Isolation of biosynthesis genes for pseudo-oligosaccharides from Streptomyces glaucescens GLA.O, and their use

The present invention relates to the isolation of genes which encode enzymes for the biosynthesis of α-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) Systematik 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 α-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 α-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 α-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 α-amylase inhibitor products, it is only the compound having the relative molecular weight of 645.5 (acarviosin containing 2 glucose units (Truscheit (1984) VIIIth 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) VIIIth 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 and Piepersberg (1992) FEMS Microbiol, Letters 90: 185-190). Since deoxysugars are a frequent constituent of natural products and antibiotics, this enzyme may possibly be a means for isolating the corresponding antibiotic biosynthesis genes. Since these genes are always present as clusters, it is sufficient to initially isolate one gene; the isolation and characterization of the adjoining DNA regions can then be undertaken subsequently.

For example a dTDP-glucose 4,6-dehydratase catalyzes a step in the biosynthesis of hydroxystreptomycin in

Streptomyces glaucescens

GLA.O (Retzlaff et al. (1993) Industrial Microorganisms. Basic and applied molecular genetics ASM, Washington D.C., USA). Further dTDP-glucose 4,6-dehydratases have been isolated from other microorganisms, for example from

Streptomyces griseus

(Pissowotzki et al. (1991) Mol. Gen. Genet. 231: 113-123),

Streptomyces fradiae

(Merson-Davies and Cundcliffe (1994) Mol. Microbiol. 13: 349-355) and

Streptomyces violaceoruber

(Bechthold, et al. (1995) Mol. Gen. Genet. 248: 610-620).

It was consequently possible to deduce the sequences for the PCR primers for amplifying a dTDP-glucose 4,6-dehydratase from the amino acid sequences of already known biosynthesis genes. For this, conserved regions in the protein sequences of these enzymes were selected and the amino acid sequences were translated into a nucleic acid sequence in accordance with the genetic code. The protein sequences were taken from the EMBL and Genbank databases. The following sequences were used:

Streptomyces griseus

; accession number: X62567 gene: strE (dated Oct. 30, 1993);

Streptomyces violaceoruber

; accession number: L37334 gene: graE (dated Apr. 10, 1995);

Saccharopolyspora etythraea

; accession number: L37354 gene: gdh (dated Nov. 9, 1994). A large number of possible primer sequences are obtained as a result of the degeneracy of the genetic code. The fact that

streptomycetes

usually contain a G or C in the third position of a codon (Wright and Bibb (1992) gene 113: 55-65) reduces the number of primers to be synthesized. These primer mixtures can then be used to carry out a PCR amplification with the DNA from strains to be investigated, with the amplification ideally leading to an amplified DNA fragment. In the case of highly conserved proteins, this fragment is of a predictable length which ensues from the distance between the primers in the nucleic acid sequence of the corresponding gene. However, an experimental mixture of this nature does not inevitably have to result in an amplificate. The primers may be too unspecific and amplify a very large number of fragments; alternatively, no PCR product is obtained if there are no complementary binding sites in the chromosome for the PCR primers which have been prepared.

The investigation of the

streptomycete

strain

Streptomyces glaucescens

GLA.O resulted in an amplified DNA fragment (acbD ) which had the expected length of 550 bp. Further investigation showed that, besides containing a dTDP-glucose 4,6-dehydratase gene for biosynthesizing hydroxystreptomycin, this strain surprisingly contains a second dTDP-glucose 4,6-dehydratase gene for biosynthesizing pseudo-oligosaccharides such as acarbose. While the two genes exhibit a high degree of homology, they are only 65% identical at the amino acid level.

The acbD probe (see Example 2 and Table 2A) was used to isolate, from

Streptomyces glaucescens

GLA.O, a 6.8 kb Pstl DNA fragment which encodes a variety of genes (acbA, acbB, acdC, acbD, acbE and acbF) which are involved in the biosynthesis of the pseudo-oligosaccharides.

Deleting the acbBCD genes (aminotransferase, acbB, dTDP-glucose synthase, acbC, dTDP-glucose 4,6-dehydratase, acbD, see Example 6) resulted in the production of a mutant of

Streptomyces glaucescens

GLA.O which no longer produces any pseudo-oligosaccharides in the production medium. The involvement of the acbBCD genes in the synthesis of pseudo-oligosaccharides was therefore verified by deleting the corresponding loci.

The two genes, i.e. dTDP-glucose synthase and dTDP-glucose 4,6-dehydratase, ought to be involved in the biosynthesis of the deoxysugar of the pseudo-oligosaccharides, as can be concluded from the function of thoroughly investigated homologous enzymes (see above). The amino-transferase (encoded by the acbB gene) is probably responsible for transferring the amino group either to the sugar residue or to the cyclitol residue. By analyzing the protein sequence of acbB, an amino acid motif was found which is involved in binding pyridoxal phosphate. This motif is typical of class III aminotransferases (EC 2.6.1.11; EC 2.6.1.13; EC 2.6.1.18; EC 2.6.1.19; EC 2.6.1.62; EC 2.6.1.64; EC 5.4.3.8). The precise enzymic function of acbB can only be elucidated by further investigation of the biosynthesis of the pseudo-oligosaccharides. acbE encodes a transcription-regulating protein which exhibits a great deal of similarity to DNA-binding proteins which possess a helix-turn-helix motif (e.g.

Bacillus subtilis

DegA, P37947: Swiss-Prot database). Thus, the transcription activator CcpA from

Bacillus subtilis

inhibits the formation of α-amylase in the presence of glucose, for example (Henkin et al. (1991) Mol. Microbiol. 5: 575-584). Other representatives of this group are proteins which recognize particular sugar building blocks and are able to exhibit a positive or negative effect on the biosynthesis of metabolic pathways. The biosynthesis of the pseudo-oligosaccharides is also regulated in

Streptomyces glaucescens

GLA.O. It was only previously possible to demonstrate the synthesis of pseudo-oligosaccharides on starch-containing media. While this method indicated that AcbE might be responsible for regulating pseudo-oligosaccharide synthesis, the precise mechanism is still not known. However, molecular biological methods can now be used to modify the gene specifically in order to obtain an increased rate of pseudo-oligosaccharide biosynthesis. Furthermore, the DNA site at which acbE binds can be identified by means of so-called gel shift assays (Miwa et al. (1994) Microbiology 140: 2576-2575). An increase in the rate at which acarbose is biosynthesized can be achieved after identifying and then modifying the promoters and other regulatory DNA regions which are responsible for the transcription of the pseudo-oligosaccharide genes.

At present, the function of acbF is still not definitely known. The corresponding gene product exhibits homologies with sugar-binding proteins such as the sugar-binding protein from

Streptococcus mutans

(MsmE; Q00749: Swissprot database), making it probable that it is involved in the biosynthesis of the pseudo-oligosaccharides. The gene product of the acbA gene exhibits homologies with known bacterial ATP-binding proteins (e.g. from

Streptomyces peucitus

DrrA, P32010: SwissProt database). The AcbA protein possesses the typical ATP/GTP binding motif, i.e. the so-called P loop. These proteins constitute an important component of so-called ABC transporters, which are involved in the active transport of metabolites at biological membranes (Higgins (1995) Cell 82: 693-696). Accordingly, AcbA could be responsible for exporting pseudo-oligosaccharides out of the cell or be involved in importing sugar building blocks for biosynthesizing α-amylase inhibitors such as maltose.

All

streptomycete

genes for biosynthesizing secondary metabolites which have so far been analyzed are arranged in a cluster. For this reason, it is to be assumed that the acarbose biosynthesis genes according to the application are also arranged in such a gene cluster. The remaining genes which are relevant for pseudo-oligosaccharide biosynthesis can therefore also be isolated by isolating the DNA regions which adjoin the 6.8 kb Pstl DNA fragment according to the invention. As has also already been mentioned above, it is readily possible to isolate homologous gene clusters from microorganisms other than

Streptomyces glaucescens

GLA.O.

The invention therefore relates to a recombinant DNA molecule which comprises genes for biosynthesizing acarbose and homologous pseudo-oligosaccharides, in particular a recombinant DNA molecule in which individual genes are arranged, with respect to their direction of transcription and order, as depicted in FIG.

3

and/or which exhibits a restriction enzyme cleavage site pattern as depicted in

FIG. 3

, and, preferably, to a recombinant DNA molecule which

(a) comprises a DNA sequence according to Table 4, or parts thereof;

(b) comprises a DNA sequence which is able to hybridize, under stringent conditions, with the DNA molecule according to (a), or parts thereof; or

(c) comprises a DNA sequence which, because of the degeneracy of the genetic code, differs from the DNA molecules according to (a) and (b) but which permits the expression of the proteins which can be correspondingly expressed using the DNA molecule according to (a) and (b), or parts thereof.

The invention furthermore relates to a recombinant DNA molecule which comprises the acbA gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 1 to 720 according to Table 4, or parts thereof; to a recombinant DNA molecule which comprises the acbB gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 720 to 2006 according to Table 4, or parts thereof; to a recombinant DNA molecule which comprises the acbC gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 2268 to 3332 according to Table 4, or parts thereof; to a recombinant DNA molecule which comprises the acbD gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 3332 to 4306 according to Table 4, or parts thereof; to a recombinant DNA molecule which comprises the acbE gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 4380 to 5414 according to Table 4, or parts thereof; and to a recombinant DNA molecule which comprises the acbF gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 5676 to 6854 according to Table 4, or parts thereof.

The invention furthermore relates to oligonucleotide primers for the PCR amplification of a recombinant DNA molecule which is as described above and which comprises genes for biosynthesizing acarbose and homologous pseudo-oligosaccharides, with the primers having, in particular, the sequence according to Table 1.

The invention furthermore relates to a vector which comprises a recombinant DNA molecule which comprises a DNA molecule as described in the penultimate and prepenultimate paragraphs, in particular which is characterized in that the vector is an expression vector and said DNA molecule is linked operatively to a promoter sequence, with the vector preferably being being suitable for expression in host organisms which are selected from the group consisting of

E. coli, Bacillus subtilis, Actinomycetales

, such as

Streptomyces, Actinoplanes, Ampullariella

and

Streptosporangium

strains,

Streptomyces hygroscopicus

var.

limoneus, Streptomyces glaucescens

and also biotechnologically relevant fungi (e.g.

Aspergillus niger, Penicillium chrysogenum

) and yeasts (e.g.

Saccharomyces cerevisiae

), with

Streptomyces glaucescens

GLA.O or

Actinoplanes

sp. being very particularly preferred. Since the operative linkage of said DNA molecule to promoter sequences of the vector is only one preferably embodiment of the invention, it is also possible for expression to be achieved using promoter sequences which are endogenous in relation to the DNA molecule, e.g. the promoters which are in each case natural, or the natural promoters which have been mutated with regard to optimizing the acarbose yield. Such natural promoters are part of the DNA molecule according to the invention.

The invention furthermore relates to a vector which comprises a DNA molecule according to the invention for use in a process for eliminating or altering natural acarbose biosynthesis genes in an acarbose-producing microorganism. Such a vector is preferably selected from the group consisting of pGM160 and vectors as described in European Patents EP 0 334 282 and EP 0 158 872.

The invention furthermore relates to a host cell which is transformed with one of the above-described DNA molecules or vectors, in particular characterized in that said host cell is selected from the group consisting of

E. coli, Bacillus subtilis, Actinomycetales

, such as

Streptomyces, Actinoplanes, Ampullariella

or

Streptosporangium

strains,

Streptomyces hygroscopicus

var.

limoneus

or

Streptomyces glaucescens

, and also biotechnologically relevant fungi (e.g.

Aspergillus niger

and

Penicillium chrysogenum

) and yeasts (e.g.

Saccharomyces cerevisiae

); it is very particularly preferred for it to be selected from the group consisting of

Streptomyces glaucescens

GLA.O and

Actinoplanes

sp.

The invention furthermore relates to a protein mixture which can be obtained by expressing the genes of the recombinant DNA molecule according to the invention, comprising genes for biosynthesizing acarbose and homologous pseudo-oligosaccharides, in particular characterized in that the DNA molecule

(a) comprises a DNA sequence according to Table 4, or parts thereof;

(b) comprises a DNA sequence which is able to hybridize, under stringent conditions, with the DNA molecule according to (a) or parts thereof; or

(c) comprises a DNA sequence which, because of the degeneracy of the genetic code, differs from the DNA molecules according to (a) and (b) but which permits the expression of the proteins which can correspondingly be expressed using the DNA molecule according to (a) and (b), or parts thereof.

The invention furthermore relates to isolated proteins which can be obtained by expressing the genes which are encoded by the DNA molecule described in the previous paragraph.

The following statements apply to all the individual genes identified within the context of the present invention and have only been brought together for reasons of clarity: the invention furthermore relates to a protein which is encoded by a recombinant DNA molecule as described in the last paragraph but one, in particular characterized in that it comprises the DNA sequence of nucleotides 1 to 720 or 720 to 2006 or 2268 to 3332 or 3332 to 4306 or 4380 to 5414 or 5676 to 6854 according to Table 4 or parts thereof; a protein is very particularly preferred which is encoded by the acbA gene or the acbB gene or the acbC gene or the acbD gene or the acbE gene or the acbF gene, and which comprises the amino acid sequence according to Table 4 or parts thereof.

The invention furthermore relates to a process for obtaining the proteins which were described above as being part of the subject-matter of the invention, which process is characterized in that

(a) the proteins are expressed in a suitable host cell, in particular which is characterized in that said host cell is selected from the group consisting of

E. coli, Bacillus subtilis, Actinomycetales

, such as

Streptomyces, Actinoplanes, Ampullariella

or

Streptosporangium

strains,

Streptomyces, hygroscopicus

var.

limoneus

or

Streptomyces glaucescens

, and also biotechnologically relevant fungi (e.g.

Aspergillus niger

and

Penicillium chrysogenum

) and yeasts (e.g.

Saccharomyces cerevisiae

); with the host cell very particularly preferably being selected from the group consisting of

Streptomyces glaucescens

GLA.O and

Actinoplanes

sp., and

(b) are isolated.

The invention furthermore relates to a process for preparing acarbose, characterized in that

(a) one or more genes of the recombinant DNA molecule which comprises a DNA sequence according to Table 4 or parts thereof or which comprises a DNA sequence which is able to hybridize, under stringent conditions, with the DNA molecule according to Table 4, or parts thereof, or which comprises a DNA sequence which, because of the degeneracy of the genetic code, differs from the DNA molecules which have just been described but which permits the expression of the proteins which can be correspondingly expressed using these DNA molecules, or parts thereof, is/are used for expression in a suitable host cell which is selected, in particular, from the same group as in the last paragraph, and

(b) the acarbose is isolated from culture supernatants of said host cell.

The invention furthermore relates to a process for preparing acarbose, characterized in that

(a) one or more genes of the recombinant DNA molecule which comprises a DNA sequence according to Table 4 or parts thereof or which comprises a DNA sequence which is able to hybridize, under stringent conditions, with the DNA molecule according to Table 4, or parts thereof, or which comprises a DNA sequence which, because of the degeneracy of the genetic code, differs from the DNA molecules which have just been described but which permits expression of the proteins which can be correspondingly expressed using the DNA molecules, or parts thereof, are eliminated in an acarbose-producing host cell, in particular

Streptomyces glaucescens

GLA.O and

Actinoplanes

sp., and

(b) the acarbose is isolated from said host cell.

In this connection, the elimination of one or more genes can be effected by means of standard molecular biological methods, for example using the above-described vectors (pGM160 and others). A gene to be eliminated could, for example, be the acbE gene, which propably has a regulatory function. Genes could likewise be eliminated with the aim of obtaining pure acarbose as the only fermentation product and no longer obtaining a mixture of homologous pseudo-oligosaccharides (see above). The elimination of said genes is preferably achieved using the vectors which have been described above for this purpose.

The invention furthermore relates to a process for preparing acarbose, characterized in that the processes for preparing acarbose which have been described in the previous two paragraphs are combined with each other, such that, therefore, one or more of said genes is/are expressed artificially and one or more of said genes is/are eliminated.

The invention furthermore relates to a process for altering the gene expression of endogenous acarbose biosynthesis genes by mutating the respective gene promoter in order to obtain improved yields of acarbose. In this context, known methods of homologous recombination can be used to introduce the mutations into the production strain to be improved. These mutations can be transitions, deletions and/or additions. An “addition” can, for example, denote the addition of one single nucleotide or several nucleotides or of one or more DNA sequences which have a positive regulatory effect and which bring about an enhancement of the expression of an endogenous gene for biosynthesizing acarbose. The converse case, i.e. the addition of a DNA sequence which has a negative regulatory effect for repressing an endogenous acarbose biosynthesis gene is also a preferred form of an addition. “Transitions” may, for example, be nucleotide exchanges which reduce or amplify the effect of regulatory elements which act negatively or positively. “Deletions” can be used to remove regulatory elements which act negatively or positively. The endogenous genes of this process are preferably present in

Actinomycetales

, such as

Streptomyces, Actinoplanes, Ampullariella

or

Streptosporangium

strains,

Streptomyces hygroscopicus

var.

limoneus

or

Streptomyces glaucescens

; very particularly, they are present in

Streptomyces glaucescens

GLA.O and

Actinoplanes

sp.

The invention furthermore relates to the use of

Streptomyces

GLA.O for obtaining acarbose.

The invention furthermore relates to the use of

Streptomyces

GLA.O for preparing mutants of this strain by the “classical route”, which mutants make it possible to achieve a more abundant production of acarbose. The methods for preparing improved natural product producers of this nature have been known for a long time and frequently make use of classical steps of mutagenesis and selection.

The invention furthermore relates to a process for completing the gene cluster for biosynthesizing acarbose and homologous polysaccharides according to Table 4, characterized in that

a) hybridization probes which are derived from the DNA molecule according to Table 4 are prepared,

b) these hybridization probes are used for the genomic screening of DNA libraries obtained from

Streptomyces glaucescens

GLA.O, and

c) the clones which are found are isolated and characterized.

The invention furthermore relates to a process for completing the gene cluster for biosynthesizing acarbose and homologous pseudo-oligosaccharides according to Table 4, characterized in that, proceeding from the recombinant DNA molecule according to Table 4,

a) PCR primers are prepared,

b) these PCR primers are used to accumulate DNA fragments of genomic DNA from

Streptomyces glaucescens

GLA.O, with these primers being combined with those primers which hybridize from sequences of the vector system employed,

c) the accumulated fragments are isolated and characterized.

The invention furthermore relates to a process for isolating a gene cluster for biosynthesizing acarbose and homologous pseudo-oligosaccharides from acarbose-producing microorganisms other than

Streptomyces glaucescens

GLA.O, characterized in that, proceeding from the recombinant DNA molecule according to claim 4,

a) hybridization probes are prepared,

b) these hybridization probes are used for the genomic or cDNA screening of DNA libraries which have been obtained from the corresponding microorganism, and

c) the clones which are found are isolated and characterized.

The invention furthermore relates to a process for isolating a gene cluster for biosynthesizing acarbose and homologous pseudo-oligosaccharides from acarbose-producing microorganisms other than

Streptomyces glaucescens

GLA.O, characterized in that, proceeding from the recombinant DNA molecule according to claim 4,

a) PCR primers are prepared,

b) these PCR primers are used for accumulating DNA fragments of gemonic DNA or cDNA from a corresponding microorganism,

c) the accumulated fragments are isolated and characterized, and

d) where appropriate, employed in a process as described in the previous paragraph.

The described processes for isolating a gene cluster for the biosynthesis of acarbose and homologous pseudo-oligosaccharides from acarbose-producing microorganisms other than

Streptomyces glaucescens

GLA.O are characterized in that the microorganisms are selected from the group consisting of

Actinomycetales

, such as

Streptomyces, Actinoplanes, Ampullariella

and

Streptosporangium

strains,

Streptomyces hygroscopicus

var.

limoneus

and

Streptomyces glaucescens

, preferably from the group consisting of

Streptomyces glaucescens

GLA.O and

Actinoplanes

sp.

The invention furthermore relates to the use of

Streptomyces glaucescens

GLA.O for isolating acarbose.

The invention will now be explained in more detail with the aid of the examples, tables and figures, without being restricted thereto.

All the plasmid isolations were carried out using a Macherey and Nagel (Düren, Germany) isolation kit (Nucleobond®) in accordance with the manufacturer's instructions. Molecular biological procedures were carried out in accordance with standard protocols (Sambrock et al. (1989) Molecular cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, USA) or in accordance with the instructions of the respective manufacturer. DNA and protein sequences were examined using Genetics Computer Group Software, Version 8 (progams: FastA, TFastA, BlastX, Motifs, GAP and CODONPREFERENCE) and the SwissProt (release 32), EMBL (release 46) and Prosite (release 12.2) databases. The molecular biological manipulation of

Streptomyces glaucescens

and

Actinoplanes

(DNA isolation and DNA transformations) were carried out as described in Hopwood et al.: Genetic Manipulation of

Streptomyces

: A Laboratory Manual. The John Innes Foundation, Norwich, UK, 1985 and Motamedi and Hutchinson: Cloning and heterologous expression of a gene cluster for the biosynthesis of tetracenomycin C, the anthracycline antitumor antibiotic of

Streptomyces glaucescens

. Proc. Natl. Acad. Sci. USA 84:4445-4449 (1987).

In general, hybridizations were performed using the “Non-radioactive DNA labeling kit” from Boehringer Mannheim (Cat. No. 1175033). The DNA was visualized using the “Luminescent Detection Kit” from Boehringer Mannheim (Cat. No. 1363514). In all the examples given in this patent application, hybridization was carried out under stringent conditions: 68° C., 16 h. 5×SSC, 0.1% N-laurylsarcosine, 0.02% SDS, 1% Blocking Reagent (Boehringer Mannheim). SSC denotes 0.15M NaCl/0.015M sodium citrate. The definition of “stringent conditions” which is given here applies to all aspects of the present invention which refer to “stringent conditions”. In this connection, the manner of achieving this stringency, i.e. the cited hybridization conditions, is not intended to have a limiting effect since the skilled person can select other conditions as well in order to achieve the same stringent conditions, e.g. by means of using other hybridization solutions in combination with other temperatures.

EXAMPLE 1

Synthesis and Sequences of the PCR Primers and Amplification of the Fragments from

S. glaucescens

GLA.O

The PCR was carried out under standard conditions using in each case 100 pmol of primer 1 and of primer 2 in 100 μl of reaction mixture

PCR buffer

1

10 μl

PCR primers

in each case 2.5 μl

dNTPs

in each case 0.2 mM

BSA (10 mg/ml)

1 μl

Template DNA

1 μg (1 μl)

Taq polymerase

2

(5 units/ml)

1.5 μl

H

2

O

to make up to 100 μl

1

Promega

2

Boehringer Mannheim

The samples are overlaid with 75 μl of mineral oil and the amplification is carried out using a Perkin Elmer TC1 DNA thermal cyler.

Parameters:

Cycles

Temperature

Duration

1

96° C.

5 min

74° C.

5 min

30

95° C.

1.5 min

74° C.

1.5 min

1

74° C.

5 min

Table 1 lists the sequences of the degenerate primers which should be used for amplifying dTDP-glucose dehydratases from different

streptomycetes

.

TABLE 1

Primer sequences for amplifying dTDP-glucose

4,6-dehydratases

Primer 1:

CSGGSGSSGCSGGSTTCATSGG

(SEQ ID NO.:1)

Primer 2:

GGGWVCTGGYVSGGSCCGTAGTTG

(SEQ ID NO.:2)

In this table, S = G or C, W = A or T, V = A or G, and Y = T or C.

EXAMPLE 2

DNA Sequences of the PCR Fragments Isolated From

Streptomyces glaucescens

GLA.O

The sequencing was performed by the dideoxy chain termination method of Sanger et al. (PNAS USA, 74: 5463-5467 (1977)). The reactions were carried out using the Auto Read Sequenzing Kit® from Pharmacia Biotech (Freiburg, Germany) in accordance with the manufacturer's instructions. An ALF DNA Sequencer® from Pharmacia Biotech (Freiburg, Germany) was used for separation and detection.

The subsequent cloning of the PCR fragments (Sure Clone Kit®, Pharmacia Biotech, Frieburg) into the

E. coli

vector pUC 18, and the sequencing of the fragment, provided support for the supposition that the fragment encoded a dTDP-glucose 4,6-dehydratase. However, 2 different genes were isolated which both exhibit high degrees of homology with dTDP-glucose 4,6-dehydratase but are not identical. In that which follows, the PCR fragments are designated acbD and HstrE.

The sequences of the isolated PCR fragments are shown in Table 2A and 2B and the homology comparison of the deduced amino acid sequences of HstrE and acbD is shown in Table 2C. The two proteins exhibit an identity of only 65%.

TABLE 2A

DNA sequence of acbD* (primer-binding

sites are underlined, (SEQ ID NO.: 3)

Primer 1

1

CC

CGGGCGGG GCGGGGTTCA TCGG

CTCCGC CTACGTCCGC CGGCTCCTGT

51

CGCCCGGGGC CCCCGGCGGC GTCGCGGTGA CCGTCCTCGA CAAACTCACC

101

TACGCCGGCA GCCTCGCCCG CCTGCACGCG GTGCGTGACC ATCCCGGCCT

151

CACCTTCGTC CAGGGCGACG TGTGCGACAC CGCGCTCGTC GACACGCTGG

201

CCGCGCGGCA CGACGACATC GTGCACTTCG CGGCCGAGTC GCACGTCGAC

251

CGCTCCATCA CCGACAGCGG TGCCTTCACC CGCACCAACG TGCTGGGCAC

301

CCAGGTCCTG CTCGACGCCG CGCTCCGCCA CGGTGTGCGC ACCCTCGTGC

351

ACGTCTCCAC CGACGAGGTG TACGGCTCCC TCCCGCACGG GGCCGCCGCG

401

GAGAGCGACC CCCTGCTCCC GACCTCGCCG TACGCGGCGT CGAAGGCGGC

451

CTCGGACCTC ATGGCGCTCG CCCACCACCG CACCCACGGC CTGGACGTCC

501

GGGTGACCCG CTGTTCGAA

C AACTACGGCC CGCACCAGTT CCC

GGG

Primer 2

TABLE 2B

DNA sequence of HstrE* (primer-binding sites are

underlined, (SEQ ID NO.: 4)

Primer 2

1

CCCC

GGGTGC TGGTAGGGGC CGTAGTTG

TT GGAGCAGCGG GTGATGCGCA

51

CGTCCAGGCC GTGGCTGACG TGCATGGCCA GCGCGAGCAG GTCGCCCGAC

101

GCCTTGGAGG TGGCATAGGG GCTGTTGGGG CGCAGCGGCT CGTCCTCCGT

151

CCACGACCCC GTCTCCAGCG AGCCGTAGAC CTCGTCGGTG GACACCTGCA

201

CGAAGGGGGC CACGCCGTGC CGCAGGGCCG CGTCGAGGAG TGTCTGCGTG

251

CCGCCGGCGT TGGTCCGCAC GAACGCGGCG GCATCGAGCA GCGAGCGGTC

301

CACGTGCGAC TCGGCGGCGA GGTGCACGAC CTGGTCCTGG CCGGCCATGA

351

CCCGGTCGAC CAGGTCCGCG TCGCAGATGT CGCCGTGGAC GAAGCGCAGC

401

CGGGGGTGGT CGCGGACCGG GTCGAGGTTG GCGAGGTTGC CGGCGTAGCT

451

CAGGGCGTCG AGCACGGTGA CGACGGCGTC GGGCGGCCCG TCCGGACCGA

501

GGAGGGTGCG GACGTAGTGC

GAGCCCATGA ACCCCGCCGC C

Primer 1

TABLE 2C

Homology comparison of the deduced amino acid sequences

of the PCR products HstrE* and acbD* (program: GAP)

Quality:

196.3

Length:

182

Ratio:

1.091

Gaps:

0

Percent similarity:

77.654

Percent identity:

65.363

PCRstrE.Pep × PCRabcD.Pep

. . . . .

1 ..AAGFMGSHYVRTLLGPDGPPDAVVTVLDALSYAGNLANLDPVRDHPRL

48

:|||:|| ||| ||:|::|.:..||||| |.|||.||.|.:||||| |

1 PGGAGFIGSAYVRRLLSPGAPGGVAVTVLDKLTYAGSLARLHAVRDHPGL

50

. . . . .

49 RFVHGDICDADLVDRVMAGQDQVVHLAAESHVDRSLLDAAAFVRTNAGGT

98

||:||:||..||| : | :|::||:|||||||||: |.:||.|||. ||

51 TFVQGDVCDTALVDTLAARHDDIVHFAAESHVDRSITDSGAFTRTNVLGT

100

. . . . .

99 QTLLDAALRHGVAPFVQVSTDEVYGSLETGSWTEDEPLRPNSPYATSKAS

148

|.|||||||||| .:|:||||||||||. |. .|.:|| |.||||.|||.

101 QVLLDAALRHGVRTLVHVSTDEVYGSLPHGAAAESDPLLPTSPYAASKAA

150

. . .

149 GDLLALAMHVSHGLDVRITRCSNNYGPYQHPG

180

:||:||| | .||||||:|||||||||.| |

151 SDLMALAHHRTHGLDVRVTRCSNNYGPHQFP.

181

in each case, upper row: SEQ ID NO.:5

in each case, lower row: SEQ ID NO.:6

EXAMPLE 3

Southern Analysis Using Chromosomal DNA From

Streptomyces glaucescens

GLA.O and the Isolated and Labeled PCR Fragments

The cells were grown in R2YENG medium and harvested for the DNA isolation after 30 h. The chromosomal DNA was isolated from

S. glaucescens

GLA.O as described in Hopwood et al. (1985) Genetic manipulations of

Streptomyces

: a laboratory manual. The John Innes Foundation, Norwich UK).

A Southern blot analysis was carried out using the

S. glaucescens

GLA.O producer strain chromosomal DNA, which was digested with Pstl, BgIII and BamHI, using the labeled probes consisting of the acbD and HstrE PCR fragments. The two PCR fragments were labeled with digoxygenin in accordance with the manufacturer's (Boehringer Mannheim; Mannheim) instructions, and a digest of the

Streptomyces glaucescens

GLA.O producer strain chromosomal DNA was separated on an agarose gel. The DNA was transferred by capillary transfer to nylon membranes and DNA regions which were homologous with the labeled probes were subsequently visualized following hybridization.

The two genes label different DNA regions (FIG.

1

and FIG.

2

), with the fragments which were labeled by HstrE having to be gene fragments from

Streptomyces glaucescens

GLA.O hydroxystreptomycin biosynthesis. While the DNA sequence is not published, the high degree of homology of the protein sequence deduced from HstrE with StrE (Pissowotzki et al. (1991) Mol. Gen. Genet. 231: 113-123) from

Streptomyces griseus

N2-3-11

streptomycin

biosynthesis (82% identity) and the concordance of the HstrE-labeled DNA fragments (

FIG. 1

) with the published restriction map of the

Streptomyces glaucescens

GLA.O hydroxystreptomycin gene cluster (Retzlaff et al. (1993) Industrial Microorganisms. Basic and applied molecular genetics ASM, Washington D.C., USA) permits this conclusion. The fragments which were labeled by the acbD probe (

FIG. 2

) belong to a DNA region which has not previously been investigated. This region encodes the enzymes for biosynthesizing the

Streptomyces glaucescens

GLA.O pseudo-oligosaccharides.

EXAMPLE 4

Cloning the 6.8 kb Pstl Fragment

Inter alia, the acbD PCR fragment labels a 6.8 kB Pstl DNA fragment (FIG.

2

). This DNA fragment was isolated as follows. The region of the gel was excised with a razor blade and the DNA was isolated from the gel using an isolation kit from Pharmacia Biotech and cloned into plasmid pUC19 which had been cut with the restriction enzyme Pstl (plasmid pacb1); this latter plasmid was then transformed into the

E. coli

strain DH5α. The individual clones were subcultured from these plates and a plasmid DNA isolation was carried out using these clones. A PCR amplification using the above-described primers 1 and 2 (Tab. 1) was carried out using the DNA from these clones (250). In this manner, the appropriate

E. coli

clone containing the 6.8 kb Pstl fragment was isolated.

EXAMPLE 5

Sequencing the Isolated 6.8 kb Pstl DNA Fragment

The DNA was digested with various restriction enzymes and individual DNA fragments were cloned into pUC19. The DNA sequence of the entire fragment, which is shown in Tab. 4 (SEQ ID NO.: 7), was then determined. The DNA sequence of the 6.8 kb Pstl fragment was only partially confirmed by supplementary sequencing of the opposing strand. Several open reading frames, encoding various proteins, were found (programs: CODONPREFERENCE and BlastX). A total of 6 coding regions was found, i.e. a gene having a high degree of homology with ATP-binding protein, acbA, an aminotransferase acbB, a dTDP-glucose synthase acbC, a dTDP-glucose dehydratase acbD, a regulatory gene having homologies with the Lacl protein family acbE, and a protein having similarities to sugar-binding proteins acbF. The sequences of the acbA and acbF genes were only determined in part. The homologies with other proteins from the databases, and the properties of the putative proteins, are summarized in Tab. 3.

FIG. 3

shows, in summary form, a restriction map of the fragment, containing the most important restriction cleavage sites mentioned in the text, and the arrangement of the identified open reading frames.

TABLE 3

Analysis of the identified open reading frames on the 6.8 kb

Pstl fragment from

Streptomyces glaucescens

GLA.O

Amino

Accession

ORF

acid

MW

FastA

§

% Identity

number

§

acbA

239

*

MalK,

E. coli

29%

P02914

acbB

429

45618

DgdA,

Burkholderia

32%

P16932

cepacia

acbC

355

37552

StrD,

Streptomyces

60%

P08075

griseus

acbD

325

35341

StrE,

Streptomyces

62%

P29782

griseus

acbE

345

36549

DegA,

Bacillus

31%

P37947

subtilis

acbF

396

*

MalE,

E. coli

22%

P02928

*incomplete open reading frame;

§

Swiss-Prot database (release 32)

EXAMPLE 6

Deletion of Genes acbBCD for Pseudo-Oligosaccharide Biosynthesis from the

Streptomyces glaucescens

GLA.O Chromosome

Evidence that the identified DNA fragment encoded pseudo-oligosaccharide biosynthesis genes was produced as follows. A 3.4 kb gene region (EcoR1/Sstl fragment b,

FIG. 3

) was replaced with the erythromycin resistance gene (1.6 kb) and cloned, together with flanking DNA regions from the 6.8 kb Pstl fragment (pacb1) into the temperature-sensitive plasmid pGM160. The plasmid was constructed as described in the following: the 2.2 kb EcoR1/HindIII fragment (c,

FIG. 3

) from plasmid pacb1 was cloned into pGEM7zf (Promega, Madison, Wis., USA; plasmid pacb2), and the 1 kb Sstl fragment from pacb1 (a,

FIG. 3

) was cloned into pUC19 (plasmid pacb3). A ligation was then carried out using the following fragments. The plasmid pGM160 (Muth et al. (1989) Mol. Gen Genet. 219:341-348) was cut with BamH/HindIII, the plasmid pacb2 was cut with Xbal/BamHI (c, FIG.

3

), the plasmid pacb3 was cut with EcoRI/HindIII (a, FIG.

3

), and the plasmid plJ4026 (Bibb et al. (1985) Gene 38:215-226) was cut with EcoRI/Xbal in order to isolate the 1.6 kb ermE resistance gene.

The fragments were ligated in a mixture and transformed into

E. coli

DH5α and selected on ampicillin. The resulting plasmid, i.e. pacb4, was isolated from

E. coli

DH5α, tested for its correctness by means of restriction digestion and then transferred by protoplast transformation into

S. glaucescens

GLA.O. The transformants were selected with thiostrepton at 27° C. in R2YENG agar. The transformants were subsequently incubated at the non-permissive temperature of 39° C. and integration of the plasmid into the genome by way of homologous recombination thereby instituted (selection with thiostrepton (25 μg/ml) and erythromycin (50 μg/ml)). Under these conditions, the only clones which can grow are those in which the plasmid has become integrated into the genome. The corresponding clones were isolated, caused to sporulate (medium 1, see below) and plated out on erythromycin-containing agar (medium 1). Individual clones were isolated once again from this plate and streaked out on both thiostrepton-containing medium and erythromycin-containing medium. The clones which were erythromycin-resistant but no longer thiostrepton-resistant were analyzed. In these clones, the acbBCD genes had been replaced with ermE. Several clones were examined and the strain

S. glaucescens

GLA.O Δacb was finally selected as the reference strain (erythromycin-resistant, thiostrepton-sensitive) for further investigation.

Medium 1

Yeast extract

4 g/L

Malt extract

10 g/L

Glucose

4 g/L

Agar

15 g/L

pH

7.2

A further experiment examined whether the corresponding strain still produced acarbose. Some clones were grown and investigated for formation of the α-amylase inhibitor in a bioassay; however, no activity was found. The mutants were subsequently further characterized by means of Southern hybridization. Integration of the ermE gene had taken place at the predicted site.

FIG. 4

shows a Southern hybridization which was carried out with the wild type and with the

Streptomyces glaucescens

GLA.O Δacb deletion mutant. The Sstl fragment from pacb3 was used as the probe. The chromosomal DNA was isolated from the wild type and mutant and digested with the enzymes Pstl and Pstl/HindIII. The fragment pattern obtained for the deletion mutant corresponds to the predicted recombination event. The wild type exhibits the unchanged 6.8 kb Pstl fragment, whereas the mutant exhibits a fragment which has been truncated by 1.8 kb (compare lanes

1

and

3

, FIG.

4

). Integration of the ermE resistance gene additionally introduced an internal HindIII cleavage site into the Pstl fragment (compare lanes

2

and

4

, FIG.

4

).

EXAMPLE 7

Inhibition of α-amylase by Acarbose

Using an enzymic test for detecting starch (TC-Starch, Boehringer-Mannheim, Cat. No. 297748), it was possible to demonstrate that the compound isolated from

Streptomyces glaucescens

GLA.O inhibits α-amylase. Test principle: starch is cleaved into D-glucose by amyloglucosidase. The glucose is then converted with hexokinase into glucose-6-phosphate and the latter is converted with glucose-6-phosphate dehydrogenase into D-gluconate-6-phosphate. This reaction produces NADPH, whose formation can be determined photometrically. Acarbose inhibits the α-amylase and thereby prevents the formation of D-glucose and ultimately the formation of NADPH as well.

EXAMPLE 8

Medium for Growing

S. glaucescens

GLA.O and Producing Acarbose

The fermentation was carried out, at 27° C. on an orbital shaker at 120 rpm, in 500 ml Erlenmeyer flasks which were fitted with side baffles and which contained 100 ml of medium 2. The fermentation was terminated after 2 or 3 days. The pseudo-oligosaccharides were detected in a plate diffusion test as described in Example 9. No α-amylase inhibitors were produced when medium 3 was used. This means that the production of the pseudo-oligosaccharides is inhibited by glucose. Other sugars, such as maltose and sucrose, or complex sugar sources (malt extract) can also come into consideration for producing pseudo-oligosaccharides using

S. glaucescens

GLA.O.

Medium 2:

Soybean flour

20 g/L

Starch

20 g/L

pH

7.2

Medium 3:

Soybean flour

20 g/L

Glucose

20 g/L

pH

7.2

EXAMPLE 9

Biotest Using

Mucor miehei

A suspension of spores of the strain

Mucor miehei

was poured into agar (medium 5) (10

5

spores/ml), and 10 ml of this mixture were in each case poured into Petri dishes. Paper test disks (6 mm diameter) were loaded with 10 μl of acarbose [lacuna] (1 mg/ml) or with a sample from an

S. glaucescens

culture and laid on the test plates. The plates were then incubated at 37° C. Inhibition halos appeared on the starch-containing medium 5. A plate which was prepared with glucose (medium 4) instead of starch was used as a control. On this medium, no inhibition halo formed around the filter disks loaded with active compound.

Media 4 and 5:

KH

2

PO

4

x 3 H

2

O

0.5 g

MgSO

4

x 7 H

2

O

0.2 g

NaCl

0.1 g

Ammonium sulfate

5 g

Yeast nitrogen base

1.7 g

Glucose (4) or starch (5)

5 g

Agar

15 g

EXAMPLE 10

Transformation of

S. glaucescens

GLA.O

Protoplasts of the

Streptomyces glaucescens

strain were isolated as described in Motamedi and Hutchinson ((1987) PNAS USA 84: 4445-4449), and the isolated plasmid DNA was transferred into the cells by means of PEG transformation as explained in Hopwood et al. ((1985) Genetic manipulations of Streptomyces: a laboratory manual. The John Innes Foundation, Norwich UK). The protoplasts were regenerated on R2YENG medium at 30° C. (Motamedi and Hutchinson (1987) PNAS USA 84: 4445-4449). After 18 h, the agar plates were overlaid with a thiostrepton-containing solution and incubated at 30° C. (final concentration of thiostrepton: 20 μg/ml).

EXAMPLE 11

Isolation of the Pseudo-Oligosaccharides from

Streptomyces glaucescens

GLA.O, HPLC Analysis and Mass Spectroscopy

Isolation

The culture broth was separated from the mycelium by filtration. The culture filtrate which has been obtained in this way is then loaded onto an XAD16 column, after which the column is washed with water and the active components are eluted with 30% methanol. The eluate was concentrated down to the aqueous phase and the latter was extracted with ethyl acetate in order to remove lipophilic impurities. The aqueous phase was then concentrated and the active components were further purified in 5% methanol using a biogel P2 column. The individual fractions are collected in a fraction collector. The individual fractions were analyzed by means of the

Mucor miehei

biotest. Active eluates were rechromatographed, for further purification, in 5% methanol on biogel P2. The material which was isolated in this way was investigated by HPLC and MS.

HPLC

Column: Nucleosil® 100 C-18

Eluent 0.1% phosphoric acid=A/acetonitrile=B

Gradient: from 0 to 100% B in 15 min

Detection: 215 nm

Flow 2 ml/min

Injection volume: 10-20 μl

Using HPLC, it was not possible to distinguish the pseudo-oligosaccharide preparation from

S. glaucescens

GLA.O from authentic acarbose. Both the retention time and the UV absorption spectrum of the two components were identical in this eluent system. The pseudo-oligosaccharide mixture was not fractionated under these conditions.

Mass spectroscopic analysis (MS)

The molecular weights and the fragmentation pattern of authentic acarbose and the pseudo-oligosaccharides isolated from

Streptomyces glaucescens

GLA.O were determined by means of electrospray MS. Analysis of the acarbose which is commercially obtainable from Bayer (Glucobay) gave a mass peak at 645.5 (acarbose). The purified samples from

S. glaucescens

GLA.O contain a mixture of different pseudo-oligosaccharides whose sugar side chains are of different lengths: 969 (acarbose+2 glucose units), 807 (acarbose+1 glucose unit), 645 (corresponds to authentic acarbose). When acarbose and the compound which is isolated from

S. glaucescens

GLA.O and which has a molecular weight of 645 are fragmented, the same molecular fragments are formed, i.e.: 145 (4-amino-4,6-deoxyglucose), 303 (Acarviosin) and 465 (303 together with one glucose unit).

Actinoplanes

sp. SE50 also produces a mixture of acarbose molecules having sugar side chains of different length (Truscheit (1984) VIIIth International Symposium on Medicinal Chemistry, Proc. Vol 1. Swedish Academy of Pharmaceutical Sciences, Stockholm, Sweden). The length of the sugar side chains can be influenced by the choice of the fermentation parameters and of the substrate in the nutrient solution.

EXAMPLE 12

Southern Hybridization Using

Actinoplanes

sp. SE50/110 (ATCC31044)

The chromosomal DNA was isolated from the strain

Actinoplanes

sp. SE50/100 and digested with restriction enzymes (Pstl and BamHI). A Southern hybridization was then carried out using a probe which encompasses the coding region of the dTDP-glucose 4,6-dehydratase acbD from

Streptomyces glaucescens

GLA.O (fragment d, FIG.

3

). The probe hybridizes with distinct bands from

Actinoplanes

sp. SE50/110 (

FIG. 5

, lanes

1

and

2

). This provides the possibility of isolating the corresponding fragments from

Actinoplanes

sp. SE50/100 and other strain lines. Whether these DNA regions are in fact involved in the biosynthesis of acarbose remains to be demonstrated in subsequent investigations. Alternatively, the PCR primers 1 and 2 (Tab. 1) could also be used for amplifying the dTDP-glucose 4,6-dehydratase from

Actinoplanes

sp.

Legends:

FIG.

1

: Southern hybridization using

S. glaucescens

GLA.O. Lane

1

: Pstl, lane

2

: BamHI, lane

3

: BgIII. The labeled PCR fragment HstrE was used as the probe. Labeling of DNA fragments which are involved in the biosynthesis of hydroxy-streptomycin.

FIG.

2

: Southern hybridization using

S. glaucescens

GLA.O. Lane

1

: Pstl, lane

2

: BamHI, lane

3

: BgIII. The labeled PCR fragment acbD was used as the probe. Labeling of DNA fragments which are involved in the biosynthesis of the pseudo-oligosaccharides.

FIG.

3

: Restriction map of the 6.8 kb Pstl fragment from

Streptomyces glaucescens

GLA.O. Open reading frames and the direction in which each is transcribed are indicated by arrows. The fragments a, b, c and d identify DNA regions which are explained in more detail in the text.

FIG.

4

: Southern hybridization using

Streptomyces glaucescens

Δacb: lane

1

: Pstl, lane

2

: Pstl/HindIII, and

Streptomyces glaucescens

GLA.O lane

3

: Pstl, lane

4

: Pstl/HindIII. The labeled 1.0 kb Sstl fragment a (

FIG. 3

) was used as the probe.

FIG.

5

: Southern hybridization using

Actinoplanes

sp. SE50/100: lane

1

: Pstl, lane

2

: BamHI and

Streptomyces glaucescens

GLA.O lane

3

: Pstl. The labeled 1.0 kb SmaI/EcoRI fragment d (dTDP-glucose 4,6-hydratase,

FIG. 3

) was used as the probe. The arrows indicate the labeled DNA fragments (BamHI: 2.1 and 0.7 kb, Pstl: ˜11-12 kb)

Tab. 4: DNA sequence of the 6.8 kb Pstl fragment from

Streptomyces glaucescens

GLA.O (SEQ ID NO.: 7). The deduced amino acid sequences (SEQ ID NO.: 8-13) of the identified open reading frames are given under the DNA sequences. Start and stop codons and potential ribosome binding sites are underlined.

acbA: SEQ ID NO.: 8

acbB: SEQ ID NO.: 9

acbC: SEQ ID NO.: 10

acbD: SEQ ID NO.: 11

acbE: SEQ ID NO.: 12

acbF: SEQ ID NO.: 13

TABLE 4

(SEQ ID NO.: 7, 8, 9, 10, 11, 12, 13)

P

s

t

I

CTGCAGGGTTCCCTGGTGCACGACCCGCCCCTGGTCGACGACCAGGGCGCTGTCGCAGAT

---------+---------+---------+---------+---------+---------+

60

GACGTCCCAAGGGACCACGTGCTGGGCGGGGACCAGCTGCTGGTCCCGCGACAGCGTCTA

Q L T G Q H V V R G Q D V V L A S D C I

-

CGCGGCGATGTCGGCGATGTCGTGGCTGGTGAGCACCACGGTGGTGCCCAGTTCCCGGTG

---------+---------+---------+---------+---------+---------+

120

GCGCCGCTACAGCCGCTACAGCACCGACCACTCGTGGTGCCACCACGGGTCAAGGGCCAC

A A I D A I D H S T L V V T T G L E R H

-

GGCGCGGTTGACCAGCCGGCGCACCGCGTCCTTCAGCACCATGTCGAGGCCGATCGTGGG

---------+---------+---------+---------+---------+---------+

180

CCGCGCCAACTGGTCGGCCGCGTGGCGCAGGAAGTCGTGGTACAGCTCCGGCTAGCACCC

A R N V L R R V A D K L V M D L G I T P

-

CTCGTCCCAGAACAGCACGGCCGGGTCGTGCAGCAGGCTCGCCGCGATCTCGGCGCGCAT

---------+---------+---------+---------+---------+---------+

240

GAGCAGGGTCTTGTCGTGCCGGCCCAGCACGTCGTCCGAGCGGCGCTAGAGCCGCGCGTA

E D W F L V A P D H L L S A A I E A R M

-

S

p

h

I

GCGCTGTCCGAGGCTGAGCTGCCGCACGGGGGTGGACCCCAGCGCGTCGATGTCGAGGAG

---------+---------+---------+---------+---------+---------+

300

CGCGACAGGCTCCGACTCGACGGCGTGCCCCCACCTGGGGTCGCGCAGCTACAGCTCCTC

R Q G L S L Q R V P T S G L A D I D L L

-

GTCCCGGAACAGGGCGAGGTTGCGCCGGTAGACCGGTCCGGGGATGTCGTAGATGCGGCG

---------+---------+---------+---------+---------+---------+

360

CAGGGCCTTGTCCCGCTCCAACGCGGCCATCTGGCCAGGCCCCTACAGCATCTACGCCGC

D R F L A L N R R Y V P G P I D Y I R R

-

K

p

n

I

CAGGATGCGGAAGGAGTCGGGTACCGACAGGTCCCACCAGAGCTGGCTGCGCTGGCCGAA

---------+---------+---------+---------+---------+---------+

420

GTCCTACGCCTTCCTCAGCCCATGGCTGTCCAGGGTGGTCTCGACCGACGCGACCGGCTT

L I R F S D P V S L D W W L Q S R Q G F

-

GACGACGCCGATCGTGCGGGCGTTGCGCTGCCGGTGCCGGTAGGGCTCCAGCCCGGCGAC

---------+---------+---------+---------+---------+---------+

480

CTGCTGCGGCTAGCACGCCCGCAACGCGACGGCCACGGCCATCCCGAGGTCGGGCCGCTG

V V G I T R A N R Q R H R Y P E L G A V

-

CGTGCAGCGGCCGGAGGTGGGGGTCATGATGCCGGTCAGCATCTTGATCGTGGTCGACTT

---------+---------+---------+---------+---------+---------+

540

GCACGTCGCCGGCCTCCACCCCCAGTACTACGGCCAGTCGTAGAACTAGCACCAGCTGAA

T C R G S T P T M I G T L M K I T T S K

-

GCCGGCTCCGTTGGCGCCGATGTAGGCGGTCTTCGTGCCGGCCGGTATCTCGAAGGAGAC

---------+---------+---------+---------+---------+---------+

600

CGGCCGAGGCAACCGCGGCTACATCCGCCAGAAGCACGGCCGGCCATAGAGCTTCCTCTG

G A G N A G I Y A T K T G A P I E F S V

-

K

p

n

I

GTCGTCGACGGCGCGCACGACGCGGTACCGGCGGGTCAGGAGGGTGGAGAGGCTGCCGAG

---------+---------+---------+---------+---------+---------+

660

CAGCAGCTGCCGCGCGTGCTGCGCCATGGCCGCCCAGTCCTCCCACCTCTCCGACGGCTC

D D V A R V V R Y R R T L L T S L S G L

-

CAGGCCGGGCTCGCGTTCGGCCAGCCGGAACTCCTTGACGAGGTGTTCGGCCACGATCAC

---------+---------+---------+---------+---------+---------+

720

GTCCGGCCCGAGCGCAAGCCGGTCGGCCTTGAGGAACTGCTCCACAAGCCGGTGCT

AGTG

*

-

L G P E R E A L R F E K V L H E A V I V

-

—————— acbA

GCGATCACCCGCTCGACGGCCGTCTCCAGCAGGCGCAGGCCCTCGTCGAGCAGCGCCTCG

---------+---------+---------+---------+---------+---------+

780

CGCTAGTGGGCGAGCTGCCGGCAGAGGTCGTCCGCGTCCGGGAGCAGCTCGTCGCGGAGC

A I V R E V A T E L L R L G E D L L A E

-

TCGAGGGTGAACGGCGGTGCCAGCCGCAGGATGTGGCCGCCCAGGGAGGTGCGCAGCCCC

---------+---------+---------+---------+---------+---------+

840

AGCTCCCACTTGCCGCCACGGTCGGCGTCCTACACCGGCGGGTCCCTCCACGCGTCGGGG

D L T F P P A L R L I H G G L S T R L G

-

S

m

a

I

AGGTCGAGGGCGGTGGTGTAGACGGCCCGGGCGGTCTCGGGGGCGGGTGCCCGGCCGACG

---------+---------+---------+---------+---------+---------+

900

TCCAGCTCCCGCCACCACATCTGCCGGGCCCGCCAGAGCCCCCGCCCACGGGCCGGCTGC

L D L A T T Y V A R A T E P A P A R G V

-

GCGTCGGTGACGAACTCCAGGCCCCACAGCAGTCCGAGGCCGCGTACCTGGCCGAGCTGG

---------+---------+---------+---------+---------+---------+

960

CGCAGCCACTGCTTGAGGTCCGGGGTGTCGTCAGGCTCCGGCGCATGGACCGGCTCGACC

A D T V F E L G W L L G L G R V Q G L Q

-

S

s

t

I

GGGAAGCGGGACTCCAGGGCGCGCAGCCGCTCCTGGATGAGCTCGCCGAGGACGCGCACG

---------+---------+---------+---------+---------+---------+

1020

CCCTTCGCCCTGAGGTCCCGCGCGTCGGCGAGGACCTACTCGAGCGGCTCCTGCGCGTGC

P F R S E L A R L R E Q I L E G L V R V

-

CGGTCGATCAGCCGGTCGCGCTCGACGACCTCCAGCGTGGCGCGGGCGGCGGCGATCCCC

---------+---------+---------+---------+---------+---------+

1080

GCCAGCTAGTCGGCCAGCGCGAGCTGCTGGAGGTCGCACCGCGCCCGCCGCCGCTAGGGG

R D I L R D R E V V E L T A R A A A I G

-

S

m

a

I

AGTGGGTTGCTCGCGTACGTCGAGGCGTACGCCCCGGGGTGGCCGCCTCCGGCCTGCGCA

---------+---------+---------+---------+---------+---------+

1140

TCACCCAACGAGCGCATGCAGCTCCGCATGCGGGGCCCCACCGGCGGAGGCCGGACGCGT

L P N S A Y T S A Y A G P H G G G A Q A

-

GCTTCCGCGCGTCCGGCCAGCACGGCGAAGGGGAATCCGCTCGCGGTGCCCTTGGACAGC

---------+---------+---------+---------+---------+---------+

1200

CGAAGGCGCGCAGGCCGGTCGTGCCGCTTCCCCTTAGGCGAGCGCCACGGGAACCTGTCG

A E A R G A L V A F P F G S A T G K S L

-

ATCGCCAGGTCCGGCTCGATGCCGAACAGTTCGCTGGCGAGGAAGGCGCCGGTGCGCCCG

---------+---------+---------+---------+---------+---------+

1260

TAGCGGTCCAGGCCGAGCTACGGCTTGTCAAGCGACCGCTCCTTCCGCGGCCACGCGGGC

M A L D P E I G F L E S A L F A G T R G

-

CCGCCGGTGAGGACCTCGTCGGCGACGAGCAGCACGCCGCCGTCCCGGCAGGCGCCGGCG

---------+---------+---------+---------+---------+---------+

1320

GGCGGCCACTCCTGGAGCAGCCGCTGCTCGTCGTGCGGCGGCAGGGCCGTCCGCGGCCGC

G G T L V E D A V L L V G G D R C A G A

-

ATCCGCTCCCAGTAGCCGGGGGGCGGCACGATGACGCCTGCCGCGCCGAGGACGGGTTCG

---------+---------+---------+---------+---------+---------+

1380

TAGGCGAGGGTCATCGGCCCCCCGCCGTGCTACTGCGGACGGCGCGGCTCCTGCCCAAGC

I R E W Y G P P P V I V G A A G L V P E

-

AAGACCAGGGCCGAGACGTTGGGCTTCTCCGCGATGTGCCGGCGCACGAGGGTCGCGCAC

---------+---------+---------+---------+---------+---------+

1440

TTCTGGTCCCGGCTCTGCAACCCGAAGAGGCGCTACACGGCCGCGTGCTCCCAGCGCGTG

F V L A S V N P K E A I H R R V L T A C

-

CGCACGTCGCACGAGGGGTACTCCAGGCCCAGGGGACAGCGGTAGCCAGTAGGGGCTGTA

---------+---------+---------+---------+---------+---------+

1500

GCGTGCAGCGTGCTCCCCATGAGGTCCGGGTCCCCTGTCGCCATCGGTCATCCCCGACAT

R V D C S P Y E L G L P C R Y G T P A T

-

GCCAGCACGCTGTTGCCGCTGAAGGCCTGGTGGCCGATGTCCCAGTGGACCAGCATCCGG

---------+---------+---------+---------+---------+---------+

1560

CGGTCGTGCGACAACGGCGACTTCCGGACCACCGGCTACAGGGTCACCTGGTCGTAGGCC

A L V S N G S F A Q H G I D W H V L M R

-

GCGCCCATGGTCTTGCCGTGGAAGCCGTGGCGCAGGGCGCAGATCCGGTTGCGGCCCGGC

---------+---------+---------+---------+---------+---------+

1620

CGCGGGTACCAGAACGGCACCTTCGGCACCGCGTCCCGCGTCTAGGCCAACGCCGGGCCG

A G M T K G H F G H R L A C I R N R G P

-

GCGGCGGTCGCCTGGACGACCCGCAGGGCGGCCTCGACCACCTCCGCGCCGGTGGAGAAG

---------+---------+---------+---------+---------+---------+

1680

CGCCGCCAGCGGACCTGCTGGGCGTCCCGCCGGAGCTGGTGGAGGCGCGGCCACCTCTTC

A A T A Q V V R L A A E V V E A G T S F

-

AAGGCGTAGGTGTCGAGCTGTTCGGGCAGCAGCCTGGCGAGCAGTTCCAGCAGGCCGGCG

---------+---------+---------+---------+---------+---------+

1740

TTCCGCATCCACAGCTCGACAAGCCCGTCGTCGGACCGCTCGTCAAGGTCGTCCGGCCGC

F A Y T D L Q E P L L R A L L E L L G A

-

CGGTCCGGCGTGGCGCTGTCGTGGACGTTCCACAGGCGGCGGGCCTGGGTGGTGAGTGCC

---------+---------+---------+---------+---------+---------+

1800

GCCAGGCCGCACCGCGACAGCACCTGCAAGGTGTCCGCCGCCCGGACCCACCACTCACGG

R D P T A S D H V N W L R R A Q T T L A

-

TCGACGACCTCCGGGTGCCCGTGGCCCAGTGACTGGGTGAGGGTCCCGGCCGCGAAGTCG

---------+---------+---------+---------+---------+---------+

1860

AGCTGCTGGAGGCCCACGGGCACCGGGTCACTGACCCACTCCCAGGGCCGGCGCTTCAGC

E V V E P H G H G L S Q T L T G A A F D

-

AGGTACTGGTTGCCGTCCAGGTCGGTCAGAACGGGACCGCGTCCCTCGGCGAAGACCCGG

---------+---------+---------+---------+---------+---------+

1920

TCCATGACCAACGGCAGGTCCAGCCAGTCTTGCCCTGGCGCAGGGAGCCGCTTCTGGGCC

L Y Q N G D L D T L V P G R G E A F V R

-

CGTCCGTGGACGGCTTCCTCGGAGGCGCCCGGCGCCAGGTGGCGGGCCTCCCGTGCCAGG

---------+---------+---------+---------+---------+---------+

1980

GCAGGCACCTGCCGAAGGAGCCTCCGCGGGCCGCGGTCCACCGCCCGGAGGGCACGGTCC

R G H V A E E S A G P A L H R A E R A L

-

TGCTGTGTCTGCCGTAAGCCTGTCATCGCTGCCTCTGCTCGTCGGACCGGCTGACGCGAT

---------+---------+---------+---------+---------+---------+

2040

ACGACACAGACGGCATTCGGACA

GTA

GCGAC

GGAG

ACGAGCAGCCTGGCCGACTGCGCTA

H Q T Q R L G T M

———————— acbB

CGCCGGCGAACTGCGTTGTGGCGCACCACGGTTGGGGCGGCTCGGCGCTGAGTCAAACAC

---------+---------+---------+---------+---------+---------+

2100

GCGGCCGCTTGACGCAACACCGCGTGGTGCCAACCCCGCCGAGCCGCGACTCAGTTTGTG

TTGAACACACACCGCTGCAAGAGTTTGCGGGTTGTTTCAGAAAGTTGTTGCGAGCGGCCC

---------+---------+---------+---------+---------+---------+

2160

AACTTGTGTGTGGCGACGTTCTCAAACGCCCAACAAAGTCTTTCAACAACGCTCGCCGGG

CGGCACTCTGGTTGAGTCGACGTGCTTACGGCGCCACCACGCCTCACGTTCGAGGAGGGA

---------+---------+---------+---------+---------+---------+

2220

GCCGTGAGACCAACTCAGCTGCACGAATGCCGCGGTGGTGCGGAGTGCAAGCTCCTCCCT

CCTGTGAGAACAAGCCCGCAGACCGACCCGCTCCCGC

GGAGG

CCGAG

GTG

AAGGCCCTGG

---------+---------+---------+---------+---------+---------+

2280

GGACACTCTTGTTCGGGCGTCTGGCTGGGCGAGGGCGCCTCCGGCTCCACTTCCGGGACC

V K A L V

-

acbC ——————

P

v

u

I

I

TCCTGGCAGGTGGAACCGGCAGCAGACTGAGGCCGTTCACCCACACCGCCGCCAAGCAGC

---------+---------+---------+---------+---------+---------+

2340

AGGACCGTCCACCTTGGCCGTCGTCTGACTCCGGCAAGTGGGTGTGGCGGCGGTTCGTCG

L A G G T G S R L R P F T H T A A K Q L

-

TGCTCCCCATCGCCAACAAGCCCGTGCTCTTCTACGCGCTGGAGTCCCTCGCCGCGGCGG

---------+---------+---------+---------+---------+---------+

2400

ACGAGGGGTAGCGGTTGTTCGGGCACGAGAAGATGCGCGACCTCAGGGAGCGGCGCCGCC

L P I A N K P V L F Y A L E S L A A A G

-

GTGTCCGGGAGGCCGGCGTCGTCGTGGGCGCGTACGGCCGGGAGATCCGCGAACTCACCG

---------+---------+---------+---------+---------+---------+

2460

CACAGGCCCTCCGGCCGCAGCAGCACCCGCGCATGCCGGCCCTCTAGGCGCTTGAGTGGC

V R E A G V V V G A Y G R E I R E L T G

-

GCGACGGCACCGCGTTCGGGTTACGCATCACCTACCTCCACCAGCCCCGCCCGCTCGGTC

---------+---------+---------+---------+---------+---------+

2520

CGCTGCCGTGGCGCAAGCCCAATGCGTAGTGGATGGAGGTGGTCGGGGCGGGCGAGCCAG

D G T A F G L R I T Y L H Q P R P L G L

-

TCGCGCACGCGGTGCGCATCGCCCGCGGCTTCCTGGGCGACGACGACTTCCTGCTGTACC

---------+---------+---------+---------+---------+---------+

2580

AGCGCGTGCGCCACGCGTAGCGGGCGCCGAAGGACCCGCTGCTGCTGAAGGACGACATGG

A H A V R I A R G F L G D D D F L L Y L

-

TGGGGGACAACTACCTGCCCCAGGGCGTCACCGACTTCGCCCGCCAATCGGCCGCCGATC

---------+---------+---------+---------+---------+---------+

2640

ACCCCCTGTTGATGGACGGGGTCCCGCAGTGGCTGAAGCGGGCGGTTAGCCGGCGGCTAG

G D N Y L P Q G V T D F A R Q S A A D P

-

CCGCGGCGGCCCGGCTGCTGCTCACCCCGGTCGCGGACCCGTCCGCCTTCGGCGTCGCGG

---------+---------+---------+---------+---------+---------+

2700

GGCGCCGCCGGGCCGACGACGAGTGGGGCCAGCGCCTGGGCAGGCGGAAGCCGCAGCGCC

A A A R L L L T P V A D P S A F G V A E

-

AGGTCGACGCGGACGGGAACGTGCTGCGCTTGGAGGAGAAACCCGACGTCCCGCGCAGCT

---------+---------+---------+---------+---------+---------+

2760

TCCAGCTGCGCCTGCCCTTGCACGACGCGAACCTCCTCTTTGGGCTGCAGGGCGCGTCGA

V D A D G N V L R L E E K P D V P R S S

-

CGCTCGCGCTCATCGGCGTGTACGCCTTCAGCCCGGCCGTCCACGAGGCGGTACGGGCCA

---------+---------+---------+---------+---------+---------+

2820

GCGAGCGCGAGTAGCCGCACATGCGGAAGTCGGGCCGGCAGGTGCTCCGCCATGCCCGGT

L A L I G V Y A F S P A V H E A V R A I

-

TCACCCCCTCCGCCCGCGGCGAGCTGGAGATCACCCACGCCGTGCAGTGGATGATCGACC

---------+---------+---------+---------+---------+---------+

2880

AGTGGGGGAGGCGGGCGCCGCTCGACCTCTAGTGGGTGCGGCACGTCACCTACTAGCTGG

T P S A R G E L E I T H A V Q W M I D R

-

GGGGCCTGCGCGTACGGGCCGAGACCACCACCCGGCCCTGGCGCGACACCGGCAGCGCGG

---------+---------+---------+---------+---------+---------+

2940

CCCCGGACGCGCATGCCCGGCTCTGGTGGTGGGCCGGGACCGCGCTGTGGCCGTCGCGCC

G L R V R A E T T T R P W R D T G S A E

-

AGGACATGCTGGAGGTCAACCGTCACGTCCTGGACGGACTGGAGGGCCGCATCGAGGGGA

---------+---------+---------+---------+---------+---------+

3000

TCCTGTACGACCTCCAGTTGGCAGTGCAGGACCTGCCTGACCTCCCGGCGTAGCTCCCCT

D M L E V N R H V L D G L E G R I E G K

-

AGGTCGACGCGCACAGCACGCTGGTCGGCCGGGTCCGGGTGGCCGAAGGCGCGATCGTGC

---------+---------+---------+---------+---------+---------+

3060

TCCAGCTGCGCGTGTCGTGCGACCAGCCGGCCCAGGCCCACCGGCTTCCGCGCTAGCACG

V D A H S T L V G R V R V A E G A I V R

-

GGGGGTCACACGTGGTGGGCCCGGTGGTGATCGGCGCGGGTGCCGTCGTCAGCAACTCCA

---------+---------+---------+---------+---------+---------+

3120

CCCCCAGTGTGCACCACCCGGGCCACCACTAGCCGCGCCCACGGCAGCAGTCGTTGAGGT

G S H V V G P V V I G A G A V V S N S S

-

GTGTCGGCCCGTACACCTCCATCGGGGAGGACTGCCGGGTCGAGGACAGCGCCATCGAGT

---------+---------+---------+---------+---------+---------+

3180

CACAGCCGGGCATGTGGAGGTAGCCCCTCCTGACGGCCCAGCTCCTGTCGCGGTAGCTCA

V G P Y T S I G E D C R V E D S A I E Y

-

ACTCCGTCCTGCTGCGCGGCGCCCAGGTCGAGGGGGCGTCCCGCATCGAGGCGTCCCTCA

---------+---------+---------+---------+---------+---------+

3240

TGAGGCAGGACGACGCGCCGCGGGTCCAGCTCCCCCGCAGGGCGTAGCTCCGCAGGGAGT

S V L L R G A Q V E G A S R I E A S L I

-

TCGGCCGCGGCGCCGTCGTCGGCCCGGCCCCCCGTCTCCCGCAGGCTCACCGACTGGTGA

---------+---------+---------+---------+---------+---------+

3300

AGCCGGCGCCGCGGCAGCAGCCGGGCCGGGGGGCAGAGGGCGTCCGAGTGGCTGACCACT

G R G A V V G P A P R L P Q A H R L V I

-

TCGGCGACCACAGCAAGGTGTATCTCACCCC

ATGA

CCACGACCATCCTCGTCACCGGCGG

---------+---------+---------+---------+---------+---------+

3360

AGCCGCTGGTGTCGTTCCACATAGAGTGGGGTACTGGTGCTGGTAGGAGCAGTGGCCGCC

M T T T I L V T G G

-

G D H S K V Y L T P *

acbD ————————

s

m

a

I

AGCGGGCTTCATTCGCTCCGCCTACGTCCGCCGGCTCCTGTCGCCCGGGGCCCCCGGCGG

---------+---------+---------+---------+---------+---------+

3420

TCGCCCGAAGTAAGCGAGGCGGATGCAGGCGGCCGAGGACAGCGGGCCCCGGGGGCCGCC

A G F I R S A Y V R R L L S P G A P G G

-

CGTCGCGGTGACCGTCCTCGACAAACTCACCTACGCCGGCAGCCTCGCCCGCCTGCACGC

---------+---------+---------+---------+---------+---------+

3480

GCAGCGCCACTGGCAGGAGCTGTTTGAGTGGATGCGGCCGTCGGAGCGGGCGGACGTGCG

V A V T V L D K L T Y A G S L A R L H A

-

GGTGCGTGACCATCCCGGCCTCACCTTCGTCCAGGGCGACGTGTGCGACACCGCGCTCGT

---------+---------+---------+---------+---------+---------+

3540

CCACGCACTGGTAGGGCCGGAGTGGAAGCAGGTCCCGCTGCACACGCTGTGGCGCGAGCA

V R D H P G L T F V Q G D V C D T A L V

-

CGACACGCTGGCCGCGCGGCACGACGACATCGTGCACTTCGCGGCCGAGTCGCACGTCGA

---------+---------+---------+---------+---------+---------+

3600

GCTGTGCGACCGGCGCGCCGTGCTGCTGTAGCACGTGAAGCGCCGGCTCAGCGTGCAGCT

D T L A A R H D D I V H F A A E S H V D

-

CCGCTCCATCACCGACAGCGGTGCCTTCACCCGCACCAACGTGCTGGGCACCCAGGTCCT

---------+---------+---------+---------+---------+---------+

3660

GGCGAGGTAGTGGCTGTCGCCACGGAAGTGGGCGTGGTTGCACGACCCGTGGGTCCAGGA

R S I T D S G A F T R T N V L G T Q V L

-

GCTCGACGCCGCGCTCCGCCACGGTGTGCGCACCTTCGTGCACGTCTCCACCGACGAGGT

---------+---------+---------+---------+---------+---------+

3720

CGAGCTGCGGCGCGAGGCGGTGCCACACGCGTGGAAGCACGTGCAGAGGTGGCTGCTCCA

L D A A L R H G V R T F V H V S T D E V

-

GTACGGCTCCCTCCCGCACGGGGCCGCCGCGGAGAGCGACCCCCTGCTTCCGACCTCGCC

---------+---------+---------+---------+---------+---------+

3780

CATGCCGAGGGAGGGCGTGCCCCGGCGGCGCCTCTCGCTGGGGGACGAAGGCTGGAGCGG

Y G S L P H G A A A E S D P L L P T S P

-

GTACGCGGCGTCGAAGGCGGCCTCGGACCTCATGGCGCTCGCCCACCACCGCACCCACGG

---------+---------+---------+---------+---------+---------+

3840

CATGCGCCGCAGCTTCCGCCGGAGCCTGGAGTACCGCGAGCGGGTGGTGGCGTGGGTGCC

Y A A S K A A S D L M A L A H H R T H G

-

CCTGGACGTCCGGGTGACCCGCTGTTCGAACAACTTCGGCCCCCACCAGCATCCCGAGAA

---------+---------+---------+---------+---------+---------+

3900

GGACCTGCAGGCCCACTGGGCGACAAGCTTGTTGAAGCCGGGGGTGGTCGTAGGGCTCTT

L D V R V T R C S N N F G P H Q H P E K

-

GCTCATACCGCGCTTCCTGACCAGCCTCCTGTCCGGCGGCACCGTTCCCCTCTACGGCGA

---------+---------+---------+---------+---------+---------+

3960

CGAGTATGGCGCGAAGGACTGGTCGGAGGACAGGCCGCCGTGGCAAGGGGAGATGCCGCT

L I P R F L T S L L S G G T V P L Y G D

-

CGGGCGGCACGTGCGCGACTGGCTGCACGTCGACGACCACGTCAGGGCCGTCGAACTCGT

---------+---------+---------+---------+---------+---------+

4020

GCCCGCCGTGCACGCGCTGACCGACGTGCAGCTGCTGGTGCAGTCCCGGCAGCTTGAGCA

G R H V R D W L H V D D H V R A V E L V

-

B

g

l

I

I

CCGCGTGTCGGGCCGGCCGGGAGAGATCTACAACATCGGGGGCGGCACCTCGCTGCCCAA

---------+---------+---------+---------+---------+---------+

4080

GGCGCACAGCCCGGCCGGCCCTCTCTAGATGTTGTAGCCCCCGCCGTGGAGCGACGGGTT

R V S G R P G E I Y N I G G G T S L P N

-

S

s

t

I

CCTGGAGCTCACGCACCGGTTGCTCGCACTGTGCGGCGCGGGCCCGGAGCGCATCGTCCA

---------+---------+---------+---------+---------+---------+

4140

GGACCTCGAGTGCGTGGCCAACGAGCGTGACACGCCGCGCCCGGGCCTCGCGTAGCAGGT

L E L T H R L L A L C G A G P E R I V H

-

CGTCGAGAACCGCAAGGGGCACGACCGGCGCTACGCGGTCGACCACAGCAAGATCACCGC

---------+---------+---------+---------+---------+---------+

4200

GCAGCTCTTGGCGTTCCCCGTGCTGGCCGCGATGCGCCAGCTGGTGTCGTTCTAGTGGCG

V E N R K G H D R R Y A V D H S K I T A

-

N

r

u

I

GGAACTCGGTTACCGGCCGCGCACCGACTTCGCGACCGCGCTGGCCGACACCGCGAAGTG

---------+---------+---------+---------+---------+---------+

4260

CCTTGAGCCAATGGCCGGCGCGTGGCTGAAGCGCTGGCGCGACCGGCTGTGGCGCTTCAC

E L G Y R P R T D F A T A L A D T A K W

-

GTACGAGCGGCACGAGGACTGGTGGCGTCCCCTGCTCGCCGCGACA

TGA

CGTCGGGCCGG

---------+---------+---------+---------+---------+---------+

4320

CATGCTCGCCGTGCTCCTGACCACCGCAGGGGACGAGCGGCGCTGTACTGCAGCCCGGCC

Y E R H E D W W R P L L A A T *

ACCGCAACCACCGGCCCCGGCCGGCACACCGCCGCCCGCGGCCGGTGGCCGGCCGGTCAG

---------+---------+---------+---------+---------+---------+

4380

TGGCGTTGGTGGCCGGGGCCGGCCGTGTGGCGGCGGGCGCCGGCCACCGGCCGGCCAGTC

*

-

CGTCCGTGAGCCGGGCGCCGGCCGCCCCGCGGGCCGGCGGCGGTGGACCCCCGGACCACC

---------+---------+---------+---------+---------+---------+

4440

GCAGGCACTCGGCCCGCGGCCGGCGGGGCGCCCGGCCGCCGCCACCTGGGGGCCTGGTGG

R G H A P R R G G R P G A A T S G R V V

-

E

c

o

R

I

AGTTCCGGCATGAAGACGAATTCGGTGCGCGGCGGCGGCGTTCCGCTCATCTCCTCCAGC

---------+---------+---------+---------+---------+---------+

4500

TCAAGGCCGTACTTCTGCTTAAGCCACGCGCCGCCGCCGCAAGGCGAGTAGAGGAGGTCG

L E P M F V F E T R P P P T G S M E E L

-

AGTGCGTCCACGGCGACCTGCCCCATCGCCTTGACGGGCTGTCTGATGGTGGTCAGGGGA

---------+---------+---------+---------+---------+---------+

4560

TCACGCAGGTGCCGCTGGACGGGGTAGCGGAACTGCCCGACAGACTACCACCAGTCCCCT

L A D V A V Q G M A K V P Q R I T T L P

-

GGGTCGGTGAAGGCCATGAGCGGCGAGTCGTCGAAGCCGACCACCGAGATGTCACCGGGA

---------+---------+---------+---------+---------+---------+

4620

CCCAGCCACTTCCGGTACTCGCCGCTCAGCAGCTTCGGCTGGTGGCTCTACAGTGGCCCT

P D T F A M L P S D D F G V V S I D G P

-

ACCGTGAGACCCCGCCGGCGCGCGGCCCGCACGGCGCCGAGGGCCATCATGTCGCTGGCG

---------+---------+---------+---------+---------+---------+

4680

TGGCACTCTGGGGCGGCCGCGCGCCGGGCGTGCCGCGGCTCCCGGTAGTACAGCGACCGC

V T L G R R R A A R V A G L A M M D S A

-

CACATGACGGCGGTGCAGCCCAGGTCGATCAGCGCGGACGCGGCGGCCTGGCCCCCCTCC

---------+---------+---------+---------+---------+---------+

4740

GTGTACTGCCGCCACGTCGGGTCCAGCTAGTCGCGCCTGCGCCGCCGGACCGGGGGGAGG

C M V A T C G L D I L A S A A A Q G G E

-

S

s

t

I

AGGGAGAACAGCGAGTGCTGCACGAGCTCCTCGGACTCCCGCGCCGACACTCCCAGGTGC

---------+---------+---------+---------+---------+---------+

4800

TCCCTCTTGTCGCTCACGACGTGCTCGAGGAGCCTGAGGGCGCGGCTGTGAGGGTCCACG

L S F L S H Q V L E E S E R A S V G L H

-

TCCCGCACGCCGGCCCGGAACCCCTCGATCTTCCGCTGCACCGGCACGAAGCGGGCGGGC

---------+---------+---------+---------+---------+---------+

4860

AGGGCGTGCGGCCGGGCCTTGGGGAGCTAGAAGGCGACGTGGCCGTGCTTCGCCCGCCCG

E R V G A R F G E I K R Q V P V F R A P

-

CCGACGGCGAGGCCGACGCGCTCGTGCCCCAGCTCCGCCAGGTGCGCCACGGCCAGGCGC

---------+---------+---------+---------+---------+---------+

4920

GGCTGCCGCTCCGGCTGCGCGAGCACGGGGTCGAGGCGGTCCACGCGGTGCCGGTCCGCG

G V A L G V R E H G L E A L H A V A L R

-

ATCGCGGCCCGGTCGTCCGGGGAGACGAAGGGTGCCTCGATCCGGGGCGAGAACCCGTTC

---------+---------+---------+---------+---------+---------+

4980

TAGCGCCGGGCCAGCAGGCCCCTCTGCTTCCCACGGAGCTAGGCCCCGCTCTTGGGCAAG

M A A R D D P S V F P A E I R P S F G N

-

ACGAGGACGAAGGGCACCTGCCGCTCGTGCAGCCGGCCGTACCGTCCGGTCTCGGCGGTG

---------+---------+---------+---------+---------+---------+

5040

TGCTCCTGCTTCCCGTGGACGGCGAGCACGTCGGCCGGCATGGCAGGCCAGAGCCGCCAC

V L V F P V Q R E H L R G Y R G T E A T

-

GTGTCCGCGTGCAGTCCGGAGACGAAGATGATGCCGGACACCCCGCGGTCCACGAGCATC

---------+---------+---------+---------+---------+---------+

5100

CACAGGCGCACGTCAGGCCTCTGCTTCTACTACGGCCTGTGGGGCGCCAGGTGCTCGTAG

T D A H L G S V F I I G S V G R D V L M

-

S

m

a

I

TCCGTGAGTTCGTCCTCGGTCGAGCCGCCCGGGGTCTGCGTGGCGAGCACGGGCGTGTAG

---------+---------+---------+---------+---------+---------+

5160

AGGCACTCAAGCAGGAGCCAGCTCGGCGGGCCCCAGACGCACCGCTCGTGCCCGCACATC

E T L E D E T S G G P T Q T A L V P T Y

-

CCCTGACGCGTGAGCGCCTGCCCCATCACCTGGGCCAGTGCGGGGAAGAAGGGGTTGTCC

---------+---------+---------+---------+---------+---------+

5220

GGGACTGCGCACTCGCGGACGGGGTAGTGGACCCGGTCACGCCCCTTCTTCCCCAACAGG

G Q R T L A Q G M V Q A L A P F F P N D

-

AGTTCGGGGGTGACCAGTCCGACCAGCTCGGCGCGGCGCTGTCGCGCCGGCTGCTCGTAG

---------+---------+---------+---------+---------+---------+

5280

TCAAGCCCCCACTGGTCAGGCTGGTCGAGCCGCGCCGCGACAGCGCGGCCGACGAGCATC

L E P T V L G V L E A R R Q R A P Q E Y

-

CCCAGCGCGTCCAGTGCGGTCAGCACCGAGTCGCGGGTGCCGGTGGCCACACCGCGCGCA

---------+---------+---------+---------+---------+---------+

5340

GGGTCGCGCAGGTCACGCCAGTCGTGGCTCAGCGCCCACGGCCACCGGTGTGGCGCGCGT

G L A D L A T L V S D R T G T A V G R A

-

S

m

a

I

CCGTTCAGCACCCGGCTGACCGTGGCCTTGCTGACGCCCGCCCGGGCTGCGATGTCGGCG

---------+---------+---------+---------+---------+---------+

5400

GGCAAGTCGTGGGCCGACTGGCACCGGAACGACTGCGGGCGGGCCCGACGCTACAGCCGC

G N L V R S V T A K S V G A R A A I D A

-

AGCCGCATGGTCATGGCAACGCACTCTACCTGTCGGGGCGTCAGGGCGTGCCCACCGCGC

---------+---------+---------+---------+---------+---------+

5460

TCGGCGTACCA

GTA

CCGTTGCGTGAGATGGACAGCCCCGCAGTCCCGCACGGGTGGCGCG

L R M T M

———————— acbE

GCGGAACCGGCGGACTGCGGGGCACGGCCCGTCCGCCGCCCACGGACCACGCGCCCGAAA

---------+---------+---------+---------+---------+---------+

5520

CGCCTTGGCCGCCTGACGCCCCGTGCCGGGCAGGCGGCGGGTGCCTGGTGCGCGGGCTTT

CGATGGCTGAAAATGCTTGCAGCAAATTGCCGCAACGTCTTTCGGCGGCTTTTCGATCCT

---------+---------+---------+---------+---------+---------+

5580

GCTACCGACTTTTACGAACGTCGTTTAACGGCGTTGCAGAAAGCCGCCGAAAAGCTAGGA

GTTACGTTCCTGGCAACCCCGGCGCCGCGCAGAAGCGGTTGGCGTGAGGCGTCCAGACCT

---------+---------+---------+---------+---------+---------+

5640

CAATGCAAGGACCGTTGGGGCCGCGGCGCGTCTTCGCCAACCGCACTCCGCAGGTCTGGA

CCGCCCGATTCCGGGATCACTCAGG

GGAG

TTCACA

ATG

CGGCGTGGCATTGCGGCCACCG

---------+---------+---------+---------+---------+---------+

5700

GGCGGGCTAAGGCCCTAGTGAGTCCCCTCAAGTGTTACGCCGCACCGTAACGCCGGTGGC

M R R G I A A T A

-

acbF ————————

CGCTGTTCGCGGCTGTGGCCATGACGGCATCGGCGTGTGGCGGGGGCGACAACGGCGGAA

---------+---------+---------+---------+---------+---------+

5760

GCGACAAGCGCCGACACCGGTACTGCCGTAGCCGCACACCGCCCCCGCTGTTGCCGCCTT

L F A A V A M T A S A C G G G D N G G S

-

K

p

n

I

GCGGTACCGACGCGGGCGGCACGGAGCTGTCGGGGACCGTCACCTTCTGGGACACGTCCA

---------+---------+---------+---------+---------+---------+

5820

CGCCATGGCTGCGCCCGCCGTGCCTCGACAGCCCCTGGCAGTGGAAGACCCTGTGCAGGT

G T D A G G T E L S G T V T F W D T S N

-

ACGAAGCCGAGAAGGCGACGTACCAGGCCCTCGCGGAGGGCTTCGAGAAGGAGCACCCGA

---------+---------+---------+---------+---------+---------+

5880

TGCTTCGGCTCTTCCGCTGCATGGTCCGGGAGCGCCTCCCGAAGCTCTTCCTCGTGGGCT

E A E K A T Y Q A L A E G F E K E H P K

-

AGGTCGACGTCAAGTACGTCAACGTCCCGTTCGGCGAGGCGAACGCCAAGTTCAAGAACG

---------+---------+---------+---------+---------+---------+

5940

TCCAGCTGCAGTTCATGCAGTTGCAGGGCAAGCCGCTCCGCTTGCGGTTCAAGTTCTTGC

V D V K Y V N V P F G E A N A K F K N A

-

CCGCGGGCGGCAACTCCGGTGCCCCGGACGTGATGCGTACGGAGGTCGCCTGGGTCGCGG

---------+---------+---------+---------+---------+---------+

6000

GGCGCCCGCCGTTGAGGCCACGGGGCCTGCACTACGCATGCCTCCAGCGGACCCAGCGCC

A G G N S G A P D V M R T E V A W V A D

-

ACTTCGCCAGCATCGGCTACCTCGCCCCGCTCGACGGCACGCCCGCCCTCGACGACGGGT

---------+---------+---------+---------+---------+---------+

6060

TGAAGCGGTCGTAGCCGATGGAGCGGGGCGAGCTGCCGTGCGGGCGGGAGCTGCTCCCCA

F A S I G Y L A P L D G T P A L D D G S

-

CGGACCACCTTCCCCAGGGCGGCAGCACCAGGTACGAGGGGAAGACCTACGCGGTCCCGC

---------+---------+---------+---------+---------+---------+

6120

GCCTGGTGGAAGGGGTCCCGCCGTCGTGGTCCATGCTCCCCTTCTGGATGCGCCAGGGCG

D H L P Q G G S T R Y E G K T Y A V P Q

-

AGGTGATCGACACCCTGGCGCTCTTCTACAACAAGGAACTGCTGACGAAGGCCGGTGTCG

---------+---------+---------+---------+---------+---------+

6180

TCCACTAGCTGTGGGACCGCGAGAAGATGTTGTTCCTTGACGACTGCTTCCGGCCACAGC

V I D T L A L F Y N K E L L T K A G V E

-

AGGTGCCGGGCTCCCTCGCCGAGCTGAAGACGGCCGCCGCCGAGATCACCGAGAAGACCG

---------+---------+---------+---------+---------+---------+

6240

TCCACGGCCCGAGGGAGCGGCTCGACTTCTGCCGGCGGCGGCTCTAGTGGCTCTTCTGGC

V P G S L A E L K T A A A E I T E K T G

-

GCGCGAGCGGCCTCTACTGCGGGGCGACGACCCGTACTTGGTTCCTGCCCTACCTCTACG

---------+---------+---------+---------+---------+---------+

6300

CGCGCTCGCCGGAGATGACGCCCCGCTGCTGGGCATGAACCAAGGACGGGATGGAGATGC

A S G L Y C G A T T R T W F L P Y L Y G

-

GGGAGGGCGGCGACCTGGTCGACGAGAAGAACAAGACCGTCACGGTCGACGACGAAGCCG

---------+---------+---------+---------+---------+---------+

6360

CCCTCCCGCCGCTGGACCAGCTGCTCTTCTTGTTCTGGCAGTGCCAGCTGCTGCTTGGGC

E G G D L V D E K N K T V T V D D E A G

-

GTGTGCGCGCCTACCGCGTCATCAAGGACCTCGTGGACAGCAAGGCGGCCATCACCGACG

---------+---------+---------+---------+---------+---------+

6420

CACACGCGCGGATGGCGCAGTAGTTCCTGGAGCACCTGTCGTTCCGCCGGTAGTGGCTGC

V R A Y R V I K D L V D S K A A I T D A

-

CGTCCGACGGCTGGAACAACATGCAGAACGCCTTCAAGTCGGGCAAGGTCGCCATGATGG

---------+---------+---------+---------+---------+---------+

6480

GCAGGCTGCCGACCTTGTTGTACGTCTTGCGGAAGTTCAGCCCGTTCCAGCGGTACTACC

S D G W N N M Q N A F K S G K V A M M V

-

TCAACGGCCCCTGGGCCATCGAGGACGTCAAGGCGGGAGCCCGCTTCAAGGACGCCGGCA

---------+---------+---------+---------+---------+---------+

6540

AGTTGCCGGGGACCCGGTAGCTCCTGCAGTTCCGCCCTCGGGCGAAGTTCCTGCGGCCGT

N G P W A I E D V K A G A R F K D A G N

-

ACCTGGGGGTCGCCCCCGTCCCGGCCGGCAGTGCCGGACAGGGCTCTCCCCAGGGCGGGT

---------+---------+---------+---------+---------+---------+

6600

TGGACCCCCAGCGGGGGCAGGGCCGGCCGTCACGGCCTGTCCCGAGAGGGGTCCCGCCCA

L G V A P V P A G S A G Q G S P Q G G W

-

GGAACCTCTCGGTGTACGCGGGCTCGAAGAACCTCGACGCCTCCTACGCCTTCGTGAAGT

---------+---------+---------+---------+---------+---------+

6660

CCTTGGAGAGCCACATGCGCCCGAGCTTCTTGGAGCTGCGGAGGATGCGGAAGCACTTCA

N L S V Y A G S K N L D A S Y A F V K Y

-

S

s

t

I

ACATGAGCTCCGCCAAGGTGCAGCAGCAGACCACCGAGAAGCTGAGCCTGCTGCCCACCC

---------+---------+---------+---------+---------+---------+

6720

TGTACTCGAGGCGGTTCCACGTCGTCGTCTGGTGGCTCTTCGACTCGGACGACGGGTGGG

M S S A K V Q Q Q T T E K L S L L P T R

-

GCACGTCCGTCTACGAGGTCCCGTCCGTCGCGGACAACGAGATGGTGAAGTTCTTCAAGC

---------+---------+---------+---------+---------+---------+

6780

CGTGCAGGCAGATGCTCCAGGGCAGGCAGCGCCTGTTGCTCTACCACTTCAAGAAGTTCG

T S V Y E V P S V A D N E M V K F F K P

-

CGGCCGTCGACAAGGCCGTCGAACGGCCGTGGATCGCCGAGGGCAATGCCCTCTTCGAGC

---------+---------+---------+---------+---------+---------+

6840

GCCGGCAGCTGTTCCGGCAGCTTGCCGGCACCTAGCGGCTCCCGTTACGGGAGAAGCTCG

A V D K A V E R P W I A E G N A L F E P

-

P

s

t

I

CGATCCGGCTGCAG

---------+---- 6854

GCTAGGCCGACGTC

I R L Q -

22 base pairs

nucleic acid

single

linear

other nucleic acid

/desc = “Primer”

1
CSGGSGSSGC SGGSTTCATS GG 22

24 base pairs

nucleic acid

single

linear

other nucleic acid

/desc = “Primer”

2
GGGWVCTGGY VSGGSCCGTA GTTG 24

546 base pairs

nucleic acid

single

linear

DNA (genomic)

3
CCCGGGCGGG GCGGGGTTCA TCGGCTCCGC CTACGTCCGC CGGCTCCTGT CGCCCGGGGC 60
CCCCGGCGGC GTCGCGGTGA CCGTCCTCGA CAAACTCACC TACGCCGGCA GCCTCGCCCG 120
CCTGCACGCG GTGCGTGACC ATCCCGGCCT CACCTTCGTC CAGGGCGACG TGTGCGACAC 180
CGCGCTCGTC GACACGCTGG CCGCGCGGCA CGACGACATC GTGCACTTCG CGGCCGAGTC 240
GCACGTCGAC CGCTCCATCA CCGACAGCGG TGCCTTCACC CGCACCAACG TGCTGGGCAC 300
CCAGGTCCTG CTCGACGCCG CGCTCCGCCA CGGTGTGCGC ACCCTCGTGC ACGTCTCCAC 360
CGACGAGGTG TACGGCTCCC TCCCGCACGG GGCCGCCGCG GAGAGCGACC CCCTGCTCCC 420
GACCTCGCCG TACGCGGCGT CGAAGGCGGC CTCGGACCTC ATGGCGCTCG CCCACCACCG 480
CACCCACGGC CTGGACGTCC GGGTGACCCG CTGTTCGAAC AACTACGGCC CGCACCAGTT 540
CCCGGG 546

541 base pairs

nucleic acid

single

linear

DNA (genomic)

4
CCCCGGGTGC TGGTAGGGGC CGTAGTTGTT GGAGCAGCGG GTGATGCGCA CGTCCAGGCC 60
GTGGCTGACG TGCATGGCCA GCGCGAGCAG GTCGCCCGAC GCCTTGGAGG TGGCATAGGG 120
GCTGTTGGGG CGCAGCGGCT CGTCCTCCGT CCACGACCCC GTCTCCAGCG AGCCGTAGAC 180
CTCGTCGGTG GACACCTGCA CGAAGGGGGC CACGCCGTGC CGCAGGGCCG CGTCGAGGAG 240
TGTCTGCGTG CCGCCGGCGT TGGTCCGCAC GAACGCGGCG GCATCGAGCA GCGAGCGGTC 300
CACGTGCGAC TCGGCGGCGA GGTGCACGAC CTGGTCCTGG CCGGCCATGA CCCGGTCGAC 360
CAGGTCCGCG TCGCAGATGT CGCCGTGGAC GAAGCGCAGC CGGGGGTGGT CGCGGACCGG 420
GTCGAGGTTG GCGAGGTTGC CGGCGTAGCT CAGGGCGTCG AGCACGGTGA CGACGGCGTC 480
GGGCGGCCCG TCCGGACCGA GGAGGGTGCG GACGTAGTGC GAGCCCATGA ACCCCGCCGC 540
C 541

180 amino acids

amino acid

linear

protein

5
Ala Ala Gly Phe Met Gly Ser His Tyr Val Arg Thr Leu Leu Gly Pro
1 5 10 15
Asp Gly Pro Pro Asp Ala Val Val Thr Val Leu Asp Ala Leu Ser Tyr
20 25 30
Ala Gly Asn Leu Ala Asn Leu Asp Pro Val Arg Asp His Pro Arg Leu
35 40 45
Arg Phe Val His Gly Asp Ile Cys Asp Ala Asp Leu Val Asp Arg Val
50 55 60
Met Ala Gly Gln Asp Gln Val Val His Leu Ala Ala Glu Ser His Val
65 70 75 80
Asp Arg Ser Leu Leu Asp Ala Ala Ala Phe Val Arg Thr Asn Ala Gly
85 90 95
Gly Thr Gln Thr Leu Leu Asp Ala Ala Leu Arg His Gly Val Ala Pro
100 105 110
Phe Val Gln Val Ser Thr Asp Glu Val Tyr Gly Ser Leu Glu Thr Gly
115 120 125
Ser Trp Thr Glu Asp Glu Pro Leu Arg Pro Asn Ser Pro Tyr Ala Thr
130 135 140
Ser Lys Ala Ser Gly Asp Leu Leu Ala Leu Ala Met His Val Ser His
145 150 155 160
Gly Leu Asp Val Arg Ile Thr Arg Cys Ser Asn Asn Tyr Gly Pro Tyr
165 170 175
Gln His Pro Gly
180

181 amino acids

amino acid

linear

protein

6
Pro Gly Gly Ala Gly Phe Ile Gly Ser Ala Tyr Val Arg Arg Leu Leu
1 5 10 15
Ser Pro Gly Ala Pro Gly Gly Val Ala Val Thr Val Leu Asp Lys Leu
20 25 30
Thr Tyr Ala Gly Ser Leu Ala Arg Leu His Ala Val Arg Asp His Pro
35 40 45
Gly Leu Thr Phe Val Gln Gly Asp Val Cys Asp Thr Ala Leu Val Asp
50 55 60
Thr Leu Ala Ala Arg His Asp Asp Ile Val His Phe Ala Ala Glu Ser
65 70 75 80
His Val Asp Arg Ser Ile Thr Asp Ser Gly Ala Phe Thr Arg Thr Asn
85 90 95
Val Leu Gly Thr Gln Val Leu Leu Asp Ala Ala Leu Arg His Gly Val
100 105 110
Arg Thr Leu Val His Val Ser Thr Asp Glu Val Tyr Gly Ser Leu Pro
115 120 125
His Gly Ala Ala Ala Glu Ser Asp Pro Leu Leu Pro Thr Ser Pro Tyr
130 135 140
Ala Ala Ser Lys Ala Ala Ser Asp Leu Met Ala Leu Ala His His Arg
145 150 155 160
Thr His Gly Leu Asp Val Arg Val Thr Arg Cys Ser Asn Asn Tyr Gly
165 170 175
Pro His Gln Phe Pro
180

6854 base pairs

nucleic acid

single

linear

DNA (genomic)

7
CTGCAGGGTT CCCTGGTGCA CGACCCGCCC CTGGTCGACG ACCAGGGCGC TGTCGCAGAT 60
CGCGGCGATG TCGGCGATGT CGTGGCTGGT GAGCACCACG GTGGTGCCCA GTTCCCGGTG 120
GGCGCGGTTG ACCAGCCGGC GCACCGCGTC CTTCAGCACC ATGTCGAGGC CGATCGTGGG 180
CTCGTCCCAG AACAGCACGG CCGGGTCGTG CAGCAGGCTC GCCGCGATCT CGGCGCGCAT 240
GCGCTGTCCG AGGCTGAGCT GCCGCACGGG GGTGGACCCC AGCGCGTCGA TGTCGAGGAG 300
GTCCCGGAAC AGGGCGAGGT TGCGCCGGTA GACCGGTCCG GGGATGTCGT AGATGCGGCG 360
CAGGATGCGG AAGGAGTCGG GTACCGACAG GTCCCACCAG AGCTGGCTGC GCTGGCCGAA 420
GACGACGCCG ATCGTGCGGG CGTTGCGCTG CCGGTGCCGG TAGGGCTCCA GCCCGGCGAC 480
CGTGCAGCGG CCGGAGGTGG GGGTCATGAT GCCGGTCAGC ATCTTGATCG TGGTCGACTT 540
GCCGGCTCCG TTGGCGCCGA TGTAGGCGGT CTTCGTGCCG GCCGGTATCT CGAAGGAGAC 600
GTCGTCGACG GCGCGCACGA CGCGGTACCG GCGGGTCAGG AGGGTGGAGA GGCTGCCGAG 660
CAGGCCGGGC TCGCGTTCGG CCAGCCGGAA CTCCTTGACG AGGTGTTCGG CCACGATCAC 720
GCGATCACCC GCTCGACGGC CGTCTCCAGC AGGCGCAGGC CCTCGTCGAG CAGCGCCTCG 780
TCGAGGGTGA ACGGCGGTGC CAGCCGCAGG ATGTGGCCGC CCAGGGAGGT GCGCAGCCCC 840
AGGTCGAGGG CGGTGGTGTA GACGGCCCGG GCGGTCTCGG GGGCGGGTGC CCGGCCGACG 900
GCGTCGGTGA CGAACTCCAG GCCCCACAGC AGTCCGAGGC CGCGTACCTG GCCGAGCTGG 960
GGGAAGCGGG ACTCCAGGGC GCGCAGCCGC TCCTGGATGA GCTCGCCGAG GACGCGCACG 1020
CGGTCGATCA GCCGGTCGCG CTCGACGACC TCCAGCGTGG CGCGGGCGGC GGCGATCCCC 1080
AGTGGGTTGC TCGCGTACGT CGAGGCGTAC GCCCCGGGGT GGCCGCCTCC GGCCTGCGCA 1140
GCTTCCGCGC GTCCGGCCAG CACGGCGAAG GGGAATCCGC TCGCGGTGCC CTTGGACAGC 1200
ATCGCCAGGT CCGGCTCGAT GCCGAACAGT TCGCTGGCGA GGAAGGCGCC GGTGCGCCCG 1260
CCGCCGGTGA GGACCTCGTC GGCGACGAGC AGCACGCCGC CGTCCCGGCA GGCGCCGGCG 1320
ATCCGCTCCC AGTAGCCGGG GGGCGGCACG ATGACGCCTG CCGCGCCGAG GACGGGTTCG 1380
AAGACCAGGG CCGAGACGTT GGGCTTCTCC GCGATGTGCC GGCGCACGAG GGTCGCGCAC 1440
CGCACGTCGC ACGAGGGGTA CTCCAGGCCC AGGGGACAGC GGTAGCCAGT AGGGGCTGTA 1500
GCCAGCACGC TGTTGCCGCT GAAGGCCTGG TGGCCGATGT CCCAGTGGAC CAGCATCCGG 1560
GCGCCCATGG TCTTGCCGTG GAAGCCGTGG CGCAGGGCGC AGATCCGGTT GCGGCCCGGC 1620
GCGGCGGTCG CCTGGACGAC CCGCAGGGCG GCCTCGACCA CCTCCGCGCC GGTGGAGAAG 1680
AAGGCGTAGG TGTCGAGCTG TTCGGGCAGC AGCCTGGCGA GCAGTTCCAG CAGGCCGGCG 1740
CGGTCCGGCG TGGCGCTGTC GTGGACGTTC CACAGGCGGC GGGCCTGGGT GGTGAGTGCC 1800
TCGACGACCT CCGGGTGCCC GTGGCCCAGT GACTGGGTGA GGGTCCCGGC CGCGAAGTCG 1860
AGGTACTGGT TGCCGTCCAG GTCGGTCAGA ACGGGACCGC GTCCCTCGGC GAAGACCCGG 1920
CGTCCGTGGA CGGCTTCCTC GGAGGCGCCC GGCGCCAGGT GGCGGGCCTC CCGTGCCAGG 1980
TGCTGTGTCT GCCGTAAGCC TGTCATCGCT GCCTCTGCTC GTCGGACCGG CTGACGCGAT 2040
CGCCGGCGAA CTGCGTTGTG GCGCACCACG GTTGGGGCGG CTCGGCGCTG AGTCAAACAC 2100
TTGAACACAC ACCGCTGCAA GAGTTTGCGG GTTGTTTCAG AAAGTTGTTG CGAGCGGCCC 2160
CGGCACTCTG GTTGAGTCGA CGTGCTTACG GCGCCACCAC GCCTCACGTT CGAGGAGGGA 2220
CCTGTGAGAA CAAGCCCGCA GACCGACCCG CTCCCGCGGA GGCCGAGGTG AAGGCCCTGG 2280
TCCTGGCAGG TGGAACCGGC AGCAGACTGA GGCCGTTCAC CCACACCGCC GCCAAGCAGC 2340
TGCTCCCCAT CGCCAACAAG CCCGTGCTCT TCTACGCGCT GGAGTCCCTC GCCGCGGCGG 2400
GTGTCCGGGA GGCCGGCGTC GTCGTGGGCG CGTACGGCCG GGAGATCCGC GAACTCACCG 2460
GCGACGGCAC CGCGTTCGGG TTACGCATCA CCTACCTCCA CCAGCCCCGC CCGCTCGGTC 2520
TCGCGCACGC GGTGCGCATC GCCCGCGGCT TCCTGGGCGA CGACGACTTC CTGCTGTACC 2580
TGGGGGACAA CTACCTGCCC CAGGGCGTCA CCGACTTCGC CCGCCAATCG GCCGCCGATC 2640
CCGCGGCGGC CCGGCTGCTG CTCACCCCGG TCGCGGACCC GTCCGCCTTC GGCGTCGCGG 2700
AGGTCGACGC GGACGGGAAC GTGCTGCGCT TGGAGGAGAA ACCCGACGTC CCGCGCAGCT 2760
CGCTCGCGCT CATCGGCGTG TACGCCTTCA GCCCGGCCGT CCACGAGGCG GTACGGGCCA 2820
TCACCCCCTC CGCCCGCGGC GAGCTGGAGA TCACCCACGC CGTGCAGTGG ATGATCGACC 2880
GGGGCCTGCG CGTACGGGCC GAGACCACCA CCCGGCCCTG GCGCGACACC GGCAGCGCGG 2940
AGGACATGCT GGAGGTCAAC CGTCACGTCC TGGACGGACT GGAGGGCCGC ATCGAGGGGA 3000
AGGTCGACGC GCACAGCACG CTGGTCGGCC GGGTCCGGGT GGCCGAAGGC GCGATCGTGC 3060
GGGGGTCACA CGTGGTGGGC CCGGTGGTGA TCGGCGCGGG TGCCGTCGTC AGCAACTCCA 3120
GTGTCGGCCC GTACACCTCC ATCGGGGAGG ACTGCCGGGT CGAGGACAGC GCCATCGAGT 3180
ACTCCGTCCT GCTGCGCGGC GCCCAGGTCG AGGGGGCGTC CCGCATCGAG GCGTCCCTCA 3240
TCGGCCGCGG CGCCGTCGTC GGCCCGGCCC CCCGTCTCCC GCAGGCTCAC CGACTGGTGA 3300
TCGGCGACCA CAGCAAGGTG TATCTCACCC CATGACCACG ACCATCCTCG TCACCGGCGG 3360
AGCGGGCTTC ATTCGCTCCG CCTACGTCCG CCGGCTCCTG TCGCCCGGGG CCCCCGGCGG 3420
CGTCGCGGTG ACCGTCCTCG ACAAACTCAC CTACGCCGGC AGCCTCGCCC GCCTGCACGC 3480
GGTGCGTGAC CATCCCGGCC TCACCTTCGT CCAGGGCGAC GTGTGCGACA CCGCGCTCGT 3540
CGACACGCTG GCCGCGCGGC ACGACGACAT CGTGCACTTC GCGGCCGAGT CGCACGTCGA 3600
CCGCTCCATC ACCGACAGCG GTGCCTTCAC CCGCACCAAC GTGCTGGGCA CCCAGGTCCT 3660
GCTCGACGCC GCGCTCCGCC ACGGTGTGCG CACCTTCGTG CACGTCTCCA CCGACGAGGT 3720
GTACGGCTCC CTCCCGCACG GGGCCGCCGC GGAGAGCGAC CCCCTGCTTC CGACCTCGCC 3780
GTACGCGGCG TCGAAGGCGG CCTCGGACCT CATGGCGCTC GCCCACCACC GCACCCACGG 3840
CCTGGACGTC CGGGTGACCC GCTGTTCGAA CAACTTCGGC CCCCACCAGC ATCCCGAGAA 3900
GCTCATACCG CGCTTCCTGA CCAGCCTCCT GTCCGGCGGC ACCGTTCCCC TCTACGGCGA 3960
CGGGCGGCAC GTGCGCGACT GGCTGCACGT CGACGACCAC GTCAGGGCCG TCGAACTCGT 4020
CCGCGTGTCG GGCCGGCCGG GAGAGATCTA CAACATCGGG GGCGGCACCT CGCTGCCCAA 4080
CCTGGAGCTC ACGCACCGGT TGCTCGCACT GTGCGGCGCG GGCCCGGAGC GCATCGTCCA 4140
CGTCGAGAAC CGCAAGGGGC ACGACCGGCG CTACGCGGTC GACCACAGCA AGATCACCGC 4200
GGAACTCGGT TACCGGCCGC GCACCGACTT CGCGACCGCG CTGGCCGACA CCGCGAAGTG 4260
GTACGAGCGG CACGAGGACT GGTGGCGTCC CCTGCTCGCC GCGACATGAC GTCGGGCCGG 4320
ACCGCAACCA CCGGCCCCGG CCGGCACACC GCCGCCCGCG GCCGGTGGCC GGCCGGTCAG 4380
CGTCCGTGAG CCGGGCGCCG GCCGCCCCGC GGGCCGGCGG CGGTGGACCC CCGGACCACC 4440
AGTTCCGGCA TGAAGACGAA TTCGGTGCGC GGCGGCGGCG TTCCGCTCAT CTCCTCCAGC 4500
AGTGCGTCCA CGGCGACCTG CCCCATCGCC TTGACGGGCT GTCTGATGGT GGTCAGGGGA 4560
GGGTCGGTGA AGGCCATGAG CGGCGAGTCG TCGAAGCCGA CCACCGAGAT GTCACCGGGA 4620
ACCGTGAGAC CCCGCCGGCG CGCGGCCCGC ACGGCGCCGA GGGCCATCAT GTCGCTGGCG 4680
CACATGACGG CGGTGCAGCC CAGGTCGATC AGCGCGGACG CGGCGGCCTG GCCCCCCTCC 4740
AGGGAGAACA GCGAGTGCTG CACGAGCTCC TCGGACTCCC GCGCCGACAC TCCCAGGTGC 4800
TCCCGCACGC CGGCCCGGAA CCCCTCGATC TTCCGCTGCA CCGGCACGAA GCGGGCGGGC 4860
CCGACGGCGA GGCCGACGCG CTCGTGCCCC AGCTCCGCCA GGTGCGCCAC GGCCAGGCGC 4920
ATCGCGGCCC GGTCGTCCGG GGAGACGAAG GGTGCCTCGA TCCGGGGCGA GAACCCGTTC 4980
ACGAGGACGA AGGGCACCTG CCGCTCGTGC AGCCGGCCGT ACCGTCCGGT CTCGGCGGTG 5040
GTGTCCGCGT GCAGTCCGGA GACGAAGATG ATGCCGGACA CCCCGCGGTC CACGAGCATC 5100
TCCGTGAGTT CGTCCTCGGT CGAGCCGCCC GGGGTCTGCG TGGCGAGCAC GGGCGTGTAG 5160
CCCTGACGCG TGAGCGCCTG CCCCATCACC TGGGCCAGTG CGGGGAAGAA GGGGTTGTCC 5220
AGTTCGGGGG TGACCAGTCC GACCAGCTCG GCGCGGCGCT GTCGCGCCGG CTGCTCGTAG 5280
CCCAGCGCGT CCAGTGCGGT CAGCACCGAG TCGCGGGTGC CGGTGGCCAC ACCGCGCGCA 5340
CCGTTCAGCA CCCGGCTGAC CGTGGCCTTG CTGACGCCCG CCCGGGCTGC GATGTCGGCG 5400
AGCCGCATGG TCATGGCAAC GCACTCTACC TGTCGGGGCG TCAGGGCGTG CCCACCGCGC 5460
GCGGAACCGG CGGACTGCGG GGCACGGCCC GTCCGCCGCC CACGGACCAC GCGCCCGAAA 5520
CGATGGCTGA AAATGCTTGC AGCAAATTGC CGCAACGTCT TTCGGCGGCT TTTCGATCCT 5580
GTTACGTTCC TGGCAACCCC GGCGCCGCGC AGAAGCGGTT GGCGTGAGGC GTCCAGACCT 5640
CCGCCCGATT CCGGGATCAC TCAGGGGAGT TCACAATGCG GCGTGGCATT GCGGCCACCG 5700
CGCTGTTCGC GGCTGTGGCC ATGACGGCAT CGGCGTGTGG CGGGGGCGAC AACGGCGGAA 5760
GCGGTACCGA CGCGGGCGGC ACGGAGCTGT CGGGGACCGT CACCTTCTGG GACACGTCCA 5820
ACGAAGCCGA GAAGGCGACG TACCAGGCCC TCGCGGAGGG CTTCGAGAAG GAGCACCCGA 5880
AGGTCGACGT CAAGTACGTC AACGTCCCGT TCGGCGAGGC GAACGCCAAG TTCAAGAACG 5940
CCGCGGGCGG CAACTCCGGT GCCCCGGACG TGATGCGTAC GGAGGTCGCC TGGGTCGCGG 6000
ACTTCGCCAG CATCGGCTAC CTCGCCCCGC TCGACGGCAC GCCCGCCCTC GACGACGGGT 6060
CGGACCACCT TCCCCAGGGC GGCAGCACCA GGTACGAGGG GAAGACCTAC GCGGTCCCGC 6120
AGGTGATCGA CACCCTGGCG CTCTTCTACA ACAAGGAACT GCTGACGAAG GCCGGTGTCG 6180
AGGTGCCGGG CTCCCTCGCC GAGCTGAAGA CGGCCGCCGC CGAGATCACC GAGAAGACCG 6240
GCGCGAGCGG CCTCTACTGC GGGGCGACGA CCCGTACTTG GTTCCTGCCC TACCTCTACG 6300
GGGAGGGCGG CGACCTGGTC GACGAGAAGA ACAAGACCGT CACGGTCGAC GACGAAGCCG 6360
GTGTGCGCGC CTACCGCGTC ATCAAGGACC TCGTGGACAG CAAGGCGGCC ATCACCGACG 6420
CGTCCGACGG CTGGAACAAC ATGCAGAACG CCTTCAAGTC GGGCAAGGTC GCCATGATGG 6480
TCAACGGCCC CTGGGCCATC GAGGACGTCA AGGCGGGAGC CCGCTTCAAG GACGCCGGCA 6540
ACCTGGGGGT CGCCCCCGTC CCGGCCGGCA GTGCCGGACA GGGCTCTCCC CAGGGCGGGT 6600
GGAACCTCTC GGTGTACGCG GGCTCGAAGA ACCTCGACGC CTCCTACGCC TTCGTGAAGT 6660
ACATGAGCTC CGCCAAGGTG CAGCAGCAGA CCACCGAGAA GCTGAGCCTG CTGCCCACCC 6720
GCACGTCCGT CTACGAGGTC CCGTCCGTCG CGGACAACGA GATGGTGAAG TTCTTCAAGC 6780
CGGCCGTCGA CAAGGCCGTC GAACGGCCGT GGATCGCCGA GGGCAATGCC CTCTTCGAGC 6840
CGATCCGGCT GCAG 6854

240 amino acids

amino acid

linear

protein

8
Val Ile Val Ala Glu His Leu Val Lys Glu Phe Arg Leu Ala Glu Arg
1 5 10 15
Glu Pro Gly Leu Leu Gly Ser Leu Ser Thr Leu Leu Thr Arg Arg Tyr
20 25 30
Arg Val Val Arg Ala Val Asp Asp Val Ser Phe Glu Ile Pro Ala Gly
35 40 45
Thr Lys Thr Ala Tyr Ile Gly Ala Asn Gly Ala Gly Lys Ser Thr Thr
50 55 60
Ile Lys Met Leu Thr Gly Ile Met Thr Pro Thr Ser Gly Arg Cys Thr
65 70 75 80
Val Ala Gly Leu Glu Pro Tyr Arg His Arg Gln Arg Asn Ala Arg Thr
85 90 95
Ile Gly Val Val Phe Gly Gln Arg Ser Gln Leu Trp Trp Asp Leu Ser
100 105 110
Val Pro Asp Ser Phe Arg Ile Leu Arg Arg Ile Tyr Asp Ile Pro Gly
115 120 125
Pro Val Tyr Arg Arg Asn Leu Ala Leu Phe Arg Asp Leu Leu Asp Ile
130 135 140
Asp Ala Leu Gly Ser Thr Pro Val Arg Gln Leu Ser Leu Gly Gln Arg
145 150 155 160
Met Arg Ala Glu Ile Ala Ala Ser Leu Leu His Asp Pro Ala Val Leu
165 170 175
Phe Trp Asp Glu Pro Thr Ile Gly Leu Asp Met Val Leu Lys Asp Ala
180 185 190
Val Arg Arg Leu Val Asn Arg Ala His Arg Glu Leu Gly Thr Thr Val
195 200 205
Val Leu Thr Ser His Asp Ile Ala Asp Ile Ala Ala Ile Cys Asp Ser
210 215 220
Ala Leu Val Val Asp Gln Gly Arg Val Val His Gln Gly Thr Leu Gln
225 230 235 240

429 amino acids

amino acid

linear

protein

9
Met Thr Gly Leu Arg Gln Thr Gln His Leu Ala Arg Glu Ala Arg His
1 5 10 15
Leu Ala Pro Gly Ala Ser Glu Glu Ala Val His Gly Arg Arg Val Phe
20 25 30
Ala Glu Gly Arg Gly Pro Val Leu Thr Asp Leu Asp Gly Asn Gln Tyr
35 40 45
Leu Asp Phe Ala Ala Gly Thr Leu Thr Gln Ser Leu Gly His Gly His
50 55 60
Pro Glu Val Val Glu Ala Leu Thr Thr Gln Ala Arg Arg Leu Trp Asn
65 70 75 80
Val His Asp Ser Ala Thr Pro Asp Arg Ala Gly Leu Leu Glu Leu Leu
85 90 95
Ala Arg Leu Leu Pro Glu Gln Leu Asp Thr Tyr Ala Phe Phe Ser Thr
100 105 110
Gly Ala Glu Val Val Glu Ala Ala Leu Arg Val Val Gln Ala Thr Ala
115 120 125
Ala Pro Gly Arg Asn Arg Ile Cys Ala Leu Arg His Gly Phe His Gly
130 135 140
Lys Thr Met Gly Ala Arg Met Leu Val His Trp Asp Ile Gly His Gln
145 150 155 160
Ala Phe Ser Gly Asn Ser Val Leu Ala Thr Ala Pro Thr Gly Tyr Arg
165 170 175
Cys Pro Leu Gly Leu Glu Tyr Pro Ser Cys Asp Val Arg Cys Ala Thr
180 185 190
Leu Val Arg Arg His Ile Ala Glu Lys Pro Asn Val Ser Ala Leu Val
195 200 205
Phe Glu Pro Val Leu Gly Ala Ala Gly Val Ile Val Pro Pro Pro Gly
210 215 220
Tyr Trp Glu Arg Ile Ala Gly Ala Cys Arg Asp Gly Gly Val Leu Leu
225 230 235 240
Val Ala Asp Glu Val Leu Thr Gly Gly Gly Arg Thr Gly Ala Phe Leu
245 250 255
Ala Ser Glu Leu Phe Gly Ile Glu Pro Asp Leu Ala Met Leu Ser Lys
260 265 270
Gly Thr Ala Ser Gly Phe Pro Phe Ala Val Leu Ala Gly Arg Ala Glu
275 280 285
Ala Ala Gln Ala Gly Gly Gly His Pro Gly Ala Tyr Ala Ser Thr Tyr
290 295 300
Ala Ser Asn Pro Leu Gly Ile Ala Ala Ala Arg Ala Thr Leu Glu Val
305 310 315 320
Val Glu Arg Asp Arg Leu Ile Asp Arg Val Arg Val Leu Gly Glu Leu
325 330 335
Ile Gln Glu Arg Leu Arg Ala Leu Glu Ser Arg Phe Pro Gln Leu Gly
340 345 350
Gln Val Arg Gly Leu Gly Leu Leu Trp Gly Leu Glu Phe Val Thr Asp
355 360 365
Ala Val Gly Arg Ala Pro Ala Pro Glu Thr Ala Arg Ala Val Tyr Thr
370 375 380
Thr Ala Leu Asp Leu Gly Leu Arg Thr Ser Leu Gly Gly His Ile Leu
385 390 395 400
Arg Leu Ala Pro Pro Phe Thr Leu Asp Glu Ala Leu Leu Asp Glu Gly
405 410 415
Leu Arg Leu Leu Glu Thr Ala Val Glu Arg Val Ile Ala
420 425

355 amino acids

amino acid

linear

protein

10
Val Lys Ala Leu Val Leu Ala Gly Gly Thr Gly Ser Arg Leu Arg Pro
1 5 10 15
Phe Thr His Thr Ala Ala Lys Gln Leu Leu Pro Ile Ala Asn Lys Pro
20 25 30
Val Leu Phe Tyr Ala Leu Glu Ser Leu Ala Ala Ala Gly Val Arg Glu
35 40 45
Ala Gly Val Val Val Gly Ala Tyr Gly Arg Glu Ile Arg Glu Leu Thr
50 55 60
Gly Asp Gly Thr Ala Phe Gly Leu Arg Ile Thr Tyr Leu His Gln Pro
65 70 75 80
Arg Pro Leu Gly Leu Ala His Ala Val Arg Ile Ala Arg Gly Phe Leu
85 90 95
Gly Asp Asp Asp Phe Leu Leu Tyr Leu Gly Asp Asn Tyr Leu Pro Gln
100 105 110
Gly Val Thr Asp Phe Ala Arg Gln Ser Ala Ala Asp Pro Ala Ala Ala
115 120 125
Arg Leu Leu Leu Thr Pro Val Ala Asp Pro Ser Ala Phe Gly Val Ala
130 135 140
Glu Val Asp Ala Asp Gly Asn Val Leu Arg Leu Glu Glu Lys Pro Asp
145 150 155 160
Val Pro Arg Ser Ser Leu Ala Leu Ile Gly Val Tyr Ala Phe Ser Pro
165 170 175
Ala Val His Glu Ala Val Arg Ala Ile Thr Pro Ser Ala Arg Gly Glu
180 185 190
Leu Glu Ile Thr His Ala Val Gln Trp Met Ile Asp Arg Gly Leu Arg
195 200 205
Val Arg Ala Glu Thr Thr Thr Arg Pro Trp Arg Asp Thr Gly Ser Ala
210 215 220
Glu Asp Met Leu Glu Val Asn Arg His Val Leu Asp Gly Leu Glu Gly
225 230 235 240
Arg Ile Glu Gly Lys Val Asp Ala His Ser Thr Leu Val Gly Arg Val
245 250 255
Arg Val Ala Glu Gly Ala Ile Val Arg Gly Ser His Val Val Gly Pro
260 265 270
Val Val Ile Gly Ala Gly Ala Val Val Ser Asn Ser Ser Val Gly Pro
275 280 285
Tyr Thr Ser Ile Gly Glu Asp Cys Arg Val Glu Asp Ser Ala Ile Glu
290 295 300
Tyr Ser Val Leu Leu Arg Gly Ala Gln Val Glu Gly Ala Ser Arg Ile
305 310 315 320
Glu Ala Ser Leu Ile Gly Arg Gly Ala Val Val Gly Pro Ala Pro Arg
325 330 335
Leu Pro Gln Ala His Arg Leu Val Ile Gly Asp His Ser Lys Val Tyr
340 345 350
Leu Thr Pro
355

325 amino acids

amino acid

linear

protein

11
Met Thr Thr Thr Ile Leu Val Thr Gly Gly Ala Gly Phe Ile Arg Ser
1 5 10 15
Ala Tyr Val Arg Arg Leu Leu Ser Pro Gly Ala Pro Gly Gly Val Ala
20 25 30
Val Thr Val Leu Asp Lys Leu Thr Tyr Ala Gly Ser Leu Ala Arg Leu
35 40 45
His Ala Val Arg Asp His Pro Gly Leu Thr Phe Val Gln Gly Asp Val
50 55 60
Cys Asp Thr Ala Leu Val Asp Thr Leu Ala Ala Arg His Asp Asp Ile
65 70 75 80
Val His Phe Ala Ala Glu Ser His Val Asp Arg Ser Ile Thr Asp Ser
85 90 95
Gly Ala Phe Thr Arg Thr Asn Val Leu Gly Thr Gln Val Leu Leu Asp
100 105 110
Ala Ala Leu Arg His Gly Val Arg Thr Phe Val His Val Ser Thr Asp
115 120 125
Glu Val Tyr Gly Ser Leu Pro His Gly Ala Ala Ala Glu Ser Asp Pro
130 135 140
Leu Leu Pro Thr Ser Pro Tyr Ala Ala Ser Lys Ala Ala Ser Asp Leu
145 150 155 160
Met Ala Leu Ala His His Arg Thr His Gly Leu Asp Val Arg Val Thr
165 170 175
Arg Cys Ser Asn Asn Phe Gly Pro His Gln His Pro Glu Lys Leu Ile
180 185 190
Pro Arg Phe Leu Thr Ser Leu Leu Ser Gly Gly Thr Val Pro Leu Tyr
195 200 205
Gly Asp Gly Arg His Val Arg Asp Trp Leu His Val Asp Asp His Val
210 215 220
Arg Ala Val Glu Leu Val Arg Val Ser Gly Arg Pro Gly Glu Ile Tyr
225 230 235 240
Asn Ile Gly Gly Gly Thr Ser Leu Pro Asn Leu Glu Leu Thr His Arg
245 250 255
Leu Leu Ala Leu Cys Gly Ala Gly Pro Glu Arg Ile Val His Val Glu
260 265 270
Asn Arg Lys Gly His Asp Arg Arg Tyr Ala Val Asp His Ser Lys Ile
275 280 285
Thr Ala Glu Leu Gly Tyr Arg Pro Arg Thr Asp Phe Ala Thr Ala Leu
290 295 300
Ala Asp Thr Ala Lys Trp Tyr Glu Arg His Glu Asp Trp Trp Arg Pro
305 310 315 320
Leu Leu Ala Ala Thr
325

345 amino acids

amino acid

linear

protein

12
Met Thr Met Arg Leu Ala Asp Ile Ala Ala Arg Ala Gly Val Ser Lys
1 5 10 15
Ala Thr Val Ser Arg Val Leu Asn Gly Ala Arg Gly Val Ala Thr Gly
20 25 30
Thr Arg Asp Ser Val Leu Thr Ala Leu Asp Ala Leu Gly Tyr Glu Gln
35 40 45
Pro Ala Arg Gln Arg Arg Ala Glu Leu Val Gly Leu Val Thr Pro Glu
50 55 60
Leu Asp Asn Pro Phe Phe Pro Ala Leu Ala Gln Val Met Gly Gln Ala
65 70 75 80
Leu Thr Arg Gln Gly Tyr Thr Pro Val Leu Ala Thr Gln Thr Pro Gly
85 90 95
Gly Ser Thr Glu Asp Glu Leu Thr Glu Met Leu Val Asp Arg Gly Val
100 105 110
Ser Gly Ile Ile Phe Val Ser Gly Leu His Ala Asp Thr Thr Ala Glu
115 120 125
Thr Gly Arg Tyr Gly Arg Leu His Glu Arg Gln Val Pro Phe Val Leu
130 135 140
Val Asn Gly Phe Ser Pro Arg Ile Glu Ala Pro Phe Val Ser Pro Asp
145 150 155 160
Asp Arg Ala Ala Met Arg Leu Ala Val Ala His Leu Ala Glu Leu Gly
165 170 175
His Glu Arg Val Gly Leu Ala Val Gly Pro Ala Arg Phe Val Pro Val
180 185 190
Gln Arg Lys Ile Glu Gly Phe Arg Ala Gly Val Arg Glu His Leu Gly
195 200 205
Val Ser Ala Arg Glu Ser Glu Glu Leu Val Gln His Ser Leu Phe Ser
210 215 220
Leu Glu Gly Gly Gln Ala Ala Ala Ser Ala Leu Ile Asp Leu Gly Cys
225 230 235 240
Thr Ala Val Met Cys Ala Ser Asp Met Met Ala Leu Gly Ala Val Arg
245 250 255
Ala Ala Arg Arg Arg Gly Leu Thr Val Pro Gly Asp Ile Ser Val Val
260 265 270
Gly Phe Asp Asp Ser Pro Leu Met Ala Phe Thr Asp Pro Pro Leu Thr
275 280 285
Thr Ile Arg Gln Pro Val Lys Ala Met Gly Gln Val Ala Val Asp Ala
290 295 300
Leu Leu Glu Glu Met Ser Gly Thr Pro Pro Pro Arg Thr Glu Phe Val
305 310 315 320
Phe Met Pro Glu Leu Val Val Arg Gly Ser Thr Ala Ala Gly Pro Arg
325 330 335
Gly Gly Arg Arg Pro Ala His Gly Arg
340 345

393 amino acids

amino acid

linear

protein

13
Met Arg Arg Gly Ile Ala Ala Thr Ala Leu Phe Ala Ala Val Ala Met
1 5 10 15
Thr Ala Ser Ala Cys Gly Gly Gly Asp Asn Gly Gly Ser Gly Thr Asp
20 25 30
Ala Gly Gly Thr Glu Leu Ser Gly Thr Val Thr Phe Trp Asp Thr Ser
35 40 45
Asn Glu Ala Glu Lys Ala Thr Tyr Gln Ala Leu Ala Glu Gly Phe Glu
50 55 60
Lys Glu His Pro Lys Val Asp Val Lys Tyr Val Asn Val Pro Phe Gly
65 70 75 80
Glu Ala Asn Ala Lys Phe Lys Asn Ala Ala Gly Gly Asn Ser Gly Ala
85 90 95
Pro Asp Val Met Arg Thr Glu Val Ala Trp Val Ala Asp Phe Ala Ser
100 105 110
Ile Gly Tyr Leu Ala Pro Leu Asp Gly Thr Pro Ala Leu Asp Asp Gly
115 120 125
Ser Asp His Leu Pro Gln Gly Gly Ser Thr Arg Tyr Glu Gly Lys Thr
130 135 140
Tyr Ala Val Pro Gln Val Ile Asp Thr Leu Ala Leu Phe Tyr Asn Lys
145 150 155 160
Glu Leu Leu Thr Lys Ala Gly Val Glu Val Pro Gly Ser Leu Ala Glu
165 170 175
Leu Lys Thr Ala Ala Ala Glu Ile Thr Glu Lys Thr Gly Ala Ser Gly
180 185 190
Leu Tyr Cys Gly Ala Thr Thr Arg Thr Trp Phe Leu Pro Tyr Leu Tyr
195 200 205
Gly Glu Gly Gly Asp Leu Val Asp Glu Lys Asn Lys Thr Val Thr Val
210 215 220
Asp Asp Glu Ala Gly Val Arg Ala Tyr Arg Val Ile Lys Asp Leu Val
225 230 235 240
Asp Ser Lys Ala Ala Ile Thr Asp Ala Ser Asp Gly Trp Asn Asn Met
245 250 255
Gln Asn Ala Phe Lys Ser Gly Lys Val Ala Met Met Val Asn Gly Pro
260 265 270
Trp Ala Ile Glu Asp Val Lys Ala Gly Ala Arg Phe Lys Asp Ala Gly
275 280 285
Asn Leu Gly Val Ala Pro Val Pro Ala Gly Ser Ala Gly Gln Gly Ser
290 295 300
Pro Gln Gly Gly Trp Asn Leu Ser Val Tyr Ala Gly Ser Lys Asn Leu
305 310 315 320
Asp Ala Ser Tyr Ala Phe Val Lys Tyr Met Ser Ser Ala Lys Val Gln
325 330 335
Gln Gln Thr Thr Glu Lys Leu Ser Leu Leu Pro Thr Arg Thr Ser Val
340 345 350
Tyr Glu Val Pro Ser Val Ala Asp Asn Glu Met Val Lys Phe Phe Lys
355 360 365
Pro Ala Val Asp Lys Ala Val Glu Arg Pro Trp Ile Ala Glu Gly Asn
370 375 380
Ala Leu Phe Glu Pro Ile Arg Leu Gln
385 390

Number	Date	Country	Kind
196 22 783	Jun 1996	DE
97/02826	May 1997	WO

Number	Date	Country
22 09 834	Sep 1973	DE
EP 0 730 029	Sep 1996	DE

Isolation of biosynthesis genes for pseudo-oligosaccharides from Streptomyces glaucescens GLA.O, and their use

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