) Systernatik der Streptomyceten (Taxonomy of the Streptomycetes). Basel, Karger Verlag) and tetracenomycin (Weber et al. (1979) Arch. Microbiol. 121: 111-116). It is known that streptomycetes are able to synthesize structurally varied natural products. However, the conditions under which these compounds are produced are frequently unknown, or else the substances are only produced in very small quantities and not detected.
The &agr;-amylase inhibitor acarbose has been isolated from a variety of Actinoplanes strains (SE50, SE82 and SE18) (Schmidt et al. (1977) Naturwissenschaften 64: 535-536). This active substance was discovered in association with screening for (&agr;-amylase inhibitors from organisms of the genera Actinoplanes, Ampullariella and Streptosporangium. Acarbose is pseudotetrasaccharide which is composed of an unusual unsaturated cyclitol unit to which an amino sugar, i.e. 4,6-dideoxy-4-amino-D-glucopyranose, is bonded. Additional (&agr;-1,4-glycosidically linked D-glucopyranose units can be bonded to the amino sugar. Thus, acarbose, for example, contains two further molecules of D-glucose. The producing strain synthesizes a mixture of pseudo-oligosaccharide products which possess sugar side chains of different lengths (Schmidt et al. (1977) Naturwissenschaften 64: 535-536). The acarbose cyclitol residue is identical to the compound valienamine, which is a component of the antibiotic validamycin A (Iwasa et al. (1979) J. Antibiot. 32: 595-602) from
Streptomyces hygroscopicus var. limoneus.
Acarbose can be produced by fermentation using an Actinoplanes strain and has achieved great economic importance as a therapeutic agent for diabetics. While Actinoplanes synthesizes a mixture of &agr;-amylase inhibitor products, it is only the compound having the relative molecular weight of 645.5 (acarviosin containing 2 glucose units (Truscheit (1984) Vlilth International Symposium on Medicinal Chemistry, Proc. Vol. 1. Swedish Academy of Pharmaceutical Sciences, Stockholm, Sweden), which is employed under the generic name of acarbose. The fermentation conditions are selected to ensure that acarbose is the main product of the fermentation. Alternatives are to use particular selectants and strains in which acarbose is formed as the main product or to employ purification processes for achieving selective isolation (Truscheit (1984) VlIlth International Symposium on Medicinal Chemistry, Proc. Vol. 1. Swedish Academy of Pharmaceutical Sciences, Stockholm, Sweden). It is also possible to transform the product mixture chemically in order, finally, to obtain the desired product acarbose.
In contrast to the genus Streptomyces, the genus Actinoplanes has not so far been investigated intensively from the genetic point of view. Methods which were established for the genus Streptomyces are not transferable, or are not always transferable, to the genus Actinoplanes. In order to use molecular biological methods to optimize acarbose production in a purposeful manner, the genes for acarbose biosynthesis have to be isolated and characterized. In this context, the possibility suggests itself of attempting to set up a host/vector system for Actinoplanes sp. However, this is very tedious and elaborate owing to the fact that studies on Actinoplanes have been relatively superficial.
The invention described in the present patent application achieves the object of cloning the biosynthesis genes for acarbose and homologous pseudo-oligosaccharides, with these genes being cloned from
Streptomyces glaucescens
GLA.O, which is a streptomycete which has been thoroughly investigated genetically (Crameri et al. (1983) J. Gen. Microbiol. 129: 519-527; Hintermann et al. (1984) Mol. Gen. Genet. 196: 513-520; Motamedi and Hutchinson (1987) PNAS USA 84: 4445-4449; Geistlich et al. (1989) Mol. Microbiol. 3: 1061-1069) and which, surprisingly, is an acarbose producer. In starch-containing medium,
Streptomyces glaucescens
GLA.O produces pseudo-oligosaccharides having the molecular weights 645, 807 and 970.
Part of the subject matter of the invention is, therefore, the isolation of the corresponding biosynthesis genes from
Streptomyces glaucescens
GLA.O and their use for isolating the adjoining DNA regions in order to complete the gene cluster of said biosynthesis genes.
The isolation of the genes for biosynthesizing pseudo-oligosaccharides, and the characterization of these genes, are of great importance for achieving a better understanding of the biosynthesis of the pseudo-oligosaccharides and its regulation. This knowledge can then be used to increase the productivity of the
Streptomyces glaucescens
GLA.O strain with regard to acarbose production by means of established classical and molecular biological methods. In addition to this, the entire gene cluster which encodes the synthesis of the pseudo-oligosaccharides, or individual genes from this gene cluster, can also be expressed in other biotechnologically relevant microorganisms in order to achieve a further increase in, or a simplification of, the preparation of pseudo-oligosaccharides such as acarbose. Specific modification of the biosynthesis genes can also be used to prepare a strain which exclusively produces acarbose having a molecular weight of 645. Since the genes for biosynthesizing antibiotics are always present in clusters and are often very strongly conserved (Stockmann and Piepersberg (1992) FEMS Microbiol. Letters 90: 185-190; Malpartida et al. (1987) Nature 314:642-644), the
Streptomyces glaucescens
GLA.O genes can also be used as a probe for isolating the acarbose-encoding genes from Actinoplanes sp., for example. The expression of regulatory genes, or of genes which encode limiting steps in the biosynthesis, can result in productivity in
Streptomyces glaucescens
GLA.O, Actinoplanes sp. or corresponding producer strains being increased. An increase in productivity can also be achieved by switching off (knocking out or mutagenizing) those acarbose biosynthesis genes which have an inhibitory effect in the biosynthesis.
One possible strategy for cloning antibiotic biosynthesis genes which have not previously been isolated is that of using gene-specific probes (Stockmann and Piepersberg (1992) FEMS Microbiol. Letters 90: 185-190; Malpartida et al. (1987) Nature 314:642-644). These probes can be DNA fragments which are P
32
-labeled or labeled in some other way; otherwise, the appropriate genes can be amplified directly from the strains to be investigated using degenerate PCR primers and isolated chromosomal DNA as the template.
The latter method has been employed in the present study. Pseudo-oligosaccharides such as acarbose contain a 4,6-deoxyglucose building block as a structural element. The enzyme dTDP-glucose 4,6-dehydratase is known to be involved in the biosynthesis of 4,6-deoxyglucose (Stockmann
Achutamurthy Ponnathapu
Aventis Pharma Deutschland GmbH
Heller Ehrman White & McAuliffe LLP
Rao Manjunath N.
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