Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• the present invention relates to a new, microbiological , method for the production of ⁇ -L- aspartyl-L-phenylalanme (Asp-Phe) from the substrates L-aspartic acid (L-Asp) and L-phenylalamne (L-Phe) .
• the present invention also relates to novel DNA fragments or combination of DNA fragments encoding a new Asp-Phe dipeptide synthetase, micro-organisms containing such DNA fragments, as well as to the new Asp-Phe dipeptide synthetases itself.
• APM ⁇ -L-aspartyl-L-phenylalanme methyl ester
• This new method thus provides a microbiological process for direct fermentation of Asp- Phe, which m a subsequent methylation step may be converted into the intense sweetener aspartame.
• Production of Asp-Phe by direct fermentation is hitherto unknown, as is non- ⁇ bosomal synthesis of this dipeptide
• the inventors thus have provided a direct microbiological method for producing the dipeptide Asp- Phe without tne need for any protecting and deprotectmg steps .
• novel non- ⁇ bosomal dipeptide synthetases which, according to the present invention, can be used for the production of Asp-Phe are also indicated hereinafter as Asp-Phe dipeptide synthetases or as Asp-Phe synthetases. It is known (for instance, from P. Zuber et al . , in " Bacill us suhtilis and other Gram-positive bacteria", Sonenshe et al . (Eds.), Am. Soc. Microbiol., Washington, DC, 1993, p.897-916) that micro-organisms can produce bioactive peptides through ⁇ bosomal and non-ribosomal mechanisms.
• bioactive peptides so far known to be synthesised non- ⁇ bosomally, are produced by a number of soil bacteria and fungi. These bioactive peptides can range from 2 to 48 residues, and are structurally diverse. They may show a broad spectrum of biological properties including antimicrobial, antiviral or antitumor activities, or immunosuppressive or enzyme-inhibiting activities. As such, these non-ribosomally synthesised bioactive peptides form a class of peptide secondary metabolites which has found widespread use medicine, agriculture, and biological research. Already more than 300 different residues so far have been found to be incorporated into these peptide secondary metabolites.
• the thiol -activated substrates may be modified (e.g. by epime ⁇ sation or N-methylation) ;
• this synthesis involves the subsequent steps of d) recognition of L-Asp and L-Phe, (ii) formation of an L- Asp- and an L-Phe-acyladenylate, (m) binding thereof to the cysteamme group of the 4 ' -PP cofactor m the respective thiolation domains, (iv) formation of the Asp-Phe dipeptide by transfer of the thioester- activated carboxyl group of L-Asp to the ammo group of L-Phe, while the condensation product remains covalently attached to the multi -enzyme complex via the 4 ' -PP cofactor m the thiolation domain of the Phe- recognismg module, and (v) release of the Asp-Phe formed.
• the substrates L-Asp and L-Phe are contacted with a non- ribosomal Asp-Phe dipeptide synthetase, in the presence of an effective amount of ATP.
• An effective amount of ATP as meant herein is an amount of ATP which ensures that the dipeptide formation takes place at a suitable rate. Usually such rate will be at least one turn-over per minute, i.e. a turn-over number (k cat ) of 1 per minute; preferably k cat is at least 10 per minute.
• k cat turn-over number
• the ATP consumed by the peptide synthesis reaction is preferably regenerated.
• the contacting of the substrates L-Asp and L-Phe with the non-ribosomal Asp-Phe dipeptide synthetase may be done any suitable way; for instance - if the Asp-Phe dipeptide synthetase is present a micro-organism - L-Asp and L-Phe may be fed into the culture medium containing said microorganism.
• micro-organisms may be used which are capable of overproducing L-Asp and/or L-Phe (e.g. from glucose), with separately feeding to the micro-organism of the ammo acid (L-Asp or L-Phe) which is not produced by the micro-organism. All these methods may be called m vivo methods. ATP may be regenerated m vivo m the Asp-Phe producing microorganism.
• L-Phe with the non-ribosomal Asp-Phe dipeptide synthetase also may be done by using the synthetase its isolated form, that is by an in vi tro method.
• ATP-regeneration is to be taken care of separately. This may be done by applying an
• ATP-regeneration system ATP-regeneration system. ATP-regeneration systems are readily available to the skilled man.
• modules have been defined as semi -autonomous units within peptide synthetases that carry all information needed for recognition, activation, and modification of one substrate. Although the modules in principle can act independently, it is generally assumed that they have to work m concert, in a template-based mode of action to achieve peptide elongation.
• tne modules of peptide synthetases are themselves composed as a linear arrangement of conserved domains specifically representing the enzyme activities involved in substrate recognition, activation, (and, optionally, as the case may be, modification) and condensation (i.e. peptide bond formation) .
• conserved domains specifically representing the enzyme activities involved in substrate recognition, activation, (and, optionally, as the case may be, modification) and condensation (i.e. peptide bond formation) .
• Two of such distinct domains, the adenylation and thiolation domains (A-domain and T-domam) , together form the smallest part of a module that retains all catalytic activities for specific activation and covalent binding of the ammo acid substrate.
• Stachelhaus et al have designated this core fragment of the modules as a "minimal module" (T. Stachelhaus et al . , J. Biol. Chem. 270, 1995, p.6163-6169) .
• minimal module refers to such combined core fragment of the modules, consisting of an adenylation domain and a thiolation domain.
• A- domain A- domain, about 550 ammo acids
• the A-domain has been shown to bear the substrate-recognition and ATP-b dmg sites and is therefore solely responsible for activation of the recognised amino acid as its acyl adenylate through ATP hydrolysis (T. Stachelhaus et al . , J. Biol. Chem. 270,
• the A-domam of a module is very important in determining the specificity of the module.
• “Specificity" of a module means that the module has a certain preference recognising one ammo acid above other ammo acids or above another ammo acid. Of course, also the concentration of each individual ammo acid present near the module may play a role. If, for instance, the concentration of a specific ammo acid is much higher than that of (most of) the other ammo acids, the requirements for specificity may be somewhat less strict .
• T- domain about 100 ammo acids; also called peptidyl carrier protein (PCP)
• PCP peptidyl carrier protein
• T-domam 4'-PP is covalently bound to the sidecham of an invariant se ⁇ ne residue located withm the highly conserved thiolation core motif (see table 1) .
• T-domam m a module of the peptide synthetase does not carry its 4'-PP cofactor, no covalent binding of the ammoacyl substrate can take place and chain elongation will be impossible.
• peptide synthetases known so far every T-domam is converted from the inactive apo form to the active holo form by transfer of the 4'-PP moiety from Coenzyme A (CoA) to the sidecham of the above mentioned se ⁇ ne residue.
• CoA Coenzyme A
• This post -translational priming of each T-domam is mediated by peptide synthetase specific members of a recently discovered enzyme superfamily, the 4'- phosphopantethe yl transferases . They utilise CoA as a common substrate, and appear to attain specificity through protem/protem interactions.
• the Asp-Phe dipeptide synthetase as used m the method of the present invention comprises two minimal modules, respectively one minimal module at its N-termmal side recognising L-Asp and another minimal module at its C- erminal side recognising L-Phe.
• Minimal module is used m the same meaning as given thereto by Stachelhaus et al . , J. Biol. Chem. 270, 1995, p.6163-6169.
• Each of these minimal modules is composed of an adenylation domain (A-domam) and a thiolation domain (T-domam) . Moreover, the two minimal modules of the
• Asp-Phe dipeptide synthetases according to the invention are connected by a so-called condensation domain, which needs to be covalently bound to the polypeptide chain of the Phe-module, namely to the N- terminal part thereof.
• the condensation domain does not need to be bound covalently to both minimal modules (i.e. to the modules recognising respectively L-Asp and L-Phe) because there is no requirement that these two minimal modules are located on a single polypeptide chain.
• the term "connected" therefore means that the condensation domain ensures that both minimal modules can operate concertedly.
• condensation domains occur as a moderately conserved region.
• the condensation domain usually consists of about 400 ammo acid residues and is known to be involved the catalysis of non- ribosomal peptide formation.
• One of the conserved core motifs contains the catalytically active histidme residue. See T. Stachelhaus et al . , J. Biol. Chem. 273, 1998, p.22773-22781.
• the Asp-Phe formed can be recovered from the reaction medium by any method available to the skilled man.
• the condensation domain in the dipeptide synthetase is connected to both minimal modules m such way that it is also covalently bound to the module recognising L-Asp.
• the condensation domain is not only bound covalently to the N-terminal end of the L-Phe recognising module, but also to the C-termmal end of the L-Asp recognising module, and forms part of a single polypeptide chain comprising the L-Asp and L-Phe recognising modules.
• a distinguishing feature of non-ribosomal peptide synthesis is the fact that the peptide formed on the template is covalently bound to the T-domam of the C-termmal module as a peptidyl- (4 ' -PP) -T-domam intermediate. Release of the peptide from this intermediate is assumed to take place either by mtermolecular attack by water, resulting net hydrolysis, or by intramolecular capture by a hydroxyl or ammo group of the peptide chain itself, giving rise to a cyclic peptide product and of the peptide synthetase the holo form.
• the first termination route yields a linear peptide with a free C-termmal carboxylic group (as should be the case for the Asp-Phe synthesis according to the present invention) .
• Asp-Phe is present an intermediate form oound to the template of tne Asp-Phe dipeptide synthetase, it is advantageous to take additional measures for enhancing the release of the Asp-Phe from said template. It is therefore particularly preferred that also a releasing factor is present for the Asp-Phe formed on the dipeptide synthetase.
• the term "releasing factor" as used here is intended to comprise any means, whether part of the synthetase or present m combination therewith, which enhance the releasing from the synthetase of the Asp-Phe formed on the synthetase.
• All known bacterial and some fungal peptide synthetase modules that incorporate the last ammo acid into the growing peptide chain show a region with a thioesterase-like function. These regions of approximately 250 ammo acids are located at the C- termmal end of the am o acid recognising modules. These thioesterase-like regions are integrated regions which exhibit homology to thioesterase-like proteins, and therefore also are referred to as the thioesterase domain ( (integrated) TE-domam) . All these integrated TE-domams contain an active site serme residue, which is part of the core motif GxSxG (see table 1) .
• the releasing factor therefore is a protein which shows thioesterase-like functions and forms an integrated domain of the dipeptide synthetase at the C- erminus thereof.
• the Asp- Phe dipeptide synthetase prior to the production of Asp-Phe, has undergone optimisation for its function by using one or more post-translational modifying activities. This is useful for achieving the most efficient non-ribosomal synthesis of Asp-Phe.
• post-translational modifying activities for efficient non-ribosomal synthesis of Asp-Phe as used here is intended to comprise any activities which modify the dipeptide synthetase after its formation thereby positively affecting its Asp-Phe synthesismg function.
• the post- translational modifying activity used is a 4 ' - phosphopantethemyl (4 ' -PP) transferase.
• the 4 ' -PP transferase provides for effective conversion of the apo- to holo-enzyme of the peptide synthetase and by loading the 4 ' -PP cofactor to the ser e side-chains the core motif of the T-domams, and thereby increases the yield of Asp-Phe m the production thereof.
• Effective conversion of apo- to holo-enzyme is provided if in each of both T-domains of the Asp-Phe dipeptide synthetase at least 10% of the apo-enzyme is converted to the holo-form.
• proteins having thioesterase Type-II-like activity are proteins with strong sequence similarities to type-II fatty acid thioesterases of vertebrate origin.
• Such non-integrated protein with thioesterase Type-II-like activity is different from the integrated thioesterase (TE-domam) .
• the genes coding for the non- integrated proteins with thioesterase Type-II-like activity can be positioned at the 5'- or 3 ' -end of the peptide synthetase encoding operon. These proteins have molecular masses of 25-29 kDa, are about 220-340 am o acid residues m length, and carry the sequence GxSxG which is presumed to form the active site. It is noticed that in almost all of the prokaryotic peptide synthetase coding operons known so far, such distinct genes have been detected.
• the Asp-Phe dipeptide synthetase is preferably present living cell-material of a micro-organism, and glucose, L-Asp and/or L-Phe are being fed to said fermentor, and the Asp-Phe formed is then recovered.
• glucose is intended to cover glucose and any otner energy source necessary for the regeneration of ATP the living cell material and for the mamtamance energy required for said living cell material.
• the glucose (or other energy source) is used as starting material for the production of any L-Asp and/or L-Phe to be produced m the living cell the course of the process of the invention.
• the Asp-Phe formed is recovered. Such recovery may take place during the process or at the end thereof.
• the living cell -material may be present m any appropriate form as available to the skilled man. For instance, whole cells may be used as such or m immobilised form.
• the micro-organism may be any kind of micro-organism wherein the Asp-Phe dipeptide syntnetases according to the invention can stably be expressed. Suitable micro-organisms are, for instance, micro-organisms which
• (a) are producing peptides via non-ribosomal synthesis, for instance, bacteria as Strepto yces species, Bacillus species, Actmomyces species, Micrococcus species, Nocardia species, or fungal species as Tolypocladiu species, Fusa ⁇ um species, Pem cillium species, Aspergillus species, and Cochliobolus species; or
• (b) are capable of producing ammo acids, m particular L-Asp and/or L-Phe, preferably on industrial scale, for instance, Esche ⁇ chia species, e.g. E. coli , and Corynebacte ⁇ um species, e.g. C. glutamicum.
• the micro-organisms may be grown, under conditions which can easily be found by the skilled man, m a fermentor, and production of the Asp-Phe then can be carried out m the same or another fermentor.
• the fermentor may be any type of fermentor or other types of (enzyme) reactor known to the skilled man.
• the micro-organism is first grown m a fermentor to reach a predetermined cell density before the expression of the Asp-Phe dipeptide synthetase is switched on and feeding of the glucose, L-Asp and/or L- Phe for the synthesis of the Asp-Phe dipeptide is started.
• growth phase and Asp-Phe synthetase productio phase are preferably uncoupled.
• uncoupling can be achieved by expressing the gene for the Asp-Phe synthetase from an mducible, tightly regulable, promoter.
• the expression of the Asp-Phe dipeptide synthetase is preferably switched on by addition of a specific chemical component (mducer) or by changing the physical conditions, e.g.
• the expression is assumed to be switched-on as compared to the non- induced state, if the expression level of the Asp-Phe dipeptide synthetase is raised at least by a factor of 10.
• the micro-organism is an L- phenylalanme producing micro-organism and, apart from required amounts of salts and trace elements etc., only glucose and L-Asp are being fed.
• L-Phe producing microorganisms are well-known. For instance, E. coli and Corynebacterium species are being used for L-Phe production.
• L- Phe dipeptide synthetase m By expressing an Asp-Phe dipeptide synthetase m such micro-organisms availability of L- Phe in the micro-organism is provided for, and only glucose, an appropriate nitrogen source, organic growth factors, salts and trace elements, etc., as well as L- Asp, should be supplied as required.
• micro-organism used is an Escherichia or a Bacillus species.
• the micro- organism used is a strain with reduced protease activity for Asp-Phe or lacking such activity towards Asp-Phe. By using such strains degradation of Asp-Phe formed is prevented. Any suitable strain which lacks protease activity (either naturally or because the activity of the protease has been lowered substantially or has been removed completely) may be used.
• the micro-organism used also contains a suitable export system for Asp-Phe formed and/or one or more suitable uptake system(s) for glucose and/or L-Asp and/or L-Phe.
• a suitable export system will ensure achieving more efficient secretion of the Asp- Phe formed.
• the secretion meant here is the secretion of Asp-Phe formed m the micro-organism into the extracellular environment .
• Efficient secretion of Asp- Phe is important for improving the recovery yield of Asp-Phe and for maintaining the activity of the Asp-Phe dipeptide synthetase at a suitable level as well as for preventing intracellular degradation of Asp-Phe.
• the down-stream processing for Asp-Phe secreted is more easy.
• m vi tro systems are characterised that the Asp-Phe dipeptide synthetase is not present living cell material; it may, however, be present any other environment, for instance m permeabilised cells, cell-free extract, or as an isolated dipeptide synthetase. In such case regeneration of ATP does not take place m living cell material used for the synthesis of Asp-Phe, and special measures for supply of ATP m an effective amount have to be taken.
• the production of Asp-Phe is carried out m vi tro m an enzyme reactor, while ATP is supplied, and L-Asp and/or L-Phe are being fed, and the Asp-Phe formed is recovered.
• the supply of ATP is provided part by an m situ ATP-regenerating system.
• ATP regenerating systems which the literature are also being referred to as ATP generating systems
• ATP regenerating systems both whole cell systems (e.g. yeast glycolysis systems) or isolated ATP regenerating enzymes, for instance adenylate kinase combined with acetate kinase, may be used.
• a very elegant ATP regeneration system has been described by T. Fu]io et al . (Biosci., Biotechnol . , Biochem. 61, 1997, p.840- 845) . They have shown the use of permeabilised
• Corynebacterium a momagenes cells for regeneration of ATP from the corresponding monophosphate (AMP) coupled to an ATP-requirmg reaction m permeabilised E. coll cells.
• AMP monophosphate
• m permeabilised E. coll cells.
• glucose can be supplied as an energy source instead of most cf the ATP.
• the ATP-regenerating system is preferably present in a permeabilised micro-organism.
• This permeabilised micro-organism present in the (enzyme) reactor used ensures that an effective amount of adenosine- triphosphate (ATP) will always be present and available during the Asp-Phe production according to the present invention.
• ATP adenosine- triphosphate
• DNA FRAGMENTS ENCODING AN Asp-Phe DIPEPTIDE SYNTHETASE, ETC.
• the present invention also relates to novel DNA fragments encoding an Asp-Phe dipeptide synthetase. These novel DNA fragments or combination of
• DNA fragments code for a non-ribosomal Asp-Phe dipeptide synthetase, which synthetase comprises two minimal modules connected by one condensation domain wherein the N-termmal module of these modules is recognising L-aspartic acid and the C-termmal module of these modules is recognising L-phenylalamne and is covalently bound at its N-termmal end to the condensation domain, and wherein each of these minimal modules is composed of an adenylation domain and a 4 ' - phosphopantetheinyl cofactor containing thiolation domain.
• DNA- fragment or combination of DNA fragments as used herein is understood to have its broadest possible meaning.
• the term first of all relates to the composite biological material (on one or more
• DNA-fragments as mentioned herein-above and coding for the minimal modules for Asp and Phe the correct order and for the condensation domain, each coding sequence being surrounded by any transcription and translation control sequences (e.g. promoters, transcription terminators) and the like which may be suitable for the expression of the Asp-Phe dipeptide synthesis g activity.
• the control sequences may be homologous or heterologous, and the promoter (s) present m the DNA may be constitutive or ducible.
• DNA-fragment as used herein is further understood to code, m addition to coding for the Asp and Phe minimal modules and the condensation domain, for the activities of the other domains, e.g. TE-domams. Furthermore, these fragments may code"'*-? ⁇ * activities which are not located on the Asp-Phe dipeptide synthetase polypeptide itself, sucn as non- mtegrated thioesterase Type-II-like proteins, and other activities co-operating concertedly with the Asp and Phe minimal modules .
• DNA- ragment as used herein is also understood to comprise gene structures comprising DNA fragments as described herein-above. More precisely, a gene structure is to be understood as being a gene and any other nucleotide sequence which carries the DNA-fragments according to the invention. Appropriate nucleotide sequences can, for example, be plasmids, vectors, chromosomes, or phages .
• the gene structures may exist either as (part of) an autonomously replicating vector in single or multicopy situation, or integrated into the chromosome m single or multicopy situation.
• the gene structure is also to be understood as being a combination of the above-mentioned gene carriers, such as vectors, chromosomes and phages, on which the DNA- fragments according to the invention are distributed.
• the Asp-Phe dipeptide synthetase encoding DNA- fragment can be introduced into the cell on a vector and the non-integrated thioesterase Type-II-like protein encoding DNA-fragment can be inserted into the chromosome.
• a further DNA-fragment can, for example, be introduced into the cell using a phage .
• the DNA-fragments according to the invention may be introduced into the micro-organism at a sufficiently high copy number, for instance of up to 50 copies.
• a sufficiently high copy number for instance of up to 50 copies.
• Specificity of a module means that the module has a certain preference m recognising one ammo acid above other ammo acids or above another ammo acid.
• homologous recombination events are used to bring about the desired changes m the genomic DNA m the native peptide producing micro-organism. Because the homologous recombination events take place m the native peptide producing micro-organism, these methods would have the advantage that ail the native nost enzymes and relevant regulatory elements are present principle .
• homologous recombination through use of the native non-ribosomal peptide producer suffers from several severe drawbacks . The most serious of these drawbacks is that it is often tedious and technically difficult, especially when applied to slow- growing micro-organisms with poorly developed transformation systems or which are lacking m other genetic tools.
• Asp-Phe dipeptide synthetase according to the invention and use thereof for the industrial production of Asp- Phe.
• Asp-Phe dipeptide synthetase can be readily obtained by use of m vi tro engineering techniques. So far no in vi tro engineering techniques for the construction of peptide synthetases have been described. Detailed protocols for the construction of Asp-Phe dipeptide synthetases according to the invention can be found m the experimental part of this application.
• the Leu minimal module encoding DNA-fragment thereof then was replaced by a DNA-fragment (obtained by PCR method) from the Bacillus brevis ATCC 8185 tyrocidme A synthetase gene coding for a Phe minimal module ( tycA) . Then an integral TE-domam was added to the C-termmal end of the Asp-Phe encoding DNA- fragment by replacement of the thiolation domain of the Phe module by a PCR-fragment coding for the srfA-C thiolation and TE domain.
• This construction was done m such a way that the DNA encoding the additional TE domain was fused in-frame with the gene encoding the Asp-Phe synthetase.
• the TE-domam forms an integrated part of the Asp-Phe synthetase produced.
• this TE-domam containing Asp-Phe synthetase will be referred to as Asp-Phe-TE.
• the encoding DNA- fragments were introduced into a suitable host microorganism.
• Suitable host micro-organisms are, for instance, E. coli and Bacillus species.
• cells were lysed and the synthetases produced were purified by IMAC (Immobilised Metal Affinity Chromatography) .
• IMAC Immobilised Metal Affinity Chromatography
• the condensation domain m the encoded dipeptide synthetase is connected to both minimal modules m such way that it is also covalently bound to the module recognising L-aspartic acid.
• DNA fragment or combination of DNA fragments encoding the dipeptide synthetase also code for a releasing factor for the Asp-Phe formed on that dipeptide synthetase.
• releasing factor is used in the same meaning as it has been used m the previous part of the specification.
• the DNA fragment or combination of DNA fragments encoding the Asp-Phe dipeptide synthetase is/are also coding for a protein which shows thioesterase-like functions and forms an integrated domain of the dipeptide synthetase at the C- termmus thereof.
• the synthetase encoding DNA fragment or combination of DNA fragments preferably also express (es) one or more post-translational modifying activities for efficient non-ribosomal synthesis of Asp-Phe on the synthetase.
• post-translational modifying activities etc. are used in the same meaning as they have been used m the previous part of the specification.
• the post-translational modifying activity expressed by the DNA fragment or combination of DNA fragments is a 4 ' - phosphopantethemyl (4 ' -PP) transferase activity.
• the formation of this activity provides for effective conversion of apo- to holo-enzyme. Effective conversion of apo- to holo-enzyme, etc. already has been explained m the previous part of the specification.
• DNA fragment or combination of DNA fragments also code(s) for a non- integrated protein with thioesterase Type-II- like activity.
• non-mtegrated protein with thioesterase Type-II-like activity is used the same meaning as it has been used the previous part of the specification.
• the invention further relates to microorganisms containing a DNA fragment or combination of DNA fragments according to the invention, and in particular to such micro-organisms which are capable of producing L-Asp and/or L-Phe.
• the microorganism is an Escherichia coli or Bacillus species.
• the present invention finally also relates to novel Asp-Phe dipeptide synthetases.
• the terms and expressions used hereinafter with respect to the Asp-Phe dipeptide synthetase all have the same meaning as explained herein-above.
• the non-ribosomal Asp-Phe dipeptide synthetases according to tne present invention are characterised m that they comprise two minimal modules connected by one condensation domain wherein the N- terminal module of these modules is recognising L- aspartic acid and the C-termmal module of these modules is recognising L-phenylalanme and is covalently bound at its N-termmal end to the condensation domain, and wherein each of these minimal modules is composed of an adenylation domain and a 4 ' - phosphopantethemyl cofactor containing thiolation domain.
• condensation domain in the dipeptide synthetases is connected to both minimal modules m such way that it is also covalently bound to the module recognising L-aspartic acid.
• the Asp-Phe dipeptide synthetase also comprises a releasing factor for the Asp-Phe formed on that dipeptide synthetase.
• the releasing factor is a protein which shows thioesterase-like functions and forms an integrated domain of the dipeptide synthetase at its C-termmus.
• a 4934 bp fragment comprising regions from the srfB locus from chromosomal Bacillus subtilis ATCC 21332 DNA was amplified (PCR) using the following primers:
• the fragment (20 ⁇ g) was digested with 1 unit of the enzymes BairiHI / Sphl (37°C, 16 h) to generate terminal restriction sites.
• Plasmid pQE70 (provided by Qiagen, D-Hilden)
• Plasmid pasp-phe-His 6 was constructed from plasmid pasp-leu-His 6 as follows. A 1894 bp chromosomal DNA-fragment from
• Bacillus brevis ATCC 8185 DNA was amplified (PCR) using the following primers:
• the fragment was digested with 1 unit of enzyme BairiHI and incubated at 30°C for 4 hours.
• Plasmid pasp-leu-His ⁇ was digested m the same way and subsequently incubated for 1 hour with 1 unit of Alkaline phosphatase.
• the vector portion (ca. 6,5 kb) was separated from other DNA fragments by agarose gel electrophoresis and repu ⁇ fied. Complete digestion was confirmed as before with linearised pasp- leu-H ⁇ s s .
• the two fragments were ligated in a equimolar ratio for 5 hours at 16°C using 1 unit of T4-l ⁇ gase enzyme. 1 ⁇ L of the ligation mixture was used for electroporation of E. coli XL1 blue competent cells.
• Transformants were selected on 2x YT agar containing Ampicillin (100 ⁇ g/mL) . Analysis of transformants revealed that 1 out of 90 clones had inserted a fragment of ca. 2000 bp . Correct insertion was confirmed using restriction enzyme digestion analysis and terminal sequencing of the insert.
• the correct clone was designated pasp-phe- His 6 .
• the peptide synthetase encoding gene on plasmid pasp-leu -His e is a hybrid gene obtained by exchanging the DNA-fragment coding for the second (Leu) minimal module (A- and T-domam) , for a DNA-fragment coding for a Phe minimal module.
• Plasmid pasp-phe-TE-H ⁇ s 6 was constructed from plasmid pasp -phe- His 6 .
• a 910 bp chromosomal DNA-fragment from Bacillus subtilis ATCC 21332 DNA was amplified (PCR) using the following primers : 5' ATA ATC GAT AAT CGC ACA AAT ATG GTC (5' TE-srfCl-
• the fragment was digested with 1 unit of enzyme Clal for 4 hours at 37°C, before adjusting buffer conditions and digesting with 1 unit of enzyme Bgll l (4 hours, 37°C) .
• Plasmid pas -phe- His 6 was digested with enzyme Clal (4 h, 37°C) and subsequently with Ba-riHI (4 h, 37°C) before the linearised plasmid was incubated for one hour with 1 unit of Alkaline phosphatase.
• the vector portion (ca. 8 kb) was separated from other DNA-fragments by agarose gel electrophoresis and repu ⁇ fied.
• the peptide synthetase encoding gene on plasmid pasp-phe-H ⁇ s 6 contains a second fusion site located between the DNA coding for the adenylation domain and thiolation domain of the second (Phe) minimal module.
• the C-termmal T-TE domains resemble the native C-terminus of the Surfactm synthetase srfC .
• Plasmid pgsp which is cased on plasmid pREP4 (obtained from Qiagen, D-Hilden) , contains the gsp gene (the 4 ' -PP transferase gene from the Gramicidin S-biosyntnesis operon from Bacillus brevis ATCC 9999) under control of the T7 promoter.
• Transformants were selected on 2x YT agar plates containing Ampicillin (100 ⁇ g/mL ) and Kanamyc (25 ⁇ g/mL) .
• Several colonies were used to inoculate 4 mL of 2x YT liquid medium (containing m addition 10 mM MgCl 2 ) and incubated at 37°C for 16 hours. These 4 mL cultures were subsequently used to inoculate 400 mL of the same medium.
• Cells were grown at 30°C m a waterbath shaker (250 rpm) . After 3-4 hours the cells reached an optical density of 0,7 (OD 6 -o nm ) and were induced by the addition of 200 ⁇ M IPTG. Cells were incubated for an additional 1,5 hours before being harvested.
• Fractions containing the recombinant proteins were detected using the Bradford reagent, by the absorption at 595 nm. These fractions were pooled and dialysed against a buffer containing 50 mM HEPES, 100 mM NaCl, 10 mM MgCl 2 and 5 mM DTE for 16 hours. After dialysation protein concentrations were again determined.
• ammo acid activation was determined indirectly by incorporation of labelled 32 PP X into ATP during reverse reaction (Lee, S.G. & Lipmann, F.; Tyrocidme synthetase system; Methods Enzymol . 43, 1975, p.585-602) .
• 20 pmol of each enzyme was incubated with 1 mM ammo acid, 1 mM ATP, 0,1 mM PP X , 50 mM HEPES, 100 mM NaCl and 10 mM MgCl 2 and 2 mCi 32 PP 1 at 37°C a total volume of 100 ⁇ L .
• Reactions were quenched after 10 mm by adding 500 ⁇ L of a solution containing 100 mM NaPP x , 560 mM perchloric acid and 1,2% (w/v) active charcoal. The mixture was centrifuged at 13000 rpm for 1 mm. The pellet was washed and resuspended twice with 1 mL H 2 0.
• ammo acid activation patterns of Asp- Phe-H ⁇ s s and Asp-Phe-TE-H ⁇ s s were found to be identical.
• Quantity of holo-enzyme formation was determined by measuring the amount of labelled Asp, Leu and Phe, that could be covalently oound to one equivalent of the purified proteins Asp-Leu-H ⁇ s s , Asp- Rhe-His s and Asp-Phe-TE-His s . (Lee, S.G ; see aoovej .
• the precipitated protein was collected by centrifugation, washed once with 500 ⁇ l of 10% aqueous TCA, then with 1 ml of a 3 : 1 (v/v) mixture of diethyl ether and ethanol, and finally with 1 ml of diethyl ether.
• the pellet was dried for 15 minutes at 37 °C, before being resuspended in 200 ⁇ l of 100 mM of an aqueous solution of potassium hydroxide under vigorous shaking.
• the mixture then was treated for 0.5 hours at 70 °C to release any thioester bound dipeptide from the enzyme.
• the solution was replenished to 1 ml with methanol. This solution was centrifuged for 30 minutes at 4 °C and the supernatant was evaporated in vacuo for 3 hours at room temperature.
• the obtained pellet was finally resuspended in 25 ⁇ l of 10% aqueous methanol .
• Phe m a buffer 50 mM HEPES, 20 mM MgCl 2 , pH 8) for 6 hours at 37 °C.
• 100 ⁇ l of n- butanol were added to precipitate the enzyme which then was removed.
• the remaining clear solution was evaporated m vacuo for 3 hours and replenished to a volume of 20 ⁇ l with 10 vol% of methanol m water.
• this volume was diluted lOx (again with 10 voi% metnanol m water) and portions thereof then were subjected to HPLC followed by photometric detection (as shown below) and to Electrospray Ionisation LC-MS analysis. Reference samples of Asp-Phe syntnesised chemically were used for comparison.
• HPLC with photometric detection a 50 ⁇ l portion of the lOx diluted sample was injected on a Chromsep Inertsil 5 ODS-3 column (250 x 3 mm; particle size 5 ⁇ ) .
• Eluents A (0.05 M aq.
• Detection was done photometrically (at 210 nm and 257 nm, with quantification at 210 nm) .
• Detection was done by total ion current (TIC) using electrospray ionisation m the positive ion mode as ionisation technique.
• the scan range was 120-300 amu with a dwell time of 1 msec.
• the retention time for Asp-Phe was 23.7 minutes (both for the sample and the reference compound) .
• Mass spectroscopy confirmed the same molecular weight (280 g/mol) for both the Asp-Phe from the sample and the reference compound, and also identical fragmentation patterns were observed. According to this technique the amount of Asp-Phe the sample was calculated to be about 20 mg/1.

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