Title of Invention


Abstract 1. An expression vector comprising isolated nucleic acid sequence represented by SEQ ID NO 1 or variants or portions thereof, the said sequence encoding for a polypeptide useful in bio-synthesis of safracin or its analogue.
Full Text FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
(See section 10, rule 13)
PHARMA MAR, S.A., of Calle de la Calera 3, Poligono Industrial de Ires Cantos, Tres Cantos, E-28760 Madrid, Spain,
The following specification particularly describes the invention and the manner in which it is to be performed.


The present invention relates to an expression vector comprising nucleic acid sequence represented by SEQ ID NO 1 or variants or portions thereof, the said sequence encoding for a polypeptide useful in synthesis of safracin or its analogue. Further, the present invention relates to a gene cluster having open reading frames which encode polypeptides directing the synthesis of a safracin molecule. More particularly, the present invention relates to the gene cluster responsible for the biosynthesis of safracin, its uses for genetic enGlneering and new safracins obtained by manipulation of biosynthesis mechanism.
Safracins, a family of new compounds with a potent broad-spectrum antibacterial activity, were discovered in a culture broth of Pseudomonas sp. Safracin occurs in two Pseudomonas sp. strains, Pseudomonas fluorescens A2-2 isolated from a soil sample collected in Tagawagun, Fukuoka, Japan (Ikeda et al. /. Antibiotics 1983,36,1279-1283; WO 82 00146 and JP 58113192) and Pseudomonas fluorescens SC 12695 isolated from water samples taken from the Raritan-Delaware Canal, near New Jersey (Meyers et al. /. Antibiot. 1983,36 (2), 190-193). Safracins A and B, produced by Pseudomonas fluorescens A2-2, have been examined against different tumor cell lines and has been found to possess antitumor activity in addition to antibacterial activity.

SafracinsA R=H ET-743
Due to the structural similarities between safracin B and ET-743 safracin offers the possibility of hemi-synthesis of the highly promising potent new antitumor agent ET-743, isolated from the marine tunicate Ecteinascidia turbinata and which is currently in Phase II clinical trials in Europe and the United States. A hemisynthesis of ET-743 has been achieved starting from safracin B (Cuevas et al. Organic Lett. 2000, 10, 2545-2548; WO 00 69862 and WO 01 87895).
As an alternative of making safracins or its structural analogs by chemical synthesis, manipulating genes of governing secondary metabolism offer a promising alternative and allows for preparation of these compounds bio synthetically. Additionally, safracin structure offers exciting possibilities for combinatorial biosynthesis.
In view of the complex structure of the safracins and the limitations in their obtention from Pseudomonas fluorescens A2-2, it would be highly desirable to understand the genetic basis of their synthesis in order to create the means to influence them in a targeted manner. This could increase the amounts of safracins being produced, because natural

production strains generally yield only low concentrations of the secondary metabolites that are of interest. It could also allow the production of safracins in hosts that otherwise do not produce these compounds. Additionally, the genetic manipulation could be used for combinatorial creation of novel safracin analogs that could exhibit improved properties and that could be used in the hemi-synthesis of new ecteinascidins compounds.
However, the success of a biosynthetic approach depends critically on the availability of novel genetic systems and on genes encoding novel enzyme activities. Elucidation of the safracin gene cluster contributes to the general field of combinatorial biosynthesis by expanding the repertoire of genes uniquely associated with safracin biosynthesis, leading to the possibility of making novel precursors and safracins via combinatorial biosynthesis.
We have now been able to identify and clone the genes of safracin ) Jbiosynthesis providing the genetic basis for improving and manipulating : in a targeted manner the productivity of Pseudomonas sp., and using genetic methods, for synthesising safracin analogues. Additionally, these genes encode enzymes that are involved in biosynthetic processes to produce structures, such as safracin precursors, that can form the basis of combinatorial chemistry to produce a wide variety of compounds. These compounds can be screened for a variety of bioactivities including anticancer activity.

Therefore in a first aspect the present invention provides a nucleic acid, suitably an isolated nucleic acid, which includes a DNA sequence (including mutations or variants thereof), that encodes non-ribosomal peptide synthetases which are responsible for the biosynthesis of safracins. This invention provides a gene cluster, suitably an isolated i gene cluster, with open reading frames encoding polypeptides to direct the ' assembly of a safracin molecule.
One aspect of the present invention is a composition including at least one nucleic acid sequence, suitably an isolated nucleic acid molecule, that encodes at least one polypeptide that catalyses at least one step of the biosynthesis of safracins. Two or more such nucleic acid sequences can be present in the composition. DNA or corresponding RNA is also provided.
In particular the present invention is directed to a nucleic acid sequence, suitably an isolated nucleic acid sequence, from a safracin gene cluster comprising said nucleic acid sequence, a portion or portions of said nucleic acid sequence wherein said portion or portions encode a polypeptide or polypeptides or a biologically active fragment of a polypeptide or polypeptides, a single-stranded nucleic acid sequence derived from said nucleic acid sequence, or a single stranded nucleic acid sequence derived from a portion or portions of said nucleic acid sequence, or a double-stranded nucleic acid sequence derived from the single-stranded nucleic acid sequence (such as cDNA from mRNA). The nucleic acid sequence can be DNA or RNA.
More particularly, the present invention is directed to a nucleic acid sequence, suitably an isolated nucleic acid sequence, which includes or comprises at least SEQ ID 1, variants or portions thereof, or at least one of

the sacA, sacB, sacC, sacC, sacD, sacE, sacF, sacG, sacH, sacH, sad, sacJ, orfl, orf2, orf3 or orf4 genes, including variants or portions. Portions can be at least 10, 15, 20, 25, 50, 100, 1000, 2500, 5000, 10000, 20000, 25000 or more nucleotides in length. Typically the portions are in the range 100 to 5000, or 100 to 2500 nucleotides in length, and are biologically functional.
Mutants or variants include polynucleotide molecules in which at least one nucleotide residue is altered, substituted, deleted or inserted. Multiple changes are possible, with a different nucleotide at 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, 200, 500 or more positions. Degenerate variants are envisaged which encode the same polypeptide, as well as non-degenerate variants which encode a different polypeptide. The portion, mutant or variant nucleic acid sequence suitably encodes a polypeptide which retains a biological activity of the respective polypeptide encoded by any of the open reading frames of the safracin gene cluster. Allelic forms and polymorphisms are embraced.
The invention is also directed to an isolated nucleic acid sequence capable of hybridizing under stringent conditions with a nucleic acid sequence of this invention. Particularly preferred is hybridisation with a translatable length of a nucleic acid sequence of this invention.
The invention is also directed to a nucleic acid encoding a polypeptide which is at least 30%, preferably 50%, preferably 60%, more preferably 70%, in particular 80%, 90%, 95% or more identical in amino acid sequence to a polypeptide encoded by any of the safracin gene cluster open reading frames sacA to sacJ and orfl to orf4 (SEQ ID 1 and genes encoded in SEQ ID 1) or encoded by a variant or portion thereof. The polypeptide suitably retains a biological activity of the respective polypeptide encoded by any of the safracin gene cluster open reading

In particular, the invention is directed to an isolated nucleic acid sequence encoding for any of SacA, SacB, SacC, SacD, SacE, SacF, SacG, SacH, Sad, SacJ, Orfl, 0rf2, Orf3 or 0rf4 proteins (SEQ ID 2-15), and variants, mutants or portions thereof.
In one aspect, an isolated nucleic acid sequence of this invention encodes a peptide synthetase, a L-Tyr derivative hidroxylase, a L-Tyr derivative methylase, a L-Tyr O-methylase, a methyl-transferase or a monooxygenase or a safracin resistance protein.
The invention also provides a hybridization probe which is a nucleic acid sequence as defined above or a portion thereof. Probes suitably comprise a sequence of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, or more nucleotide residues. Sequences with a length on the range 25 to 60 are preferred. The invention is also directed to the use of a probe as defined for the detection of a safracin or ecteinascidin gene. In particular, the probe is used for the detection of genes in Ecteinascidia turbinata.
In a related aspect the invention is directed to a polypeptide encoded by a nucleic acid sequence as defined above. Full sequence, variant, mutant or fragment polypeptides are envisaged.
In a further aspect the invention is directed to a vector, preferably an expression vector, preferably a cosmid, comprising a nucleic acid sequence encoding a protein or biologically active fragment of a protein, wherein said nucleic acid is as defined above.
In another aspect the invention is directed to a host cell transformed with one or more of the nucleic acid sequences as defined above, or a

vector, an expression vector or cosmid as defined above. A preferred host cell is transformed with an exogenous nucleic acid comprising a gene cluster encoding polypeptides sufficient to direct the assembly of a safracin or safracin analog. Preferably the host cell is a microorganism, more preferably a bacteria.
The invention is also directed to a recombinant bacterial host cell in which at least a portion of a nucleic acid sequence as defined above is disrupted to result in a recombinant host cell that produces altered levels of safracin compound or safracin analogue, relative to a corresponding nonrecombinant bacterial host cell.
The invention is also directed to a method of producing a safracin compound or safracin analogue comprising fermenting, under conditions and in a medium suitable for producing such a compound or analogue, an organism such as Pseudomonas sp, in which the copy number of the safracin genes/cluster encoding polypeptides sufficient to direct the assembly of a safracin or safracin analog has been increased.
The invention is also directed to a method of producing a safracin compound or analogue comprising fermenting, under conditions and in a medium suitable for producing such compound or analogue, an organism such as Pseudomonas sp in which expression of the genes encoding polypeptides sufficient to direct the assembly of a safracin or safracin analogue has been modulated by manipulation or replacement of one or more genes or sequence responsible for regulating such expression. Preferably expression of the genes is enhanced.
The invention is also directed to the use of a composition including at least one isolated nucleic acid sequence as defined above or a modification thereof for the combinatorial biosynthesis of non-ribosomal

peptides, diketopiperazine rings and safracins.
In particular the method involves contacting a compound that is a substrate for a polypeptide encoded by one or more of the safracin biosynthesis gene cluster open reading frames as defined above with the polypeptide encoded by one or more safracin biosynthesis gene cluster open reading frames, whereby the polypeptide chemically modifies the compound.
In still another embodiment, this invention provides a method of producing a safracin or safracin analog. The method involves providing a microorganism transformed with an exogenous nucleic acid comprising a safracin gene cluster encoding polypeptides sufficient to direct the assembly of said safracin or safracin analog; culturing the bacteria junder conditions permitting the biosynthesis of safracin or safracin analog; and isolating said safracin or safracin analog from said cell.
The invention is also directed to any of the precursor compounds P2, P14, analogs and derivatives thereof and their use in the combinatorial biosynthesis non-ribosomal peptides, diketopiperazine rings and safracins.
Additionally, the invention is also directed to the new safracins obtained by knock out safracin P19B, safracin P22A, safracin P22B, safracin D and safracin E, and their use as antimicrobial or antitumor agents, as well as their use in the synthesis of ecteinascidin compounds.
The invention is also directed to new safracins obtained by directed biosynthesis as defined_above, and their use as antimicrobial or antitumor agents, as well as their use in the synthesis of ecteinascidin compounds. In particular the invention is directed to safracin B-ethoxy and safracin A-ethoxy and their use.

In one aspect, the present invention enables the preparation of structures related to safracins and ecteinascidins which cannot or are difficult to prepare by chemical synthesis. Another aspect is to use the knowledge to gain access to the biosynthesis of ecteinascidins in Ecteinasddia turbinata, for example using these sequences or parts as probes in this organism or a putative symbiont.
More fundamentally, the invention opens a broad field and gives access to ecteinascidins by genetic enGlneering.
Fig. 1: Structural organization of the chromosomal DNA region cloned in pL30p cosmid. The region of P. fluorescens A2-2 DNA, containing the safracin gene cluster, is shown. Both, sacABCDEFGH and sacIJ, gene operons and the modular organization of the peptide synthetases deduced from sacA, sacB and sacC are illustrated. The following domains are indicated: C: condensation; T: thiolation; A: adenylation and Re: reductase. Location of other genes present in pL30p cosmid (orf 1 to orf4) as well as their proposed function is shown.
Fig. 2: Conserved core motifs between NRPSs. Conserved amino acid sequences in SacA, SacB and SacC proteins and their comparison with its homologous sequences from Myxococcus xanthus DM50415.
Figure 3. NRPS biosynthesis mechanism proposed for the formation of the Ala-Gly dipeptide. Step a*, adenylation of Ala; b*, transfer to the 4'-phosphopantetheinyl arm; c*, transfer to the waiting/elongation site; d*, adenylation of the Gly; e*, transfer to the 4'-phosphopantetheinyl arm; f*,

condensation of the elongation chain on the 4'-phosphopantetheinyl arm with the starter chain at the waiting/elongation site; g*, Ala-Gly dipeptide attached to the phosphopantetheinyl arm of SacA and h*, transfer of the elongated chain to the following waiting/elongation site.
Fig. 4: Cross-feeding experiments. A. Scheme of A2-2 DNA fragments cloned in pBBRl-MCS2 vector and products obtained in the heterologous host. B. HPLC profile of safracin production in wild type strain versus sacF mutant. The addition of P2 precursor to the sacF mutant, provided both in trans and synthetically, yield safracin B production. SfcA, safracin A and SfcB, safracin B.
Fig. 5: Scheme of the safracin biosynthesis mechanism and biosynthetic intermediates. Single enzymatic steps are indicated by a continuous arrow and multiple reactions steps are indicated by discontinuous arrows.
Fig. 6: Safracin gene disruptions and compounds produced. A. Gene disruption and precursor molecules synthesized by the mutants constructed. Gene marked with an asterisk does not belong to the safracin cluster. Inactivation of genes orf 1, orf 2, orf 3 and orf 4 has demonstrated to have no effect over safracin production. B. HPLC profile of safracin production in wild type strain and in sacA, sad and sacJ mutants. Structure of the different molecules obtained is shown.
Fig. 7: Structure of the different molecules obtained by gene disruption. Inactivation of SacJ protein (a) yields P22B, P22A and P19 molecules, whereas gene disruption of sacl (b), produces only P19 compound. The sad disruption, together with the sacJ reconstructed expression, produces two new safracins: safracin D (possible precursor for ET-729 hemi-synthesis) and safracin E (c).

Fig. 8: Addition of specific designed 'unnatural' precursors (P3). Chemical structure of the two molecules obtained by addition of P3 compound to the sacF mutant.
Fig. 9: Scheme of the gene disruption event through simple recombination, using an homologous DNA fragment cloned into pK18:MOB (an integrative plasmid in Pseudomonas).
Non ribosomal peptide synthetases (NRPS) are en2ymes responsible for the biosynthesis of a family of compounds that include a large number of structurally and functionally diverse natural products. For example, peptides with biological activities provide the structural backbone for compounds that exhibit a variety of biological activities such as, antibiotics, antiviral, antitumor, and immunosuppressive agents (Zuber et al. Biotechiology of Antibiotics 1997 (W. Strohl, ed.), 187-216 Marcel dekker, Inc., N.Y; Marahiel et al. Chem. Rev. 1997, 97, 2651-2673).
Although structurally diverse, most of these biologically active peptides share a common mechanistic scheme of biosynthesis. According to this model, peptide bond formation takes place on multienzymes designated peptides synthetases, on which amino acid substrates are activated by ATP hydrolysis to the corresponding adenylate. This unstable intermediate is subsequently transferred to another site of the multienzymes where it is bound as a thioester to the cysteamine group of an enzyme-bound 4'-phosphopantetheninyl (4'-PP) cofactor. At this stage, the thiol-activated substrates can undergo modifications such as epimerisation or N-methylation. Thioesterified substrate amino acids are then integrated into the peptide product through a step-by-step elongation by a series of

transpeptidation reactions. With this template arrangement in peptide synthetases, the modules seem to operate independently of one another, but they act in concert to catalyse the formation of successive peptide bonds (Stachelhaus et al. Science 1995, 269, 69-72; Stachelhaus et al. Chem. Biol. 1996, 3, 913-921). The general scheme for non-ribosomal peptide biosynthesis has been widely reviewed (Marahiel et al. Chem. Rev. 1997, 97, 2651-2673; Konz and Marahiel, Chem, and Biol 1999, 6, R39-R48; Moffit and Neilan, FEMS Microbiol. Letters 2000, 191, 159-167).
A large number of bacterial operons and fungal genes encoding peptide synthetases have recently been cloned, sequenced and partially characterized, providing valuables insights into their molecule architecture (Marahiel, Chem and Biol 1997, 4, 561-567). Different cloning strategies were used, including probing of expression libraries by antibodies raised against peptide synthetases, complementation of deficient mutants, and the use of designed oligonucleotides derived from amino acid sequences of peptide synthetase fragments.
Analysis of the primary structure of these genes revealed the presence of distinct homologous domains of about 600 amino acids. This specific functional domains consist of at least six highly conserved core sequences of about three to eight amino acids in length, whose order and location within all known domains are very similar (Kusard and Marahiel, Peptide Research 1994, 7, 238-241). The used of degenerated oligonucleotides derived from the conserved cores opens the possibility of identifying and cloning peptide synthetases from genomic DNA, by using the polymerase chain reaction (PCR) technology (Kusard and Marahiel, Peptide Research 1994, 7, 238-241; Borchert et al. FEMS Microbiol Letters 1992,92,175-180).
The structure of safracin suggests that this compound is synthesized

by a NRPS mechanism. The cloning and expression of the non-ribosomal peptide synthetases and the associated tailoring enzymes from Pseudomonas fluorescens A2-2 safracin cluster would allow production of unlimited amounts of safracin. In addition, the cloned genes could be used for combinatorial creation of novel safracin analogs that could exhibit improved properties and that could be used in the hemi-synthesis of new ecteinascidins. Moreover, cloning and expressing the safracin gene cluster in heterologous systems or the combination of safracin gene cluster with other NRPS genes could result in the creation of novel drugs with improved activities.
The present invention provides, in particular, the DNA sequence encoding NRPS responsible for biosynthesis of safracin, i.e., safracin synthetases. We have characterized a 26,705 bp region (SEQ ID NO:l) from Pseudomonas fluorescens A2-2 genome, cloned in pL30P cosmid and demonstrated, by knockout experiments and heterologous expression, that this region is responsible for the safracin biosynthesis. We expressed the pL30P cosmid in two strains of Pseudomonas sp., which do not produce safracin, and the result was a production of safracin A and B at levels of a 22%, for P. fluorescens (CECT 378), and 2%, for P. aeruGlnosa (CECT 110), in comparison with P. fluorescens A2-2 production. The predicted amino acids sequences of the various peptides encoded by this DNA sequence is shown in SEQ ID NO:2 through SEQ ID NO: 15 respectively.
The gene cluster for safracin biosynthesis derived from P. fluorescens A2-2, is characterized by the presence of several open reading frames (ORF) that are organized in two divergent operons (Fig. 1), an eight genes operdh {sacABCDEFGH) and a two genes operon {sacIJ), preceded by well-conserved putative promoters regions that overlap. The safracin biosynthesis gene cluster is present in only one copy in P. fluorescens A2-2 genome.

propose that the Ala would be first charged on the phosphopantetheinyl arm of SacA (Fig. 3 a* and b*) before being transferred to a waiting position, a condensation domain, located in N-terminal of sacA (Fig. 3, c*). The Gly adenylate would then be charged on the same phosphopantetheinyl arm (Fig. 3, d* and e*), positioned to the elongation site, and elongation would occur (Fig. 3, f*). The arm of the first module would at this stage be charged with a Ala-Gly dipeptide (Fig. 3, g*). We proposed that the dipeptide would then be transferred on a waiting position in the second phosphopantetheinyl arm (Fig. 3, h*), located in SacB, to continue the synthesis of the safracin tetrapeptide basic skeleton. An alternative biosynthesis mechanism could be the direct incorporation of a dipeptide Ala-Gly into SacA. In this case, the dipeptide could be oriGlnated from the activity of highly active peptidyl transferase ribozyme family (Sun et al, Chem. and Biol 2002, 9, 619-626) or from the activity of bacterial proteolysis.
And fourthly, although in most of the prokaryotic peptide synthetases the thioesterase moiety, which appears to be responsible for the release of the mature peptide chain from the enzyme, is fused to the C-terminal end of the last amino acid binding module (Marahiel et al. Chem. Rev. 1997, 97, 2651-2673), in the case of safracin synthetases, the TE domain is missing. Probably, in the safracin synthesis after the last elongation step, the tetrapeptide could be released by an alternative strategy for peptide-chain termination that also occurs in the saframycin synthesis (Pospiech et al. Microbiol 1996, 142, 741-746). This particular termination strategy is catalysed by a reductase domain at the carboxy-terminal end of the SacC peptide synthetase which catalyses the reductive cleavage of the associated T-domain-tethered acyl group, releasing a linear aldehyde.
Our cross feeding experiments indicate that the last two amino acids incorporated into the safracin molecule are two L-Tyr derivatives called P2

(3-hydroxy-5-methyl-0-methyltyrosine) (Fig. 4, S), instead of two L-Tyr as it is proposed to occur in saframycin synthesis. First, the products of two genes (sacF and sacG), similar to bacterial methyltransferases, have shown to be involved in the O, C-methylation of L-Tyr to produce P14 (3-methyl-Omethyltyrosine), precursor of P2. A possible mechanism could envisage that the O-methylation occurs first and then the C-methylation of the amino acid derivative is produced. Secondly, P2, the substrate for the peptide synthetases SacB and SacC, is formed by the hydroxylation of P14 by SacD (Fig. 4, 5).

P-14 P-2
Apart from the safracin biosynthetic genes, in the sacABCDEFGH operon there are also found two genes, sacE and sacH, involved in an unknown function and in the safracin resistance mechanism, respectively. We have demonstrated that sacH gene codes for a protein that when is heterologous expressed, in different Pseudomonas strains, a highly increase of the safracin B resistance is produced. SacH is a putative transmembrane protein, that transforms the C21-OH group of safracin B into a C21-H group, to produce safracin A, a compound with less antibiotic and antitumoral activity. Finally, even though still is unknown about the putative function of SacE, homologous of this gene have been found close

to various secondary metabolites biosynthetic gene clusters in some microorganisms genomes, suggesting a conserved function of this genes in secondary metabolite formation or regulation.
In the sacIJ operon, the deduced amino acid sequences encoded by sacl and sacJ strongly resemble gene products of methyltransferase and hydroxylase/monoxygenase, respectively. Our data reveals that Sacl is the enzyme responsible for the iV-methylation present in the safracin structure, and that SacJ is the protein which makes an additional hydroxylation on one of the L-Tyr derivative incorporated into the tetrapeptide to produce the quinone structure present in all safracin molecules. N-N-Methylation is one of the modifications of nonribosomally synthesized peptides that significantly contributes to their biological activity. Except for saframycin (Pospiech et al. Microbiol 1996, 142, 741-746), that is produced by bacteria and is N-methylated, all the N-methylated nonribosomal peptides .known are produced by fungi or actinomycetes and, in most of the cases, the responsible for the N-methylation is a domain which reside in the nonribosomal peptide synthetase.
Table I. Summary of safracin biosynthetic and resistance genes identified in cosmid pL30P.

ORF Protein Proposed function Position Amino acids Molecular
nam name start-stop bp weight
Peptide synthetase 3052-6063 1004 110.4
Peptide synthetase 6068-9268 1063 117.5
Peptide synthetase 9275-13570 1432 157.3
L-Tyr derivative 13602-14651 350 39.2
sacA SacA
sacB SacB sacC SacC sacD SacD

sacE SacE Unknown 14719-14901 61 6.7
sacF SacF L-Tyr derivative methylase 14962-16026 355 39.8
sacG SacG L-Tyr O-methylase ' 16115-17155 347 38.3
sacH SacH Resistance protein 17244-17783 180 19.6
sacl Sad methyl-transferase 2513-1854 220 24.2
sacJ SacJ monooxygenase 1861-355 509 55.3
The safracin putative synthetic pathway, with indications of the specific amino acid substrates used for each condensation reaction and the various post-condensation activities, is shown in Fig. S.
To further evaluate the role of safracin biosynthetic genes, we constructed knock out mutants of each of the genes of the safracin cluster (Fig. 6). The disruption of the NRPSs genes {sacA, sacB and sacQ as well as sacD, sacF and sacG, resulted in safracin and P2 non producing mutants. Our results indicate that the genes from sacA to sacH are part of the same genetic operon. As a consequence of the sacl and sacJ gene disruptions three new molecules were oriGlnated, P19B, P22A and P22B (Fig. 6).

The production of P22A and P22B (Fig. 7a*) by sacJ mutant demonstrated that the role of the SacJ protein is to produce the additional hydroxylation of the left L-Tyr derivatives amino acid of the safracin, the one involved in the quinone ring. The production of P19B (Pig. 7b*) by sad mutant, a safracin like molecule where the N-methylation and the quinone ring are missing, confirms that Sacl is the N-methyltransferase enzyme and suggests that sadJ is a transcriptional operon. The production of P19B also by sacJ mutant (Fig. 7a*) suggests that probably the N-methylation occurs after the quinone ring has been formed. Even though these new structures have no interesting antimicrobial activity on B. subtilis or no high citotoxic activity on cancer cells, they can serve as interesting new precursors for the hemisynthesis of new active molecules. As far as structure activity is concerned, the observation that P19B, P22A and P22B appear to loose their activity, suggests that the lost of the quinone ring from the safracin structure is directly related with the lost of activity of the safracin family molecules.
The safracin D and safracin E are safracin B and safracin A like
The disruption of sad gene with the reconstitution of the sacJ gene expression resulted in the production of two new safracins. The two antibiotics produced, at levels of production as high as the levels of safracin A/safracin B production in the wild type strain, have been named as safracin D and safracin E (Fig. 7c*).

molecules, respectively, where the N-methylation is missing. Both, safracin D and safracin E have been shown to possess the same antibacterial and antitumoral activities as safracin B and safracin A, respectively. Apart from its high activities properties, antibacterial and antitumoral, safracin D could be used in the hemi-synthesis of the ecteinascidin ET-729, a potent antitumoral agent, as well as in the hemi-synthesis of new ecteinascidins.
A question arises concerning the role of the aminopeptidase-like protein coded by a gene located at 3'site of the safracin operon. The insertional inactivation of orfl (PM-S1-14) showed no effect on safracin A/safracin B production. Because of its functionality properties it remains unclear if this protein could play some role in the safracin metabolism. The other genes present in the pL30P cosmid (or/2 to orf4) will have to be studied in more detail.
Another aspect of the invention is that provides the tools necessary for the production of new specific designed "unnatural" molecules. The addition of a specific modified P2 derivative precursor named P3, a 3-hydroxy-5-methyl-O-methylryrosine, to the sacE mutant yields two "unnatural" safracins that incorporated this specific modified precursor, safracin A(OEt) and safracin B(OEt) (Fig. 8).

The two new safracins are potent antibiotic and antitumoral compounds. The biological activities of safracin A(OEt) and Safracin B(OEt) are as potent as the activities of safracin A and safracin B, respectively. These new safracins could be the source for new potent antitumoral agents, as well as a source of molecules for the hemi-synthesis of new ecteinascidins.
In addition, the genes involved in safracin synthesis could be combined with other non ribosomal peptide synthetases genes to result in the creation of novel "unnatural" drugs and analogs with improved activities.
Example 1: Extraction of nucleic acid molecules from Pseudomonas fluorescens A2-2
Bacterial strains
Strains of Pseudomonas sp. were grown at 27°C in Luria-Bertani (LB) broth (Ausubel et al. 1995, J. Wiley and Sons, New York, N.Y). E. coli strains were grown at 37°C in LB medium. Antibiotics were used at the following concentrations: ampicillin (50 ng/ml), tetracycline (20 p-g/ml) and kanamycin (50 ug/ml).
Table II. Strains used in this invention.

Code Genotype
PM-S1 -001 P. fluorescens A2-2 wild type
PM-S1-002 sacA-
PM-S1-003 sacB
PM-S 1-004 sacC
PM-S 1-005 sacJ-
PM-S1-006 sacf
PM-S1-007 sad- with sacj expression reconstitution
PM-S 1-008 sacF-
PM-S1-009 sacG-
PM-S1- 010 sacD-
PM-S1- 014 orfl-
PM-S1-015 A2-2 + pLAFR3
PM-S1- 016 A2-2 + pL30p
PM-19- 001 P. fluorescens CECT378 + pLAFR3
PM-19- 002 P. fluorescens CECT378 + pL30p
PM-19- 003 P. fluorescens CECT378 + pBBRl-MCS2
PM-19- 004 P. fluorescens CECT378 + pB5H83
PM-19- 005 P. fluorescens CECT378 + pB7983
PM-19- 006 P. fluorescens CECT378 + pBHPT3
PM-16- 001 P. aeruGlnosa CECT110 + pLAFR3
PM-16- 002 P. aeruGlnosa CECT110 + pL30p
PM-17- 003 P. putida ATCC12633+ pBBRl-MCS2
PM-17- 004 P. putida ATCC 12633+ pB5H83
PM-17- 005 P. putida ATCC12633+ pB7983
PM-18- 003 P. stutzeri ATCC 17588+ pBBRl-MCS2
PM-18- 004 P. stutzeri ATCC 17588+ pB5H83
PM-18- 005 P. stutzeri ATCC 17588+ pB7983
DNA manipulation
Unless otherwise noted, standard molecular biology techniques for in vitro DNA manipulations and cloning were used (Sambrook et al. 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory).

DNA extraction
Total DNA from Pseudomonas fluorescens A2-2 cultures was prepared as reported (Sambrook et al. 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory).
Computer analysis
Sequence data were compiled and analysed using DNA-Star software package.
Example 2: Identification of NRPS genes responsible for safracin production in Pseudomonas fluorescens A2-2.
Primer design
Marahiel et al (Marahiel et al Chem. Rev. 1997, 97, 2651-2673) previously reported highly conserved core motifs of the catalytic domains of cyclic and branched peptide synthetases. Based on multiple sequence alignments of several reported peptide synthetases the conserved regions A2, A3, A5, A6, A7 and A8 of adenylation and T of thiolation modules were targeted for the degenerate primer design (Turgay and Marahiel, Peptide Res. 1994, 7, 238-241). The wobble positions were designed in respect to codon preferences within the selected modules and the expected high G/C content of Pseudomonas sp. All oligonucleotides were obtained from ISOGEN (Bioscience BV). A PCR fragment was obtained when degenerate oligonucleotides derived from the YGPTE (A5 core) and LGGXS (T core) sequences were used. These oligonucleotides were denoted PS34-YG and PS6-FF, respectively.

Table III. PCJR primers designed for this study.

Primer designation
orientation Sequence Length
PS34-YG (forward) 5'- TAYGGNCCNACNGA -3' 14-mer
PS6-FF (reverse) 5'-TSNCCNCCNADNTCRAARAA-3' 20-mer
PCR conditions for amplification of DNA from P. fluorescens A2-2
A fragment internal to nonribosomal peptide synthetases (NRPS) was amplified using PS-34-YG and PS6-FF oligonucleotides and P. fluorescens A2-2 chromosomal DNA as template. Reaction buffer and Taq polymerase from Promega were used. The cycling profile performed in a Personal thermocycler (Eppendorf) consists on: 30 cycles of 1 min at 95°C, 1 min at 50°C, 2 min at 72°C. PCR products were on the expected size (750 bp aprox.) based on the location of the primers within the NRPS domains of other synthetase genes.
DNA cloning
PCR amplification fragments were cloned into pGEM-Teasy vector according to the manufacturer (Qiagen, Inc., Valencia, CA). In this way, cloned fragments are flanked by two EcoRI restriction sites, in order to facilitate subsequent subclonig in other plasmids (see below). Since NRPSs enzymes are modular, clones from the degenerated PCR primers represents a pool of fragments from different domains.
DNA sequencing
All sequencing was performed using primers directed against the cloning vector, with an ABI Automated sequencer (Perkin-Elmer). Cloned DNA sequences were identified using the BLAST server of the National

Center for Biotechnology Information accessed over the Internet (Altschul et al., Nucleic Acids Res. 1997, 25, 3389-3521). All of the sequences have signature regions for NRPSs and show high similarity in BLAST searches to bacterial NRPS showing that they are in fact of peptide oriGln. Moreover, a probable domain similarity search was performed using the PROSITE (European Molecular Biology Laboratory, Heidelberg, Germany) web server.
Gene disruption ofPseudomonas fluorescens A2-2
In order to analyse the function of the genes cloned, these genes were disrupted through homologous recombination (Fig. 9). For this purpose, recombinant plasmids (pG-PS derivatives) harbouring the NRPS gene fragment were digested with EcoKL restriction enzyme. The resulting fragments belonGlng to the gene to be mutated were cloned into the pK18mob mobilizable plasmid (Schafer et al Gene 1994, 145, 69-73), a chromosomal integrative plasmid able to replicate in B. coli but not in Pseudomonas strains. Recombinant plasmids were introduced first in E. coli S17-XPIR strain by transformation and then in P. fluorescens A2-2 through biparental conjugation (Herrero et al, J Bacteriol 1990, 172, 6557-6567). Different dilutions of the conjugation were plated onto LB solid medium containing ampicillim plus kanamycin and incubated overnight at 27°C. Kanamycin-resistant transconjugants, containing plasmids integrated into the genome via homologous recombination, were selected.
Biological assay (biotest)for safracin production
Strains P. fluorescens A2-2 and its derivatives were incubated in 50 ml baffled erlenmeyer flasks containing fermentation medium with the corresponding antibiotics. Initially, SA3 fermentation medium was used (Ikeda Y. J. Ferment Technol 1985, 63, 283-286). In order to increase the productivity of the fermentation process statistical-mathematical methods like Plackett-Burman designed was used to select nutrients and response surface optimisation techniques were tested (Hendrix C. Chemtech 1980,

10, 488-497) in order to determine the optimum level of each key independent variable. Experiments to improve the culture conditions like incubation temperature and agitation have also been done. Finally a highly safracin B producer medium named 16B (152 g/1 of mannitol, 35g/l of G20-25 yeast, 26 g/1 of CaC03 , 14 g/1 of ammonium sulphate, 0.18 g/1 of ferric chloride, pH 6.5) was selected.
The safracin production was assay testing the capacity of inhibition a Bacillus subtilis solid culture by 10 μl of the supernatant of a 3 days Pseudomonas sp. culture incubated at 27°C (Alijah et al. Appl Microbiol Biotechnol 1991, 34, 749-755). P. fluorescens A2-2 cultures produce inhibition zones of 10-14 mm diameter while non-producing mutants did not inhibit B. subtilis growth. Three isolated clones had the safracin biosynthetic pathway affected. In order to confirm the results, HPLC analysis of safracin production was performed.
HPLC analysis of safracin production.
The supernatant was analysed by using HPLC Symmetry C-18. 300A, 5 urn , 250 x 4.6 mm column (Waters) with guard-column (Symmetry C-18, 5jim 3.9 x 20 mm, Waters). An ammonium acetate buffer (10 mM, 1% Diethanolamine, pH 4.0)- acetonitrile gradient was the mobile phase. Safracin was detected by absorption at 268 nm. In Fig. 6, HPLC profile of safracin and safracin precursors produce by P. fluorescens A2-2 strain and different safracin-like structures produced by P. fluorescens mutants are shown.
Example 3. Cloning and sequence analysis of safracin cluster
Inverse PCR and phage library hybridisation

Southern hybridisation on mutant chromosomal DNAs verified the correct gene disruption and demonstrated that the peptide synthethase fragment cloned into pK18mob plasmid was essential for the production of safracin. Analysis of the non safracin producers mutants obtained demonstrated that all of them had a gene disruption into the same gene, sacA.
Icverse, PCR from genomic DNA. and screening of a. phage, library of P. fluorescens A2-2 genomic DNA revealed the presence of additional genes flanking sacA gene, probably involved in safracin biosynthesis.
The GenBank accession number for the nucleotide sequence data of the P. fluorescens A2-2 safracin biosynthetic cluster is AY061859.
Cosmid library construction and heterologous expression
To determine whether safracin cluster was able to confer safracin biosynthetic capability to a non producer strain, it was cloned into a wide range cosmid vector (pLAFR3, Staskawicz B. et ah J Bacteriol 1987, 169, 5789-5794) and conjugated to a different Pseudomonas sp collection strains.
To obtain a clone containing the whole cluster, a cosmid library was constructed and screened. For this purpose, chromosomal DNA was partially digested with the restriction enzyme Psfl, the fragments were dephosphorylated an4 ligated into the PstI site of cosmid vector pLAFR3. The cosmids were packaged with Gigapack III gold packaGlng extracts (Stratagene) as manufacturer's recommendations. Infected cells of strain XL 1-Blue were plated on LB-agar supplemented with 50 μg/ml of tetracycline. Positives clones were selected using colony hybridization with a DIG-labeled DNA fragment belonGlng to the 3'-end of the safracin cluster. In order to ensure the cloning of the whole cluster, a new colony hybridization with a 5'-end DNA fragment was done. Only cosmid pL30p showed multiple hybridizations with DNA probes. To confirm the accurate cloning, PCR amplification and DNA-sequencing with DNA oligonucleotides

belonGlng to the safracin sequence were carried out. The size of the insert of pL30P was 26,705 bp . The pL30p clone DNA was transformed into E. coli S17XPIR and the resulting strain were conjugated with the heterologous Pseudomonas sp. strains. The pL30p cosmid was introduced into P. fluorescens CECT378 and P. aeruGlnosa CECT110 by biparental conjugation as described above. Once a clone encoding the whole cluster was identified, it was determined whether the candidate was capable of producing safracin. Safracin production in the conjugated strains was assessed by HPLC analysis and biological assay of broth cultures supernatants as previously described.
The strain P. fluorescens CECT378 expressing the pL30p cosmid (PM-19-002) was able to produce safracin in considerable amounts, whereas safracin production in P. aeruGlnosa CECT110 strain expressing pL30P (PM-16-002) was 10 times less than the CECT378. Safracin production in these strains was about 22 % and 2 % of the total production in comparison with the natural producer strain.
Genes involved in the formation of safracin. Sequence analysis of sacABCDEFGH and sacIJ operons
Computer analyses of the DNA sequence of pL30P revealed 14 ORFs (Fig. 1). A potential ribosome binding site precedes each of the ATG start codons.
In the sacABCDEFGH operon, three very large ORFs, sacA, sacB and sacC (positions 3052 to 6063, 6080 to 9268 and 9275 to 13570 of the P. fluorescens A2-2 safracin sequence SEQ ID NO:l, respectively) can be read in the same direction and encode the putative safracin NRPSs: SacA (1004 amino acids, Mr 110452), SacB (1063 amino acids, Mr 117539) and SacC (1432 amino acids, Mr 157331). The three NRPSs genes contain the domains resembling amino acid activating domains of known peptide synthetases. Specifically, the amino acid activating domains from these NRPS genes are very similar to three of the four amino acid activating

domains (Gly, Tyr and Tyr) found in the Myxococcus xanthus saframycin NRPSs (Pospiech et al. Microbiology 1995, 141, 1793-803; Pospiech et al. Microbiol 1996, 142, 741-746). In particular, SacA (SEQ ID NO:2) shows 33% identity with saframycin Mxl synthetase B protein (SafB) from M. xanthus (NCBI accession number U24657), whereas SacB (SEQ ID NO:3) and SacC (SEQ ID NO:4) share, respectively, 39% and 41% identity with saframycin Mxl synthetase A (SafA) from M. xanthus (NCBI accession number U24657). The Fig. 2 shows a comparison among SacA, SacB y SacC and the different amino acid activating domains of saframycin NRPS. Downstream sacC five small ORFs reading in the same direction as the NRPSs genes exist (Fig.l). The first one, sacD (position 13602 to 14651 of P. fluorescens A2-2 safracin sequence), encodes a putative protein, SacD (350 amino acids, Mr 39187; SEQ ID NO:5), with no similarities in the GeneBank DB. The next one, sacE (position 14719 to 14901 of P. fluorescens A2-2 safracin sequence), encodes a small putative protein called SacE (61 amino acids, Mr 6729; (SEQ ID NO:6)), which shows some similarity with proteins of unknown function in the databases (ORF1 from Streptomyces viridochromogenes (NCBI accession number Y17268; 44% identity) and MbtH from Mycobacterium, tuberculosis (NCBI accession number Z95208; 36% identity). The third ORF, sacF (position 14962 to 16026 of P. fluorescens A2-2 safracin sequence), encodes a 355-residue protein with a molecular weigh calculated of 39,834 (SEQ ID NO:7). This protein most closely resembles hydroxyneurosporene methyltransferase (CrtF) from Chloroflexus aurantiacus (NCBI accession number AF288602; 25% identity). The nucleotide sequence of the fourth ORF, sacG (position 16115 to 17155 of P. fluorescens A2-2 safracin sequence), predicted a gene product of 347 amino acids having a molecular mass of 38,22 kDa (SEQ ID NO: 8). The protein, called SacG, is similar to bacterial O-methyltransferases, including O-dimethylpuromycin-O-methyltransferase (DmpM) from Streptomyces anulatus (NCBI accession number P42712; 31% identity). A computer search also shows that this protein contains the

three sequence motifs found in diverse S-adenosylmethionine-dependent methytransferases (Kagan and Clarke, Arch. Biochem. Biophys. 1994, 310, 417-427). The fifth gene, sacH (position 17244 to 17783 of P. fluorescens A2-2 safracin sequence), encodes a putative protein SacH (180 amino acids, Mr 19632; (SEQ ID NO:9). A computer search for similarities, between the deduced amino acid sequence of SacH and other protein sequences, revealed identity with some conserved hypothetical proteins of unknown function, which contains a well conserved transmembrane motif and a dihydrofolate reductase-like active site (Conserved hypothetical protein from Pseudomonas aeruGlnosa PAOl, NCBI accession number P3469; 35% identity).
Upstream sacABCDEFGH operon, reading in opposite sense, a two genes operon, sacIJ, is located. The sad gene (position 2513 to 1854) encodes a 220-amino acids protein (Mr 24219; (SEQ ID NO: 10) that most closely resembles ubiquinone/manequinone methyltrasnferase from Thermotoga maritime (NCBI accession number AE001745; 32% identity). The sacJ gene (position 1861 to 335) encodes a 509-amino acid protein (SEQ ID NO: 11), with a molecular mass of 55341 Da, similar to bacterial monooxygenases/hydroxylases, including putative monooxygenase from Bacillus subtilis (NCBI accession number Y14081; 33% identity) and Streptomyces coelicolor (NCBI accession number AL109972; 29% identity).
SacABCDEFGH and sacIJ operons are transcribed divergently and are separated by 450 bp approximately. Both operons are flanked by residual transposase fragments.
Related safracin cluster genes
A putative ORF (orfl; position 18322 to 19365 of P. fluorescens A2-2 safracin sequence) located at the 3'-end of the safracin sequence has been

found {Fig. 1). ORFl protein (SEQ ID NO: 12) shows similarity with aminopeptidases from the Gene Bank DataBase (peptidase M20/M25/M40 family from Caulobacter crescentus CB15; NCBI accession number NP422131; 30% identity). Using the strategy described in Example 2, the gene disruption of orfl do not affect safracin production in P. fluorescens A2-2.
At the 3'-end of the safracin sequence cloned in pL30p cosmid, three putative ORFs (orf2, orf3 and orf4), were found. Reading in opposite direction than sacABCDEFGH operon, or/2 gene (position 22885 to 21169 of SEQ ID NO:l) codes for a protein, ORF2 (SEQ ID NO: 13), with similarities to Aquifex aeolicus HoxX sensor protein (NCBI accession number NCO00918.1; 35% identity), whereas or/3 gene (position 23730 to 23041 of SEQ ID NO:l) codes for ORF3 protein (SEQ ID NO: 14) which shares 44% identity with a glycosil transferase related protein from Xanthomonas axonopodis pv. Citri str. 306 (NCBI accession number NP642442).
The third gene is located at the 3'-end of SEQ ID NO:l (position 25037 to 26095). This gene, named orf4 (position 2513 to 1854), encodes a protein, ORF4 (SEQ ID NO: 15), that most closely resembles to a hypothetical isochorismatase family protein YcdL from Escherichia coli. (NCBI accession number P75897; 32% identity).
Presumably, these three genes would not be involve in the safracin biosynthetic pathway, however, future gene disruption of these genes will confirm this assumption.
The different DNA sequences found are listed at the end of the description.
Example 4. Functional analysis of the safracin loci and search for

possible precursors
Since the pathway for synthesis of safracin in P. fluorescens A2-2 is at present unknown, the inactivation of each of the genes described in Example 3 would permit fundamental studies on the mechanism of safracin biosynthesis in this strain.
In order to analyze the functionality of each particular protein in the safracin production pathway, disruption of each particular gene of the cluster, but sacE, was performed. All of the genetic mutants were obtained following the disruption strategy previously described.
Figure 6 is a summary of the different mutants constructed in this invention as well as a summary of the compounds produced by the mutants as a consequence of the gene disruption. In the wild type strain both safracin A and B and other compounds, P2 and P14, were clearly detected by HPLC (see Fig. 6,WT). The gene disruption of the sacA (PM-S1-002), sacB (PM-S1-003), sacC (PM-S1-004), sacD (PM-S1-010), sacF (PM-Sl-008), and sacG (PM-S 1-009), genes generated mutants that were unable to produce neither safracin A and safracin B, nor the precursor compounds with retention times beneath 15 min, P2 and P14 respectively. The structure elucidation of P14 and P2 revealed that P14 is a 3-methyl-O methyl tyrosine, where as P2 is a 3-hydroxy-5~methyl-0-methyl tyrosine. Because of the small size of the sacE gene, the sacE- mutant was not possible to be obtained by gene disruption, but deletion of this gene is in process. The overexpression of SacE protein, in trans, had no effect on safracin B/A production. The sacl- mutants (PM-S1-006) produced P2, P14 and significant amount of a compound called P19B (Fig. 6; Fig7b*). Structure elucidation of P19B revealed that this compound is a safracin-like molecule in which the N-Met and one of the OH from the quinone ring are missing. In the sacJ- mutants (PM-S1-005), P2, P14, P19B and two new compounds called P22A and P22B were obtained (Fig. 6; Fig. 7a*).

Structure elucidation of P22A and P22B revealed that they are safracin A and safracin B like molecules, respectively, without one of the -OH group from the quinone ring. The biological assay of the sac? and the sacJ-mutants extracts revealed very low activity against Bacillus subtilis.
The disruption of sacl gene with the reconstitution of the sacJ gene expression resulted in a new safracins producer mutant, PM-S1-007. The two antibiotics produced, at levels of production as high as the levels of safracin A and safracin B in the wild type strain, have been named as safracin D and safracin E (Fig. 7c*). The safracin D and safracin E are safracin B and safracin A like molecules, respectively, where the N-methylation is missing.
These results strongly suggest that i) sacA, sacB and sacC genes encode for the safracin NRPSs; ii) sacD, sacF and sacG genes are responsible for the transformation of L-Tyr into the L-Tyr derivative P2 and iii) sacl and sacJ are responsible for the tailoring modifications that convert P19 and P22 into safracin.
Characterization of Natural Precursors: p. 14

Pseudomonas fluorescens A2-2 (wild type) (PM-S1-001)
Fermentation conditions:
Seed medium YMP3 containing 1% glucose; 0.25% beef extract; 0.5% bacto-peptone; 0.25% NaCl; 0,8% CaC03 was inoculated with 0.1% of a frozen vegetative stock of the microorganism, and incubated on a rotary-shaker (250 rpm) at 27°C. After 30h of incubation, the 2% (v/v) seed culture was transferred into 2000 ml Erlenmeyer flasks containing 250 ml of the M-16B production medium, composed of 15.2 % mannitol; 3.5 % Dried brewer's yeast; 1.4 % (NH4)2 S04; 0.001%; FeCb; 2.6 % COaCa. The temperature of the incubation was 27°C from the inoculation till 40 hours and then, 24°C to final process (71 hours). The pH was not controlled. The agitation of the rotatory shaker was 220 rpm with 5 cm eccentricity.
After 71 hours of incubation, 2 Erlenmeyer flasks were pooled and the 500 ml of fermentation broth was clarified by 7.500 rpm centrifugation during 15 minutes. 50 grams of the resin XAD-16 (Amberlite) were added to the supernatant and mixed during 30 minutes at room temperature. Then, the resin was recovered from the clarified broth by filtration. The resin was washed twice with distilled water and extracted with 250 ml of isopropanol (2-PrOH). The alcohol extract was dried under high vacuum till obtention of 500 mg crude extract. This crude was dissolved in methanol and purified by chromatographic column using Sephadex LH-20 and methanol as mobile phase. The P-14 compound was eluted and dried as a 15 mg yellowish solid. The purity was tested by analytical HPLC and 1H NMR.

P-14 was also isolated in a similar way from cultures of the sacJ- mutant (PM-S 1-005), using semipreparative HPLC as the last step in the purification process.
Biological activities: NO ACTIVE
Spectroscopic data:
ESMS m/z254 (C11H14N03Na2+), 232 (CnHisNOsNa-), 210 (M+H+). 1H RMN (300 MHz, CD3OD): 7.07 (d, J=8.1 Hz, H-9), 7.06 (s, H-5), 6.84 (d, J=8.1 Hz, H-8), 3.79 (s, H-ll), 3.72 (dd, J=8.7, 3.9 Hz, H-2), 3.20 (dd, J=14.4, 3.9 Hz, H-3a), 2.91 (dd, J=14.4, 8.9 Hz, H-3b), 2.16 (s, H-10). 13C RMN (75 MHz, CD3OD): 174.1 (C-l), 158.6 (C-7), 132.5 (C-5), 128.9 (C-9), 128.5 (C-4), 128.0 (C-6), 111.4 (C-8), 57.6 (C-2), 55.8 (C-ll), 37.4 (C-3), 16.3 (C-10)

Pseudomonas fluorescens A2-2 (wild type) (PM-S 1-001)

Fermentation conditions:
The same process than P-14
Similar procedure as the P-14, except in the Sephadex chromatography, where the fractions containing P-2 have eluted later. A semi-preparative HPLC step (Symmetry Prep C-18 column, 7.8 x 150 mm, AcONH4 10 mM pH=3/CH3CN 95:5 held for 5 min and then gradient from 5 to 6.8 % of CH3CN in 3 min) has been necessary to purify the P-2. Also this compound has been isolated from the fermentation broth of the Pseudomonas putida ATCC12633+pB5H83 (PM-17-004) as result of heterologous expression.
Biological activities: NO ACTIVE
Spectroscopic data:
ESMS m/z 226 [M+H]+; 1H RMN (CD3OD, 300 MHz): 6.65 (d, J- 1.8 Hz, H-5), 6.59 (d, J= 1.8 Hz, H-9), 3.72 (s, H-ll), 3.71 (dd, J= 9.0, 4.2 Hz, H-2), 3.16 (dd, J= 14.4, 4.2 Hz, H-3a), 2.83 (dd, J= 14.4, 9.0 Hz, H-3b), 2.22 (s, H-10); we RMN (DMSO, 75 MHz): 170.88 (s, C-l), 150.025 (s, C-7), 144.56 (s, C-8), 132.28 (s, C-4), 130.36 (s, C-6), 121.73 (d, C-5), 115.55 (d, C-9), 59.06 (q, 7-OMe), 55.40 (d, C-2), 36.21 (t, C-3), 15.86 (q, 6-Me).
Characterization of Safracins like compounds obtained by knock out COMPOUND P-22B

sac J- mutant from P.fluorescens A2-2 (PM-S1 -005)
Fermentation conditions:
50 liters of the SAM-7 medium (50 1) composed of dextrose (3.2%), mannitol (9.6%), dry brewer's yeast (2%), ammonium sulphate (1.4%), potassium secondary phosphate (0.03%), potassium chloride (0.8%), Iron (III) chloride 6-hydrtate (0.001%), L-tyrosine (0.1%), calcium carbonate (0.8%), poly- (propylene glycol) 2000 (0.05%) and antifoam ASSAF 1000 (0.2%) was poured into a jar-fermentor (BioenGlneering LP-351) with 75 1 total capacity and, after sterilization, sterile antibiotics (amplicillin 0.05 g/1 and kanamycin 0.05 g/1) were added. Then, it was inoculated with seed culture (2%) of the mutant strain PM-S 1-005. The fermentation was carried out during 71 h. under aerated and agitated conditions (1.0 1/1/min and 500 rpm). The temperature was controlled from 27°C (from the inoculation till 24 hours) to 25°C (from 24h to final process). The pH

was controlled at pH 6.0 by automatic feeding of diluted sulphuric acid from 22 hours to final process.
The whole broth was clarified (Sharpies centrifuge). The pH of the clarified broth was adjusted to pH 9.0 by addition of NaOH 10% and extracted with 25 litres of ethyl acetate. After 20' mixing, the two phases were separated. The organic phase was frozen overnight and then, filtered for removing ice and evaporated to a greasy dark green extract (65.8 g). This extract was mixed with 500 ml hexane (250 ml two times) and filtered for removing hexane soluble impurities. The remaining solid, after drying, gave a 27.4 g of a dry green-beige extract.
This new extract was dissolved in methanol and purified by a Sephadex LH-20 chromatography (using methanol as mobile solvent) and the safracins-like compounds were eluted in the central fractions {Analyzed on TLC conditions: Silica normal phase, mobile phase: EtOAaMeOH 5:3. Aprox. Rf valor: 0.3forP-22B, 0.25 P-22A and 0.1 for P-19).
The pooled fractions, (7,6g) containing the three safracin-like compound were purified by a Silica column using a mixture of EtOAc:MeOH from 50:1 to 0:1. and other chromatographic system (isocratic CHCl3:MeOH:H20:AcOH 50:45:5:0.1). Compounds P22-A, P22-B and P19-B were purified by reversed-phase HPLC (SymmetryPrep C-18 column 150 x 7.8 mm, 4 mL/min, mobile phase: 5 min MeOH:H20 (0.02 % TFA) 5:95 and gradient from MeOH:H20 (0.02 % TFA) 5:95 to MeOH 100 % in 30 min).
Biological activities ofsafracin P-22B

Antimicrobial activity: On solid medium
Bacillus subtilis. lO μg/disk (6mm diameter): 10 mm inhibition zone
Spectroscopic data:
HRFABMS m/z 509.275351 [M-H20+H]+ (calcd for C28H37N4O5 509.276396 A 1.0 mmu); LRFABMS using m-NBA as matrix m/z (rel intensity) 509 [M-H20+H]+ (5), 460 (2.7), 391 (3).
1H NMR (CD3OD, 500 MHz): 6.70 (s, H-15), 6.52 (s, H-5), 4.72 (bs, H-ll), 4.66 (d, J= 2.0 Hz, H-21), 4.62 (dd, J= 8.4, 3.7 Hz, H-1), 3.98 (bd, J= 7.6 Hz, H-13), 3.74 (s, 7-OMe), 3.71 (s, 17-OMe), 3.63 (m, overlapped signal, H-25), 3.62 (m, overlapped signal, H-3), 3.30 (m, H-22a), 3.29 (m, H-14a), 3.18 (d, J= 18.6 Hz, H-14b), 2.90 (m, H-4a), 2.88 (m, H-22b), 2.76 (s, 12-NMe), 2.30 (s, 16-Me), 2.22 (m, H-4b), 1.16 (d, J= 7.4 Hz, H-26);
13C NMR (CD3OD, 125 MHz): 170.75 (s, C-24), 149.24 (s, C-18), 147.54 (s, C-8), 145.95 (s, C-7), 145.82 (s, C17), 133.93 (s, C-16), 132.31 (s, C-9), 131.30 (s, C-6), 128.95 (s, C-20), 121.93 (d, C-15), 121.76 (d, C-5), 121.44 (s, C-10), 112.45 (s, C-19), 92.87 (d, C-21), 60.86 (q, 7-OMe), 60.76 (q, 17-OMe), 59.39 (d, C-ll), 57.96 (d, C-13), 55.51 (d, C-l), 54.29 (d, C-3), 50.08 (d, C-25), 45.55 (t, C-22), 40.43 (q, 12-NMe), 32.56 (t, C-4), 25.84 (t, C-14), 17.20 (q, C-26), 16.00 (q, 16-Me), 15.81 (q, 6-Me).


The same as for P-22B
Fermentation conditions: The same as for P-22B
The same as for P-22B
Biological activities ofsafracin P-22A Antitumor activities

Antimicrobial activity: On solid medium
Bacillus subtilis. lO ug/disk (6mm diameter): NO ACTIVE

Spectroscopic data:
HRFABMS m/z 511.290345 [M+H]+ (calcd for C28H39N4O5 511.292046 A 1.7 mmu); LRFABMS using m-NBA as matrix m/z (rel intensity) 511 [M+H]+ (61), 409 (25), 391 (4); 1H NMR (CD3OD, 500 MHz): 6.68 (s, H-15), 6.44 (s, H-5), 3.71 (s, 7-OMe), 3.67 (s, 17-OMe), 2.72 (s, 12-NMe), 2.28 (s, 16-Me), 2.20 (s, 6-Me), 0.87 (d, J- 7.1 Hz, H-26);

The same as for P-22B
Fermentation conditions: The same as for P-22B
The same as for P-22B

Biological activities ofsafracin P-19B Antitumor activities
Antimicrobial activity: On solid medium
Bacillus subtilis. lOpg/disk (6mm diameter): NO ACTIVE
Spectroscopic data:
HRFABMS m/z 495.260410 [M-H20+H]+ (calcd for C27H35N4O5 495.260746 A 0.3 mmu); LRFABMS using m-NBA as matrix m/z (rel intensity) 495 [M-H20+H]+ (13), 460 (3), 391 (2); *H NMR (CD3OD, 500 MHz): 6.67 (s, H-15), 6.5 (s, H-5), 3.73 (s, 7-OMe), 3.71 (s, 17-OMe), 2.29 (s, 16-Me), 2.24 (s, 6-Me), 1.13 (d, J = 7.1 Hz, H-26);

New Safracin compounds obtained by knock out SAFRACIN D

sac F with sacJ expression reconstitution from P.fluorescens A2-2 (PM-S1-
Fermentation conditions:
50 litres of the SAM-7 medium (50 1) composed of dextrose (3.2%), mannitol (9.6%), dry brewer's yeast (2%), ammonium sulphate (1.4%), potassium secondary phosphate (0.03%), potassium chloride (0.8%), Iron (III) chloride 6-hydrtate (0.001%), L-tyrosine (0.1%), calcium carbonate (0.8%), poly- (propylene glycol) 2000 (0.05%) and antifoam ASSAF 1000 (0.2%) was poured into a jar-fermentor (BioenGlneering LP-351) with 75 1 total capacity and, after sterilization, sterile antibiotics (arnplicillin 0.05 g/1 and kanamycin 0.05 g/1) were added. Then, it was inoculated with seed culture (2%) of the mutant strain PM-S1-007. The fermentation was carried out during 89 h. under aerated and agitated conditions (1.0

1/1/min and 500 rpm). The temperature was controlled from 27°C (from the inoculation till 24 hours) to 25°C (from 24h to final process). The pH was controlled at pH 6.0 by automatic feeding of diluted sulphuric acid from 27 hours to final process.
The cultured medium (45 1) thus obtained was, after removal of cells by centrifugation, adjusted to pH 9.5 with diluted sodium hydroxide, extracted with 25 liter of ethyl acetate twice. The mixture was carried out into an agitated-vessel at room temperature for 20 minutes. The two phases were separated by a liquid-liquid centrifuge. The organic phases were frozen at -20°C and filtered for removing ice and evaporated until obtention of a 35g. oil-dark-crude extract. After a 5 1. hexane triturating, the extract (12.6g) was purified by a flash-chromatographic column (5.5 cm diameter, 20 cm length) on silica-normal phase, mobile phase: Ethyl acetate: MeOH: 1 L of each 1:0; 20:1; 10:1; 5:1 and 7:3, 250 ml- fractions were eluted and pooled depending of the TLC (Silica-Normal, EtOAc.MeOH 5:2, Safracin D Rf 0.2, safracin E 0.05). The fraction containing impure safracin D and E was evaporated under high vacuum (2.2 g). An additional purification step was necessary to separate D and E on similar conditions (EtOAc:MeOH from 1:0 to 5:1), from this, the fractions containing safracin D and E are separate and evaporated and further purification by Sephadex LH-20 column chromatography eluted with methanol.
The safracins D and E obtained were independent precipitated from CH2CI2 (80 ml) and Hexane (1500 ml) as a green/yellowish-dried solid (800 mg safracin D) and (250 mg safracin E).
Biological activities Safracin D Antitumor screening:

Antimicrobial activity: On solid medium
Bacillus subtilis. lO μg/disk (6mm diameter): Inhibition zone: 15 mm
Spectroscopic data
ESMS: m/z 509 [M-H20+H]+; 1H NMR (CDCb, 300 MHz): 6.50 (s, C-15), 4.02 (s, OMe), 3.73 (s, OMe), 2.22 (s, Me), 1.85 (s, Me), 0.80 (d, J= 7.2 Hz); "C NMR (CDCb, 75 MHz): 186.51, 181.15, 175.83, 156.59, 145.09, 142.59, 140.78, 137.84, 131.20, 129.01, 126.88, 121.57 (2 x C), 82.59, 60.92, 60.69, 53.12, 21.40, 50.68, 50.22, 48.68, 40.57, 29.60, 25.01, 21.46, 15.64, 8.44.

The same than safracin D
Fermentation conditions: The same batch as safracin D
See safracin D conditions
Biological activities Safracin E Antitumor screening:

Antimicrobial activity: On solid medium
Bacillus subtilis. lO μg/disk (6mm diameter): 9.5 mm inhibition zone
Spectroscopic data
ESMS: m/z 511 [M+H]+; 1H NMR (CDCl3, 300 MHz): 6.51 (s, C-15), 4.04 (s, OMe), 3.75 (s, OMe), 2.23 (s, Me), 1.89 (s, Me), 0.84 (d, J = 6.6 Hz); ™C NMR (CDCb, 75 MHz): 186.32, 181.28, 175.83, 156.43, 145.27, 142.75, 141.05, 137.00, 132.63, 128.67, 126.64, 122.00, 120.69, 60.69, 60.21, 59.12, 58.04, 57.89, 50.12, 49.20, 46.72, 39.88, 32.22, 25.33, 21.29, 15.44, 8.23.

Example 5. Cross-feeding experiments
Heterologous expression of safracin biosynthetic precursors genes for P2 and PI 4 production
In the attempt to shed light on the mechanism of the P2 and PI4 biosynthesis we have cloned and expressed the downstream NRPS genes to determine their biochemical activity.
To overproduce P14, sacEFGH genes were cloned (pB7983) (Fig. 4).
To overproduce P2 in a heterologous system, sacD to sacH genes were
cloned (pB5H83)(Fig. 4). For this purpose we PCR amplified fragments
harboring the genes of interest using oligonucleotides that contain a Xbal
restriction site at the 5' end. Oligonucleotides PFSC79 (5'-
GGTCTAGATAACAGCCAACAAACATA-3') were used to amplify sacE to
sacH genes; and oligonucleotides 5HPT1-XB (5'-
GGTCTAGATAACAGCCAACAAACATA-3') were used to amplify sacD to sacH genes. The PCR fragments digested with Xbal were cloned into the Xbal restriction site of the pBBRl-MCS2 plasmid (Kovach et al, Gene 1994, 166, 175-176). The two plasmids, pB7983 and pB5H83, were introduce separately into three heterologous bacteria P. fluorescens (CECT 378), P. putida (ATCC12633) and P. stutzeri (ATCC 17588) by conjugation (see table II). When culture broth of the fermentation of the transconjugant strains was checked by HPLC analysis, big amounts of PI4 compound was visualized in the three strains containing pB7983 plasmid, whereas big amounts of P2 and some P14 product were observed when pB5H83 plasmid was expressed in the heterologa bacteria.

As it was shown in Example 4, the sacF- (PM-S1-008) and sacG-(PM-S1-009) mutants were not able to produce neither safracins nor P2 and P14 compounds. The addition of chemically synthesized P2 to these mutants during their fermentation yields safracin production.
Moreover, the co-cultivation of an heterologous strain of P. stutzeri (ATCC 17588) harboring plasmid pB5H83 (PM-18-004), which expression produces P2 and PI4, with either one of the two mutants sacF- and sacG-resulted in safracin production. The co-cultivation of an heterologous strain P. stutzeri (ATCC 17588) harboring plasmid pB7983 (PM-18-005), which expression produces only P14, with either one of the two P. fluorescens A2-2 mutants mentioned before resulted in no safracin production at all. These results suggest that P14 is transformed into P2, a molecule that can easily be transported in and out through the Pseudomonas sp. cell wall and which presence it is absolutely necessary for the biosynthesis of safracin.
Example 6. Biological production of new "unnatural" molecules
The addition of 2g/L of an specific modified P2 derivative precursor, P3, a 3-hydroxy-5-methyl-0-methyltyrosine, to the sacF mutant (PM-S1-008) fermentation yielded two "unnatural" safracins that incorporated the modified precursor P3 in its structure, Safracin A(OEt) and Safracin B(OEt).
SAFRACIN B-Etoxi (Safracin B (OEt))

saf F - mutant from P.fluorescens A2-2 (PM-S 1-008)
Fermentation conditions:
Seed medium containing 1% glucose; 0.25% beef extract; 0.5% bacto-peptone; 0.25% NaCl; 0,8% CaC03 was inoculated with 0.1% of a frozen vegetative stock of the microorganism, and incubated on a rotary shaker (250 rpm) at 27°C. After 30h of incubation, the 2% (v/v) seed culture of the mutant PM-S 1-008 was transferred into 2000 ml Erlenmeyer flasks containing 250 ml of the M-16 B production medium, composed of 15.2 % mannitol; 3.5 % Dried brewer's yeast; 1.4 % (NH4)2 0.001%; FeCl3; 2.6 % C03Ca and 0.2% P3 (3-hydroxy-5-methyl-0-methyltyrosine) The temperature of the incubation was 27°C from the inoculation till 40 hours and then, 24°C to final process (71 hours). The pH was not controlled. The agitation of the rotatory shaker was 220 rpm with 5 cm eccentricity.
4 x 2000/250 ml Erlenmeyer flasks were joined together (970 ml),
centrifuged (12.000 rpm, 4°C, 10', J2-21 Centrifuge BECKMAN) to remove

cells. The clarified broth (765 ml) was adjusted to pH 9.0 by NaOH 10%. Then, the alkali-clarified broth was extracted with 1:1 (v/v) EtOAc (x2). The organic phase was evaporated under high vacuum and a greasy-dark extract was obtained (302 mg).
This extract was washed by an hexane trituration for removing impurities and the solids were purified by a chromatography column using Silica normal-phase and a mixture of Ethyl Acetate: Methanol (from 12:1 to 1:1). The fractions were analyzed under UV on TLC (Silica 60, mobile phase EtOAc:MeOH 5:4. Rf 0.3 (Safracin B-OEt and 0.15 Safracin A-OEt). From this, safracins B OEt (25 mg) and safracin A OEt (20 mg) were obtained.
Biological activities of safracin B (OEt) Antitumor activities

Antimicrobial activity: On solid medium
Bacillus subtilis. lOpig/disk (6 mm diameter): 17,5 mm inhibition zone
Spectroscopic data:
ESMS: m/z 551 [M-H20+H]+; 1H NMR (CDCL3 300 MHz): 6.48 (s, H-15), 2.31 (s, 16-Mc), 2.22 (s, 12-NMe), 1.88 (s, 6-Me), 1.43 (t, J= 6.9 Hz, Me-Etoxy), 1.35 (t, J= 6.9 Hz, Me-Etoxy), 0.81 (d, J= 7.2 Hz, H-26)

SAFRACIN A-Etoxi (Safracin A (OEt))

The same as for Safracin B (OEt)
Fermentation conditions:
The same as for Safracin B (OEt)
4 x 2000/250 ml Erlenmeyer flasks were joined together (970 ml),
centrifuged (12.000 rpm, 4°C, 10', J2-21 Centrifuge BECKMAN) to remove
cells. The clarified broth (765 ml) was adjusted to pH 9,0 by NaOH 10%.
Then, the alkali-clarified broth was extracted with 1:1 (v/v) EtOAc (x2). The
organic phase was evaporated under high vacuum and a greasy-dark
extract was obtained (302 mg).
This extract was washed by an hexane trituration for removing impurities
and the solids were purified by a chromatography column using Silica
normal-phase and a mixture of Ethyl Acetate: Methanol (from 12:1 to 1:1).
The fractions were analysed under UV on TLC (Silica. 60, mobile phase

EtOAc:MeOH 5:4. Rf 0.3 Safracin B-OEt and 0.15 Safracin A-OEt). From this, safracins B OEt (25 mg) and safracin A OEt (20 mg) were obtained.
Biological activities of safracin A (OEt): Antitumor activities

Antimicrobial activity: On solid medium
Bacillus subtilis. lOjjg/disk (6 mm diameter): 10 mm inhibition zone
Spectroscopic data:
ESMS: m/z 553 [M+H]+; 1H NMR (CDC13, 300 MHz): 6.48 (s, H-15), 2.33 (s, 16-Me), 2.21 (s, 12-NMe), 1.88 (s, 6-Me), 1.42 (t, J = 6.9 Hz, Me-Etoxy), 1.34 (t, J= 6.9 Hz, Me-Etoxy), 0.8 (d, J= 6.9 Hz, H-26)

Example 7. Enzymatic transformation of Safracin B into Safracin A
In order to assay the enzymatic activity of conversion of safracin B into safracin A, a 120 hours fermentation cultures (see conditions in Example.2.Biological assay (biotest) for safracin production) of different strains were collected and centrifuged (9.000 rpm x 20 min.). The strains assayed were P. fhiorescens A2-2, as wild type strain, and P.fluorescens CECT378 + pBHPT3 (PM-19-006), as heterologous expression host. Supernatant were discarded and cells were washed (NaCl 0.9 %) twice and resuspended in 60 ml phosphate buffer 100 mM pH 7.2. 20 ml from the cell suspension was distributed into three Erlenmeyer flask:
A. Cell suspension + Safracin B (400 mg/L)
B. Cell suspension heated at 100 °C during 10 min. + Safracin B
(400 mg/L) (negative control)
C. Cell suspension without Safracin B (negative control)
The biochemical reaction was incubated at 27 °C at 220 rpm and samples were taken every 10 min. Transformation of safracin B into safracin A was followed by HPLC. The results clearly demonstrated that the gene cloned in pBHPT3, sacH, codes for a protein responsible for the transformation of safracin B into safracin A.
Based on this results we did an assay to find out if this same enzyme was able to recognize a different substrate such as ecteinascidin 743 (ET-743) and transform this compound into Et-745 (with the C-21 hydroxy missing). The experiment above was repeated to obtain Erlenmeyer flasks containing:
A. Cell suspension + ET-743 (567 mg/L aprox.)

B. Cell suspension heated at 100 °C during 10 min. + ET-743(567
mg/L) (negative control)
C. Cell suspension without ET-743 (negative control)
The biochemical reaction was incubated at 27 °C at 220 rpm and samples were taken at o, 10 min, lh, 2h, 3h, 4h, 20h, 40h, 44h, 48h. Transformation of ET-743 into ET-745 was followed by HPLC. The results clearly demonstrated that the gene cloned in pBHPT3, sacH, codes for a protein responsible for the transformation of Et-743 into Et-745. This demonstrates that this enzymes recognizes ecteinascidin as substrate and that it can be used in the biotransformation of a broad range of structures.
Pharma Mar
The gene cluster involved in safracin biosynthesis and its uses for genetic enGlneering aaa 15
Patentln version 3.1
Lenght: 26705
Type DNA
Organism:Pseudomonas fluorescens A2-2
LOCATION: (6080)..(9268)
OTHER INFORMATION: non ribosomal peptide synthetase gene

non ribosomal peptide synthetase gene

Hypothetical protein gene

Hypothetical protein gene

methyl-transferase protein gene

methyl-transferase protein gene

hypothetical protein gene

Complementary, methyl-transferase protein gene

Complementary, mono-oxygenase protein gene

aminoacid peptidase-like protein

complementary; hox-like regulator protein

complementary; glycosil transferase-like protein

isochorismatase-like protein

SEQUENCE 1 ctgcaggtgg tttgcgcgcg gaagacccgc cactgccggt gcgctcgttt gaattgcaca 60
tggcgtggcg tgggtcgcag gataatgatc cgggggagcg gtggttgcgg tcgcggattc 120
agatgttttt tggcgacccc gatagccttt aattaaactc cactaaaatc ggcgattgca 180
gagcctgagt acaacacggc tactggactg aagtgggcgc atcgtgccgc atagccatag 240
tgatctcggt gtgtctcgcc atgtcccggc ccaggtcgta ggtcatgctc ttgcgcattg 300
ccagcatctt cgtgctccct tgccagctgt ttcaggtcag gctctgacgc gcggatttag 360
aatcgtccag cagccactca cccaagcgct ccttggccaa ggtcgatttt ttaccgaccc 420
agatcaccac gccatccggc ctgacgatga ctccctcgcc agcggacaag ctgttattgt 480
gcgaatcctc gcagatagat gccgtttgca accccggaaa atcacgtcga agatcggcgg 540
ccagtgcttt ggcacgatga tgtaccagga caaaccgccc tgctcgcagc aactgggtca 600
agctctgtcg tggtaatcgc tcaccctcgg gaagcaggct caacaggggt aaacgtcggc 660
ccaccaagcg atgatcgcct cggcgccgca cactgtcata gcgcacgccc tctcccgcca 720
gtgcactgac caccttctgc gccacatacg gggcccgtgt cgcctgcaag ccgatccagt 780
gaatcagacg gcctataggg ccggaagccg tattgaatcg gaaaagcaga tccgtgttgc 840
gcaaggctgc cgccgcaata ggcctacgct cggcctcgta actctccaga agatccatcg 900
gcaatgtggc ctgtatcaca cccgccagct tccaggcgag gttcgccgcg tcaccgatcc 960
ccatctgcaa accttgcccc ccggcgggga cgtgggtgtg agcagcatct cccagcagaa 1020
ataccctccc ctggcgataa tgagtcgcca ggcgctgctg gctgcggtaa cgagcgctcc 1080

acagcacctg cgccaatccg aaatcggttc ccagaatatc tttcatccct ccggcaattt 1140
cctcgtgggt gaccggctgt ttgaccggag tatccatacg ttcgttgtct tcgatactga 12 00
cgcgataact gccatcgggt aatggaaaca gggcaaccag acccctggag accgaccttg 1260
catggactgc aggtgaaggc ggatttctca agacaacgtc cgccaccacc aacgaatgct 132 0
tgtagtcctg cccgacaaac gaaatattga ggagttggcg gacagaactg ttgaccccat 1380
cagcccccag cacccagtcg tagcggcttt gctgcacgct gccggtctcg ctgtgttcca 144 0
gggttacctc aacgtgtaaa tcgccggcat ccagagcctt tagcgcatac ccacgcttca 1500
gattcacccc cttgcgattg acccaatcag tcaacaccga ctcggtctga gactgtggga 1560
tgatcaccat gtggggatac tcacaaggga gtttggaaaa tgagagcgtt cggcccgcct 1620
tgtcacccaa cggcgcgctt gcccagacga tcccccgacg tatcatctca tctgccacgc 1680
cccaggcatt gagcaactcc agggtcacgg gctccaagcc aaaggcccgg gaatggggag 1740
aggcagccgg tcttttatcg atgagatcaa ccgctatacc caactcggcc agagctgcag 1800
ccacagccaa cccgacgggc cccgccccca cgaccaggac ctgtttattt ttaacgacca 1860
tgatcaccca cctctccaca gcaggggcgg aacgtggcga caatgacgtg ctggtcggtg 1920
acgctcgttt ccatgctctg cagcccggcc gtttgcgcga gctggattac ctcatgctct 1980
gatcgataat ctgccagtga gcccagcagc cagttgatca atgtggtcaa tccccagccc 2040
gcaggcaggt tctccaccag gcagaaaagg ccctgaggct tgagcacgcg acggacctca 2100
ttcaggctga cagacttgtc agcccaatgg ccgaacgaca tcgagcacac caccagatcc 2160
atgctttgcg aggggaatgg caaggcttcg gcaactcctt tgacgaacga ggcgaaggga 222 0
cgtcgtttgg cggcctcgtc gaccatgccc tgagccgggt cgacgccttc gaagcgcgcc 22 80
tcaggccaca gagcgaacat gcgttcgatc aatgcgccgg taccacaacc gatgtccaga 2340
acacgctccg gtctcgaggt gcacatccat cggctcaaca ttcgcagaca gtcgtcatgg 2400
gcctggctca gtttggtacc gtacttctct tcatacgtcg gcgcgatacg gctgaacgtc 2460
cgcacaaaac ctggatttcc attgcctttc ccgccagaaa atacgttaga cattattgaa 2520
catccatata tcaacagtta tccgccaagg accatagtag agaaaatcca tcccatccaa 258 0
ataaaaatta aataagtggg gctaaccgca atccagggaa actctgaaaa ggcccgctac 2 640
ttgtcgacgc ggctgtctgg aggccgcata gttactgaac ttactattaa aagactgggc 2 700
tttttcagag ccccaccgga tgttggctcc ttgtccatca tttcgggggc actgtaacat 2760
tctgttacct ggctatcgct tgacttttaa tctgaacggg caattatagg tctaaccgca 282 0

acagccccac ggcgcttaag ttcgaaaaaa
aataaggggc actttacaag ccgcataaga
cacgacaagt aaaaaacact tgtccaatac
cgtcaaaaag ttctctgctt acagcagtct
gaagtcgctt ttcatgtgat cacccatctt
agagtggtcg agcgacatgc gtctttacgg
tggattgaac aagccccacc gcaacaacga
gcatcgaccg atgcactgct ggcgccgagc
ttgttccgcg ccgaagtcgt tgagcgcagc
catcacatca tcgccgacct gtggtctgtc
tgtatggacc gctccagcat caccctggcg
gagttctggc ggcaccaaat gtcacaggac
gaacagcaca cggaccgccg catggtgctg
gctgacctgg cccgcctggc cacagcctgc
gcacaagtat tggcgctgtc cagaatcggc
ttccatggcc gcaacagggg caacaaggat
gtgcctttcg atgtcagcga atgcagcgtg
ctggatgagg cctcaaaagc cagcgtcggt
acgccgctgg gatgggctgc gaccgccccg
ccaggcatgc caagaggatt ggcggcggct
gagatggcgc tgaccgcgga acaggcaccg
ctgctgacgc gccacgacgg caagctgcat
cccggttggc tggcagaggc gttagccaga
cgtgatccac aggccagact gtcagccttg
ccgagccaag cgcggccggc gcctgcgtca
gtcgccatca cgccggacaa gcccgcgctg
gaattggcca gtcgagtcgc caggctctcg
gaacagaccc tggcaatact cctgcctcgc

gtagctgcac cttgctcaac tgcatcttgt 2880
cataaatttt atgctgactc cccaaacaag 2940
caaggagggt atacggtgca agcttcttta 3000
atcgatccca gccagccagg catgttactt 3060
tctacttcgc agttggtatc gcgtatcgag 3120
cagcgctttg tcatgcgcaa tggcacttac 3180
cgctactgcg tggtacgcac ctatgatgaa 3240
cgcgagcaca tcggggttga gtctgagcgt 3300
gacggacaac gctacttggt cttccgaatt 3360
ggcctcctga ttcgagactt tgccgaagac 3420
tcaagaccga ttgccccgtt gatcgaccct 3480
actccgtttt ccttgcccat ggcctccctg 3540
tcttcgttcg ttattgatca ggagagcagc 3600
gcggtaaccc cgtacaccgt aatgctcgcc 3660
cagagtggcc gtctgtcact tgcggtgacg 3720
gcggtaggtt acttcgccaa tacgcttgcc 3780
ggcgagtttg tcaaacgcac cgccaagcgc 3840
gccggttatc ccgaattggc agagttcatg 3900
accaatgcgg tgatttacca gcaggatatg 3960
ctgctgggat tgggcacggt gcagttgggc 4020
cccagcatcg gcccgtttgc cactgcgctg 4080
ggccgggtcg aggtcgatcc tgcgcagcat 4140
cagttcgctg tgatcctgcg ggaaatggtg 4200
ccagcgtgcc tgttacacca accaaaatac 4260
gaaacattgg tagccacctt tctccggcaa 4320
cgtacgccgc aggccagcat cagctatagc 4380
gcagccttgc gcgtacgcgg cttcaaacct 4440
gatatcaatc tggtacccgc tctgctggcg 4500

atcatggcct gcggtggcag ttatgtgcca ctcagtgacg cgaaccccgc cgaactcaac 4560
cgttcgattc tgaccagggc ccgttgccgc gcgattctca cggatcagga gggtttgacc 4520
cgtttcgctc acttggcgcc ctgctggtcc ttgagcgacc tgctgtcgat gcccgacgcc 46 80
ccgctgcagg accagtccaa gcttcaagcc aaggcctata tcctatttac cagcggctcc 4740
accggtgaac caaaaggcgt ggcgatcacc catgctaatg ccgccaacct gctgcgttgg 4 800
gcggctctcg attgtggccc cgagtacctg gcgcaaacac tggcggcaac ccccactacg 4860
ttcgatcttt cgattttcga gatgtttgct ccccttatgg tcggtggctg cgtacagccc 4920
gtttcctcgg tcatggcgct gatcgacaat ccggccctgc taaagggcac aacactgatc 4980
aatacggtgc cgtcggtggc cgacgctttg ttgcagcatg atgtactggt gccttccttg 504 0
cgcatgctca acctcgcggg agaacccctg aaccgggatc tttacctgcg gcttcaggca 5100
aaactgaccg ccacacgcat cgtcaacctc tacggcccga cggaaacaac aacctattcc 5160
accgccctgg tgatcgagcc cgcacaacaa gagatcacca tcggttttcc actgtatggc 522 0
acctgggtgg atgtcgttga tcaaaacatg caaagcgtcg gtatcggtgt acctggcgag 5280
ttgatcattc atggacacgg cgtggcgcaa ggctatgtca gcgaccccgt gcgtagcgcc 5340
gcttctttcc tgccggcatc cgatggcttg cgttgctacc gcacgggaga ccgtgtccgc 5400
tggttgcccg atggccgcct ggactttatc ggtcgagagg atgatcaggt caaagttcgc 5460
ggtttccggg tcgagttggg gcctgttcag gcggcactgc atgccattga gacgattcat 5520
gaatccgcag tagtcgttgt gccgaaaggg cagcagcgca gcatcgtggc gttcatcgtc 5580
ctcaaagcgc cgagcgaaga tgaagcggtg cagcgcaata acatcaaaca acacttactc 5640
ggcgtactcc cctattacgc actaccggac aagtttattt ttgttaaagc actgccaaga 5700
aacacacatg gaaaaatcga cagaacgctg ctcttgcaac atgagcccca gactgagcaa 5760
gaaagcgcca tgcgagatgc gaccgacgtc gaacatcgca tcgccaactg ctggcaaacc 5820
atcatcggac accccgtcca actccacgaa aacttcctgg acattggcgg ccactcgctt 5880
tcgcttacgc atttaacggg cctactgaga aaagaattta atattcatat ttctctacac 5940
gacctctgga tcaggccaac catagaacaa caggccgact tcattcataa gttgcaaaat 6000
tcggtattga caaaacctgc cgccgcgcca atcccgcgac ttgaccgaaa gatctctcat 6060
cattaatcag gagtaccgca tgagcgtcga tacatgcagg actgcaactt tccctgcgtc 612 0
atacggccag gaacagatct ggtttctgaa cgaactaaac ccgcactctc aactggctta 618 0
taccctggcg atgaaagtat ctatcgccgg gaaattgaac acactgcggt tgcagcgtgc 6240

ggtcaaccaa gtggtggcct cccaggaaat tttgagaaca tcattcgcct ataaaaacca 6300
gaagttgagc caggtcattt caccctccgc gacactgccc attcgcagcg cgcactgcat 6360
tgacgatgta cctgggctgc aacgcctgat caacatggaa gcccagcgtg gctggtcgct 642 0
gagcagcgcg ccactgtacc gcttgctgct gataaaaacc ggcgaccagc aacatgagct 6480
ggtcatctgc acccaccata tcgtctgcga tggcatctcg ctgcaactgc tgctgcaaaa 6540
aatagtcagc gcctatcaag gccaaagcga tgggcgggtg ctcacaagtc cggatgaaga 6600
gaccctgcaa ttcgtcgatt atgcggcctg gtcaaggcag cacgaatatg ccggtctcga 6660
gtactggcgc cagcaactgg ccgacgcccc gacaatcctg gatatttcga caaaaaccgg 6720
ccgaagtgag caacagacat ttctcggcgc gcgaattccc gtcgagttca gccaccacca 6780
atggcaagca ttgcgccaga tattcagacc ccagggtatc tcctgcgcgg cggtgttcct 6840
ggcggcctac tgcgtcgtcc tgcaccgcct ggccgagcag gacgacattc tgatcgggct 6900
gccaacttca aatcgcctgc gtccggagtt ggcacaggtg atcggctacc tgtccaatct 6960
gtgcgtgttt cgcagccagt atgctcacga ccagagcgtc acagactttc ttcaacaggt 7020
tcaattgacc ttacccaact tgatcgagca cggggagacg cctttccagc aagtactgga 7 080
aagtgttgag catacccggc aagccggtgt gacgccgttg tgccaagtac tgtttggtta 7140
tgagcaggac gttcgacgca cgctggatat cggcgacctg caattgacgg tctcggatgt 7200
ggacacgggg gccgcacgcc tggatctatc gctgttcttg ttcgaggacc acgaactcaa 7260
cgtttgcggt tttctggaat atgccacgga ccgtatcgac gccgcatctg cgcaaaacat 7320
ggtgcgcatg ctcagcagcg tgctacgcga gttcgttgcg gcgccgcagg cgccgctcag 73 80
cgaagtacag ctgggggcgg cggattccca agcccagaca cctgcgatcg caccagcatt 744 0
cccaagcgtg ccggctcgtc tgttcgcctt ggcagacagt caccccaatg cgaccgcgct 7500
gcgtgacgag caaggtgaac tgacctatgc gcaagtttgc caacagattc tgcaggcagc 7560
ggccactctg cgagcccagg gggcgaaacc tggaaccctg atcgcggtca tcggcgagcg 762 0
cggtaacccc tggttgatcg ccatgttggc gatctggcaa gtcggcggta tctatgtgcc 7680
attgtccaag gacctgcccg aacagcgcct gcaaggcatc ctggcggaac tcgaaggggc 7740
catactgatt accgacgaca ccacgccgga acgcttccgg caacgtgtga cgctgcccat 7800
gcacgcctta tgggccgatg gcgcaacgca fccacgagcgg cagacgacgg acgccagccg 7860
gctgtctggc tacatgatgt acacctcggg atcgaccggt aaaccgaaag gcgtgcatgt 7920

cagccaggcc aacctggtcg cgaccctgag cgcattcggc cagctgctgc aggtgaaacc 7980
cagcgatcgg atgctcgcac tgacgacctt ctccttcgac atttcgctgc tcgagctgct 8040
gcttcccctg gtccagggcg ccagcgtgca aatcgctgtc gcacaggctc aacgcgatgc 8100
ggaaaagctc gcgggctatc tcgcagaccc tcggatcacg cttgttcagg ccacaccggt 8160
gacctggaga ctattactgt cgacaggctg gcagccacgg gaaagcctga ccctgctgtg 8220
cggtggcgaa gcgctgccac aggatctggc ggacaggttg tgcttgccgg gcatgacctt 8280
gtggaacctc tacggcccca ccgaaacaac aatctggtcc acggcctgcc gcctgcaacc 8340
gggtgcgccg gtgcaactgg gccatcccat tgcaggtacg caaatagccc tggtggatcg 84 00
gaacctgcgc agcgtgccca gaggggttat cggtgaactg ctgatttgcg gccccggcgt 8460
cagccagggc tactatcgca acccggttga aacagccaag cggttcgtac cggacccgca 8520
tggttcaggt aagcgcgcct atctgaccgg cgaccggatg cgcatgcagc aggatggttc 85 80
gctggcctat atcggccgac gtgacgacca gatcaagctg cgcggccacc gtatcgagct 8640
gggagagatc gagacagcgt tgcgaaaact gcccggcgta cgggatgctg ccgcccaact 8700
ccatgaccag gacccaagtc gaggcataca ggcctttgtc cagctttgcg caacggtcga 8760
tgagagcctc atcgatatag gccagtggct ggaaacactg cgccaaacgc tgcctgaggc 8 820
gtggctgcct actgagtatt acaggatcga tggcatccct cttacctaca acggcaaacg 8 880
cgacaggaag cgcctcctgc accaggccgt caggctgcaa acactcagtc tgagggtggc 8940
tcccagcagt gacaccgaga cccgggtgca gcagatctgg tgcgagctgc tcggtctcga 9000
ggatatcggc gttacggatg attttttcca gttaggcggc cactccattc tggtggcgcg 9060
catggtcgag cgcatcgaaa ccgcgtttgg acggcgcgta cctatcgcag atatctattt 912 0
ttctccgacg atcgcccgtg tggcggcgac gctggactcc atgacatttg aacaaggact 9180
ggccgcacac agcgtgaaag gcgattggga gttcaccgcc atcagccttc aacacaacgc 924 0
cgacagcaca gccgccgctc aggagagatg aatcatgcac agccccacta tcgatacttt 9300
cgaggccgca ctgcgctcat tgcccgctgc ccgcgacgca cttggtgcct atcccttgtc 93 60
cagcgaacaa aagcgcctct ggttactggc ccaactggcg ggcacggcaa cgttgccggt 9420
aacggtgcgt tatgcattca ccggcacggt ggaccttgct gtcgtgcagc agaacctgag 948 0
cgcgtggatc gcacacagcg agtccttacg cagccttttc gtcgaagtac tggaacgccc 954 0
cgtcaggctt ctgatgccta cgggcctggt gaaactggag tacttcgatc gcccgccatc 9600
cgatgccgat atggccgagc tcataggcgc cgcctttgaa ctcgacaaag ggccgttgct 9660

gcgtgcgttc atcactcgaa ccgctgcaca acagcatgaa ttgcatctgg tcggccatcc $720 tattgtcgtg gacgaacctt ccctgcagcg cattgcccaa accctcttcc agaccgaacc 978 0 cgatcatcag taccccgccg tcggtgcgat cgccgaggta ttccagcgcg aacagacact 9840 ggcacaggat gcgcaaatca ccgaacaatg gcagcaatgg ggaataggcc ttcaggcgcc 9900 tgcggcaacc gaaattccga ccgaaaaccc ccgccccgct atcaagggct cagatcgtca 9960
agtacatgaa gcccttactg catggggcga ccaacccgta gcagaggccg aaattgtcag 10020
cagttggctg accgtgctga tgcgctggca gggatcgcaa* tcggcgcttt gcgcaatcaa 10080
ggtgcgcgac aaggcgcatg ccaacttgat cggcccactg caaacctacc tgccggtccg 10140
cgttgatatg ccggatggca gcaccctggc acaactgcga ctccaggtgg aggaacagct 102 00
caatggcaac gaccatccgt ccttttccac gctgctggaa gtttgcccac caaagcggga 10260
cctgagtcgc accccctact tccaaaccgg cctgcagttc attgcgcacg atgttgaaca 10320
gcgcgacttc catgccggca acttgacacg cctgccaacg aagcagccaa gcagcgacct 103 80
tgacctgttc atttcctgct gggtaagcga cggcacgctt ggcctgacgc tggattatga 10440
ttgcgccgtg ctgaattcga gccaggtcga ggttctggcc caggcgctca tcagcgtatt 10500
gtcagcgccc ggtgaacagc caatcgcaac cgttgcgctg atgggccagc aaatgcagca 10560
aaccgtcctg gctcaggccc acggcccccg cacgacgccg ccgcaactga cactgaccga 10620
atgggtcgcc gccagcacgg aaaaatcccc gctggcggtt gcggtgatcg accacggcca 10680
gcagctcagc tatgcagagt tatgggcaag agctgcactg gtagcggcga acatcagcca 1074 0
gcatgtggca aagcctcgga gcatcatcgc tgtagcactg cccagatcgg ctgaatttat 10800
tgcagcgctg ctgggggtag tgcgagcagg tcatgcgttc ttgcccatcg atccccgcct 10860
gcccaccgac cgcatccagt tcctgattga aaacagtggc tgtgagttgg tcattacctc 10920
tgatcagcaa tccgtggagg gttggccgca ggtcgccagg atacgaatgg aggcgcttga 10980
tccagacatt cgctgggtgg cgccgacggg gctcagccac agcgatgccg cctacctgat 11040
ctatacctcc ggcagcaccg gcgttccgaa gggagtcgtt gtcgagcacc ggcaagtagt 11100
gaataacatc ttgtggcggc aacgaacctg gccgctgacg gcacaggaca acgtgctgca 11160
taaccattcg ttcagcttcg atcccagcgt ctgggcgttg ttctggccgc tgctgaccgg 11220
tggcaccata gtgctggcgg atgtcagaac catggaggac agcaccgccc tcctcgacct 11280
gatgatccgc catgatgtca gcgttctggg tggcgtaccg agccttctcg gtacgctgat 11340

gctggtggcg ctacggggtt tcttgcccat gggcatcgtg ccggattacc cacgcatctt 13140
cgacatcgtg cccgtggact aygtcgccgc ggcgatcgtg cacatatcga tgcaaccgca 13200
gggcagggac aaattcttcc acctgttcaa cccggcgccg gtcaccatcc gccagttctg 13260
cgactggatt cgcgaattcg gttacgagtt caagttggtc gacttcgaac acggtcggca 13320
gcaggcattg agcgtaccgc ccgggcacct gctgtacccg ttggtccccc tgatcaggga 13380
tgccgatccg ctgccccacc gcgcgctgga ccctgactac atccatgaag tgaaccccgc 13440
actggaatgc aagcaaacct tagagctgct ggcctcctcg gacatcaccc tgtcgaaaac 13500
cacaaaggct tacgcgcaca caattttgcg ctacctgata gacaccggct tcatggccaa 13560
gcctggcgtg tagcggattg agcacaaaca ggacgaatat catggaatcg atagcctttc 13620
ccattgcaca taagcccttc atcctgggct gtccggaaaa cctgccggcc accgagcggg 13680
cgcttgcccc ttctgcggcg atggcgcggc aggttttgga gtacctcgaa gcgtgccccc 13740
aggcgaaaaa cctcgagcag tacctcggga cgctgcgtga agtcctggcg cacctgcctt 13800
gtgcttccac cggactgatg accgatgatc cacgggaaaa ccaggaaaac cgcgacaacg 13860
atttcgcctt cggtattgaa cgacaccagg gcgacactgt gaccctgatg gtcaaggcca 13920
cccttgatgc agcgattcaa acgggcgagt tggtccaacg cagcggcact agcctggatc 13980
actcggagtg gagcgacatg atgtcagtcg cacaggtgat tctgcagacg attgccgacc 14040
ctcgggttat gcccgaatcc cgtttgacgt tccaggcacc gaaaagcaag gtcgaagaag 14100
atgaccagga cccgctgcga cgctgggtgc gtggccacct gctgttcatg gtcctgtgcc 14160
aaggcatgag cctgtgtacc aacctcctga tcagcgcggc ccacgacaag gacctcgaac 14220
tggcgtgtgc acaggccaat cgcctgattc aactgatgaa catctcgcgc atcacgcttg 14280
agtttgcaac cgacctgaac tcacaacagt acgtcagcca gattcgcccg acgctcatgc 14340
cgscgatcgc gccgcccaag atgagtggca tcaactggcg tgaccatgtg gtgatgattc 14400
gttggatgcg ccagtccacc gatgcctgga acttcattga gcaggcctac cctcaactgg 14460
ctgaacgtat gcgaaccaca ttggcgcagg tctacagcgc tcatcggggg gtctgcgaaa 14520
agttcgtagg cgaagaaaac accagtttgt tggccaagga aaacgccact aatacggccg 14580
gccaggtgtt ggaaaacctg aagaaatcga gattgaaata cctc'aagaca aaaggttgcg 14640
ccggtgcggg ataagccctg actgcgcctc gcccccatca aaaccggact gatattcggg 14700
aaaacaaagg agagaagcat gccgacattt ctgggagacg acgacgcagt gccatgcgtg 14760

gtcgtcgtta acgccgacaa acactattcg atttggccaa gcgcgagaga cattccatca 14 820
ggttggtccg aagaaggatt caaggggtca cgttcagact gcttggaaca tatcgcgcaa 14 8 80
atctggccag agccgacggc atagatacaa cgtgatgcaa aaaatgcggg aaacatcaac 1494 0
taaccaaagc aaggaagaaa aatgacttca actcatcgca ccactgatca agtcaagcct 15000
gctgttctgg atatgccagg cctgtcgggc attcttttcg gccacgccgc attccaatac 15060
ctgcgggcca gctgcgaatt ggatctgttc gagcatgtcc gcgacctgcg cgaagccacc 15120
aaggagagca tcagcagccg actgaagttg caggaacgcg ccgccgatat tctgctgctg 151B0
ggcgcgacct ccctgggcat gctggtcaag gaaaacggca tctaccgcaa tgccgatgtg 15240
gttgaggatt tgatggccac ggacgactgg caacgtttca aggataccgt ggcctttgaa 15300
aactatatcg tctatgaagg gcagctggac tttaccgagt ccctgcagaa aaacactaac 15360
gtcggccttc agcgtttccc gggcgaaggg cgggacctct atcaccgcct gcaccagaat 15420
cctaagctgg aaaacgtgtt ctaccgctac atgcgctcgt ggtctgaact ggccaaccag 15480
gacctggtca agcacctcga cctgtcgcgc gtgaaaaaat tgctcgacgc gggtggcggt 15540
gatgcggtca acgccatcgc cctggccaaa cacaatgagc aactgaacgt aacggtactg 15600
gatatcgaca actccattcc ggtcactcag ggcaaaatca atgattccgg gctcagccac 15660
cgggtgaaag cccaggcatt ggatatcctg caccaatcct tccctgaagg ttacgactgc 15720
attctcttcg cccaccaatt ggtgatatgg accctcgaag aaaacaccca catgctgcgc 15780
aaggcctacg atgcgctacc agaaggcgga cgcgtggtca tcttcaactc catgtccaac 15840
gatgaaggcg acggcccggt catggccgca ctggacagcg tctactttgc ctgtctaccc 15900
gccgagggcg gcatgatcta ttcctggaaa cagtatgagg tctgcctggc ggaagccggc 1596 0
ttcaaaaacc ccgtacgcac cgcgattcca ggctggaccc cacacggcat catcgtggcc 16020
tacaagtaat tttgcctcct ccgcccctac tggggccgga ggagtcattt caacatttgc 16080
gtcattgacg ccacctggcg atagggacac ccacatggca cgttcacccg agacaaatag 16140
tgcgatgccg caacagataa gacagctttt atacagccaa ctgatttcgc aatcgattca 16200
aaccttctgt gaactgcgcc tgcctgatgt tctgcaagca gctggccagc ctacctccat 16260
cgaacggctt gctgagcaga cacacactca tatcagcgcc ctgtcacgct tgttgaaagc 1632 0
gttgaaacca ttcgggctag tgaaagaaac cgacgaaggt ttttccttga ccgatctcgg 163 80
cgccagtctg acccacgacg cctttgcttc cgctcaaccc agtgctttgt tgatcaatgg 16440
tgaaatgggc caagcctggc gtggcatggc gcagacaatc cgaaccggtg aatccagctt 16500

caagatgtac tatggcatca gcctgttcga gtattttgaa cagcacccgg aacgccgggc 16560
catttttgac cgttcccaag acatgggact ggacctggag atcccggaaa tcctggagaa 1662 0
catcaacctg aatgacggtg agaacattgt cgatgtaggg ggtggttcag ggcatttgct 16680
gatgcacatg ctggacaagt ggccagaaag cacaggcata cttttcgact tacccgtcgc 1674 0
ggcaaaaatc gcgcagcaac atctgcacaa atctggaaaa gcaggctgct ttgaaatcgt 16800
cgcaggggat tttttcaaga gcctgccgga cagtggcagc gtttacttgc tgtcccatgt 16860
cttgcacgac tggggcgacg aagactgcaa ggccattttg gccacctgcc ggcggagcat 16920
gccggacaat gcgctgttgg ttgtagtgga cttggtgatt gaccagagtg aaagtgccca 16980
gcccaacccc acgggcgcaa tgatggatct ttacatgctg tccttgttcg gtatcgccgg 1704 0
aggcaaagag cgcaacgagg atgaattcag aaccctcatt gaaaacagcg gcttcaacgt 17100
caaacaggtg aagcgcctgc caagtggaaa cggcatcatc ttcgcctacc caaaataaat 17160
gatcctcatt gcccctcgcc actttccagg ggggctattt tattctcggg tgattccccc 17220
cctaatgatt acaaggaaga cacatgtcga cgctggttta ctacgtagca gcaaccctgg 17280
atggttatat cgccactcaa caacacaaac tggattggct ggagaacttt gccctggggg 17340
atgacgcaac ggcctatgay gatttttatc agacgatcgg agcagtggtc atgggatcgc 17400
agacctatga atggatcatg tcgaacgctc ccgatgactg gccctaccag gacgtacccg 17460
cctttgtcat gagcaaccgg gatctgtcag cccccgccaa tttggatatc accttcttac 1752 0
gcggcgatgc cagtgccatc gcggtcaggg ccaggcaagc ggcgaagggc aagaatgtct 17580
ggctggtcgg tggcggcaaa acggcggcct gttttgccaa cgcaggggaa ttacagcagc 17640
tgttcatcac cactattcca acctttatcg gcaccggcgt tccggtactg cccgtagacc 17700
gcgcgcttga agtggttctc agagaacaac gcacgctgca gagcggtgcc atggaatgca 17760
tcctggacgt gaaaaaagcg gattaacgtc tacaagacaa tcgtgtatcg aaactcgcaa 17820
cgtccaaacc caagggaaaa accagtgaag cgattggtat tgagtttatg tttgttggct 17880
gttatcgctc tcgccagtgt tcaaggaata aggatggtga aacccgccgc cctgacagcc 17940
gccgatgctc gcgatatcgg ctatctgaat gtacgcgata gcctttccgt cattgccgcc 18000
gccccccacc ccaccgcctc acctcgccag gccgttgtca ggcattattt gcgggaaacg 18 060
attgcgggca tgggttacca ggtggttgag caaccctttc tatttaccat cgagagcatg 1812 0
gtgaaccggc agaaaaccct ctatgccgag ttgaacgagc agcagcgcca agcgttcgat 18180

gctgagctgg cccgggtggg cgcggacagt tttgaaaaag aagtgcggat tcgctccggc 18240
ctactggaag gcgacagcgg ccagggaacc aacttgatag cctcacaccg cgtaccggga 18300
gcgaccgcga cggtcctgtt catggcgcat tacgacagcg tcggcaccgc tcccggtgcc 18360
agtgacgatg gcatggccgt cgcctcgata ctccaactga tgcgggaaac cataacccgc 18420
agcgatgcca aaaataacgt tgtctttcta ctcrccgatg gcgaagaact gggcttgctc 184 80
ggagcggagc actacgtctc gcagctcagt acgcctgaac gtgaagccat ccgcctggtg 18540
ttgaactttg aagcccgggg taaccagggc atccctttac tgttsgagac atcccagaag 18600
gactacgccc tgatcaggac tgttaacgca ggggttcggg acatcatatc cttctcattc 18660
acgcccttga tttacaatat gctacaaaac gacaccgact ttacggtgtt caggaaaaag 18720
aacatsgcgg ggttgaattt tgcagtcgtg gagggttttc agcactacca ccacatgags 18780
gacaccgtgg agaaccttgs gccagagacc ttgtttcgct accaaaagac agtgcgtgaa 18 840
gtgggcaacc actttatcca gggtatcgac ctctcctccc tgagtgctga tgaggacgca 18900
acctatttcc cactgccagg cggcacgctg ttggtactca acttacccac cctgtatgcg 18960
ctgggcatgg gctcgttcgt gctctgcggt ctttgggcgc aacgctgccg cactcgccga 19020
cagcatcagg gcaagaattg cgtactgcgc cccatggcta ttgccctgct cggcattgcc 19080
tgcgcagcac ttgtattcta cgtcccgagc attgcctatc tattcgtcat ccccagtctg 1914 0
cttctggctt gcgccatgtt gtcgcgaagc ctctttatct cctattcgat catgctgctg 192 00
ggcgcttatg cctgcgggat actctacgcg cctatcgtct acctgatttc atcaggcctt 192 60
aaaatgccgt tcattgccgg ggtcattgca ctactcccgc tctgcctgct ggccgtggga 1932 0
ctggccggcg tcatcgcacg atcgagagac tgtcgaacct gcgactagca agacccgata 193 80
aaacgtcgct tcaaacgcca gatgacgtgc ctcgtcagcc aggcgtggaa ccatctggtg 1944 0
gcggcaaatg tgcataaggt gggaacgcag agcgcccgct gcaacacgcc caccccaagc 19500
accgcgcctc aacggataat caggctcaag ggaattccac cttgcaacct gaaagagcaa 19560
tcgagcgccc gtcggacaca acaaactgat caccgtcaat tcgggcaagg agcaatccac 19620
gggcttttgc tccaacctca actccctttg aaaaatcagc cggccacaat ttgcccctac 196 8 0
cctttcagga tatcctcgat aagcgtttta tcagaacagc gaaaaaccac ttcaagttcg 19740
tgtacttttt actgcgatct gcgatcgctc ccatggtaca araatgacag atgggaagat 19800
cgctttaata cctactctct cacctgagaa aaagtaacca ccgggccgta ttcctgatca 19860
gacactatcg cctgcacaca aaatttcttc tctggaaact tactcagcaa caccatcctc 19920

catgactgag caataaggtt gcacagttgt
cagcctgcaa aaacatcata caggtgcaca
tcacgattac aagactgcat ccaactggag
aacacgcccg actcgtaaac aacaaagtct
tcttcaggat gtacaacatc actaataaaa
cattgggact tccagcgctc gtatttggga
ttcatagcac tgttcctgca ctgatgcctt
tcgatggctc attgttcatc aagaaaaaaa
cggcgcgttg agaacagtga aaagtcactc
acaagatacc cagtattcag cttttcaatg
tcgacctctt cagcaaactc atcgccgtag
ggtttcaaag ccgaattgaa tgagccgatc
agttcgggtt cactgaacag gtgcagcatt
gccttgaaga gcgaatacca atcgacgctc
ccaggaacca cctgcccccg gacattgcgc
accgcctttt ttctgcgcca ggcaaagtcc
cgtatggccg cctttgatgc ccagcacgct
ccgaatttat ccgcattgtg cgacacctgc
aatgacgaaa tgaacgcctc tccaacaccc
gtaatttgca catagggctt cataaatcgt
gtatatccaa agagctctcc cacgttactc
cgcctcacgc gccaccgccc acggcgccat
cttgagcacg aaattgcggc atttctcggc
ctgcatctgc gccagctcgg cttcacggca
tttacgggcg cgagccacgg cgtacttttc
cagccaacgg ctgaaggcgt gcgggcagcg
ctcggcttgc aaggcactga tcggcaggca
gccaacggcg cgtggcaggc tgtaggtcca

tcataaacag cttcatcgac tctatcgctg 19980
tgattcaaaa ccttctcaac cgccaccaca 20040
aatgcttcaa cagtaaatcc gctttccaga 20100
ggaaacaaca cggtggaaaa caccaggaca 20160
caagactcaa ccaactcgga ttgatccttc 20220
gacctgacag ggtctgtcgt cctgattatt 20280
ttggcgattt tttgtttgag cgagaatcga 20340
ttctctcgaa acagactctt aagctcggcc 20400
gccacatcct gaatattctc tgtgaccctc 20460
ggaaagcctg acgcagcaat ccgctcaaca 20520
aacatagccc aaccgatatt gggcacgtcc 20580
tgaaaacttt tatatttctc atgcgggcta 20640
ccgagttgag gaggaaaaat ttcacaccat 20700
ttgctactga ccgagcggta tgtgattgcg 20760
gccgtgtgga tgacgctccc acagccttta 20820
aggtagaagt ccgataatgc gccgttaaac 20880
tcactcgcgg ccacgcccat gaaaggctct 20940
tctggaacca gtaactcccc ttcccgactt 21000
caaccgatag tttcagactt cgtcctgatt 21060
caaagtctcg tcaattcacg ggtgacacaa 21120
atcgcatcga gtctatcaac caaccaacgc 21180
caaccgctgc ggggtctggc aggttttgcg 21240
gaattggtgg cggttgtgca ccatgtccaa 21300
gcgtgtaatc tggtcgatgt ccaacgccgc 21360
atcggttaac gcgctgcttg cctgctgcat 21420
cgggccgatg ccctgaacca gcccgtattg 21480
ggcgtcggtg agttgatggg cgacttcact 21540
gtattcggag ccgtacaggc ccatggtttt 21600

gtaatgcggg ttgagtacca cgctctcgcg ggccaatacg atgtctgcgg ccagcgccag 21660
cattacacca ccggcgccgg cgctgccggt caggccgctg atcaccagtt gccgggccgt 21720
gagcagttcg tggcacacat cgtacgatgg cctgaatgtt ggcccaggct tccagccccg 21780
gcactggggc ggcctggatg acgttgaggt gcacaccatt ggaaaagctg ccgcgcccgc 21840
ccttgatcac cagcacttgg gtgtcccgcg tcttggccca gcgcaacgcc gccaccagtc 21900
gctggcactg ctcggtgctc atggcgccgt tgtagaactc aaaggtgagt tcaccgacat 21960
ggccggcttc gcgatagcga atcggttgat aggcttgctc atcgaacatt tgattggcga 22020
tcgagctgtc cagcacggga atatccgcca gtgcttccgc cagcacgtgg cgggccggca 22080
gcttgaaggt ctcctccccc ggccgggctt tgcgtttgag cgagccgatc cacaggctct 22140
gatcaccggc cgccaccagc accgcgtcgt cctgcaccgc gaggatctca cccggtgtgc 22200
cgtggcgcgc atccaggtgc gcgtcgtaca ggtaatactg cccgccctgg atactggcca 22260
gcacaccggg ctggccatcg gctgcgtcga tgcagcgttt gatgaagcgt gcgcaatcgt 22320
accaactgaa ggtgcgatca gcctgtgtca tgttcggctg caaacgcccg attacgtggg 22380
cttgggtgta atcgagcggc accgggacga aaacccgggc gaacttttcc accacgtcgc 22440
ggatgcaata gagggcggcg tcactcaccg cgccgttgta cagctcggat ttgcgcacat 22500
cggcaggcat gtcgaattca caggtcgacc agatcggccc ggcgtccatt tcctccaccg 22560
cctgcaaagc cgtgacgccc cagcggccga cctgctggct gatggcccag tccagcgcgc 22620
tggcaccacg gtcgccgacg atgcccggat ggataatcac cacagggcgc tcaaggttgc 22680
tccaaagttg ctgtggcaca cggtctttca gaaaggggca gatcaccagg tcggcgtctg 22740
aatcctcgat ctgctggcac accaaggctg gatcggtgaa cagaacaacg ctgggcgcgt 22800
gccccgactg gcgtaaatcc agccaggccc gctgggtcaa accgttgaac gccgacgcta 22860
acacgatgat cttcaatgac cgcatggctg actcatcctt gagaatgcgc ggccagaggt 22920
gctccttgag ccctccctgg cctttgatgg aagtacaagg atagttggcg tgccaggcag 22980
gctacctgat caggatcaat cttgtgtcag cgagtgcttg aacgtaggcg cctgcgttca 23040
accaataggc gcatggsctg gcgagtgctc ccgcgtgccc tctgccacaa gggacgccag 23100
gtattcgcca aacccgccgc gacacttgta gtcccgacgc gcactggtga ccacgggatt 23160
ggtcgccgtc cacacgatcc gcgcgccaat cctctcgagg tcggccacca actgcacatc 23220
ttcatgggca accaaatgct ggaacccacc cgcgtttcga taggcatccg cactcaagcc 23280
caggttggca ccgtgtatat ggcggtggtt ctcggtgaac tgatacaact caaggtagcg 23340

cgaacgaacc gattcaccgt actcgctcca gctgtccacc tcgacggttc cgcacaccgc 23400
atcggcgcca aagccgatct gacgcaccag ccagtcgrcg ggcacaactg tgtcagcgtc 23460
ggtgaatgcc agccactggg cgccgacttc aagcaatcgc tccgcgccca aggccctggc 23520
cttgcccaca tttcgaacgc tcacctcaag cgtkgcgaca cccatggccg acacgcgcgt 23580
ggcggtctcg tccgaacacg catccagcac caccagcaat tggacctgtt ggtgtgccag 23640
agccggatga gcaatggcgc gctggatgga ggcgaggcag gcactgatgt gccgttcttc 23700
gttatgggca ggtatcacta tccctatcat tgacgttccc tctaccaggc aaagtgtcta 23760
cagctatcga ccgggccgtg aggcagaagg tttaaacaat ctgaaggcgc cgccaaacaa 23820
tgacgtgaga caggtcgcag tgattaaacg gaacgtcaca ggcgccacag gctcagatgg 23880
tttacgtgtt tgatgcacgg atgaacccgc cattcctaca aacaggtcag ccatcatgtc 23940
taacgattat caaggtatcg ccagtgtcat cacggcttct cgtcacatgg gtacagactc 24000
ggatgaacgc cttaatgaga cggtaaatat tcaattgacc tgcagcggta aaccaacgat 24060
tgcgcggttg agtttcgaca ccccgcttca atggcccggc caccccaact ttgtgctgat 24120
caacctgccg gacggttcat cggtgggtgg tgtgattgcc gaaattgaaa agtcgaccga 24180
tggcccgggt tgggtgacgt ttacggtgga tgactgaggt cttcccaaca ggcttcaaat 24240
cacctccagg cggctgcctc gaatgagaca cacaggccag taatcgagac gcacagacaa 24300
gcctattttc gcagatacat tttgtaacgt cctatgattg acgcttgctc gaatcaccgc 24360
agggattggg tggcgtgtgt ttatcacgcc cttgaatccg cagcgaaaaa tgattcgagt 24420
tcagcgaaca attcgattgg gacaaacaaa aggatgcggg ctatgtcatt gcgtaattta 244B0
tctttattgg tcaccacact ggcgctgttt aagtggggtg taatgcgctc gcggggcaaa 24540
acccaacatg ctcagtgatg atgacgtgaa atcccaaagc gccggcgcac tgggctatgc 24600
cccgacagac ctgagcatcg tcaaccgtcg aaccgaaggc accaacacct acgtgctgct 24660
taaaaccaac gacaacaagc agttcaactg cattatcaac ggaggcaata tcctgacctt 24720
cggtatgtcc aacccgcctt cgtgtgcgaa gaaaggtgaa cagatcaaga gtggcccgtt 24780
cgggagctga tctgtcgctg gaaaaaaggg ccaggccacc tctaagaacg gaggcctggc 24840
ccttttttat tcgctcagat gagtttaaaa gacaagatat cgggcagctg ggctccggcc 24900
cgttcagtct gggcacccca cacaaaatgc tcagcgacta cttggccgtc gccgcacacc 24960
gtttaacggg tgcgacctac agcgtcrccc tggttgaagg cagcaacgaa taaaccctat 25020

tgatcggaga gcgaccatgc acccgcataa aaccgcgatt gtcttgattg aataccagaa 25080
cgacttcacc acccccggcg gcgtgttcca tgacgctgtg aaagacgtca tgcaaacgtc 2514 0
caacatgctg gcgaataccg ccaccacgat tgagcaggcc cgcaagctgg gcgtgaagat 252 00
catccactta cccatccgct ttgccgacgg ctacccagag ctgaccctgc gctcatacgg 25260
cattctcaaa ggcgtcgccg acggcagcgc gtttcgtgcc ggcagctggg gcgccgagat 2532 0
caccgacgcg ctgaaacgcg accccaccga tattgtgatc gaaggcaaac gcggcctgga 25380
tgctttcgcc accaccgggc tggacctggt gctgcgcaac aatggcatcc agaacctggt 25440
tgtcgcaggt ttcctgacta actgctgcgt tgaaggcacg gttcgatccg gttacgagaa 25500
aggttatgac gtggtgacct tgaccgactg caccgcgaca ttcagtgatg aacaacagcg 25560
cgcagccgag cagtttacgt tgccgatgtt tttcgcaaac cctgcaacac accgcgtttc 2562 0
tgcaagcact gaacgccgga taaaaaaagc ggcggactcc tgccgagtcg ccgctttttt 25680
agtgcttggg tcattcggtt ggcgcgtact gcatttcgcc gttcccaaac gaccagtctt 25740
cgcgcttcac gtccaccagg ctgatccaca cgtcttcctt gcgcagcccg gtcttggcat 25800
ggatgccgtc ggcgatgaac ttatagaaag cctttttcac gtcaatgctg cgcccggcgt 25860
tccacgtgac ttggataaac acgatcttgg gtgtgtaagt gacgccaaga tacccggccg 2592 0
ccgggtaaac cagctcatcc ttggcatggc ggttgatgat ctggaatttg tcgtgctcag 25980
gcacgttggc ccacactggt catcgcggcg tacacgacat caccgatggc cgtcgcggtt 26040
tcagtggaag tgtcggcggc gaggtcgatt cgaactaaag gcatggacaa atccttagtg 26100
attttcagct gaaaatgggc gtgtggctca cacactcgcg ccaaccgggc aacttgcgcc 26160
aggccaacga gttgctggcc cagggagttg ccgacggttt gcgctagtgc gccgcgaaac 26220
ttcggcattt gacgcatcgg tgaatggctg accggatgtc agtgcttatt gacctgaata 262 80
tagactgccg tgcacagacc aatcaaacaa ataccggcga tgtagtaagc ggcgcccatc 26340
tgactgtatt gaagcaatag agtaacgacc atcggcgtca ggccaccgaa tacggcgtac 26400
gacaagttgt aggaaaatga caagccggaa aaccgcacta ccggtggaaa ggcacgcacc 26460
atcacagcag gggctgcgcc tatcgcgccg acaaaaaaac cggtaagtga atagagtgga 26520
accagccatt gcgggtgcgt ttcaagcgtc ttgaacaaga gcagtgcgct gaacagaagc 26580
atgacgctgc cgatcatcaa tacccarccc gcactgaaat gatcggccag tttcccggcg 2664 0
atcacgcaac caacactcaa rrcacrcaat agcgagactg ttggcctgca aggcttgcgc 26700
tgcag 26705

Lenght: 1004
Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met Leu Leu Glu Val Ala Phe His Val He Thr His Leu Ser Thr Ser
15 10 15
Gln Leu Val Ser Arg He Glu Arg Val Val Glu Arg His Ala Ser Leu
20 25 30
Arg Gln Arg Phe Val Met Arg Asn Gly Thr Tyr Trp He Glu Gln Ala
35 40 45
Pro Pro Gln Gln Arg Arg Tyr Cys Val Val Arg Thr Tyr Asp Glu Ala
50 55 60
Ser Thr Asp Ala Leu Leu Ala Pro Ser Arg Glu His He Gly Val Glu
65 70 75 80
Ser Glu Arg Leu Phe Arg Ala Glu Val Val Glu Arg Ser Asp Gly Gln
85 90 95
Arg Tyr Leu Val Phe Arg He His His He He Ala Asp Leu Trp Ser
100 105 110
Val Gly Leu Leu He Arg Asp Phe Ala Glu Asp Cys Met Asp Arg Ser
115 120 125
Ser He Thr Leu Ala Ser Arg Pro He Ala Pro Leu He Asp Pro Glu
130 135 140
Phe Trp Arg His Gln Met Ser Gln Asp Thr Pro Phe Ser Leu Pro Met
145 150 155 160
Ala Ser Leu Glu Gln His Thr Asp Arg Arg Met Val Leu Ser Ser Phe
165 170 175
Val He Asp Gln Glu Ser Ser Ala Asp Leu Ala Arg Leu Ala Thr Ala
180 185 190

Cys Ala Val Thr 195

Pro Tyr Thr Val 200

Met Leu Ala Ala

Gln Val Leu Ala 205

Leu Ser Arg Ilee 210

Gly Gln Ser Gly

Arg Leu Ser Leu 220

Ala Val Thr Phe

His Gly Arg Asn 225

Arg Gly Asn Lys 230

Asp Ala Val Gly 235

Tyr Phe Ala Asn 240

Thr Leu Ala Val

Pro Phe Asp Val 245

Ser Glu Cys Ser 250

Val Gly Glu Phe 255

Val Lys Arg Thr 260

Ala Lys Arg Leu

Asp Glu Ala Ser 265

Lys Ala Ser Val 270

Gly Ala Gly Tyr 275

Pro Glu Leu Ala 280

Glu Phe Met Thr

Pro Leu Gly Trp 285

Ala Ala Thr Ala 290

Pro Thr Asn Ala 295

Val He Tyr Gln 300

Gln Asp Met Pro

Gly Met Pro Arg 305

Gly Leu Ala Ala 310

Ala Leu Leu Gly 315

Leu Gly Thr Val 320

Gln Leu Gly Glu

Met Ala Leu Thr 325

Ala Glu Gln Ala 330

Pro Pro Ser He 335

Gly Pro Phe Ala 340

Thr Ala Leu Leu

Leu Thr Arg His 345

Asp Gly Lys Leu 350

His Gly Arg Val 355

Glu Val Asp Pro 360

Ala Gln His Pro

Gly Trp Leu Ala 365

Glu Ala Leu Ala 370

Arg Gln Phe Ala 375

Val He Leu Arg 380

Glu Met Val Arg

Asp Pro Gln Ala 385

Arg Leu Ser Ala 390

Leu Pro Ala Cys 395

Leu Leu His Gln 400

Pro Lys Tyr Pro

Ser Gln Ala Arg 405

Pro Ala Pro Ala 410

Ser Glu Thr Leu 415

Thr Leu Ile Asn Thr Val Pro Ser Val Ala Asp Ala Leu Leu Gln His
645 650 655
Asp Val Leu Val Pro Ser Leu Arg Met Leu Asn Leu Ala Gly Glu Pro
660 665 670
Leu Asn Arg Asp Leu Tyr Leu Arg Leu Gln Ala Lys Leu Thr Ala Thr
675 680 685
Arg Ile Val Asn Leu Tyr Gly Pro Thr Glu Thr Thr Thr Tyr Ser Thr
690 695 700
Ala Leu Val He Glu Pro Ala Gln Gln Glu Ile Thr Ile Gly Phe Pro
705 710 715 720
Leu Tyr Gly Thr Trp Val Asp Val Val Asp Gln Asn Met Gln Ser Val
725 730 735
Gly Ile Gly Val Pro Gly Glu Leu Ile Ile His Gly His Gly Val Ala
740 745 750
Gln Gly Tyr Val Ser Asp Pro Val Arg Ser Ala Ala Ser Phe Leu Pro
755 760 765
Ala Ser Asp Gly Leu Arg Cys Tyr Arg Thr Gly Asp Arg Val Arg Trp
770 775 780
Leu Pro Asp Gly Arg Leu Asp Phe Ile Gly Arg Glu Asp Asp Gln Val
785 790 795 800
Lys Val Arg Gly Phe Arg Val Glu Leu Gly Pro Val Gln Ala Ala Leu
805 810 815
His Ala Ile Glu Thr Ile His Glu Ser Ala Val Val Val Val Pro Lys
820 825 830
Gly Gln Gln Arg Ser Ile Val Ala Phe He Val Leu Lys Ala Pro Ser
835 840 845
Glu Asp Glu Ala Val Gln Arg Asn Asn Ile Lys Gln His Leu Leu Gly
850 855 860Val Leu Pro Tyr Tyr Ala Leu Pro Asp Lys Phe Ile Phe Val Lys Ala
865 870 875 880
Leu Pro Arg Asn Thr His Gly Lys Ile Asp Arg Thr Leu Leu Leu Gln
885 890 895
His Glu Pro Gl Thr Glu Gl Glu Ser Ala Met Arg Asp Ala Thr Asp
900 905 910
Val Glu His Arg Ila Asn Cys Trp Gln Thr Ile Ile Gly His Pro
915 920 925
Val Gln Leu His Glu Asn Phe Leu Asp Ile Gly Gly His Ser Leu Ser
930 935 940
Leu Thr His Leu Thr Gly Leu Leu Arg Lys Glu Phe Asn Ile His Ile
945 950 955 960
Ser Leu His Asp Leu Trp Ile Arg Pro Thr Ile Glu Gln Gln Ala Asp
965 970 975
Phe Ile His Lys Leu Gln Asn Ser Val Leu Thr Lys Pro Ala Ala Ala
980 965 990
Pro Ile Pro Arg Leu Asp Arg Lys Ile Ser His His
995 1000
Lenght: 1062
Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met Ser Val Asp Thr Cys Arg Thr Ala Thr Phe Pro Ala Ser Tyr Gly
15 10 15
Gln Glu Gln ILe Trp Phe Leu Asn Glu Leu Asn Pro His Ser Gln Leu
20 25 30
Ala Tyr Thr Leu Ala Met Lys Val Ser Ile Ala Gly Lys Leu Asn Thr
35 40 45
Leu Arg Leu Gln Arg Ala Val Asn Gln Val Val Ala Ser Gln Glu Ile
50 55 60

Leu Arg Thr Ser Phe Ala Tyr Lys Asn Gln Lys Leu Ser Gln Val Ile
65 70 75 80
Ser Pro Ser Ala Thr Leu Pro Ile Arg Ser Ala His Cys Ile Asp Asp
85 90 95
Val Pro Gly Leu Gln Arg Leu Ile Asn Met Glu Ala Gln Arg Gly Trp
100 105 110
Ser Leu Ser Ser Ala Pro Leu Tyr Arg Leu Leu Leu Ile Lys Thr Gly
115 120 125
Asp Gln Gln His Glu Leu Val Ile Cys Thr His His Ile Val Cys Asp
130 135 140
Gly Ile Ser Leu Gln Leu Leu Leu Gln Lys Ile Val Ser Ala Tyr Gln
145 150 155 160
Gly Gln Ser Asp Gly Arg Val Leu Thr Ser Pro Asp Glu Glu Thr Leu
165 170 175
Gln PIle Val Asp Tyr Ala Ala Trp Ser Arg Gln His Glu Tyr Ala Gly
180 185 190
Leu Glu Tyr Trp Arg Gln Gln Leu Ala Asp Ala Pro Thr Ile Leu Asp
195 200 205
Ile Ser Thr Lys Thr Gly Arg Ser Glu Gin Gin Thr Phe Leu Gly Ala
210 215 220
Arg Ile Pro Val Glu Phe Ser His His Gin Trp Gin Ala Leu Arg Gin
225 230 235 240
Ile Phe Arg Pro Gln Gly Ile Ser Cys Ala Ala Val Phe Leu Ala Ala
245 250 255
Tyr Cys Val Val Leu His Arg Leu Ala Glu Gln Asp Asp Ile Leu Ile
260 265 270
Gly Leu Pro Thr Ser Asn Arg Leu Arg Pro Glu Leu Ala Gln Val Ile
275 280 285

IGly Tyr Leu Ser 290

Asn Leu Cys Val

Phe Arg Ser Gln 300

Tyr Ala His Asp

Gln Ser Val Thr 305

Asp Phe Leu Gln 310

Gln Val Gln Leu 315

Thr Leu Pro Asn 320

Leu lie Glu His

Gly Glu Thr Pro 325

Phe Gln Gln Val 330

Leu Glu Ser Val 335

Glu His Thr Arg 340

Gln Ala Gly Val

Thr Pro Leu Cys 345

Gln Val Leu Phe 350

Gly Tyr Glu Gln 355

Asp Val Arg Arg 360

Thr Leu Asp lie

Gly Asp Leu Gln 365

Leu Thr Val Ser 370

Asp Val Asp Thr 375

Gly Ala Ala Arg 380

Leu Asp Leu Ser

Leu Phe Leu Phe 385

Glu Asp Glu Leu 390

Asn Val Cys Gly 395

Phe Leu Glu Tyr 400

Ala Thr Asp Arg

lie Asp Ala Ala 405

Ser Ala Gln Asn 410

Met Val Arg Met 415

Leu Ser Ser Val 420

Leu Arg Glu Phe

Val Ala Ala Pro 425

Gln Ala Pro Leu 430

Ser Glu Val Gln 435

Leu Gly Ala Ala 440

Asp Ser Gln Ala

Gln Thr Pro Ala 445

lie Ala Pro Ala 450

Phe Pro Ser Val 455

Pro Ala Arg Leu 460

Phe Ala Leu Ala

Asp Ser His Pro 465

Asn Ala Thr Ala 470

Leu Arg Asp Glu 475

Gln Gly Glu Leu 4B0

Thr Tyr Ala Gln

Val Cys Gln Gln 4B5

lie Leu Gln Ala 490

Ala Ala Thr Leu 495

Arg Ala Gln Gly 500

Ala Lys Pro Gly

Thr Leu lie Ala 505

Val lie Gly Glu 510

Arg Gly Asn Pro

Trp Leu lie Ala

Met Leu Ala lie

Trp Gln Val Gly

515 520 525
Gly lie Tyr Val Pro Leu Ser Lys Asp Leu Pro Glu Gln Arg Leu Gln
530 535 540
Gly lie Leu Ala Glu Leu Glu Gly Ala lie Leu lie Thr Asp Asp Thr
545 550 555 560
Thr Pro Glu Arg Phe Arg Gln Arg Val Thr Leu Pro Met His Ala Leu
565 570 575
Trp Ala Asp Gly Ala Thr His His Glu Arg Gln Thr Thr Asp Ala Ser
580 585 590
Arg Leu Ser Gly Tyr Met Met Tyr Thr Ser Gly Ser Thr Gly Lys Pro
595 600 605
Lys Gly Val His Val Ser Gln Ala Asn Leu Val Ala Thr Leu Ser Ala
610 615 620
Phe Gly Gln Leu Leu Gln Val Lys Pro Ser Asp Arg Met Leu Ala Leu
625 630 635 640
Thr Thr Phe Ser Phe Asp lie Ser Leu Leu Glu Leu Leu Leu Pro Leu
645 650 655
Val Gln Gly Ala Ser Val Gln lie Ala Val Ala Gln Ala Gln Arg Asp
660 665 670
Ala Glu Lys Leu Ala Gly Tyr Leu Ala Asp Pro Arg lie Thr Leu Val
675 680 685
Gln Ala Thr Pro Val Thr Trp Arg Leu Leu Leu Ser Thr Gly Trp Gln
690 695 700
Pro Arg Glu Ser Leu Thr Leu Leu Cys Gly Gly Glu Ala Leu Pro Gln
705 710 715 720
Asp Leu Ala Asp Arg Leu Cys Leu Pro Gly Met Thr Leu Trp Asn Leu
725 730 735
Tyr Gly Pro Thr Glu Thr Thr lie Trp Ser Thr Ala Cys Arg Leu Gln
740 745 750

Pro Gly Ala Pro 755

Val Gln Leu Gly 760

His Pro Ile Ala

Gly Thr Gln Ile 765

Ala Leu Val Asp 770

Arg Asn Leu Arg 775

Ser Val Pro Arg 780

Gly Val Ile Gly

Glu Leu Leu He 785

Cys Gly Pro Gly 790

Val Ser Gln Gly

Tyr Tyr Arg Asn BOO

Pro Val Glu Thr

Ala Lys Arg Phe 805

Val Pro Asp Pro 810

His Gly Ser Gly 815

Lys Arg Ala Tyr 820

Leu Thr Gly Asp

Arg Met Arg Met 825

Gln Gln Asp Gly 830

Ser Leu Ala Tyr 835

Ile Gly Arg Arg 840

Asp Asp Gln Ile

Lys Leu Arg Gly 845

His Arg Ile Glu 850

Leu Gly Glu Ile 855

Glu Thr Ala Leu 860

Arg Lys Leu Pro

Gly Val Arg Asp 865

Ala Ala Ala Gln B70

Leu His Asp Gln 875

Asp Pro Ser Arg 880

Gly Ile Gln Ala

Phe Val Gln Leu 885

Cys Ala Thr Val 890

Asp Glu Ser Leu 895

Ile Asp Ile Gly 900

Gln Trp Leu Glu

Thr Leu Arg Gln 905

Thr Leu Pro Glu 910

Ala Trp Leu Pro 915

Thr Glu Tyr Tyr 920

Arg Ile Asp Gly

Ile Pro Leu Thr 925

Tyr Asn Gly Lys 930

Arg Asp Arg Lys 935

Arg Leu Leu His 940

Gln Ala Val Arg

Leu Gln Thr Leu 945

Ser Leu Arg Val 950

Ala Pro Ser Ser 955

Asp Thr Glu Thr 960

Arg Val Gln Gln

Ile Trp Cys Glu 965

Leu Leu Gly Leu 970

Glu Asp Ile Gly 975

Val Thr Asp Asp PIle Phe Gln Leu Gly Gly His Ser Ile Leu Val Ala
980 985 990
Arg Met Val Glu Arg Ile Glu Thr Ala Phe Gly Arg Arg Val Pro Ile
995 1000 1005
Ala Asp Ile Tyr Phe Ser Pro Thr Ile Ala Arg Val Ala Ala Thr
1010 1015 1020
Leu Asp Ser Met Thr Phe Glu Gln Gly Leu Ala Ala His Ser Val
1025 1030 1035
Lys Gly Asp Trp Glu Phe Thr Ala Ile Ser Leu Gln His Asn Ala
1040 1045 1050
Asp Ser Thr Ala Ala Ala Gln Glu Arg
1055 1060
Length: 1432
Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met His Ser Pro Thr Ile Asp Thr Phe Glu Ala Ala Leu Arg Ser Leu
15 10 15
Pro Ala Ala Arg Asp Ala Leu Gly Ala Tyr Pro Leu Ser Ser Glu Gln
20 25 30
Lys Arg Leu Trp Leu Leu Ala Gln Leu Ala Gly Thr Ala Thr Leu Pro
35 40 45
Val Thr Val Arg Tyr Ala Phe Thr Gly Thr Val Asp Leu Ala Val Val
50 55 60
Gln Gln Asn Leu Ser Ala Trp Ile Ala His Ser Glu Ser Leu Arg Ser
65 70 75 80
Leu Phe Val Glu Val Leu Glu Arg Pro Val Arg Leu Leu Met Pro Thr




Gly Leu Val Lys Leu Glu Tyr Phe Asp Arg Pro Pro Ser Asp Ala Asp
100 105 110
Met Ala Glu Leu Ile Gly Ala Ala Phe Glu Leu Asp Lys Gly Pro Leu
115 120 125
Leu Arg Ala Phe He Thr Arg Thr Ala Ala Gln Gln His Glu Leu His
130 135 140
Leu Val Gly His Pro Ile Val Val Asp Glu Pro Ser Leu Gln Arg Ile
145 150 155 160
Ala Gln Thr Leu Phe Gln Thr Glu Pro Asp His Gln Tyr Pro Ala Val
165 170 175
Gly Ala Ile Ala Glu Val Phe Gln Arg Glu Gln Thr Leu Ala Gln Asp
180 185 190
Ala Gln Ile Thr Glu Gln Trp Gln Gln Trp Gly Ile Gly Leu Gln Ala
195 200 205
Pro Ala Ala Thr Glu Ile Pro Thr Glu Asn Pro Arg Pro Ala Ile Lys
210 215 220
Gly Ser Asp Arg Gln Val His Glu Ala Leu Thr Ala Trp Gly Asp Gln
225 230 235 240
Pro Val Ala Glu Ala Glu Ile Val Ser Ser Trp Leu Thr Val Leu Met
245 250 255
Arg Trp Gln Gly Ser Gln Ser Ala Leu Cys Ala Ile Lys Val Arg Asp
260 265 270
Lys Ala His Ala Asn Leu Ile Gly Pro Leu Gln Thr Tyr Leu Pro Val
275 280 285
Arg Val Asp Met Pro Asp Gly Ser Thr Leu Ala Gln Leu Arg Leu Gln
290 295 300
Val Glu Glu Gln Leu Asn Gly Asn Asp His Pro Ser Phe Ser Thr Leu
305 310 315 320

Leu Glu Val Cys

Pro Pro Lys Arg 325

Asp Leu Ser Arg 330

Thr Pro Tyr Phe 335

Gln Thr Gly Leu 340

Gln Phe Ile Ala

His Asp Val Glu 345

Gln Arg Asp Phe 350

His Ala Gly Asn 355

Leu Thr Arg Leu 360

Pro Thr Lys Gln

Pro Ser Ser Asp 365

Leu Asp Leu Phe 370

Ile Ser Cys Trp 375

Val Ser Asp Gly 380

Thr Leu Gly Leu

Thr Leu Asp Tyr 385

Asp Cys Ala Val 390

Leu Asn Ser Ser 395

Gln Val Glu Val 400

Leu Ala Gln Ala

Leu Ile Ser Val 405

Leu Ser Ala Pro 410

Gly Glu Gln Pro 415

Ile Ala Thr Val 420

Ala Leu Met Gly

Gln Gln Met Gln 425

Gln Thr Val Leu 430

Ala Gln Ala His

Gly Pro Arg Thr 440

Thr Pro Pro Gln

Leu Thr Leu Thr 445

Glu Trp Val Ala 450

Ala Ser Thr Glu 455

Lys Ser Pro Leu 460

Ala Val Ala Val

lie Asp His Gly 465

Gln Gln Leu Ser 470

Tyr Ala Glu Leu 475

Trp Ala Arg Ala 480

Ala Leu Val Ala

Ala Asn Ile Ser

Gln His Val Ala 490

Lys Pro Arg Ser 495

Ile Ile Ala Val 500

Ala Leu Pro Arg

Ser Ala Glu Phe 505

Ile Ala Ala Leu 510

Leu Gly Val Val 515

Arg Ala Gly His 520

Ala Phe Leu Pro

Ile Asp Pro Arg 525

Leu Pro Thr Asp 530

Arg Ile Gln PIle 535

Leu Ile Glu Asn 540

Ser Gly Cys Glu

Leu Val Ile Thr Ser Asp Gln Gln Ser Val Glu Gly Trp Pro Gln Val
545 550 555 560
Ala Arg Ile Arg Met Glu Ala Leu Asp Pro Asp Ile Arg Trp Val Ala
565 570 575
Pro Thr Gly Leu Ser His Ser Asp Ala Ala Tyr Leu Ile Tyr Thr Ser
580 585 590
Gly Ser Thr Gly Val Pro Lys Gly Val Val Val Glu His Arg Gln Val
595 600 605
Val Asn Asn Ile Leu Trp Arg Gln Arg Thr Trp Pro Leu Thr Ala Gln
610 615 620
Asp Asn Val Leu His Asn His Ser Phe Ser Phe Asp Pro Ser Val Trp
625 630 635 640
Ala Leu Phe Trp Pro Leu Leu Thr Gly Gly Thr lle Val Leu Ala Asp
645 650 655
Val Arg Thr Met Glu Asp Ser Thr Ala Leu Leu Asp Leu Met Ile Arg
660 665 670
His Asp Val Ser Val Leu Gly Gly Val Pro Ser Leu Leu Gly Thr Leu
675 680 685
Ile Asp His Pro Phe Ala Asn Asp Cys Arg Ala Val Lys Leu Val Leu
690 695 700
Ser Gly Gly Glu Val Leu Asn Pro Glu Leu Ala His Lys Ile Gln Lys
705 710 715 720
Val Trp Gln Ala Asp Val Ala Asn Leu Tyr Gly Pro Thr Glu Ala Thr
725 730 735
lle Asp Ala Leu Tyr PIle Ser Ile Asp Lys Asn Ala Ala Gly Ala Ile
740 745 750
Pro Ile Gly Tyr Pro lle Asp Asn Thr Asp Ala Tyr Ile Val Asp Leu
755 760 765

Asn Leu Asn Pro Val Pro Pro Gly Val Pro Gly Glu Ile Met Leu Ala
770 775 780
Gly Gln Asn Leu Ala Arg Gly Tyr Leu Gly Lys Pro Ala Gln Thr Ala
785 790 795 600
Gln Arg Phe Leu Pro Asn Pro Phe Gly Asn Gly Arg Val Tyr Ala Thr
805 810 815
Gly Asp Leu Gly Arg Arg Trp Ser Ser Gly Ala Ile Ser Tyr Leu Gly
820 825 830
Arg Arg Asp Gln Gln Val Lys Ile Arg Gly His Arg Ile Glu Leu Asn
835 840 845
Glu Val Ala His Leu Leu Cys Gln Ala Leu Glu Leu Lys Glu Ala Ile
850 855 860
Val Phe Ala Gln His Ala Gly Thr Glu Gln Ala Arg Leu Val Ala Ala
865 870 875 880
Ile Glu Gln Gln Pro Gly Leu His Ser Glu Gly Ile Lys Gln Glu Leu
885 890 895
Leu Arg His Leu Pro Ala Tyr Leu Ile Pro Ser Gln Leu Leu Leu Leu
900 905 910
Asp Glu Leu Pro Arg Thr Ala Thr Gly Lys Val Asp Met Leu Lys Leu
915 920 925
Asp Gln Leu Ala Ala Pro Gln Leu Asn Asp Ala Gly Gly Thr Glu Cys
930 935 940
Arg Ala Pro Arg Thr Asp Leu Glu Gln Ser Val Met Thr Asp Phe Ala
945 950 955 960
Gln Val Leu Gly Leu Thr Ala Val Thr Pro Asp Thr Asp Phe Phe Glu
965 970 975
Gln Gly Gly Asn Ser Ile Leu Leu Thr Arg Leu Ala Gly Thr Leu Ser
980 985 990
Ala Lys Tyr Gln Val Gln lle Pro Leu His Glu PIle Phe Leu Thr Pro




Thr Pro Ala Ala Val Ala Gln Ala Ile Glu Ile Tyr Arg Arg Glu
1010 1015 1020
Gly Leu Thr Ala Leu Leu Ser Arg Gln His Ala Gln Thr Leu Glu
1025 1030 1035
Gln Asp Ile Tyr Leu Glu Glu His Ile Arg Pro Asp Gly Leu Pro
1040 1045 1050
His Ala Asn Trp Tyr Gln Pro Ser Val Val Phe Leu Thr Gly Ala
1055 1060 1065
Thr Gly Tyr Leu Gly Leu Tyr Leu Ile Glu Gln Leu Leu Lys Arg
1070 1075 1080
Thr Thr Ser Arg Val Ile Cys Leu Cys Arg Ala Lys Asp Ala Glu
1085 1090 1095
His Ala Lys Ala Arg Ile Leu Glu Gly Leu Lys Thr Tyr Arg Ile
1100 1105 1110
Asp Val Gly Ser Glu Leu His Arg Val Glu Tyr Leu Thr Gly Asp
1115 1120 1125
Leu Ala Leu Pro His Leu Gly Leu Ser Glu His Gln Trp Gln Thr
1130 1135 1140
Leu Ala Glu Glu Val Asp Val Ile Tyr His Asn Gly Ala Leu Val
1145 1150 1155
Asn Phe Val Tyr Pro Tyr Ser Ala Leu Lys Ala Thr Asn Val Gly
1160 1165 1170
Gly Thr Gln Ala Ile Leu Glu Leu Ala Cys Thr Ala Arg Leu Lys
1175 1180 1185
Ser Val Gln Tyr Val Ser Thr Val Asp Thr Leu Leu Ala Thr His
1190 1195 1200
Val Pro Arg Pro Phe Ile Glu Asp Asp Ala Pro Leu Arg Ser Ala
1205 1210 1215

Val Gly Val Pro Val Gly Tyr Thr Gly Ser Lys Trp Val Ala Glu
1220 1225 1230
Gly Val Ala Asn Leu Gly Leu Arg Arg Gly lle Pro Val Ser lle
1235 1240 1245
Phe Arg Pro Gly Leu lle Leu Gly His Thr Glu Thr Gly Ala Ser
1250 1255 1260
Gln Ser lle Asp Tyr Leu Leu Val Ala Leu Arg Gly Phe Leu Pro
1265 1270 1275
Met Gly lle Val Pro Asp Tyr Pro Arg lle Phe Asp lle Val Pro
1280 1285 1290
Val Asp Tyr Val Ala Ala Ala lie Val His lie Ser Met Gln Pro
1295 1300 1305
Gln Gly Arg Asp Lys Phe Phe His Leu Phe Asn Pro Ala Pro Val
1310 1315 1320
Thr lle Arg Gln Phe Cys Asp Trp lle Arg Glu Phe Gly Tyr Glu
1325 1330 1335
Phe Lys Leu Val Asp Phe Glu His Gly Arg Gln Gln Ala Leu Ser
1340 1345 1350
Val Pro Pro Gly His Leu Leu Tyr Pro Leu Val Pro Leu Ile Arg
1355 1360 1365
Asp Ala Asp Pro Leu Pro His Arg Ala Leu Asp Pro Asp Tyr Ile
1370 1375 1380
His Glu Val Asn Pro Ala Leu Glu Cys Lys Gln Thr Leu Glu Leu
1385 1390 1395
Leu Ala Ser Ser Asp Ile Thr Leu Ser Lys Thr Thr Lys Ala Tyr
1400 1405 1410
Ala His Thr Ile Leu Arg Tyr Leu Ile Asp Thr Gly PIle Met Ala
1415 1420 1425

Lys Pro Gly Val 1430
Lenght: 350
Organism: Pseudomonas fluorescens A2-2
Met Glu Ser Ile Ala Phe Pro Ile Ala His Lys Pro Phe Ile Leu Gly
15 10 15
Cys Pro Glu Asn Leu Pro Ala Thr Glu Arg Ala Leu Ala Pro Ser Ala
20 25 30
Ala Met Ala Arg Gln Val Leu Glu Tyr Leu Glu Ala Cys Pro Gln Ala
35 40 45
Lys Asn Leu Glu Gln Tyr Leu Gly Thr Leu Arg Glu Val Leu Ala His
50 55 60
Leu Pro Cys Ala Ser Thr Gly Leu Met Thr Asp Asp Pro Arg Glu Asn
65 70 75 80
Gln Glu Asn Arg Asp Asn Asp Phe Ala Phe Gly Ile Glu Arg His Gln
85 90 95
Gly Asp Thr Val Thr Leu Met Val Lys Ala Thr Leu Asp Ala Ala Ile
100 105 110
Gln Thr Gly Glu Leu Val Gln Arg Ser Gly Thr Ser Leu Asp His Ser
115 120 125
Glu Trp Ser Asp Met Met Ser Val Ala Gln Val Ile Leu Gln Thr Ile
130 135 140
Ala Asp Pro Arg Val Met Pro Glu Ser Arg Leu Thr Phe Gln Ala Pro
145 150 155 160
Lys Ser Lys Val Glu Glu Asp Asp Gln Asp Pro Leu Arg Arg Trp Val
165 170 175

Arg Gly His Leu Leu Phe Met Val Leu Cys Gln Gly Met Ser Leu Cys
180 185 150
Thr Asn Leu Leu lle Ser Ala Ala His Asp Lys Asp Leu Glu Leu Ala
195 200 205
Cys Ala Gln Ala Asn Arg Leu lle Gln Leu Met Asn Ile Ser Arg Ile
210 215 220
Thr Leu Glu Phe Ala Thr Asp Leu Asn Ser Gln Gln Tyr Val Ser Gln
225 230 235 240
Ile Arg Pro Thr Leu Met Pro Ala Ile Ala Pro Pro Lys Met Ser Gly
245 250 255
Ile Asn Trp Arg Asp His Val Val Met Ile Arg Trp Met Arg Gln Ser
260 255 270
Thr Asp Ala Trp Asn Phe Ile Glu Gln Ala Tyr Pro Gln Leu Ala Glu
275 280 285
Arg Met Arg Thr Thr Leu Ala Gln Val Tyr Ser Ala His Arg Gly Val
290 295 300
Cys Glu Lys Phe Val Gly Glu Glu Asn Thr Ser Leu Leu Ala Lys Glu
305 310 315 320
Asn Ala Thr Asn Thr Ala Gly Gln Val Leu Glu Asn Leu Lys Lys Ser
325 330 335
Arg Leu Lys Tyr Leu Lys Thr Lys Gly Cys Ala Gly Ala Gly
340 345 350
Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met Pro Thr Phe Leu Gly Asp Asp Asp Ala Val Pro Cys Val Val Val
15 10 15


Val Asn Ala Asp Lys His Tyr Ser Ile Trp Pro Ser Ala Arg Asp Ile
20 25 30
Pro Ser Gly Trp Ser Glu Glu Gly Phe Lys Gly Ser Arg Ser Asp Cys
35 40 45
Leu Glu His Ile Ala Gln Ile Trp Pro Glu Pro Thr Ala
50 55 60
Lenght: 355
Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met Thr Ser Thr His Arg Thr Thr Asp Gln Val Lys Pro Ala Val Leu
15 10 15
Asp Met Pro Gly Leu Ser Gly Ile Leu Phe Gly His Ala Ala Phe Gln
20 25 30
Tyr Leu Arg Ala Ser Cys Glu Leu Asp Leu Phe Glu His Val Arg Asp
35 40 45
Leu Arg Glu Ala Thr Lys Glu Ser Ile Ser Ser Arg Leu Lys Leu Gln
50 55 60
Glu Arg Ala Ala Asp Ile Leu Leu Leu Gly Ala Thr Ser Leu Gly Met
65 70 75 80
Leu Val Lys Glu Asn Gly lle Tyr Arg Asn Ala Asp Val Val Glu Asp
85 90 95
Leu Met Ala Thr Asp Asp Trp Gln Arg Phe Lys Asp Thr Val Ala Phe
100 105 110
Glu Asn Tyr Ile Val Tyr Glu Gly Gln Leu Asp Phe Thr Glu Ser Leu
115 120 125
Gln Lys Asn Thr Asn Val Gly Leu Gln Arg Phe Pro Gly Glu Gly Arg
130 135 140

Asp Leu Tyr His Arg Leu His Gln Asn Pro Lys Leu Glu Asn Val Phe
145 150 155 160
Tyr Arg Tyr Met Arg Ser Trp Ser Glu Leu Ala Asn Gln Asp Leu Val
165 170 175
Lys His Leu Asp Leu Ser Arg Val Lys Lys Leu Leu Asp Ala Gly Gly
180 185 190
Gly Asp Ala Val Asn Ala lle Ala Leu Ala Lys His Asn Glu Gln Leu
195 200 205
Asn Val Thr Val Leu Asp lle Asp Asn Ser lle Pro Val Thr Gln Gly
210 215 220
Lys lle Asn Asp Ser Gly Leu Ser His Arg Val Lys Ala Gln Ala Leu
225 230 235 240
Asp Ile Leu His Gln Ser Phe Pro Glu Gly Tyr Asp Cys Ile Leu Phe
245 250 255
Ala His Gln Leu Val Ile Trp Thr Leu Glu Glu Asn Thr His Met Leu
260 265 270
Arg Lys Ala Tyr Asp Ala Leu Pro Glu Gly Gly Arg Val Val Ile Phe
275 280 285
Asn Ser Met Ser Asn Asp Glu Gly Asp Gly Pro Val Met Ala Ala Leu
290 295 300
Asp Ser Val Tyr Phe Ala Cys Leu Pro Ala Glu Gly Gly Met Ile Tyr
305 310 315 320
Ser Trp Lys Gln Tyr Glu Val Cys Leu Ala Glu Ala Gly Phe Lys Asn
325 330 335
Pro Val Arg Thr Ala Ile Pro Gly Trp Thr Pro His Gly Ile Ile Val
340 345 350
Ala Tyr Lys 355

Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met Ala Arg Ser Pro Glu Thr Asn Ser Ala Met Pro Gln Gln lle Arg
15 10 15
Gln Leu Leu Tyr Ser Gln Leu lle Ser Gln Ser Ile Gln Thr Phe Cys
20 25 30
Glu Leu Arg Leu Pro Asp Val Leu Gln Ala Ala Gly Gln Pro Thr Ser
35 40 45
Ile Glu Arg Leu Ala Glu Gln Thr His Thr His Ile Ser Ala Leu Ser
50 55 60
Arg Leu Leu Lys Ala Leu Lys Pro Phe Gly Leu Val Lys Glu Thr Asp
65 70 75 80
Glu Gly Phe Ser Leu Thr Asp Leu Gly Ala Ser Leu Thr His Asp Ala
85 90 95
Phe Ala Ser Ala Gln Pro Ser Ala Leu Leu Ile Asn Gly Glu Met Gly
100 105 110
Gln Ala Trp Arg Gly Met Ala Gln Thr Ile Arg Thr Gly Glu Ser Ser
115 120 125
Phe Lys Met Tyr Tyr Gly Ile Ser Leu Phe Glu Tyr Phe Glu Gln His
130 135 140
Pro Glu Arg Arg Ala Ile Phe Asp Arg Ser Gln Asp Met Gly Leu Asp
145 150 155 160
Leu Glu lle Pro Glu Ile Leu Glu Asn Ile Asn Leu Asn Asp Gly Glu
165 170 175
Asn Ile Val Asp Val Gly Gly Gly Ser Gly His Leu Leu Met His Met
180 185 190

Leu Asp Lys Trp Pro Glu Ser Thr Gly Ile Leu Phe Asp Leu Pro Val
195 200 205
Ala Ala Lys Ile Ala Gln Oln His Leu His Lys Ser Gly Lys Ala Gly
210 215 220
Cys Phe Glu Ile Val Ala Gly Asp Phe Phe Lys Ser Leu Pro Asp Ser
225 230 235 240
Gly Ser Val Tyr Leu Leu Ser His Val Leu His Asp Trp Gly Asp Glu
245 250 255
Asp Cys Lys Ala Ile Leu Ala Thr Cys Arg Arg Ser Met Pro Asp Asn
260 265 270
Ala Leu Leu Val Val Val Asp Leu Val Ile Asp Gln Ser Glu Ser Ala
275 280 285
Gln Pro Asn Pro Thr Gly Ala Met Met Asp Leu Tyr Met Leu Ser Leu
290 295 300
Phe Gly Ile Ala Gly Gly Lys Glu Arg Asn Glu Asp Glu PIle Arg Thr
305 310 315 320
Leu Ile Glu Asn Ser Gly Phe Asn Val Lys Gln Val Lys Arg Leu Pro
325 330 335
Ser Gly Asn Gly Ile Ile Phe Ala Tyr Pro Lys
340 345
Lenght: 180
Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met Ser Thr Leu Val Tyr Tyr Val Ala Ala Thr Leu Asp Gly Tyr Ile
15 10 15
Ala Thr Gln Gln His Lys Leu Asp Trp Leu Glu Asn Phe Ala Leu Gly
20 25 30

Asp Asp Ala Thr Ala Tyr Asp Asp Phe Tyr Gln Thr lle Gly Ala Val
35 40 45
Val Met Gly Ser Gln Thr Tyr Glu Trp Ile Met Ser Asn Ala Pro Asp
50 55 SO
Asp Trp Pro Tyr Gln Asp Val Pro Ala Phe Val Met Ser Asn Arg Asp
65 70 75 80
Leu Ser Ala Pro Ala Asn Leu Asp Ile Thr PIle Leu Arg Gly Asp Ala
85 90 95
Ser Ala Ile Ala Val Arg Ala Arg Gln Ala Ala Lys Gly Lys Asn Val
100 105 110
Trp Leu Val Gly Gly Gly Lys Thr Ala Ala Cys Phe Ala Asn Ala Gly
115 120 125
Glu Leu Gln Gln Leu Phe Ile Thr Thr Ile Pro Thr Phe Ile Gly Thr
130 135 140
Gly Val Pro Val Leu Pro Val Asp Arg Ala Leu Glu Val Val Leu Arg
145 150 155 160
Glu Gln Arg Thr Leu Gln Ser Gly Ala Met Glu Cys Ile Leu Asp Val
165 170 175
Lys Lys Ala Asp 180
Length: 220
Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met Ser Asn Val Phe Ser Gly Gly Lys Gly Asn Gly Asn Pro Gly Phe
15 10 15
Val Arg Thr Phe Ser Arg Ile Ala Pro Thr Tyr Glu Glu Lys Tyr Gly
20 25 30
Thr Lys Leu Ser Gln Ala His Asp Asp Cys Leu Arg Met Leu Ser Arg




Trp Met Cys Thr Ser Arg Pro Glu Arg Val Leu Asp Ile Gly Cys Gly
50 55 60
Thr Gly Ala Leu Ile Glu Arg Met Phe Ala Leu Trp Pro Glu Ala Arg
65 70 75 80
Phe Glu Gly Val Asp Pro Ala Gln Gly Met Val Asp Glu Ala Ala Lys
85 90 95
Arg Arg Pro Phe Ala Ser Phe Val Lys Gly Val Ala Glu Ala Leu Pro
100 105 110
Phe Pro Ser Gln Ser Met Asp Leu Val Val Cys Ser Met Ser Phe Gly
115 120 125
His Trp Ala Asp Lys Ser Val Ser Leu Asn Glu Val Arg Arg Val Leu
130 135 140
Lys Pro Gln Gly Leu Phe Cys Leu Val Glu Asn Leu Pro Ala Gly Trp
145 150 155 160
Gly Leu Thr Thr Leu Ile Asn Trp Leu Leu Gly Ser Leu Ala Asp Tyr
165 170 175
Arg Ser Glu His Glu Val Ile Gln Leu Ala Gln Thr Ala Gly Leu Gln
180 185 190
Ser Met Glu Thr Ser Val Thr Asp Gln His Val Ile Val Ala Thr Phe
195 200 205
Arg Pro Cys Cys Gly Glu Val Gly Asp His Gly Arg
210 215 220
Length: 509
Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met Val Val Lys Asn Lys Gln Val Leu Val Val Gly Ala Gly Pro Val

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

Glu lle Ala Gly

Gly Met Lys Asp 245

Ile Leu Gly Thr 250

Asp Phe Gly Leu 255

Ala Gln Val Leu 260

Trp Ser Ala Arg

Tyr Arg Ser Gln 265

Gln Arg Leu Ala 270

Thr His Tyr Arg 275

Gln Gly Arg Val 280

Phe Leu Leu Gly

Asp Ala Ala His 285

Thr His Val Pro 290

Ala Gly Gly Gln 295

Gly Leu Gln Met 300

Gly Ile Gly Asp

Ala Ala Asn Leu 305

Ala Trp Lys Leu 310

Ala Gly Val Ile 315

Gln Ala Thr Leu 320

Pro Met Asp Leu

Leu Glu Ser Tyr 325

Glu Ala Glu Arg 330

Arg Pro Ile Ala 335

Ala Ala Ala Leu 340

Arg Asn Thr Asp

Leu Leu Phe Arg 345

Phe Asn Thr Ala 350

Ser Gly Pro lle 355

Gly Arg Leu lle 360

His Trp Ile Gly

Leu Gln Ala Thr 365

Arg Ala Pro Tyr 370

Val Ala Gln Lys 375

Val Val Ser Ala 380

Leu Ala Gly Glu

Gly Val Arg Tyr 385

Asp Ser Val Arg 390

Arg Arg Gly Asp 395

His Arg Leu Val 400

Gly Arg Arg Leu

Pro Leu Leu Ser

Leu Leu Pro Glu 410

Gly Glu Arg Leu 415

Pro Arg Gln Ser 420

Leu Thr Gln Leu

Leu Arg Ala Gly 425

Arg Phe Val Leu 430

Val His His Arg 435

Ala Lys Ala Leu 440

Ala Ala Asp Leu

Arg Arg Asp Phe 445

Pro Gly Leu Gln 450

Thr Ala Ser Ile 455

Cys Glu Asp Ser 460

His Asn Asn Ser

Leu Ser Ala Gly Glu Gly Val Ile Val Arg Pro Asp Gly Val Val Ile
465 470 475 480
Trp Val Gly Lys Lys Ser Thr Leu Ala Lys Glu Arg Leu Gly Glu Trp
485 490 495
Leu Leu Asp Asp Ser Lys Ser Ala Arg Gln Ser Leu Thr
500 505
Organism: Pseudomonas fluorescens A2-2
Met Ala His Tyr Asp Ser Val Gly Thr Ala Pro Gly Ala Ser Asp Asp
15 10 15
Gly Met Ala Val Ala Ser Ile Leu Gln Leu Met Arg Glu Thr Ile Thr
20 25 30
Arg Ser Asp Ala Lys Asn Asn Val Val Phe Leu Leu Ala Asp Gly Glu
35 40 45
Glu Leu Gly Leu Leu Gly Ala Glu His Tyr Val Ser Gln Leu Ser Thr
50 55 60
Pro Glu Arg Glu Ala Ile Arg Leu Val Leu Asn Phe Glu Ala Arg Gly
65 70 75 80
Asn Gln Gly Ile Pro Leu Leu Phe Glu Thr Ser Gln Lys Asp Tyr Ala
85 90 95
Leu Ile Arg Thr Val Asn Ala Gly Val Arg Asp Ile Ile Ser Phe Ser
100 105 110
Phe Thr Pro Leu Ile Tyr Asn Met Leu Gln Asn Asp Thr Asp Phe Thr
115 120 125
Val Phe Arg Lys Lys Asn Ile Ala Gly Leu Asn Phe Ala Val Val Glu
130 135 140

Gly Phe Gln His Tyr His His Met Ser Asp Thr Val Glu Asn Leu Gly
145 150 155 160
Pro Glu Thr Leu Phe Arg Tyr Gln Lys Thr Val Arg Glu Val Gly Asn
165 170 175
His Phe Ile Gln Gly Ile Asp Leu Ser Ser Leu Ser Ala Asp Glu Asp
180 185 190
Ala Thr Tyr Phe Pro Leu Pro Gly Gly Thr Leu Leu Val Leu Asn Leu
195 200 205
Pro Thr Leu Tyr Ala Leu Gly Met Gly Ser Phe Val Leu Cys Gly Leu
210 215 220
Trp Ala Gln Arg Cys Arg Thr Arg Arg Gln His Gln Gly Lys Asn Cys
225 230 235 240
Val Leu Arg Pro Met Ala Ile Ala Leu Leu Gly Ile Ala Cys Ala Ala
245 250 255
Leu Val Phe Tyr Val Pro Ser Ile Ala Tyr Leu Phe Val Ile Pro Ser
260 265 270
Leu Leu Leu Ala Cys Ala Met Leu Ser Arg Ser Leu Phe Ile Ser Tyr
275 280 285
Ser Ile Met Leu Leu Gly Ala Tyr Ala Cys Gly Ile Leu Tyr Ala Pro
290 295 300
Ile Val Tyr Leu Ile Ser Ser Gly Leu Lys Met Pro Phe Ile Ala Gly
305 310 315 320
Val Ile Ala Leu Leu Pro Leu Cys Leu Leu Ala Val Gly Leu Ala Gly
325 330 335
Val Ile Ala Arg Ser Arg Asp Cys Arg Thr Cys Asp
340 345

Lenght: 572
Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met Arg Ser Leu Lys ILe ILe Val Leu Ala Ser Ala Phe Asn Gly Leu
15 10 15
Thr Gln Arg Ala Trp Leu Asp Leu Arg Gln Ser Gly His Ala Pro Ser
20 25 30
Val Val Leu Phe Thr Asp Pro Ala Leu Val Cys Gln Gln ILe Glu Asp
35 40 45
Ser Asp Ala Asp Leu Val Ile Cys Pro Phe Leu Lys Asp Arg Val Pro
50 55 60
Gln Gln Leu Trp Ser Asn Leu Glu Arg Pro Val Val Ile Ile His Pro
65 70 75 80
Gly Ile Val Gly Asp Arg Gly Ala Ser Ala Leu Asp Trp Ala Ile Ser
85 90 95
Gln Gln Val Gly Arg Trp Gly Val Thr Ala Leu Gln Ala Val Glu Glu
100 105 110
Met Asp Ala Gly Pro Ile Trp Ser Thr Cys Glu PIle Asp Met Pro Ala
115 120 125
Asp Val Arg Lys Ser Glu Leu Tyr Asn Gly Ala Val Ser Asp Ala Ala
130 135 140
Leu Tyr Cys Ile Arg Asp Val Val Glu Lys Phe Ala Arg Val Phe Val
145 150 155 160
Pro Val Pro Leu Asp Tyr Thr Gln Ala His Val Ile Gly Arg Leu Gln
165 170 175
Pro Asn Met Thr Gln Ala Asp Arg Thr Phe Ser Trp Tyr Asp Cys Ala
180 185 190
Arg Phe Ile Lys Arg Cys Ile Asp Ala Ala Asp Gly Gln Pro Gly Val




Leu Ala Ser Ile 210

Gln Gly Gly Gln 215

Tyr Tyr Leu Tyr 220

Asp Ala His Leu

Asp Ala Arg His 225

Gly Thr Pro Gly 230

Glu Ile Leu Ala 235

Val Gln Asp Asp 240

Ala Val Leu Val

Ala Ala Gly Asp 245

Gln Ser Leu Trp 250

Ile Gly Ser Leu 255

Lys Arg Lys Ala 260

Arg Pro Gly Glu

Glu Thr Phe Lys 265

Leu Pro Ala Arg 270

His Val Leu Ala 275

Glu Ala Leu Ala 280

Asp Ile Pro Val

Leu Asp Ser Ser 285

Ile Ala Asn Gln 290

Met Phe Asp Glu 295

Gln Ala Tyr Gln 300

Pro Ile Arg Tyr

Arg Glu Ala Gly 305

His Val Gly Glu 310

Leu Thr Phe Glu 315

Phe Tyr Asn Gly 320

Ala Met Ser Thr

Glu Gln Cys Gln 325

Arg Leu Val Ala 330

Ala Leu Arg Trp 335

Ala Lys Thr Arg 340

Asp Thr Gln Val

Leu Val Ile Lys 345

Gly Gly Arg Gly 350

Ser Phe Ser Asn 355

Gly Val His Leu 360

Asn Val Ile Gln

Ala Ala Pro Val 365

Pro Gly Leu Glu 370

Ala Trp Ala Asn 375

Ile Gln Ala Ile 380

Tyr Asp Val Cys

His Glu Leu Leu 385

Thr Ala Arg Gln 390

Leu Val Ile Ser 395

Gly Leu Thr Gly 400

Ser Ala Gly Ala

Gly Gly Val Met 405

Leu Ala Leu Ala 410

Ala Asp Ile Val 415

Leu Ala Arg Glu 420

Ser Val Val Leu

Asn Pro His Tyr 425

Lys Thr Met Gly 430

Leu Tyr Gly Ser Glu Tyr Trp Thr Tyr Ser Leu Pro Arg Ala Val Gly
435 440 445
Ser Glu Val Ala His Gln Leu Thr Asp Ala Cys Leu Pro Ile Ser Ala
450 455 460
Leu Gln Ala Glu Gln Tyr Gly Leu Val Gln Gly Ile Gly Pro Arg Cys
465 470 475 480
Pro His Ala Phe Ser Arg Trp Leu Met Gln Gln Ala Ser Ser Ala Leu
485 490 495
Thr Asp Glu Lys Tyr Ala Val Ala Arg Ala Arg Lys Ala Ala Leu Asp
500 505 510
Ile Asp Gln Ile Thr Arg Cys Arg Glu Ala Glu Leu Ala Gln Met Gln
515 520 525
Leu Asp Met Val His Asn Arg His Gln Phe Ala Glu Lys Cys Arg Asn
530 535 540
Phe Val Leu Lys Arg Lys Thr Cys Gln Thr Pro Gln Arg Leu Met Ala
545 550 555 560
Pro Trp Ala Val Ala Arg Glu Ala Ala Leu Val Gly
565 570
Lenght: 230
Type: PRT
Organism: Pseudomonas fluorescens A2-2
Met Ile Gly Ile Val Ile Pro Ala His Asn Glu Glu Arg His Ile Ser
15 10 15
Ala Cys Leu Ala Ser Ile Gln Arg Ala Ile Ala His Pro Ala Leu Ala
20 25 30
His Gln Gln Val Gln Leu Leu Val Val Leu Asp Ala Cys Ser Asp Glu
35 40 45

Thr Ala Thr Arg Val Ser Ala Met Gly Val Ala Thr Leu Glu Val Ser
50 55 60
Val Arg Asn Val Gly Lys Ala Arg Ala Leu Gly Ala Glu Arg Leu Leu
65 70 75 80
Glu Val Gly Ala Gln Trp Leu Ala Phe Thr Asp Ala Asp Thr Val Val
85 90 95
Pro Ala Asp Trp Leu Val Arg Gln ILe Gly Phe Gly Ala Asp Ala Val
100 105 110
Cys Gly Thr Val Glu Val Asp Ser Trp Ser Glu Tyr Gly Glu Ser Val
115 120 125
Arg Ser Arg Tyr Leu Glu Leu Tyr Gln Phe Thr Glu Asn His Arg His
130 135 140
ILe His Gly Ala Asn Leu Gly Leu Ser Ala Asp Ala Tyr Arg Asn Ala
145 150 155 160
Gly Gly Phe Gln His Leu Val Ala His Glu Asp Val Gln Leu Val Ala
165 170 175
Asp Leu Glu Arg ILe Gly Ala Arg ILe Val Trp Thr Ala Thr Asn Pro
180 185 190
Val Val Thr Ser Ala Arg Arg Asp Tyr Lys Cys Arg Gly Gly Phe Gly
195 200 205
Glu Tyr Leu Ala Ser Leu Val Ala Glu Gly Thr Arg Glu His Ser Pro
210 215 220
Ala His Ala Pro ILe Gly
225 230
Lenght: 348
Type. PRT
Organism: Pseudomonas fTuorescens A2-2

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

Ile Ser Pro Phe Pro Asn Asp Gln Ser Ser Arg Phe Thr Ser Thr Arg
225 230 235 240
Leu Ile His Thr Ser Ser Leu Arg Ser Pro Val Leu Ala Trp Met Pro
245 250 255
Ser Ala Met Asn Leu Lys Ala Phe Phe Thr Ser Met Leu Arg Pro Ala
260 265 270
Phe His Val Thr Trp Ile Asn Thr Ile Leu Gly Val Val Thr Pro Arg
275 280 285
Tyr Pro Ala Ala Gly Thr Ser Ser Ser Leu Ala Trp Arg Leu Met Ile
290 295 300
Trp Asn Leu Ser Cys Ser Gly Thr Leu Ala Thr Leu Val Ile Ala Ala
305 310 315 320
Tyr Thr Thr Ser Pro Met Ala Val Ala Val Ser Val Glu Val Ser Ala
325 330 335
Ala Arg Ser Ile Arg Thr Lys Gly Met Asp Lys Ser
340 345

Comparison of new dehydroxysafracins with safracin A.

We Claim:
1. An expression vector comprising isolated nucleic acid sequence represented by SEQ ID NO 1 or variants or portions thereof, the said sequence encoding for a polypeptide useful in bio-synthesis of safracin or its analogue.
2. An expression vector as claimed in claim 1, wherein the vector is a cosmid.
3. A host cell transformed with an expression vector encoding polypeptides sufficient to direct the synthesis of a safracin or safracin analogue wherein the expression vector comprising nucleic acid sequence represented by SEQ ID NO 1 or variants or portions thereof.
4. A host cell as claimed in claim 3, wherein the host cell is a microorganism
5. A host cell as claimed in claim 4, wherein the micro-organism is bacteria.
6. A recombinant bacterial cell wherein a nucleic acid sequence represented by SEQ ID NO 1 or a portion thereof is disrupted to enable the cell to produce altered levels of safracin or its analogues, relative to corresponding non-recombinant bacterial cell.
7. A recombinant cell as claimed in claim 6, wherein the disrupted nucleic acid sequence is endogenous.
8. A method of producing a bio-systhesized safracin compound or safracin analogue, said method comprising fermenting by an organism in which the copy number of the gene cluster as represented by SEQ ID NO. 1 has been increased and wherein the method comprises of:
a. inoculating a mutant strain in a suitable medium as herein described,
b. incubating the medium for a period in the range of 50 to 1 00 hours at a
controlled temperature such as herein described,
c. transferring the culture obtained from step (b) to a fermentor, and
d. fermenting under agitated and aerated conditions for a period of 20 to
75 hours to produce said safracin.
9. A method as claimed in claim 8, wherein mutant strain is a mutated Pseudomonas sp.
10. A method as claimed in claim 8, wherein the temperature from inoculation till 24 hours is about 27°C.
11. A method as claimed in claim 8, wherein the temperature for incubation is maintained at about 25°C beyond 24 hours.

12. A bio-synthesized safracin compound oblained by a method as claimed in claim 8.
13. A bio-synthesized safracin compound as claimed in claim 12, wherein the compound is selected from any of:


14. A pharmaceutical composition comprising a compound as claimed in claims 12 or 1 3 and a pharmaceutically acceptable diluent, carrier or excipient.

Dated this 9th day of June, 2005


00600-mumnp-2005-sequence listing.doc

600-mumnp-2005-cancelled pages(19-06-2007).pdf






600-mumnp-2005-form 1(13-06-2005).pdf

600-mumnp-2005-form 1(19-06-2007).pdf

600-mumnp-2005-form 1(20-09-2005).pdf

600-mumnp-2005-form 18(19-12-2005).pdf

600-mumnp-2005-form 2(granted)-(19-06-2007).doc

600-mumnp-2005-form 2(granted)-(19-06-2007).pdf

600-mumnp-2005-form 26(13-06-2005).pdf

600-mumnp-2005-form 3(09-06-2005).pdf

600-mumnp-2005-form 3(13-09-2005).pdf

600-mumnp-2005-form 5(13-06-2005).pdf


Patent Number 210475
Indian Patent Application Number 600/MUMNP/2005
PG Journal Number 43/2007
Publication Date 26-Oct-2007
Grant Date 05-Oct-2007
Date of Filing 13-Jun-2005
Name of Patentee PHARMA MAR S.A
# Inventor's Name Inventor's Address
PCT International Classification Number C12N15/52, 9/00 9/02
PCT International Application Number PCT/GB2003/005563
PCT International Filing date 2003-12-19
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0229793.5 2002-12-20 GB