CA2364569A1 - Non-ribosomal peptide synthetases, method for producing same and the use thereof - Google Patents

Abstract

The present invention relates to a method for producing modified non-ribosom al peptide synthetases (NRPS). The invention also relates to the use thereof fo r producing native or artificial peptides or proteinogenes. In particular, a desired peptide can be obtained by means of a novel non-ribosomal code.</SDO AB>

Description

Description NON-RIBOSOMAL PEPTIDE SYNTHETASES, METHOD FOR THE
PRODUCTION THEREOF AND USE THEREOF
The present invention relates to a method for producing modified non-ribosomal peptide synthetases (NRPS) and to the use thereof for producing native or artificial peptides and proteinogens.
Non-ribosomal peptide synthetases (NRPS or peptide synthetases) are modular enzymes with complex structures and important biological functions. Numerous peptides of pharmaceutical and biotechnological interest - in particular such as biosurfactants, siderophores, antibiotics, cytostatics, immunosuppressives or antitumor agents - are biosynthesized by large enzyme complexes, the "NRPS"
(Marahiel et al. (1997), Chem. Rev. 97: p. 2651-2673).
This likewise includes known pharmaceuticals such as cyclosporin A and vancomycin. The wide variety of bioactive peptides produced in this way are a result of the great structural variety of NRPS. In addition to the 20 proteinogenic amino acids, NRPS often form particular residues, such as, for example, a-hydroxyamino acids or non-proteinogenic amino acids.
The individual residues are linked together via peptide bonds or the formation of esters and lactones.
Furthermore, the residues may be further modified by N-methylation, heterocyclic ring formation or epimerization. Many of the peptides are synthesized by natural NRPS are additionally cyclized by ester or peptide bonds . Generally it can be said that NRPS play a key part in the synthesis of complex peptidic biocompounds.
It was found that the multifunction protein templates are essentially composed in a modular way. Modular means the catalytic unit which incorporates a specific Confirmation copy n-_ 2 _ amino acid into the product (peptide) in (Marahiel et al. (1997), Chem. Rev. 97: p. 2651-2673).
The individual modules are composed of "domains" which in each case are responsible for a particular reaction.
The adenylation domain (A domain) determines entering of the substrate into the peptide in that the A domain selects and adenylates the substrate, usually an amino acid. The activated amino acid is then bound via a thioester bond to the cofactor 4'-phosphopantethein of a thiolation domain (T). From there, the aminoacyl or peptidyl residues are condensed to a neighboring ring module. This reaction is catalyzed by the condensation domain (C domain). These three domains, C, A and T
domain, form the base unit of the multimodular NRPS.
The last NRPS module normally contains a termination domain (Te domain) which is responsible for liberation of the synthesis product. The sequence of the modules within the NRPS determines the structure of the peptide.
At positions at which a modified amino acid is incorporated, modification domains are incorporated into the appropriate module.
EP 0789078 A2 merely suggested the exchange of domains for varying known compounds; only technical teaching regarding the principle of controlling the peptide chain termination reaction can be found there.
In detail: an adenylation (A) domain, for example, recognizes and activates its cognate substrate amino acid as enzyme-associated aminoacyladenylate with simultaneous ATP hydrolysis. These relatively unstable intermediates are then stabilized by transferring a 4'-phosphopantethein cofactor (4'-PAN) which is covalently linked to a thiolation (T) domain onto the thiol group.
With participation of condensation (C) domains and in ' WO 00/52152 PCT/EP00/01652 an ordered sequence of transpeptidations of the amino acids activated in this way, a successive chain growth then takes place until the peptide product is finished.
Modification of the incorporated monomers (e. g.
epimerization or N-methylation) or of the peptide main chain (e.g. acylation or glycosylation) can additionally alter the structure of the product formed.
Functionalizations of this kind are catalyzed by special domains or polyketide synthase (PKS) modules within the NRPS matrix.
It was shown that A domains via their substrate specificity and relative sequence within a specific NRPS system determine the primary structure (sequence) of the non-ribosomal peptide formed. Solving the crystal structure of two members of the superfamily of adenylate-forming enzymes, which were luciferase from photinus pyralis [Conti et et., 1996, Structure 4: 287-298] and the A domain of gramicidin S synthetase 1 (denoted PHeA below) from Bacillus brevis [Conti et al . , 1997, EMBO J. 16 : 4174-4183 ] , made it possible to show that both enzymes have a decidedly homologous folding topology, although they are only 16o identical with respect to their primary structure. The structure of PheA (figure lA), which was determined in the presence of its substrates ATP and phenylalanin, allowed first insights into the molecular mechanisms of substrate recognition and substrate activation.
Sequence comparisons of NRPS A domains identified highly conserved sequence regions, the "core motifs", which appear within the A domains with virtually unchanged locations and amino acid sequence. The previous mutuational analyses and now also the crystal structure of PheA makes it possible to show that the overwhelming majority of said core sequences is essentially involved in ATP binding and ATP hydrolysis.
Moreover, some core motifs have also functions in binding the substrate amino acid. Thus, for example, the a-amino group and a-carboxyl group of the substrate amino acid L-phenylalanin in PheA are electrostatically coordinated by residues Asp235 (Core A4) and Lys 517 (Core A10), respectively [Marahiel et al., 1997, Chem.
Rev. 97: 2651-2673]. The actual substrate specificity-mediating PheA region, however, is located in an approx. 100 amino acid-long region which is less conserved within the group of A domains. This region forms the binding pocket for the side chain of the phenylalanin substrate (figure 1B), which on one side is composed of residues A1a236, I1e330 and Cys331, and on the other side of residues A1a332, A1a301, I1e299 and Thr278. Both sides of the pocket are separated by the indole ring of Trp239, which is located at the bottom of the pocket.
From previous in silico studies (computer simulations) on NRPS A domains it was possible to conclude that the substrate specificity-determining region of A domains is presumably located between core motifs A3 and A6.
This region is comparatively less conserved, whereas for domains of identical substrate specificity, however, an increased homology relation has been postulated. Accordingly, domains which activate the same substrates should appear in groups in phylogenetic trees. Detailed analyses, however, were unable to verify correlations of this kind. On the contrary, it became obvious that the evolutionary origin (host organism) of the A domains has a greater influence on their clustering than their substrate specificity.
In order to derive the consensus sequences, we determined via sequence comparisons with PheA and luciferase the corresponding ten residues of the putative binding pockets of 160 A domains accessible in public sequence databases. For each A domain only these ten residues were used below as independent amino acid sequence, in order to carry out alignments and phylogenetic studies using the laser gene MegAlign program (available from e.g. GATC, GmbH, Fritz-Arnold Str. 23, D-78467 Konstanz, Germany). By means of these studies a phylogenetic family tree was obtained (figure 2), in which a cluster of the 10 amino acid-long sequences appears, according to the specificity of their original domains. This result unambiguously indicates that the selected residues are indeed involved in substrate recognition. Based on these studies, it is possible to explain discrepancies between postulated and observed substrate specificities of A domains and to predict the specificities of NRPS
systems.
The object of the present invention was a method for modifying A domains for the purpose of providing and producing appropriately modified non-ribosomal peptide synthetases for the production of novel peptides. The invention allows the specific production of a desired non-ribosomal peptide.
Surprisingly, based on the phylogenetic studies, it was possible to derive consensus sequences which are to be understood as the non-ribosomal code of the NRPS
(table 1). Similar to the ribosomal code, this code is degenerated (redundant), and it was possible, for example, to identify up until now four different condons for Leu-activating domains, three condons for Val- activating domains and two for Cys-activating domains. It can thus be assumed that also for other substrates a plurality of strategies for substrate recognition exist, which can be determined with the aid of the inventive method.
Therefore, the invention likewise relates to the non-ribosomal code represented in table 1 and coding for a non-ribosomal peptide.

' WO 00/52152 PCT/EP00/01652 Based on the "condon" determined by sequence comparisons, it is possible to predict the general shape of substrate binding pockets. The putative binding pockets for the substrate amino acids Asp, Orn and Val are depicted as examples in figure 3. Whereas Asp235 and Lys517 mediate in all A domains key interactions with the amino and carboxyl groups of the substrate and are therefore highly conserved, the remaining residues are thought to determine to a large extent the specificity for a particular amino acid side chain. The corresponding residues (pos.236 to 331) in the three examples stated support this theory and perfectly reflect the requirements regarding polarity and size of the activated substrates.
(1) Asp activation ('Asp' condon): the basic His322 residue (possibly also Lys278) establishes a polar interaction with the acidic side chain, whereas the Leu236 residue locks the binding pocket for uptake of longer-chain substrates (e. g. Glu).
(2) Orn activation ('Orn(2)' condon): the acidic residues G1u278 and Asp331 seem to play a key part in the recognition of the large basic side chain.
(3) Val activation ('Val(3)' condon): the entire binding pocket is composed of hydrophobic residues. The relatively large space requirement arising from branching of the ~ position is taken into account by using (1) in the upper part smaller side groups and (2) in the lower part more spacious residues which extend the binding pockets.
In addition to the degeneracy, there is further homogeneity to the ribosomal code in the occurrence of flexible (Wobble-like) positions. As shown in table l, such wobble-like residues which have increased flexibility within particular condons have positions , _ 278, 299 and 331. A general observation is that binding pockets which recognize relatively small amino acids have a higher flexibility in positions near the bottom of the pocket, whereas for large substrates greater variability in the upper part is observed (see table 1).
The results of the in silico studies which are summarized in figure 3 and table 1, show that the individual constituents of a binding pocket have different significance for the development of a particular substrate specificity. In order to confirm this observation, the residues of 160 binding pockets were studied in more detail. The results depicted in figure 4 and table 2 show that the ten constituents of a binding pocket can be divided into three groups.
Their uneven variability probably reflects their different importance in mediating substrate specificity:
(1) 'Invariant' positions (pos. 235 and 517):
Asp235 is absent merely in A domains which activate substrates without a-amino group. Examples for this are, for example, the a-amino adipate-activating domains of the AcvA-synthetases or the carboxy acid-activating domains of the enterobactin (Escherichia coli) and Yersiniabactin system (Yersinia pestis). In contrast, Lys517 is absolutely invariant and binds both the a-carboxyl group of the cognate amino acid and 04' and 05' of the ATP/AMP ribose unit and, as a result, presumably both substrates are brought into a position of optimal interaction. Both residues thus mediate important interaction with the (substantially) invariable a-amino and a-carboxyl groups of the substrate, and this makes it possible to explain their own invariance.
,y g _ (2) 'Partially variant' positions (pos.236, 301 and 330): these residues will exhibit only low variability within all domains studied and in 93% of all cases hydrophobic amino acids are formed in these positions. With respect to the low flexibility of these positions and to the disproportionately low presence of charged or polar side chains, an essential importance in mediating substrate specificity can be excluded.
(3) 'Highly variant' positions (pos. 239, 278, 299, 322 and 331): these positions have the highest flexibility in relation to the amino acids used. Apart from pos. 229 and 331 which generally seem very adaptive and wobble-like (cf. table 1), all positions are predestined to establish substrate specificity.
With respect to their relative location within the binding pocket and to the data shown in table 1+2 and figure 3, it can be assumed that position 322 has a greater influence on smaller substrate residues, whereas positions 239 and 278 influence rather larger residues. This hypothesis is supported, for example, by the condons of the Asp- (His322), Glu- (Lys239) and Orn- (G1u278) activating A domains (table 1+2). In all examples mentioned, the polarity of the recognized substrate and 'highly variant' positions of the binding pocket are perfectly correlated (cf. table 2; in dark gray background).
The altered DNA is chromosomally or extrachromosomally inserted into the host organism according to methods known per se to the skilled worker (Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989), Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory Press).
The present invention therefore relates to the embodiments listed in the patent claims.

. _ For experimental confirmation, we mutated a plurality of the specificity-mediating amino acid constituents of the PheA binding pocket and studied the mutants for resulting alteration in (1) the rate of ATP-pyrophosphate exchange (enzyme activity) and (2) activation of noncognate amino acids (enzyme specificity). With regard to the latter point, it should not go unmentioned that the wild type protein from the outset showed significant activation of the noncognate substrates Trp (180) and Leu(7%). The results of our PheA mutagenesis experiments are summarized in table 3 and can be generalized as follows:
(1) The substitution with slightly larger amino acids was inevitably at the expense of the available space in the binding pocket. This caused a decrease in the ATP-pyrophosphate exchange rate of these mutants, whereas their specificity for the cognate substrate phenylalanin increased (e. g. T278M, T278Q, A301V, A322S
and C331L) . In all cases it was possible to reduce the site specificity for the noncognate substrates Trp and Leu by a factor 20.
(2) The exact opposite was observed for exchanges with slightly smaller groups, which slightly expanded the binding pocket. In these mutants the catalytic activity of the wild type protein would be preserved, while the ability to discriminate against noncognate substrates was reduced up to 5 fold (e. g. A301G, A322G and I330V).
(3) The mutation of the 'highly variant' side chains (pos. 29, 278, 322 and 331) caused up to 5 fold loss in enzyme activity (e. g. W239L, T278M and C331L).
Introduction of polar or charged side groups changed the overall polarity of the binding pocket, as a result of which the corresponding enzyme was virtually completely inactivated (e. g. A322D, A322K and A322N).

(4) The observed increase in activation of a particular noncognate substrate was predictable, owing to the specificity code (table 1).
The activity and specificity for Phe in the mutant PheA(I330V), for example, remained unaffected, but this construct exhibited a significant increase in activation of the noncognate substrates Trp and Tyr.
Interestingly, all Trp and Tyr activating domains have a valine at position 330. It was possible to observe a similar connection also for mutations in direction of the 'Leu(4)' condon (table 1), as shown below.
'Phe' and 'Leu(4)' condon (table 1) are about 600 homologous and the main differences affect positions 278, 301 and 322 (note: two of the three positions are 'highly variant' groups). Accordingly, it was assumed and subsequently also confirmed experimentally that point mutations in direction of the 'Leu(4) condon (~ T278M and A301G) show an increased leucine activation (table 3). Interestingly, the catalytic efficiency of mutant PheA(T278M) for unspecific activation of L-Leu is virtually unaffected (same Vmax), while at the same time the activity for the cognate substrates (D- and L-Phe) decreased about 3 fold. The exact opposite was observed for mutant PheA(A301G). Here, the Vmax for phenylalanine activation remained unaffected, while at the same time the catalytic activation of L-Leu increased virtually two fold. All these changes in substrate specificity and substrate activity were significant, reproducible and distinctly above the standard deviation. How only three differences in the condon make a distinction between phenylalanine and leucine possible, is still unclear at the moment, but at least for pos. 301 it can be assumed that this discrimination takes place here owing to differences in the spatial expansion of a CH
(Phe) versus a CH3 group (Leu) in the 8 position of the corresponding amino acid. All domains which activate substrates with sterically demanding y or 8 positions have the smallest possible side group (Gly) in position 301 (table 2 and 3).
In order to change the PheA substrate specificity completely to leucine, we constructed the double mutant PheA(T278M/A301G). The condon of this mutant shows a similarity of approx. 80o to 'Leu(4)' condon (cf.
table 1) and can be found in the phylogenetic tree in immediate proximity to Leu-activating domains (figure 2); medium gray background). As shown in figure 5A, this construct indeed preferably activates Leu, in fact with a catalytic efficiency which is in every way equal to the wild type enzyme and even exceeds the activity of the latter (activity of the mutant for L-Leu: kcat/Km = 86 mM-1 min-l, wild type activity for L-Phe: 69 mM-1 min-l; table 4). An at least 30 fold increase in L-Leu specific activation efficiency was detected. Astoundingly, the double mutant was only slightly adversely affected in the activation of D-Phe, whereas the catalytic efficiency for activation of the other stereoisomers, L-Phe, experienced a significant, approx. 7 fold decrease (figure 5A and table 4). In contrast, it was possible to show for the wild type protein PheA that the interactions of the protein with the two ligands, L-and D-Phe, are nearly identical. As shown in figure 1B, the benzyl ring of the side group of D-Phe is rotated by about 30° relative to the L-Phe ring and, as a result, the relative position of the (3-C atom but not of the a-C atom is slightly shifted.
According to the method of the invention, we altered the specificity of an A domain whose crystal structure has not yet been solved. The starting point of this experiment was the finding that the determined condons of Asp- and Asn-activating domain are very similar (approx. 80~; cf. table 1) and that the main differences affect positions 322 (His vs. Glu; a 'highly variant' group) and 330 (Val vs. Ile; a 'partially variant' group). We therefore chose the Asp-activating A domain of the comprehensively characterized surfactin biosynthesis complex [Cosmina, P. et al. 1993, Mol. Microbiol. 8:821-831] and tried to alter its specificity toward asparaaine. For the wild type protein AspA it was possible to confirm a specific activation of L-Asp, but not of L-Asn with moderate activity (wild type activity for L-Asp:
kcat/Km = 10 mM-1 min-1; cf. figure 5B and table 4). In contrast, the point mutant AspA(H322E) does create high selectivity for L-Asn (figure 5B), although this change in specificity was linked to a decrease in the catalytic activity (factor 10) (activity of the mutant for L-Asn: kcat/Km = 1 mM-1 min 1; table 4). Using the construct AspA(H322E) it was thus possible to show that a complete change in substrate specificity can be achieved by introducing a single point mutation.
Incidentally, the mutant AspA(H322E) showed in addition also about a 5 fold increase in activation efficiency for aliphatic Asn analogs of similar size, such as Ile, Leu and Val, for example.
It was possible to further increase the about 70 fold increase in the efficiency of L-Asn-specific activation by introducing an I1e330Va1 point mutation. At the same time, a catalytic activity of the now Asn activating domain, which was in the same order of magnitude [lacuna] of the original AspA domain, was achieved hereby.
Analysis of the condons of Glu and Gln-activating domains showed that also in these domains very similar condons are formed (table 1). The main difference affects position 239 (Lys vs. Gln; a 'highly variant' group). We therefore chose the Glu-activating domain of the surfactin biosynthesis complex (SrfA-Al) and tried to alter the specificity toward glutamine. For the wild type protein GluA it was possible to confirm a specific activation of L-Glu, but not of L-Gln, with moderate activity (kcat/Km = 25 mM-1 min-1). In contrast, the point mutant GluA(K239Q) does create high selectivity for L-Gln with, at the same time, only slightly reduced catalytic activity (kcat/Km = 20 mM-1 min-1). Owing to the condons of the mutant and Gln domains, this result was to be expected and shows that using the construct GluA(K239Q) it was also possible to achieve a complete change in substrate specificity by introducing a single point mutation.
It is shown below that the altered substrate specificity of an A domain can also make use of in vivo synthesis of a peptide with altered primary structure.
To this end, the gene fragment of the Asn-specific double mutant AspA(I330V/H322E) was transferred by way of example into the chromosome of the surfactin producer B. subtilis ATCC 21332 by means of homologous recombination. This integration was carried out with the aid of a two-stage recombination method [Schneider, A. et al. 1998, Mol. Gen. Genet. 257:308-318] and led to substitution of aspA' with aspA(H322A/I330V)' in the srfA-B-gene of the surfactin biosynthesis operon. The identity of this clone, B. subtilis Asn-5, was verified by PCT amplification of the mutated asp(H322E/I330V)' gene fragment from the chromosome thereof and subsequent DNA sequence analysis of the amplificate.
The biosurfactant surfactin is a lipoheptapeptide with a variable ~3-hydroxyfatty acid part (6-9 methylene groups), which is produced at the transition to the stationary growth phase and is secreted into the culture medium. Accordingly, the modified lipoheptapeptides were detected by fermenting the B.
subtilis strains ATCC 21332 and Asn-5 and preparing wild type surfactin and [Asn-5] surfactin from their culture supernatant. Subsequent HPLC-MS analysis showed that compared to the wild type, [Asn-5) surfactin (1) was produced in the same quantity, (2) had a longer retention time, and (3) had a mass difference 1 Da in ESIMS analysis. This mass difference was significant, and in negative ion mode it was possible to determine ion series with increments of 14 (variable fatty acid part) for the wild type m/z 992 to 1034 and for the mutant at m/z 991 to 1033. This finding, together with the reduced hemolytic activity of [Asn-5] surfactin, can be regarded as an unambiguous indication of substitution of a carboxyl (Asp-5; wild type) with a carbamide group (Asn-5; mutant) in surfactin. In summary, these results show that it was possible by specifically altering the substrate specificity of an A
domain in a catalytic NRPS matrix to alter the corresponding peptide product in vivo, too.
The inventive method for the specific synthesis of peptides basically looks as follows:
with the aid of the method of the invention it is possible, starting from a naturally occurring, functionally active NRPS system, to specifically alter within the A domains each amino acid position of the non-ribosomal product peptide formed by specific point mutations. To this end, the DNA of the A domain to be altered is firstly amplified from the chromosome of the producer organism and cloned into an E. coli His-tag expression vector. With the aid of this construct it is then possible to produce sufficient amounts of functionally active A domain protein, to purify said protein and to study it in vitro for its substrate specificity. Subsequently it is then possible to specifically introduce into the recombinant A domain DNA point mutations which lead to an alteration in substrate specificity according to table 1). Said change is verified by initially overexpressing in E.

' WO 00/52152 PCT/EP00/01652 . - 15 -coli, purifying and studying .with respect to their substrate specificity the mutated A domains. If hereby the expected specificity change can be confirmed, the mutated A domain DNA is replaced with the native A
domain DNA in the chromosome of the producer organism via homologous recombination. The thus generated producer strain with mutated A domain can then be used for producing the altered non-ribosomal peptide.
The method of the invention may also be used to influence the specificity and/or activity of known biologically active peptides. To this end, based on the code found, particular A domains are altered such that the peptides synthesized with the aid of the altered NRPS have the desired properties. By way of example, mention must be made here of the improvement in solubility, which may occur by replacing hydrophobic with hydrophilic amino acids and vice versa.
The results of the invention also allow a specific prediction of substrate specificities of already sequenced NRPS genes whose function, however, has not yet been determined. This becomes more and more important in view of the amount of sequences which steadily increases due to various genome sequencing projects. Using the method described here it is thus possible, for example, to predict from the DNA sequence of an NRPS cluster the putative structure of the product peptide formed. Thus, subsequent structure-function analysis of such genes becomes much easier.
The substrate specificity of an NRPS A domain with known DNA sequence is derived by carrying out first a sequence comparison with the PheA DNA sequence. This makes it possible to identify the corresponding ten residues of the putative binding pocket of the A domain to be studied. Subsequently, these ten residues are then used as independent amino acid sequences in alignments and phylogenetic studies together with the ten residues of the binding pocket of all other known A
domains. This results in a strict classification of the squences with respect to substrate specificity of the A
domain members. Thus it is possible to determine the substrate specificity of the A domain to be studied via the grouping behavior.
With application of the findings of the invention it becomes also possible to synthesize NRPS which are not derived from natural synthetases from the known DNA
sequences for particular A, C and T domains and, where appropriate, for other domains, as described in Mahariel et al.(supra).
The invention is illustrated in more detail by the following examples without being restricted thereto.
Examples Example 1 PCR amplification and cloning of PheA and AspA mutants.
All PheA mutants were generated by directed mutagenesis in the plasmid pPheA by means of inverse PCR. PCR
amplification of the entire plasmid was carried out using the 'Expand Long-Range PCR' system (Boehringer Mannheim; Mannheim, Germany, catalogue No.: 1681842) following the manufacturer's protocol. The following 5' phosphorylated oligonucleotides (MWG-Biotech, Ebersberg, Germany) were used (the in each case mutated condons are underlined): (Seq ID-N0:1) 3'A236: 5'-ATC
AAA AGA GAT GCT GGC A-3' ; ( Seq I D-NO : 2 ) 5' -A2 36L : 5' -TTA TCT GTA TGG GAG ATG TTT ATG-3'; (Seq ID-NO.: 3) 3'-W-239: 5' -TAC AGA TGC ATC AAA AGA G-3' ; (Seq ID-NO: 4 ) 5'-W239G: 5'-GGA GAG ATG TTT ATG GCT TTG TTA AC-3';
(Seq ID-NO.: 5) 5'-W239L: 5'-TTA GAG ATG TTT ATG GCT
TTG TTA-3'; (Seq ID-NO: 6) 3'-T278: 5'-AAT AAC AGT GAT
TTC CTT TTG G-3'; (Seq ID-N0: 7) 5'-T278M: 5'-ATG CTG

CCA CCT ACC TAT GTA GTT-3'; (Seq ID-NO: 8) 5'-T278Q:
5' - CAG CTG CCA CCT ACC TAT GTA GTT-3' ; ( Seq I D-NO : 9 ) 3'-1299: 5'-TAA CGT TTG TAT CGA TAA AAT AC-3'; (Seq ID-N0: 10) 5'-1299T: 5'-ACT ACA GCA GGC TCA GCT AC-3';
(Seq ID-N0: 11) 3'-A301G: 5'-TCC TGT AAT TAA CGT TTG
TAT CG-3'; (Seq ID-NO: 12) 3'-A301V: 5'-AAC TGT AAT TAA
CGT TTG TAT CG-3' ; ( Seq I D-NO : 13 ) 5' -A301 : 5' -GGC TCA
GCT ACC TCG CCT-3'; (Seq ID-NO: 14) 3'-A322: 5'-AAT GTA
AGT TAC TTT CTC CTT C-3'; (Seq ID-NO: 15) 5'-A322D: 5'-AAT GAT TAT GGC CCT ACG GAA ACA-3'; (Seq ID N0: 16) 5'-A322E: 5' -AAT GAA TAT GGC CCT ACG GAA ACA-3' ; (Seq ID-NO: 17) 5'-A322G: 5'-AAT GGC TAT GGC CCT ACG GAA ACA-3'; (Seq ID-NO: 18) 5'A322I: 5'-AAT ATT TAT GGC CCT ACG
GAA ACA-3'; (Seq ID-NO: 19) 5'-A322K: 5'-AAT AAG TAT
GGC CCT ACG GAA ACA-3'; (Seq ID-NO: 20) 5'-A322N: 5'-AAT TTG TAT GGC CCT ACG GAA ACA-3'; (Seq ID-N0: 21) 5'-A322Q: 5' -AAT CAG TAT GGC CCT ACG GAA ACA-3' ; (Seq ID-N0: 22) 5'-A322S: 5'-AAT AGC TAT GGC CCT ACG GAA ACA-3'; (Seq ID-NO: 23) 3'-I330: 5'-AGT TGT TTC CGT AGG GC-3'; and (Seq ID-NO: 24) 5'-I330V: 5'-GTT TGT GCG ACT
ACA TGG G-3'.
The PCT amplificates were purified using the 'QIAquick-spin PCT purification' system (Qiagen; Hilden, Germany, Catalogue No.: 28104), their cohesives ends were made blunt-ended and subsequently intramolecularly ligated.
The addition of the restriction endonuclease Dpnl (Amersham/Buchler; Braunschweig, Germany; Order No.:
E0302Z) which requires a methylated recognition sequence allowed specific degradation of the PCR matrix (pPheA; purified from a dam+ Escherichia coli strain) and thus avoiding false-positive clones. For all DNA
manipulations and preparation of the recombinant plasmid from E. coli XL-1-blue (Stratagene, Heidelberg, Germany, Order No.: 200268) the standard methods were used [Sambrook et al., 1989, Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Cloning of the PCT

amplificates made it possible to obtain the following plasmids: pPheA(A236L) (by using oligonucleotides Seq ID-N0: 1 and Seq ID-NO: 2), pPheA(W239G) (Seq ID-NO: 3 plus Seq ID-N0: 4), pPheA(W239L) (Seq IDN-N0: 3 plus Seq ID-N0: 5), pPheA(T278M) (Seq ID-N0:6 plus Seq ID-N0: 7) , pPheA (T278Q) (Seq ID-N0: 6 plus Seq ID-N0: 8) , pPheA(I299T) (Seq ID-NO: 9 plus Seq ID-N0: 10), pPheA(A301G) (Seq ID-N0: 11 plus Seq ID-NO: 13), pPheA(A301V) (Seq ID-N0: 12 plus Seq ID-N0: 13), pPheA(A322D) (Seq ID-N0: 14 plus Seq ID-NO: 15), pPheA(A322E) (Seq ID-NO: 14 plus Seq ID-NO: 16), pPheA(A322G) (Seq ID-N0: 14 plus Seq ID-N0: 17), pPheA(A322I) (Seq ID-N0: 14 plus Seq ID-NO: 18), pPheA(A322K) (Seq ID-N0: 14 plus Seq ID-NO: 19), pPheA(A322N) (Seq ID-N0: 14 plus Seq ID-NO: 20), pPheA(A322Q) (Seq ID-N0: 14 plus Seq ID-N0: 21), pPheA(A322S) (Seq ID-NO: 14 plus Seq ID-NO: 22), and pPheA(1330V) (Seq ID-NO: 23 plus Seq ID-N0: 24). The identity of all constructs was checked and confirmed by DNA sequencing reactions in an 'ABI prism 310 Genetic Analyzer' (ABI: Weiterstadt, Germany, Order No.: 310-A) .
The DNA fragment coding for the aspartate-activating A
domain from SrfA-B was amplified from the chromosomal DNA of Bacillus subtilis JH642 by means of PCR using the following oligonucleotides: (Seq ID-N0: 25) 5'-AspA: 5'-AAT CCA TGG CGA ACG TTC GGC TGT CTG-3' and (Seq ID-N0:26) 3'-AspA: 5'-AAT GGA TCC GGC CAA GGC CTT
GCC-3'. The resulting PCR amplificate was purified (see above), digested with restriction enzymes Ncol and BamHI (Amersham/Buchler; Braunchschweig, Germany; Order No.: E11602 and ElOlOY) and cloned into the 'His-tag' vector pQE60 (Qiagen; Hilden, Germany, Order No.: 33603) prepared in the same way. This cloning provided plasmid pAspA that was able to be used as template for generating pAspA(H322E) by means of inverse PCR (see above): (Seq ID-N0:27) 5'-AspA(H322E):

5'-TAA TGA GTA CGG CCC GAC AGA AGC-3' (Seq ID-N0:28) and 3'-AspA(H322E): 5'-ATA AAT TCG GTA TGT CCA TAC-3'.
The resulting plasmid pAspA(H322E) subsequently served as template for generating pAspA(H322E/I330V) by means of inverse PCR using the following primers(Seq ID-N0:29) 5'-AspA(I330V): 5'- TAC CGG CCC ACA GAA GCA ACG
GTC GGC-3' and (Seq ID-N0:30) 3'-AspA(I330V): 5'-CTG
TGG GCC CGT ACT CAT TAA TAA ATT CGG-3'. The identity of all constructs was again checked and confirmed by DNA
sequencing reactions. The DNA fragment coding for the glutamate-activating A domain from SrfA-A was amplified from the chromosomal DNA of Bacillus subtilis JH642 by means of PCR using the following oligonucleotides: (Seq ID-N0:31) 5'-GluA: 5'-TAT GGA TCC ATT GAT GAA TTA ACA
CTG-3' and (Seq ID-N0:32) 3'-GluA: 5'-TAT GGA TCC GAT
TGC TTT TTC AGT -3' . The resulting PCR amplificate was purified (see above), digested with the restriction enzyme BamHI (Amersham/Buchler; Braunschweig, Germany, Order No.: ElOlOY) and cloned into the 'His-tag' vector pQE60 (Qiagen; Hilden, Germany, Order No.. 33603) cut with BgIII (Amersham/Buchler; Braunschweig, Germany, Order No.: E1021Y). This cloning provided plasmid pGluA
that was able to be used subsequently as template for generating pGluA(K239Q) by means of inverse PCR (see above): (Seq ID-N0:33) 5'-GluA(K239Q): 5'-GTG CAG CAA
ATC TTC GCG TCG CTT C-3' and (Seq ID-N0:34) 3'-GluA(K239Q): 5'-TGA CGC ATC AAA GTG GAA C-3'. The identity of all constructs was again checked and confirmed by DNA sequencing reactions.
Example 2 Expression and purification of adenylation domains (wild type and mutants).
The expression of the (mutated) gene fragments and purification of the resulting His6 fusion proteins was carried out as described elsewhere [Mootz et al., 1997, J. Bacteriol. 178:,6843-6850]. Ligation into the BamHI
cleavage side of pQE60 led in each case to a fusion of . - 20 -amino acid sequence "GSRSHHHHHH" to the carboxy terminus of the particular recombinant protein. By means of SDS-PAGE it was possible to show that most proteins could be purified to near homogeneity by means of Ni2+affinitiy chromatography; however, two constructs were insoluble (cf. table 3). Fractions containing the particular recombinant protein were combined and dialyzed against assay puffer (50 mM
HEPES, pH 8.0, 100 mM sodium chloride, 10 mM magnesium chloride, 2 mM dithioerythritol (DTE) and 1 mM EDTA).
After adding loo glycerine (v/v) it was possible to store the proteins at -80°C without any detectable loss in their catalytic activity. The protein concentration of the various solutions was determined with the aid of the calculated extinction coefficients at 280 nm (A280nm): 64060 M-1 cm-1 for PheA and all PheA mutants except PheA(W239Xaa), 58370 M-1 cm-1 for PheA(W239Xaa), for 39780 M-1 cm-1 for AspA and AspA(H322E) and 35490 M-1 cm-1 for GluA and GluA(K239Q).
Example 3 ATP pyrophosphate exchange reaction The ATP-pyrophosphate exchange reaction was carried out in order to [lacuna] both the catalytic activity and the specificity of the purified recombinant A domains [Mootz & Marahiel, 1997, J. Bacteriol. 178: 6843-6850].
In this connection, specificity was checked in each case using all 20 proteinogeneic amino acids and in addition L-ornithine and D-phenylalanine. The reaction mixtures contained (final volume: 100 ~L): 50 mM HEPES, pH 8.0, 100 mM sodium chloride, 10 mM magnesium chloride, 2 mm DTE, 1 mM EDTA, 0 to 2 mM amino acid, and 250 nM enzyme. The various reactions were initiated by adding 2 mM ATP, 0.2 mM sodium pyrophosphate and 0.15 ~Ci (16.06 Ci/mmol) of sodium-[32P]pyrophosphate (NEN/DuPont; Germany; Order No.: NEX019) and incubated at 37°C for 10 min. Subsequently it was possible to stop the exchange reactions by adding 0.5 mL of Stop solution: 1.20 (w/v) activated carbon, 0.1 M sodium pyrophosphate and 0.35 M perchloric acid. The activated carbon was removed by centrifugation, washed once with lml of bidistilled water and resuspended in 0.5 mL of water. After addition of 3.5 mL of scintillation liquid (Rotiscint Eco Plus; Roth; Art. No.: 0016.2), it was possible to determine the carbon-bound radioactivity in a scintillation analyzer (1900CA Tri-Carb 'liquid scintillation analyzer'; Packard).
Example 4 Computer analysis for determination of the putative substrate binding pockets.
Protein sequences of 160 A domains were obtained from publically accessible databanks (e. g.
http//www.ncbi.nlm.nih.gov/Entrez/nucleotide.html) and reduced to the region of approx. 100 amino acids between the core motifs A4 and A5 [Marahiel et al., 1997, Chem. Rev. 97: 2651-2673]. These sequences were then aligned with respect to sequence homologies by means of the MegAlign subprogram of the DNAStar program package, using the 'clustal' method with its default settings. The sole purpose of this first alignment was to make the subsequent classification of the constituents of the putative substrate binding pockets easier. In the end, it was possible to make this classification by taking into account their position, relative to the highly conserved sequence motifs (core motifs A4 and A5 and motifs 'TPS' and 'GE'; structural 'anchor'). All sequences were then trimmed to the determined constituents of the binding pockets and new alignments and phylogenetic studies were carried out using these regions of merely ten amino acids. Each method and each set of parameters made it possible to achieve grouping of the coding sequences according to the substrate specificity of their original domains.
The phylogenetic tree shown in figure 2 was determined using the method of Jotun Hein, with application of following parameters: gap penalty 12, gap length penalty 6, and Ktuple 2.
Example 5 Integration of the AspA(H322E/I330V)-encoding gene fragment into the B. subtilis AS20 chromosome.
In order to prove the potential of ali methods for in vivo synthesis of specifically modified peptide antibiotics, the coding gene fragment of the AspA
double mutant (Asp(H322E/I331V) was introduced into the srfA biosynthesis operon of B. subtilis [Cosmina, P. et al. 1993, Mol. Microbiol. 8:821-831]. For the construction of the integration plasmid required for this, it was necessary first to amplify the appropriate srfA-B gene fragment which codes for the Asp-activating module (base pair 14195-18200; [Cosmina, P. et al.
1993, Mol. Microbiol. 8:821-831], from the B. subtilis ATCC 21332 chromosome by means of PCR (underlined:
restriction endocnuclease cleavage sites): (Seq ID-NO: 35) 5'-homo Asp(ClaI): 5'-TAA ATC GAT GGA GGC TGC
CAA GG-3' ; and ( Seq I D-NO : 3 6 ) 3' -homo Asp ( Spe I ) : 5' -TAA ACT AGT CAG TAA ATC CGC CCA GT-3'. The resulting amplificate was purified, digested with restriction enzymes ClaI and SpeI (Amersham/Buchler; Braunschweig, Germany; Order No.: E11034Y and E1086Z) and cloned into the pBluescript SK(-) vector prepared in the same way (Stratagene, Heidelberg, Germany, Order No.: 212206).
This cloning provided plasmid pSK-homoAsp from which subsequently an approx. 1.3 kb aspA' fragment (base pair 15212-16559; [Cosmina, P. et al. 1993, Mol.
Microbiol. 8:821-831]) was excised using restriction endonuclease EcoRI and PstI (Amersham/Buchler;
Braunschweig, Germany; Order No.: E1040Y and E1073Y) and was replaced with the complementary twice mutated fragment from pAspA(H322E/I33V). The identity of the resulting integration plasmid (pSK-homoAsp(H322E/I330V) was confirmed by DNA sequencing.

The mutated aspA' fragment was integrated into the surfactin biosynthesis operon by using a proven co-transformation technique ('congression') [Schneider, A. et al. 1998, Mol. Gen. Genet. 257:308-318]. The host used was the B. subtilis strain AS20 [Schneider, A. et al. 1998, Mol. Gen. Genet. 257:308-318] in which the aspA' fragment (base pair: 15245-17177; [Cosmina, P. et al. 1993, Mol. Microbiol. 8:821-831] was deleted and substituted with the chloramphenicol transferase gene.
B. subtilis AS20 was made competent for DNA uptake and cotransformed with the integration vector pSK-homoAsp(H322E/I330V) and the helper plasmid pNEXT33A
[Schneider, A. et al. 1998, Mol. Gen. Genet. 257:308-318]. The helper plasmid mentioned last here mediated first a positive selection via its neomycin resistance gene and it was subsequently possible to study the clones obtained for the loss of their chloramphenicol cassette and thus integration of the aspA(H322E/I330V)' gene fragment. PCR amplification of this region from the chromosome of the clones selected, and subsequent DNA sequencing made it possible to verify the identity of the constructed clone B. subtilis Asn-5.
Example 6 Surfactin preparation and analysis The surfactin derivative from B. subtilis Asn-5 was prepared according to a standard method [Schneider, A.
et al. 1998, Mol. Gen. Genet. 257:308-318]. For analysis, the methanolic solution obtained was fractionated by means of an 'HP1100 Series LC/MSD' HPLC
system using a PepRPC HR 5/5 column column (Pharmacia, Freiburg, Germany, Order No.: 18-0383-O1)-. The individual components were separated at a flow rate of 0 . 7 mL min-1 by applying a linear gradient of from 35 0 to 70o acetonitrile (v/v) in O.lo trifluoroacetic acid (v/v). Detection was carried out at a wavelength of 214 nm and by mass-spectrometric online analysis of the eluted substances. As a control, a preparation from the wild type surfactin producer B. subtilis ATCC 21332 was worked up and analyzed analogously.
Description. of the figures:
Figure 1: structural basis for recognition and activation of phenylalanine (from Conti et al. [Conti et al., 1997, EMBO J. 16: 4174-4183]). (A) The ribbon diagram shows that PheA comprises two folding domains, a large N-terminal (bottom) and a smaller C-terminal (top) domain. AMP and the substrate amino acid phenylalanine are shown in black. (B) The binding pocket for phenylalanine is formed by ten amino acids.
Asp235 and Lys517 mediate electrostatic interactions (dashed lines) with the a-amino and a-carboxyl group of the substrate. The actual binding pocket which makes specific recognition of the phenylalanine side group possible is formed by A1a236, I1e330 and Cys331 on the one side and A1a322, A1a301, I299 and Thr278 on the other side. Both sides are kept separate from one another by the Trp239 indole ring at the bottom end.
This architecture allows the PheA binding pocket to take up both stereoisomers, L-Phe (light gray) and D-Phe (dark gray) without any detectable change in conformation.
Figure 2: Phylogenetic tree constructed using the ten specificity-mediating amino acids of the A domains. The phylogenetic study displays a grouping of the determined condons, according to the specificity of their original domains (light gray boxes). This confirms the correctness of the concept of structural homogeneity between A domains and shows that the ten amino acids determined in each case indeed form the condon for recognition of the cognate substrate amino acid. Examples are given which show that the specificity of newl~r detected domains is predictable by means of this technique and that the grouping is WO 00/52152 ' PCT/EP00/01652 controlled by the experimentally observed specificity (dark gray background). This is also true for the constructed mutants PheA(T278M/A301G) and AspA(H322E) which show Leu and Asn specificity in the ATP-pyrophosphate exchange reaction (medium gray background).
Figure 3: Diagrammatic representation of the postulated binding pockets of three adenylation domains. The determined condons of three different substrates ('Asp', 'Orn(2) and 'Val(3)'; cf. table 1) were projected into the representation of the PheA binding pocket shown in figure 1B. Aliphatic (light gray), polar (hatched), acidic (white) and basic (dark gray) side groups are shown diagrammatically. In all three cases, Asp235 and Lys517 mediate interactions with the a-amino and a,-carboxyl group of the particular substrate, whereas all other residues Xaa236 to Xaa331) mediate the actual recognition of the substrate side chain. A perfect correlation between the polarity of the binding pocket and the substrate can be observed for all examples shown.
Figure 4: Observed variability of the constituents of various substrate binding pockets. The constituents amino acids of the various positions in 160 postulated substrate binding pockets were studied for their variability. The proportional distribution of the nature of the substrates in the A domains studied is depicted in the medium gray box. The light gray boxes which are connected with each position of the binding pocket indicated the number of the observed various residues (frequency: =lo) and the proportional occurrence of hydrophobic, polar, acidic and basic side groups at these positions. Owing to the results shown, the ten constituent amino acids can be classified into three subgroups: (1) positions 235 (Asp: acid, white) and 517 (Lys: basic, dark gray) are 'invariant'. (2) Positions 236, 301 and 330 are merely 'partially variant'. They indicate a subaverage occurrence of charged and polar side groups and the overwhelming majority (93%) of the A domains studied uses aliphatic residues (gray) at these positions. Positions 239, 278, 299, 322 and 331 can be regarded as 'highly variant';
said positions show a high degree of variability with respect to the use of different amino acids.
Figure 5: Specific alteration of PheA and AspA
substrate specificities. The substrate specificity of wild type (white) and mutants (various shades of gray) were studied with the aid of ATP pyrophosphate exchange reactions. The substrates used are shown on the x-axis and the observed maximum of the activity of each protein was set to 100%. (A) Point mutations in PheA in direction of the 'Leu(4)' condon (T278M and A301G) slightly increase the side specificity for Leu, whereas Phe is still the preferred substrate. The corresponding double mutant (PheA(T278M/A301G); dark gray) on the other hand preferably activates Leu with a catalytic activity which is equal in every way to the efficiency of the wild type enzyme PheA for Phe. (B) An H322E
point mutation in AspA was sufficient to shift the substrate specificity of the mutant completely from Asp to Asn. The observed activation patterns of the mutants are consistent with the position of their condons in the phylogenetic tree shown in figure 2 (medium gray background).
Figures 6-9 represent tables 1-4.

SEQUENCE LISTING
c110> Hasahiel. Staehalholz, Hootz,~Koaz ~120~ Novel non-ribosomal peptide synlholeses.
(orthe woduction Iheroof and use Ihsreof <130> 99F060 <li0> 19909196.3-44 <1q1> 1999-03-83 <160> 36 <t70> Patentln var. 3.1 <210> 1 <211> 18 < 212 > D1TA
<213> 8acillua bzevis <400> 1 ateaaaagag atgctggc <210> 2 <211> 2q <Z12> DNA
<213> Bacillus brevW
<qoo> z ttatctqtat gggagatQtt tatg <210> 3 <2:1> .9 <2I2> DNA
<213> Hac=llus brevis <400> 3 ta:aqatqca tcaaaagag 19 <210> q <2I:> 2fi <212> DNA
<213> Bacillus brevis <100> 4 ggagagatgt ttatggcttt gttaac ~ 26 <210> 5 <211> 29 <212> DNA
t213> Bacillus brevis <400> 5 ttagaqatgt ttatggcttt gtta 2q c210> 6 <211> 22 <212 > DNA
<213> Bacillus brevis <400> fi aataacagtg atttcctttt gg 22 <210> 7 <211> 24 <212> D:IA
<2I3> Bacillus brevis cq00> 7 atgctgccac ctacctatgt agtt 2q <zla> a <22I> 24 <212> DNA
<213> Bacillus brevis <400> B
cagctgccac ctaccta:gt agtt 2q <?10>
<2I1> 23 <212> DNA
<2I3> Bacillus bravis <900> 9 caacg~ttgc atcgataaaa tac 23 <210> 10 ' <211> 20 <212> DNA
<213> Bacillus brevis c4oo> Io aetacagcaq gctcagctac 20 <21C> 11 <211> 23 <212> Dt~
<213> Bacillus brevis <400> 1:
tcctgtaatt aacgtttgta tcg 23 <210> 12 <211> 23 <212> DI4A
<213> Bacillus brevta <400> 12 aactgtaatt aacgtttgta tcg 23 <210> 13 <211> I8 <212> DNA
<2I3> 9ac~.llus brevis <400> 13 ggctcagcta cctcgcc: 18 <210> 14 <~11> 22 <212> DNR
<213> eac~llua brevia <400> 14 aatgtaagt~ actttctcct t~ 22 <210> 15 <21I> 24 <212> Dt~1 <213> Bacillus brcvis <400> is aatget~atq gccctaegga aaea 24 <210> 16 <211> 24 <2.2> Dt~
<213> Bacillus brevis <400> 16 aatgaatatg gccctacgga aaca 29 <210> 1~
<211> 24 <212> DhA
<213> Bacillus brcvi~
<400> 17 aatggctatg gccctacgga aaca 24 <210> I8 <211> 24 <212> DNA
<213> Bacillus brevis <40Q> 18 aatatttatg gccctacgga aaca 24 <2;0> 19 <2:1> 24 <212 > DPIA
c213> Bacillus brevis <4~J0> 19 aataagtatg gccctacgga aaca 24 <210> 20 <211> 24 <212> DNA
<2I3> Bacillus brevis <4oc> 20 aatttgtatq qccctacgga aaca ~ 2a <210> 21 <~11> 24 <212> DNA
<213> Bacillus brevis <400> 21 aatcagtatq gccctacqqa aaca 24 <210> 22 <211> 24 <212> DNA
<213> bacillus brevis <400> 22 aatagctatg gccctacgga aaca 24 <210> 23 <211> I'~
<2I2> DNA
<213> Bacillus blevia <40C> 23 agttgtttcc gtaqggc <210> 2a <211> 19 <2i2> DNA
<213> Bacillus brevis <400> 24 gtttgtgcga ctacatggg 19 <210> 25 <211 ~ 2~.
<212> DNA
<213> Bacillus subti-is <400> 25 aatccatggc gaacgttcqg ctgtctg 27 <210> 26 -<211> 24 ' <212> DNA
<213> Bacillus subtilis .:900> is aatggatccg gccaaggcct tgcc 24 <ZIO> 27 <211> 24 <212> DNR
<213> Bacillus subtilis <400> 27 taatgagtae ggcecgaeeg aagc 24 <ZIO> 28 <211> 21 <212> DNA
c213> Bacillus subtilis t400> 28 ataaattcqg tatgtccata c 21 <210> 29 <211> :7 <2;2> DNA
c2I3> 9acillus subtilss <400> 29 taccggccca cagaagcaac ggtcggc 27 <210> 30 <2I1> 30 < Z 12 > Dt~P.
<213> Bacillus suDtilis <400> 30 ctgtgggccc gtaeteatta ataaattcgg 30 <210> 3:
<2:1> 27 <212> DNA
<213> Bacillus subtilis <400> 31 catgqatcca ttgatgaatt aacactg <210> 32 <211> 24 <Z 12 > DIdA
<2i3> Bacillus aubtilis <400> 32 tatggatecg attgcttttt cagt <210> 33 <211> 25 <212 > DttA
<213> Bacillus subtilis <400> 33 gtgcagcaae tcttcgcgtc gcttc 25 <210> 34 <21i> 19 <212> DNA
<213> Bacillus suDtilis <400> 34 tgacqcatu aagtggaac 19 <210> 35 <~lI> 23 <212> DNA
<213> Bacillus aubtllis <400> 35 taaatcgatg gaggctgcca aqg 23 <2I0> 36 <211> 20 <212> DNA
<Z13> Bacillus subtilis <400> 36 taaactagtc agtaaatccg - 20

Claims (16)

claims:

1. A method for the specific non-ribosomal synthesis of peptides of a desired structure, which comprises altering in a given DNA sequence coding for a non-ribosomal peptide synthetase one or more A domain-encoding sections according to the non-ribosomal code presented in table 1) so that the expression product of the altered DNA sequence expresses the peptide of the desired structure.

2. The method as claimed in claim 1, wherein in at least one A domain to be altered no more than 10, preferably no more than 6, particularly preferably no more than 3, and very particularly preferably one amino acid is exchanged.

3. The method as claimed in claim 1, wherein A
domain-encoding sections are specifically added to or removed from the given DNA sequence for the purpose of producing longer or shorter peptides.

4. The method as claimed in at least one of claims 1 to 3, wherein the synthesized peptides have antibiotic, immunosuppressive, cytostatic, antiviral, anthelmintic, fungicide or surface-active action.

5. The method as claimed in at least one of claims 1 to 4, wherein at least one altered non-ribosomal peptide synthetase is contained in a host organism.

6. The method as claimed in claim 5, wherein the host organism is selected from the group consisting of bacteria, fungi, and yeasts, and bacilli, actinomycetes, myxobacteria, pseudomonads and Escherichia coli are particularly preferred, and Bacillus subtilis is very particularly preferred.

7. The method as claimed in claim 5, wherein the host organism is a plant.

8. The method as claimed in claim 5, wherein the host organism is a mammal.

9. The method as claimed in claim 8, wherein the host organism is a human.

10. The method as claimed in at least one of claims 1 to 4, wherein the altered a non-ribosomal peptide synthetase is used in isolated form (in vitro).

11. The method as claimed in claim 10, wherein the altered non-ribosomal peptide synthetase is immobilized.

12. The method as claimed in claim 11, wherein the altered non-ribosomal peptide synthetase is bound to a support.

13. A DNA sequence which codes for a non-ribosomal peptide synthetase in which one or more A domains were altered on the basis of the non-ribosomal code in table 1 such that the expression product of the altered DNA sequence expresses the peptide of a predetermined structure which preferably does not correspond to a natural peptide.

14. The DNA sequence as claimed in claim 13, in which in at least one A domain to be altered no more than 10, preferably no more than 6, particularly preferably no more than 3, and very particularly preferably one amino acid is exchanged.

15. The use of the DNA sequence as claimed in either of claims 13 or 14 for the synthesis of peptides in any of the methods as claimed in claims 1 to 11.

16. A peptide obtainable on the basis of the non-ribosomal code according to table 1, wherein table 1 is part of the claim.