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Patent 2365594 Summary

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(12) Patent Application: (11) CA 2365594
(54) English Title: MICROBIOLOGICAL PRODUCTION METHOD FOR .ALPHA.-L-ASPARTYL-L-PHENYLALANINE
(54) French Title: METHODE DE PRODUCTION MICROBIOLOGIQUE D'.ALPHA.-L-ASPARTYL-L-PHENYLALANINE
Status: Deemed Abandoned and Beyond the Period of Reinstatement - Pending Response to Notice of Disregarded Communication
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/52 (2006.01)
  • C07K 5/06 (2006.01)
  • C12N 1/21 (2006.01)
  • C12N 9/00 (2006.01)
  • C12N 15/09 (2006.01)
  • C12N 15/62 (2006.01)
  • C12P 21/02 (2006.01)
(72) Inventors :
  • DOEKEL, SASHA (Germany)
  • MARAHIEL, MOHAMED ABDALLA (Germany)
  • QUAEDFLIEG, PETER JAN LEONARD MARIO
  • SONKE, THEODORUS
(73) Owners :
  • HOLLAND SWEETENER COMPANY V.O.F.
(71) Applicants :
  • HOLLAND SWEETENER COMPANY V.O.F.
(74) Agent: SMART & BIGGAR LP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2000-03-28
(87) Open to Public Inspection: 2000-10-05
Availability of licence: N/A
Dedicated to the Public: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/NL2000/000206
(87) International Publication Number: WO 2000058478
(85) National Entry: 2001-09-27

(30) Application Priority Data:
Application No. Country/Territory Date
99200954.8 (European Patent Office (EPO)) 1999-03-29
99203518.8 (European Patent Office (EPO)) 1999-10-26

Abstracts

English Abstract


The present invention relates to a new, microbiological method for the
production of .alpha.-L-aspartyl-L-phenylalanine (Asp-Phe) from the substrates
L-aspartic acid (L-Asp) and L-phenylalanine (L-Phe) wherein the substrates are
contacted, in the presence of ATP, with a non-ribosomal dipeptide synthetase
comprising two minimal modules connected by one condensation domain wherein
the N- resp. C-terminal modules are recognising L-Asp and L-Phe, respectively,
and the latter module is covalently bound at its N- terminal 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, and that the Asp-Phe formed is recovered. 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.


French Abstract

La présente invention concerne une nouvelle méthode microbiologique de production d'.alpha.-L-aspartyl-L-phénylalanine (Asp-Phe) à partir des substrats acide L-aspartique (L-Asp) et L-phénylalanine (L-Phe) dans laquelle les substrats sont mis en contact, en la présence d'ATP, avec une synthétase dipeptidique non ribosomique contenant deux modules minimaux reliés par un domaine de condensation dans lequel les modules C-terminaux N-resp reconnaissent L-Asp et L-Phe respectivement, et le dernier module est lié de manière covalente au niveau de son extrémité N-terminale au domaine de condensation, chacun de ces modules minimaux étant composés d'un domaine d'adénylation et d'un domaine de thiolation contenant un cofacteur 4'-phosphopantétheinyle, et le Asp-Phe formé est récupéré. La présente invention concerne également de nouveaux fragments d'ADN ou une combinaison de fragemts d'ADN codant une nouvelle synthétase dipeptidique Asp-Phe, des micro-organismes contenant ces fragments d'ADN, ainsi que les nouvelles synthétases dipeptidiques Asp-Phe elles-mêmes.

Claims

Note: Claims are shown in the official language in which they were submitted.


CLAIMS
1. Method for the microbiological production of .alpha.-L-
aspartyl-L-phenylalanine (Asp-Phe) from the substrates
L-aspartic acid (L-Asp) and L-phenylalanine (L-Phe)
characterised in that the substrates are contacted, in
the presence of an effective amount of adenosine-
triphosphate (ATP), with a non-ribosomal dipeptide
synthetase comprising two minimal modules connected by
one condensation domain wherein the N-terminal module
of these modules is recognising L-aspartic acid and
the C-terminal module of these modules is recognising
L-phenylalanine and is covalently bound at its N-
terminal 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, and that the .alpha.-
L-aspartyl-L-phenylalanine (Asp-Phe) formed is
recovered.
2. Method for the production of Asp-Phe according to
claim 1, characterised in that the condensation domain
in the dipeptide synthetase is connected to both
minimal modules in such way that it is also covalently
bound to the module recognising L-aspartic acid.
3. Method for the production of Asp-Phe according to
claim 1 or 2, characterised in that also a
thioesterase-like releasing factor is present for the
Asp-Phe formed on the dipeptide synthetase.

4. Method for the production of Asp-Phe according to any
of claims 1 to 3, characterised in that the
thioesterase-like releasing factor forms an integrated
domain of the dipeptide synthetase at the C-terminus
thereof.
5. Method for the production of Asp-Phe according to any
of claims 1 to 4, characterised in that also a non-
integrated protein with thioesterase Type-II-like
activity is present together with the dipeptide
synthetase.
6. Method for the production of Asp-Phe according to any
of claims 1 to 5, characterised in that the dipeptide
synthetase is present in living cell-material of a
micro-organism, and that glucose, L-Asp and/or L-Phe
are being fed to said fermentor, and that the Asp-Phe
formed is recovered.
7. Method for the production of Asp-Phe according to claim
6 characterised in that the micro-organism is first
grown in 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.
8. Method for the production of Asp-Phe according to claim
7, characterised in that the micro-organism is an L-
phenylalanine producing micro-organism and that only
glucose and L-Asp are being fed.
9. Method for the production of Asp-Phe according to claim
8, characterised in that the micro-organism is an
Escherichia or Bacillus species.

10. Method for the production of Asp-Phe according to any
of claims 6 to 9, characterised in that the micro-
organism used is a strain with reduced protease
activity for Asp-Phe or lacking such activity towards
Asp-Phe.
11. Method for the production of Asp-Phe according to any
of claims 1 to 5, characterised in that the production
of Asp-Phe is carried out in vitro in an enzyme
reactor, while ATP is supplied, and L-Asp and/or L-Phe
are being fed, and the Asp-Phe formed is recovered.
12. Method for the production of Asp-Phe according to claim
11, characterised in that the supply of ATP is
provided in part by an in situ ATP-regenerating
system.
13. Method for the production of Asp-Phe according to claim
12, characterised in that the ATP-regenerating system
is present in a permeabilised micro-organism.
14. A DNA fragment or a combination of DNA fragments coding
for a non-ribosomal Asp-Phe dipeptide synthetase,
which synthetase comprises two minimal modules
connected by one condensation domain wherein the N-
terminal module of these modules is recognising L-
aspartic acid and the C-terminal module of these
modules is recognising L-phenylalanine and is
covalently bound at its N-terminal 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.

15. A DNA fragment coding for an Asp-Phe dipeptide
synthetase according to claim 14, characterised in
that the condensation domain in the encoded dipeptide
synthetase is connected to both minimal modules in
such way that it is also covalently bound to the
module recognising L-aspartic acid.
16. A DNA fragment according to claim 14 or 15, or a
combination of DNA fragments according to claim 14,
characterised in that the DNA fragment or combination
of DNA fragments encoding the dipeptide synthetase
also code for a thioesterase-like releasing factor for
the Asp-Phe formed on that dipeptide synthetase.
17. A DNA fragment or a combination of DNA fragments
according to claim 16, characterised in that the
thioesterase-like releasing factor-forms an integrated
domain of the dipeptide synthetase at the C-terminus
thereof.
18. A DNA fragment or a combination of DNA fragments
according to any of claims 14 to 17, characterised in
that it/they also code for a non-integrated protein
with thioesterase Type-II-like activity.
19. A recombinant micro-organism containing a DNA fragment
or a combination of DNA fragments according to any of
claims 14-18.
20. A micro-organism according to claim 19 wherein the
micro-organism is capable of producing L-Asp and/or L-
Phe.
21. A micro-organism according to claim 20 wherein the
micro-organism is an Escherichia coli or Bacillus
species.

22. Asp-Phe dipeptide synthetase characterised in that it
comprises two minimal modules connected by one
condensation domain wherein the N-terminal module of
these modules is recognising L-aspartic acid and the
C-terminal module of these modules is recognising L-
phenylalanine and is covalently bound at its N-
terminal 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.
23. Asp-Phe dipeptide synthetase according to claim 22
characterised in that the condensation domain in the
dipeptide synthetase is connected to both minimal
modules in such way that it is also covalently bound
to the module recognising L-aspartic acid.
24. Asp-Phe dipeptide synthetase according to claim 22 or
23, characterised in that the dipeptide synthetase
also comprises a thioesterase-like releasing factor
for the Asp-Phe formed on that dipeptide synthetase.
25. Asp-Phe dipeptide synthetase according to claim 24,
characterised in that the thioesterase-like releasing
factor forms an integrated domain of the dipeptide
synthetase at its C-terminus.

Description

Note: Descriptions are shown in the official language in which they were submitted.


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MICROBIOLOGICAL PRODUCTION METHOD FOR
a-L-ASPARTYL-L-PHENYLALANINE
FIELD OF THE INVENTION
The present invention relates to a new,
microbiological, method for the production of a-L-
aspartyl-L-phenylalanine (Asp-Phe) from the substrates
L-aspartic acid (L-Asp) and L-phenylalanine (L-Phe).
The present invention also relates to novel DNA
fragments or combination of DNA fragments encoding a new
Asp-Phe dipeptide s~rnthetase, micro-organisms containing
such DNA fragments, as well as to the new Asp-Phe
dipeptide synthetases itself.
BACKGROUND OF THE INVENTION
a-L-Aspartyl-L-phenylalanine (hereinafter
also referred to as Asp-Phe) is an important dipeptide,
inter alia used for the production of a-L-aspartyl-L-
phenyialanine methyl ester (hereinafter also referred
to as APM). APM is known to be a high intensity
artificial sweetener, having a sweetness which is about
200x as potent as the sweetness of sucrose. The (3-form
of APM, as well as the stereoisomers of APM wherein one
or both of the amino acids are in the D-configuration,
do not have sweet properties. APM is used for the
sweetening of various edible materials.
Various production methods of APM exist;
present routes may be divided into chemical and
biochemical/microbiological (in particular, er_zymatic)

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routes. In the ways of producing APM by using known
peptide synthesis technia_ues tedious and expensive
processes have to be performed in order to achieve
selective a-L,L-coupling, involving intensive protecting
and deprotecting of a-amino, carboxyl and side chain
groups. Fermentative routes, on the other hand, in
general are cheap and intrinsically they display
enantio- and regioselectivity. Therefore, fermentative
routes have been considered to be promising alternatives
for the above-mentioned chemical and biochemical
synthesis routes. As can be seen from EP-A-0036258, it
has so far been deemed unsuited to produce the dipeptide
Asp-Phe in a micro-organism as part of the micro-
organism's own protein producing processes;
theoretically such production might be achieved by
inserting in the DNA of a micro-organism the nucleotide
base sequences GAC or GAT (being known to be a codon for
L-Asp) and TTT or TTC (being known to be a codon for L-
Phe), preceded and followed by appropriate processing or
termination codons in the correct reading frame, and
under appropriate control. It therefore has been
attempted in EP-A-0036258 to achieve the synthesis of
Asp-Phe indirectly through prior production of protein
segments of the formula (Asp-Phe)n, where n is a large
number; this has been done by inserting into a cloning
vehicle a synthesised DNA-fragment coding for such poly-
(Asp-Phe) protein. However, such ribosomal fermentative
route is still tedious and economically unattractive.
Major drawbacks are lying in the recovery of the Asp-Phe
dipeptide from the polypeptide. Similar drawbacks can be
attributed to a method, as described by Choi, S.-Y. et
al. in J. Microbiol. Biotechnol., 2, 1992, p.1-6,
wherein a polypeptide comprising segments of the
tripeptide sequence Asp-Phe-Lys is synthesised.

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Therefore still need exists for finding a direct
fermentative route to Asp-Phe. Direct fermentation of
Asp-Phe is hitherto unknown.
DESCRIPTION OF THE INVENTION
METHOD FOR THE PRODUCTION OF ASP-PHE:
Surprisingly, inventors now found a new, and
promising alternative microbiological method for the
production of a-L-aspartyl-L-phenylalanine (Asp-Phe)
from the substrates L-aspartic acid (L-Asp) and L-
phenylalanine (L-Phe) wherein the substrates are
contacted, in the presence of an effective amount of
adenosine-triphosphate (ATP), with a non-ribosomal
dipeptide synthetase comprising two minimal modules
connected by one condensation domain wherein the N-
terminal module of these modules is recognising L-
aspartic acid and the C-terminal module of these
modules is recognising L-phenylalanine and is
covalently bound at its N-terminal 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, and that the a-L-aspartyl-L-phenylalanine (Asp-
Phe) formed is recovered.
This new method thus provides a
microbiological process for direct fermentation of Asp-
Phe, which in 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-ribosomal synthesis of this
dipeptide. The inventors thus have provided a direct
microbiological method for producing the dipeptide Asp-
Phe without the need for any protecting and deprotecting

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steps.
The novel ncn-ribosomal dipeptide
synthetases which, acccrding 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 "Bacillus subtilis and other
Gram-positive bacteria", Sonenshein et al. (Eds.), Am.
Soc. Microbiol., Washingtor~, DC, 1993, p.897-916) that
micro-organisms can produce bioactive peptides through
ribosomal and non-ribosomal mechanisms. The bioactive
peptides so far known to be synthesised non-
ribosomally, 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 in medicine,
agriculture, and biological research. Already more than
300 different residues so far have been found to be
incorporated into these peptide secondary metabolites.
However, until now not a single non-ribosomally formed
peptide has been identified having (as a part of its
peptide sequence) the dipeptide Asp-Phe in it, nor has
the dipeptide Asp-Phe itself been identified as a non-
ribosomally synthesised product.
According to the present invention Asp-Phe
can now be produced nor.-ribosomally, and novel non-
ribosomal Asp-Phe synthetases can be used for the
synthesis of Asp-Phe. Hereinafter, in the part of the
specification dealing witr. the DNA-fragments encoding

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the novel Asp-Phe synthetases, it will be elucidated in
more detail how these novel Asp-Phe synthetases can be
obtained and have been made available in the context of
the present invention. For better understanding of the
present invention, first, however, some general
background as to non-ribosomal peptide synthesis is
presented.
In non-ribosomal synthesis of peptides
known so far generally a multiple carrier thiotemplate
mechanism is involved (T. Stein et al., J. Biol. Chem.
271, 1996, p.15428-15435). According to this model,
peptide bond formation takes place on multi-enzyme
complexes which are named peptide synthetases and which
comprise a sequence of amino acid recognising modules.
On the peptide synthetases a series of enzymatic
reactions take place which ultimately lead to the
formation of a peptide by sequential building-in of
amino acids, in an order predetermined by the order of
modules recognising the cognate amino acids, into the
peptide. This series of enzymatic reactions includes,
schematically:
1. recognition of the amino acid substrates;
2. activation of said recognised amino acid
substrates to their aminoacyl-adenylates (that is,
the aminoacyl adenosine-monophosphate; aa-AMP) at
the expense of Mgz+-ATP (adenylation);
3. binding of the aminoacyl-adenylates in the form of
their more stable thioesters to the cysteamine
group of the enzyme-bound 4'-phosphopantetheinyl
(4'-PP) cofactors (thiolation). The ATP consumed
in the adenylation reaction is hereby released in
the monophosphate form (AMP);
4. depending of the peptide to be synthesised non-
ribosomally, the thiol-activated substrates may be

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modified (e. g. by epimerisation or N-methylation);
5. formation of the peptide product by N to C
stepwise integration of the thioesterified
substrate amino acids (modified, as the case may
S be) into the growing peptide;
6. releasing the peptide formed non-ribosomally from
the template.
Assuming this general scheme also to be
correct for the novel non-ribosomal synthesis of Asp-
Phe according to the present invention, this means that
this synthesis involves the subsequent steps of (i)
recognition of L-Asp and L-Phe, (ii) formation of an L-
Asp- and an L-Phe-acyladenylate, (iii) binding thereof
to the cysteamine group of the 4'-PP cofactor in the
respective thiolation domains, (iv) formation of the
Asp-Phe dipeptide by transfer of the thioester-
activated carboxyl group of L-Asp to the amino group of
L-Phe, while the condensation product remains
covalently attached to the multi-enzyme complex via the
4~-PP cofactor in the thiolation domain of the Phe-
recognising module, and (v) release of the Asp-Phe
formed.
According to the present invention 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 . a . a turn-over number (k~at) of 1 per
minute; preferably k~at is at least 10 per minute. In
order to enable an economically attractive process the
ATP consumed by the peptide synthesis reaction is
preferably regenerated.

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The contacting of the substrates L-Asp and
L-Phe with the non-ribosomal Asp-Phe dipeptide
synthetase may be done in any suitable way; for
instance - if the Asp-Phe dipeptide synthetase is
present in a micro-organism - L-Asp and L-Phe may be
fed into the culture medium containing said micro-
organism. Alternatively 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 amino acid (L-Asp or L-Phe) which
is not produced by the micro-organism. All these
methods may be called in vivo methods. ATP may be
regenerated in vivo in the Asp-Phe producing micro-
organism.
The contacting of the substrates L-Asp and
L-Phe with the non-ribosomal Asp-Phe dipeptide
synthetase also may be done by using the synthetase in
its isolated form, that is by an in vitro method. In
such in vitro methods ATP-regeneration is to be taken
care of separately. This may be done by applying an
ATP-regeneration system. ATP-regeneration systems are
readily available to the skilled man.
Protein chemical studies and recent
progress in cloning and sequencing of genes encoding
peptide synthetases of bacterial and fungal origin have
made it clear that the known peptide synthetases have a
highly conserved and ordered structure composed of so-
called modules. These 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 in concert, in
a template-based mode of action to achieve peptide

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elongation.
In general, the modules of peptide
synthetases, each module being about 1000-1400 amino
acids in length (i.e., the modules have molecular
weights in the range of 120-160 kDa), 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
l0 condensation (i.e. peptide bond formation). Two of such
distinct domains, the adenylation and thiolation
domains (A-domain and T-domain), together form the
smallest part of a module that retains all catalytic
activities for specific activation and covalent binding
of the amino 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).
The term "minimal module" as used here
therefore, according to said definition, refers to such
combined core fragment of the modules, consisting of an
adenylation domain and a thiolation domain.
Some highly conserved core motifs of
adenylation and thiolation domains, as known to exist
in peptide synthetases, are listed in table 1, together
with some highly conserved core motifs of condensation
and thioesterase domains (which will be addressed in
more detail in later parts of this specification).
The so-called "adenylation domain" (A-
domain, about 550 amino acids) is an essential region
of each module. The A-domain has been shown to bear the
substrate-recognition and ATP-binding sites and is
therefore solely responsible for activation of the
recognised amino acid as its acyl adenylate through ATP

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hydrolysis (T. Stachelhaus et al., J. Biol. Chem. 270,
1995, p.6163-6169).
Table ~ Highly conserved core motifs of catalytic
domains of known peptide synthetases
Source: M. Marahiel et al., Chem.Rev. 97, 1997, p.2651-
2673
Domain Cores) Consensus sequence
Note: Former
nomenclature
is given in
brackets
Adenylation A1 L(TS)YxEL
A2 (core 1) LKAGxAYL(VL)P(LI)D
A3 (core 2) LAYxxYTSG(ST)TGxPKG
A4 FDxS
A5 NxYGPTE
A6 (core 3) GELxIxGxG(VL)ARGYL
A7 (core 4 ) Y (RK) TGDL
AS (core 5) GRxDxQVKIRGxRIELGEIE
A9 LpxYM ( I V ) P
A10 NGK(VL)DR
Thiolation T (core 6) DxFFxxLGG(HD)S(LI)
Condensation C1 SxAQxR(LM)(WY)xL
C2 RHExLRTxF
C3 (His) MHHxISDG(WV)S
C4 YxD(FY)AVW
C5 (IV) GxFVNT (QL) (CA)
xR
C6 (HN) QD (YV) PFE
C7 RDxSRNPL
Thioesterase TE G(HY)SxG
I
Very recently the first 3D structure of an
adenylation domain of a peptide synthetase (PheA from
GrsA) has been reported (E. Conti et al., EMBO J. 16,

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1997, p.4174-4183). This structure shows that almost
all highly conserved core motifs are positioned around
the active site where the substrates are bound. The
main residues involved in building the substrate-
binding pocket could also be assigned; they were found
to be located between core motifs A3 and A6 and were
not highly conserved.
The A-domain 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 in recognising one amino acid above
other amino acids or above another amino acid. Of
course, also the concentration of each individual amino
acid present near the module may play a role. If, for
instance, the concentration of a specific amino acid is
much higher than that of (most of) the other amino
acids, the requirements for specificity may be somewhat
less strict.
The so-called "thiolation domain" (T-
domain, about 100 amino acids; also called peptidyl
carrier protein (PCP)) is a domain located directly
downstream of the adenylation domain. It forms an
integral part of peptide synthetases, and is the site
of 4'-PP cofactor binding and substrate acylation.
within the T-domain, 4'-PP is covalently bound to the
sidechain of an invariant serine residue located within
the highly conserved thiolation core motif (see
table 1). If the T-domain in a module of the peptide
synthetase does not carry its 4'-PP cofactor, no
covalent binding of the aminoacyl substrate can take
place and chain elongation will be impossible.
It has been found that in peptide
synthetases known so far, every T-domain is converted
from the inactive apo form to the active bolo form by

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transfer of the 4'-PP moiety from Coenzyme A (CoA) to
the sidechain of the above mentioned serine residue.
This post-translational priming of each T-domain is
mediated by peptide synthetase specific members of a
recently discovered enzyme superfamily, the 4'-
phosphopantetheinyl transferases. They utilise CoA as a
common substrate, and appear to attain specificity
through protein/protein interactions.
The Asp-Phe dipeptide synthetase as used in
the method of the present invention comprises two
minimal modules, respectively one minimal module at its
N-terminal side recognising L-Asp and another minimal
module at its C-terminal side recognising L-Phe. The
term "minimal module" is used in 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-domain) and a thiolation
domain (T-domain).
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,
however, 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.
In known peptide synthetases the
condensation domains occur as a moderately conserved

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region. The condensation domain (conserved regicn)
usually consists of about 400 amino acid residues and
is k~.own to be involved in the catalysis of non-
ribosomal peptide formation. One of the conserved core
S motifs contains the catalytically active histidine
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.
It is preferred, that the condensation
domain in the dipeptide synthetase is connected to both
minimal modules in such way that it is also covalently
bound to the module recognising L-Asp. In such case the
condensation domain is not only bound covalently to the
N-terminal end of the L-Phe recognising module, but
also to the C-terminal 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-domain of
the C-terminal module as a peptidyl-(4~-PP)-T-domain
intermediate. Release of the peptide from this
intermediate is assumed to take place either by
intermolecular attack by water, resulting in net
hydrolysis, or by intramolecular capture by a hydroxyl
or amino group of the peptide chain itself, giving rise
to a cyclic peptide product and of the peptide
synthetase in the bolo form. The first termination
route yields a linear peptide with a free C-terminal
carboxylic group (as should be the case for the Asn-Phe
synthesis according to the present invention).
Because the Asp-Phe is present in an

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intermediate form bound to the template of the 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 in 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 amino acid
into the growing peptide chain show a region with a
thioesterase-like function. These regions of
approximately 250 amino acids are located at the C-
terminal end of the amino 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-domain). All these integrated
TE-domains contain an active site serine residue, which
is part of the core motif GxSxG (see table 1).
Recent work has given support for the
2S theory that these integrated TE-domains are involved in
the termination of the chain elongation and the product
release. For instance, deletion of the complete TE-
domain from the surfactin synthase led to a 97%
reduction of the in vivo surfactin production
(Schneider A., et al., Arch. Microbiol., 169, 1998,
p.404-410). Furthermore, it has been shown that
replacing the integrated TE-domain from the C-terminta
of module 7 of the surfactin synthase to the C-terminal
ends of modules 4 and 5, resulted in the formation of

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the corresponding lipotetra- ar~d pentapeptide. Also in
this study the removal of the integrated TE-domain led
to an almost complete reduction of peptide synthesis
(Ferra F de, et al., J. Biol. Chem., 272, 1997,
p.25304-25309).
In a particularly preferred embodiment of
the invention the releasing factor therefore is a
protein which shows thioesterase-like functions and
forms an integrated domain of the dipeptide synthetase
at the C-terminus thereof.
In addition it is preferred that 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.
The term "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
synthesising function.
In particular, in the production of Asp-Phe
according to the present invention the post-
translational modifying activity used is a 4'-
phosphopantetheinyl (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 serine side-chains in
the core motif of the T-domains, and thereby increases
the yield of Asp-Phe in the production thereof.
Effective conversicn of apo- to bolo-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

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to the holo-form.
it is particularly preferred that in the
production of Asp-Phe according to the invention also a
non-integrated protein with thioesterase Type-II-like
activity is present together with the dipeptide
synthetase. As meant herein 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-domain).
Recent work (Schneider et al., Arch. Microbiol. (1998),
169, 404-410) has shown that deletion of a gene
encoding such non-integrated protein with thioesterase
Type-II-like activity from the surfactin synthase
operon leads to an 84% reduction of peptide production.
It is suggested that the non-integrated protein with
thioesterase Type-II-like activity enhances production
of non-ribosomal peptides, possibly by reactivation
through liberation of mischarged modules that are
blocked with an incorrect aminoacyl group or an
undesired acyl group at the 4'-PP cofactor.
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 amino
acid residues in 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.
In the production of Asp-Phe according to
the present invention the Asp-Phe dipeptide synthetase

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is preferably present in 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. As used in this context the term
"glucose" is intended to cover glucose and any other
energy source necessary for the regeneration of ATP in
the living cell material and for the maintainance
energy required for said living cell material. The
glucose (or other energy source), moreover, is used as
starting material for the production of any L-Asp
and/or L-Phe to be produced in the living cell in the
course of the process of the invention.
The skilled man, of course, will be aware
that the feeding of glucose, L-Asp and/or L-Phe is to
be done under appropriate conditions of temperature and
pH, including as required the presence of an
appropriate nitrogen source, salts, trace elements, and
other organic growth factors as vitamins and amino
acids, etc. to the fermentor or other type of (enzyme)
reactor which is used for the production of Asp-Phe.
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 in
any appropriate form as available to the skilled man.
For instance, whole cells may be used as such or in
immobilised form. The micro-organism may be any kind of
micro-organism wherein the Asp-Phe dipeptide
synthetases 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 Streptomyces species,
Bacillus species, Actinomyces species, Micrococcus
species, Nocardia species, or fungal species as

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Tolypocladium species, Fusarium species, Penicillium
species, Aspergillus species, and Cochlio.bolus species;
or
(b) are capable of producing amino acids, in particular
L-Asp and/or L-Phe, preferably on industrial scale, for
instance, Escherichia species, e.g. E.coli, and
Corynebacterium species, e.g. C.glutamicum.
The micro-organisms may be grown, under
conditions which can easily be found by the skilled
man, in a fermentor, and production of the Asp-Phe then
can be carried out in the same or in another fermentor.
As meant herein the fermentor may be any type of
fermentor or other types of (enzyme) reactor known to
the skilled man.
In the method for the production of Asp-Phe
according to the present invention it is preferred that
the micro-organism is first grown in 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.
The skilled man can easily determine the
growth of the micro-organism, e.g. by measuring its
optical density (O.D.), and find the most appropriate
level of cell density. To prevent any negative effect
on the growth of the micro-organism, growth phase and
Asp-Phe synthetase productio phase are preferably
uncoupled. Such uncoupling can be achieved by
expressing the gene for the Asp-Phe synthetase from an
inducible, tightly regulable, promoter. The expression
of the Asp-Phe dipeptide synthetase is preferably
switched on by addition of a specific chemical
component (induces) or by changing the physical

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conditions, e.g. the temperature, pH or dissolved
oxygen pressure, after a predetermined level of cell
density has been reached. 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.
Then also the feeding of substrates, etc.
in amounts as required, is started, and production of
Asp-Phe starts.
Most preferably the micro-organism is an L-
phenylalanine 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 micro-
organisms are well-known. For instance, E. coli and
Corynebacterium species are being used for L-Phe
production. By expressing an Asp-Phe dipeptide
synthetase in 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.
In particular the micro-organism used is an
Escherichia or a Bacillus species.
Best results are obtained if 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.
Moreover, if the synthesis of Asp-Phe
occurs in a micro-organism, also additional measures
can be taken for improving the permeation of the Asp-

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Phe formed into the reaction medium outside the micro-
organism and recovering to Asp-Phe therefrom after
separation of the micro-organism from the reaction
medium. Similarly, also additional measures may be
taken for improving the intake of glucose and/or L-Asp
and/or L-Phe.
In an even more preferred embodiment of the
invention the micro-organism used also contains a
suitable export system for Asp-Phe formed and/or one or
more suitable uptake systems) for glucose and/or L-Asp
and/or L-Phe. Using 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 in 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.
Moreover, the down-stream processing for Asp-Phe
secreted is more easy.
Similarly, the presence of suitable uptake
systems) will improve the coupling efficiency to Asp-
Phe.
In the foregoing paragraphs in vivo non-
ribosomal synthesis methods for Asp-Phe have been
described. They all are characterised in that living
cell material is used and ATP regeneration takes place
in this cell material. The present invention can also
be carried out in vitro. As used herein, in vitro
systems are characterised in that the Asp-Phe dipeptide
synthetase is not present in living cell material; it
may, however, be present in any other environment, for
instance in permeabilised cells, cell-free extract, or

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as an isolated dipeptide synthetase. In such case
regeneration of ATP does not take place in living cell
material used for the synthesis of Asp-Phe, and special
measures for supply of ATP in an effective amount have
to be taken.
In a preferred embodiment of the invention,
the production of Asp-Phe is carried out in vitro in an
enzyme reactor, while ATP is supplied, and L-Asp and/or
L-Phe are being fed, and the Asp-Phe formed is
recovered.
In order to improve the economic
feasibility of the process it is particularly preferred
to increase the yield of Asp-Phe per mole of ATP
supplied for the synthesis of Asp-Phe. This can be
achieved by in situ ATP regeneration from the AMP
formed out of the ATP in the consecutive adenylation
and thiolation reactions.
Therefore, in a preferred mode of the
invention, the supply of ATP is provided in part by an
in situ ATP-regenerating system.
Various ATP regenerating systems (which in
the literature are also being referred to as ATP
generating systems) are known to the skilled man. As
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. Fujio et
al. (Biosci., Biotechnol., Biochem. 61, 1997, p.840-
845). They have shown the use of permeabilised
Corynebacterium ammoniagenes cells for regeneration of
ATP from the corresponding monophosphate (AMP) coupled
to an ATP-requiring reaction in permeabilised E. coli
cells. In this elegant way (cheap) glucose can be

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supplied as an energy source instead cf most cf the
ATP.
Therefore, 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.

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DNA FRAGMENTS ENCODING AN Asp-Phe DIPEPTIDE SYNTHETASE,
ETC.
The present invention also relates to novel
DNA fragments encoding an Asp-Phe dipeptide synthetase.
S 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-terminal module of these modules is
recognising L-aspartic acid and the C-terminal module
of these modules is recognising L-phenylalanine and is
covalentlv bound at its N-terminal 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.
The term "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 in 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 synthesising
activity. The control sequences may be homologous or
heterologous, and the promoters) present in the DNA may
be constitutive or inducible.
The term "DNA-fragment" as used herein is
further understood to code, in addition to coding for
the Asp and Phe minimal modules and the condensation
domain, for the activities of the other domains, e.g.

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TE-domains. Furthermore, these fragments may code'~b~~'
activities which are not located on the Asp-Phe
dipeptide synthetase polypeptide itself, such as non-
integrated thioesterase Type-II-like proteins, and other
activities co-operating concertedly with the Asp and Phe
minimal modules.
The term "DNA-fragment" 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 in 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. For example, 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. In addition, a further
DNA-fragment can, for example, ire introduced into the
cell using a phage. These examples are not intended to
exclude other combinations of DNA-fragment
distributions from the invention. 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.

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A detailed discussion of the Asp-Phe
dipeptide synthetase and the two minimal modules
comprised therein already has been given in the
preceding parts of this patent application.
The construction of the DNA fragments
according to the present invention is not self-evident,
although terms like "module", "domains", etc.
misleadingly might suggest that much is known about the
functional boundaries thereof. Such detailed information
which would enable rational design of (mutant, non-
natural) peptide synthetases, however, is not yet
available. Nevertheless, recently various techniques for
construction of mutant peptide synthetases have been
described in literature. Methods for construction of
mutant peptide synthetases described in literature are:
De Ferra et al. (J. Biol. Chem., 272, 1997,
p.25304-25309) have described a method for producing
truncated peptides of a predicted sequence by
replacement of the integral TE-domain of the surfactin
synthetase from the C-terminal module to the C-terminal
end of different internal modules. This technique alone,
however, is not suitable for the construction of an Asp-
Phe dipeptide synthetase module because the natural
order of two consecutive Asp and Phe modules is not
known to exist in nature (neither at the N- or C-
terminal end of any natural synthetase, nor as an
internal sequence of any naturally occurring
synthetase).
Another in vivo technique described for
engineering peptide synthetases is the so-called
programmed alteration within the primary structure of a
peptide product. Basis for this method is the
replacement of one module by another on the genetic
level. This technique has been described in general by

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A. Schneider et al., Mol. Gen. Genet., 257, 1998,
p.308-318). In this way amino acid-activating minimal
modules could be exchanged successfully in vivo between
mufti-modular peptide synthetases of heterologous
S origin, and micro-organisms could be obtained which
indeed produce non-ribosomal peptides with a different
primary structure from the peptides produced without
such alteration.
If this technique would be applied for the
construction of an Asp-Phe dipeptide synthetase, in
principle two options would be available, each starting
from a dipeptide synthetase, namely having a sequence
of two modules comprising either (i) Asp and XXX, the
latter representing any other amino acid than Phe, or
(ii) YYY and Phe, the former representing any other
amino acid than Asp. In those dipeptide synthetases the
DNA coding for the XXX module should be replaced by DNA
coding for the Phe module, or the DNA coding for the
YYY module by DNA coding for the Asp module.
Other methods for construction of peptide
synthetases have been described in EP-A-0637630. In
said patent application a method is suggested whereby,
next to alteration of the substrate specificity by
substitution of (part of) modules, also modules can be
deleted or inserted into the synthetase chain.
"Specificity" of a module means that the module has a
certain preference in recognising one amino acid above
other amino acids or above another amino acid.
It is a distinctive feature of the above
peptidase engineering methods that homologous
recombination events are used to bring about the
desired changes in the genomic DNA in the native
peptide producing micro-organism. Because the
homologous recombination events take place in the

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native peptide producing micro-organism, these methods
would have the advantage that ail the native host
enzymes and relevant regulatory elements are present in
principle.
However, 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 in other
genetic tools. Other drawbacks are that the native non-
ribosomal peptide producing micro-organisms often do not
have a history of safe use on industrial scale, are no
production organisms for L-Phe and/or L-Asp, and have
unknown fermentation characteristics. Moreover, all
these methods result in cells having only a single copy
of the DNA-fragment coding for the desired peptide
synthetase. Therefore, none of these in vivo engineering
methods are suitable for the preparation of the novel
Asp-Phe dipeptide synthetase according to the invention
and use thereof for the industrial production of Asp-
Phe.
The present inventors now have found that
the Asp-Phe dipeptide synthetase can be readily obtained
by use of in vitro engineering techniques. So far no in
vitro 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 in
the experimental part of this application.
The Asp-Phe dipeptide synthetase encoding
DNA-fragment can be constructed in vitro from an Asp-XXX
or YYY-Phe (with XXX and YYY having the meaning as

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_ 27
described above) dipeptide synthetase encoding DNA-
fragment (or partial sequences for such synthetase
encoding fragments as occurring in a naturally existing
peptide synthetase). This was accomplished starting from
an Asp-Leu (Leu = leucine) dimodular peptide synthetase
encoding DNA-fragment which was obtained from the
Bacillus subtilis ATCC 21332 surfactin synthetase A gene
(srfA-B) by PCR method. 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 tyrocidine A synthetase gene coding for a Phe
minimal module (tycA). Then an integral TE-domain was
added to the C-terminal 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 in
such a way that the DNA encoding the additional TE
domain was fused in-frame with the gene encoding the
Asp-Phe synthetase. As a result the TE-domain forms an
integrated part of the Asp-Phe synthetase produced. In
the experimental. part of this application this TE-domain
containing Asp-Phe synthetase will be referred to as
Asp-Phe-TE.
After the construction, the encoding DNA-
fragments were introduced into a suitable host micro-
organism. Suitable host micro-organisms are, for
instance, E. coli and Bacillus species. After
cultivation of these micro-organisms under inducing
conditions, cells were lysed and the synthetases
produced were purified by IMAC (Immobilised Metal
Affinity Chromatography). The purified enzyme
preparations were used for d'_fferert experiments to
prove the formation of Asp-Phe.

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Preferred DNA fragments
The following preferred aspects of the DNA
fragments encoding the Asp-Phe dipeptide synthetase
according to the invention closely correspond to tre
aspects discussed in the previous parts of this
specification regarding the preferred methods for the
production of Asp-Phe.
For the DNA fragments encoding the Asp-Phe
dipeptide synthetase according to the invention it is
especially preferred that the condensation domain in
the encoded dipeptide synthetase is connected to both
minimal modules in such way that it is also covalently
bound to the module recognising L-aspartic acid.
In particular it is preferred that the 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.
The term "releasing factor" is used in the same meaning
as it has been used in the previous part of the
specification.
In a more particularly preferred embodiment
of the present invention, 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-
terminus thereof. For an explanation of the terms
"integrated domain" etc., reference is made to earlier
parts of the present application.
In addition, the synthetase encoding DNA
fragment or combination of DNA fragments preferably
also expresses) one or more post-translational
modifying activities for efficient non-ribosomal
synthesis of Asp-Phe on the synthetase. The terms

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"post-translational modifying activities", etc. are
used in the same meaning as they have been used in the
previous part of the specification.
In particular, the post-translational
modifying activity expressed by the DNA fragment or
combination of DNA fragments is a 4'-
phosphopantetheinyl (4'-PP) transferase activity. The
formation of this activity provides for effective
conversion of apo- to bolo-enzyme. Effective conversion
of apo- to holo-enzyme, etc. already has been explained
in the previous part of the specification.
It is particularly preferred that the DNA
fragment or combination of DNA fragments also codes)
for a non-integrated protein with thioesterase Type-II-
like activity. The term "non-integrated protein with
thioesterase Type-II-like activity" is used in the same
meaning as it has been used in the previous part of the
specification.
Micro-organisms
The invention further relates to micro-
organisms 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. In particular, the micro-
organism is an Escherichia coli or Bacillus species.
Asp-Phe dipeptide synthetases
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 meanir_g as
explained herein-above.

CA 02365594 2001-09-27
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- 30 -
The non-ribosomal Asp-Phe dipeptide
synthetases according to the present invention are
characterised in that they comprise two minimal modules
connected by one condensation domain wherein the N-
S terminal module of these modules is recognising L-
aspartic acid and the C-terminal module of these
modules is recognising L-phenylalanine and is
covalently bound at its N-terminal 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.
In particular, the condensation domain in
the dipeptide synthetases is connected to both minimal
modules in such way that it is also covalently bound to
the module recognising L-aspartic acid.
Preferably, the Asp-Phe dipeptide
synthetase also comprises a releasing factor for the
Asp-Phe formed on that dipeptide synthetase.
Most preferably, the releasing factor is a
protein which shows thioesterase-like functions and
forms an integrated domain of the dipeptide synthetase
at its C-terminus.
The invention hereinafter now will be
clarified further in the experimental part, but will in
no way be restricted to the experiments shown. Amino
acids used all were enantiomerically pure L-amino acids.
EXPERIMENTAL PART
General procedures
Standard molecular cloning techniques such
as DNA isolation, gel electrophoresis, enzymatic
restriction modifications of nucleic acids, E.coli

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- 31 -
transformation etc., were performed as described by
Sambrcok et al., 1989, "Molecular Cloning: a iabcratory
manual", Cold Spring Harbor Laboratories, Cold Spring
Harbor, New York and Innis et al., 1990, "PCR
S protocols, a guide to methods and applications"
Academic Press, San Diego. Synthetic oligo
deoxynucleotides were obtained from MWG-Biotech AG, D-
Ebersberg. DNA sequence analyses were performed on an
Applied Biosystems ABI 310 genetic analyzer, according
to supplier's instructions. Sequencing reactions were
carried out by the chain termination method with dye-
labelied dideoxy terminators from the PRISM ready
Reaction DyeDeoxy Terminator cycle sequencing kit with
AmpliTaq FS polymerase (Applied Biosystems).
Construction of plasmid gasp-1eu-His6
A 4934 by fragment comprising regions from
the srfB locus from chromosomal Bacillus subtilis ATCC
21332 DNA was amplified (PCR) using the following
primers:
5' TAA GCA TGC TGC TTT CAT CTG CAG AAA C (S' asp-1eu-
SphI- srfB2), and
3' AAT GGA TCC TTC GGC ACG CTC TAC (3' asp-Ieu-BamHI-
srfB3 ) .
Correct size of the amplified fragment was
confirmed by agarose gel electrophoresis.
The fragment (20 fig) was digested with 1
unit of the enzymes BamHI/SphI (37°C, 16 h) to generate
terminal restriction sites.
Plasmid pQE70 (provided by Qiagen, D-Hilden)
(10 fig) was digested with the same enzymes and
subsequently incubated for 1 hour with 1 unit Alkaline
Phosphatase (37°C). Complete digestion was confirmed by

CA 02365594 2001-09-27
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- 32 -
transforming 1 ~.L of the linearised pl.asmid DNA into
competent cells of E.coli XL1 blue. Tre two fragments
were subsequently ligated in a ligation reaction (10 uL)
in a vector/insert ratio of 1:3 with 1 unit of T4-DNA-
lipase enzyme (16°C, 16 h).
1 ~L of the ligation mixture was used to transform 40 uL
competent cells of E.coli XL1 blue (Stratagene, D-
Heidelberg) by electroporation. The transformants were
selected on 2x YT agar plates containing Ampicillin (100
~g/mL). Analysis of 48 transformants resistant to
ampicillin revealed that 4 of them had inserted a ca.
5000 by fragment. Correct insertion was confirmed using
restriction enzyme digestion analysis and terminal
sequencing of the insert. A correct clone designated
gasp-leu-His6 was used for further investigations.
Construction of plasmid pasp-phe-His6
Plasmid gasp-phe-His6 was constructed from
plasmid pasp-1eu-Hiss as follows.
A 1894 by chromosomal DNA-fragment from
Bacillus brevis ATCC 8185 DNA was amplified (PCR) using
the following primers:
S' ATT TGG TCA CCA ATC TCA TCG ACA A (S' BstEII-TycA-
NLID), and
2S S' ATA GGA TCC TGT ATT CGT AAA GTT TTT C (3'-PheAT-
BamHI ) .
Correct size of the fragment was confirmed
using agarose gel electrophoresis.
The fragment was digested with 1 unit of
enzyme BamHI and incubated at 30°C for 4 hours.
Subsequently 1 unit of enzyme BstEII was added and
incubated for another 4 hours at 60°C.
Plasmid pasp-1eu-Hiss was digested in the

CA 02365594 2001-09-27
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- 33 -
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 repurified. Complete
digestion was confirmed as before with linearised pasp-
leu-His6. The two fragments were ligated in a equimolar
ratio for S hours at 16°C using 1 unit of T4-ligase
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-
His6. In contrast to the peptide synthetase encoding
gene on plasmid pasp-leu-Hiss, the peptide synthetase
encoding gene on plasmid gasp-phe-His6 is a hybrid gene
obtained by exchanging the DNA-fragment coding for the
second (Leu) minimal module (A- and T-domain), for a
DNA-fragment coding for a Phe minimal module.
Construction of plasmid pasp-phe-TE-His6
Plasmid gasp-phe-TE-His6 was constructed
from plasmid pasp-phe-His6.
A 910 by chromosomal DNA-fragment from
Bacillus suhtilis ATCC 21332 DNA was amplified (PCR)
using the following primers:
S' ATA ATC GAT AAT CGC ACA AAT ATG GTC (5' TE-srfCl-
ClaI) and
3' ATA AGA TCT AAC AAC CGT TAC GGT TTG TGT (3' int TE-
srfC l - Bg1 I I ) .

CA 02365594 2001-09-27
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- 34 -
Correct size of the fragment was confirmed
using agarose gel electrophoresis.
The fragment was digested with 1 unit of
enzyme ClaI for 4 hours at 37°C, before adjusting buffer
conditions and digesting with 1 unit of enzyme BglII (4
hours, 37°C).
Plasmid gasp-phe-His6 was digested with enzyme ClaI (4
h, 37°C) and subsequently with BamHI (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 repurified.
Control of complete digestion, ligation,
electroporation and selection of transformants was
established as described before.
Two of the analysed transformants were shown
to contain the desired DNA-fragment. Correct insertion
of the 900 by fragment was confirmed by restriction
enzyme analysis and terminal sequencing of the insert.
A correct clone was designated pasp-phe-TE-
His6. In contrast to the peptide synthetase encoding
gene on plasmid pasp-phe-His6, the peptide synthetase
encoding gene on plasmid pasp-phe-TE-His6 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-terminal T-TE domains
resemble the native C-terminus of the Surfactin
synthetase srfC.
Expression of the peptide synthetases asp-1eu-His6, asp-
phe-His6 and asp-phe-TE-Hiss
1 ~L of each constructed plasmid were
transformed in E.coli BL21/pgsp competent cells. Strain
BL21 1DE3 was obtained from Stratagene, D-Heidelberg.

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- 35 -
Plasmid pgsp, which is based on plasmid pREP4 (obtained
from Qiagen, D-Hilden), contains the gsp gene (the 4~-PP
transferase gene from the Gramicidin S-biosynthesis
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 Kanamycin
(25 ~g/mL). Several colonies were used to inoculate 4 mL
of 2x YT liquid medium (containing in addition 10 mM
MgClz) 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 in a waterbath
shaker (250 rpm). After 3-4 hours the cells reached an
optical density of 0, 7 (OD6oonm) and were induced by the
addition of 200 ~M IPTG. Cells were incubated for an
additional 1,5 hours before being harvested.
Expression of recombinant proteins was
confirmed by SDS-PAGE comparing protein samples taken at
the time of induction and 1,5 hours later.
In crude cell extracts from BL21/pgsp/pasp-
Ieu-His6 and BL21/pgsp/pasp-phe-His6 expression of an
inducible protein of ca. 180 kDa was confirmed. From
crude cell extracts of BL21/pgsp/pasp-phe-TE-His6
expression of an inducible protein of ca. 200 kDa could
be shown.
From cultures expressing the correct
recombinant proteins glycerol stocks were prepared and
stored at -80°C.
Purification of the recombinant proteins Asp-Leu-Hiss,
Asp-Phe-His6 and Asp-Phe-TE-His6
800 mL cultures of BL21/pgsp/pasp-leu-His6,
BL21/pgsp/pasp-phe-His6 and BL21/pgsp/pasp-phe-TE-Hiss

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- 36 -
treated as described in "Expression of the peptide
synthetases ..." were centrifuged at 5000 rpm for S
minutes and resuspended in 30 mL/L culture of buffer A
(50 mM HEPES, 300 mM NaCl, pH 8,0). Cell suspensions
were used directly or were stored at -20 °C till usage.
Cell lysis was established using two French press
passages at a working pressure of 12000 psi.
Directly after cell lysis PMSF was added to
a final concentration of 1 mM. After centrifugation of
the cell lysates at 10000 rpm for 30 minutes, the
supernatar~t was combined with 1% (v/v) buffer B (50 mM
HEPES, 300 mM NaCl, 250 mM Imidazol, pH 8,0). Protein
solutions were applied on a Ni2+-NTA-agarose column
(Qiagen, D-Hilden) previously equilibrated with 1°s (v/v)
buffer B. Flow rate was 0,75 mL/min. After the non-His6-
tagged proteins had passed through the column, it was
washed with 1% buffer B for another 10 min before a
linear gradient was applied (30 min to 30% B, an
additional 10 min to 100% B). All three proteins eluted
at a concentration of about 5% buffer B (15 mM Imidazol)
and were collected as 2 mL fractions.
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 MgCl2 and S mM DTE for 16 hours.
After dialysation protein concentrations were again
determined.
Till further usage proteins were stored at -
20°C after addition of glycerol to 10% (v/v).
From 1 L culture approximately 5 mg of each
pure recombinant protein could be obtained. Grade of
purification was estimated to be 95o by SDS-PAGE.

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Analysis of enzymatic activity
ATP-PPi-exchange reaction:
Specificity of amino acid activation was
determined indirectly by incorporation of labelled 32PPi
into ATP during reverse reaction (Lee, S.G. & Lipmann,
F.; Tyrocidine synthetase system; Methods Enzymol. 43,
1975, p.S85-602). For this purpose 20 pmol of each
enzyme was incubated with 1 mM amino acid, 1 mM ATP, 0,1
mM PPi, SO mM HEPES, 100 mM NaCl and 10 mM MgClz and 2
mCi 32PPi at 37°C in a total volume of l00 ~,L. Reactions
were quenched after 10 min by adding S00 ~L of a
solution containing 100 mM NaPPi, 560 mM perchloric acid
and 1,2% (w/v) active charcoal. The mixture was
centrifuged at 13000 rpm for 1 min. The pellet was
washed and resuspended twice with 1 mL H20.
Incorporation of labelled ATP (adsorbed to
the charcoal) was detected by measuring radioactivity of
the precipitate.
Asp-Leu-His6 was shown to activate Asp and Leu
exclusively. K~, values for Asp and ATP were determined to
be 3,5 mM and 0,9 mM respectively. KM values for Leu and
ATP were detected to be 0,3 mM and 0,6 mM, respectively.
Asp-Phe-His6 and Asp-Phe-TE-His6 were shown
to activate both Phe and Asp. The KM value for Phe was
determined to be about 50 ~.M.
The amino acid activation patterns of Asp-
Phe-His6 and Asp-Phe-TE-Hiss were found to be identical.
Covalent binding of amino acids to the Asp-Phe
dipeptide synthetase:
Quantity of bolo-enzyme formation was
determined by measuring the amount of labelled Asp, Leu

CA 02365594 2001-09-27
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- 38 -
and Phe, that could be covalently bound to one
equivalent of the purified proteins Asp-Leu-Hiss, Asp-
Phe-Hi s6 and Asp-Phe-TE-Hiss. (Lee, S.G. ; see above) .
SO pmol of each enz~lrme was incubated ~,aith 2
mM ATP, SO mM HEPES, 100 mM NaCl, 10 mM MgCl2 and 100
pmol of 14C labelled amino acid (Asp and Leu or Asp and
Phe respectively) at 37°C. After 30 min the reaction was
quenched by the addition of 1 mL of 10% TCA and 5 mg/mL
BSA and subsequently stored at 0°C for another 30 min.
The protein precipitates were collected by
centrifugation (30 min at 13000 rpm) and washed twice
with 10% TCA. The washed precipitates were dissolved in
50s performic acid and used to measure incorporation of
labelled amino acid.
Asp-Leu-His6 could be labelled with Asp and
Leu to a degree of approximately 20-25%. Asp-Phe-His6
and Asp-Phe-TE-His6 could be labelled with Asp to a
degree of only 10-150. The incorporation of Phe reached
a level of approximately 500.
Indirect proof of formation of the dipeptides Asp-Leu
and Asp-Phe:
Covalent binding of the constituent amino
acids to the dipeptide synthetases was shown by kinetic
experiments using radioactively labelled Asp. In a
first assay 0,85 ~M Asp-Leu-Hiss were incubated with 2
mM ATP, 2, 8 ~,M Asp (14C, 56 nCi) in a buffer (50 mM
HEPES, 10 mM MgClz, 100 mM NaCl, pH 8,0) at 37 °C. At
regular time intervals samples were taken and treated
as indicated in the previous paragraphs; radioactivity
(of Asp covalently bound to the enzyme) was measured in
each sample. After 4 minutes. the amount of
incorporated labelled Asp started levelling-off to
reach a maximum a few minutes later if no second amino

CA 02365594 2001-09-27
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- 39 -
acid was added. If, however, 1,5 uM of Leu were added
after 4 minutes, a further strong, but temporary,
increase of radioactivity was observed, which sharply
decreased after about 5 minutes to a level below the
maximum observed when no second amino acid was added.
In another experiment 0,5 ~M Asp-Phe-His6
were incubated in the same way as Asp-Leu-His6 before.
In this case the addition of 0,1 mM Phe after about 5
minutes resulted in a similar temporary increase of
covalently bound radioactive Asp, as described above.
However, the addition of Leu instead of Phe did not
lead to such temporary increase.
This clearly shows that peptide bond
formation takes place on each of these peptide
synthetases. Furthermore, the results show that the
peptides formed on the peptide synthetases are being
released therefrom.
Direct proof of formation of the dipeptide Asp-Phe:
Formation of the dipeptide Asp-Phe was proven
by a series of experiments, using different analytical
techniques, namely thin layer chromatography (TLC) with
radio-active detection, high performance liquid
chromatography and mass spectroscopy.
In a first set of experiments (method to
determine Asp-Phe released from the dipeptide
synthetase) 100 pmol of Asp-Phe-His6 were incubated in a
total volume of 200 ~.1 with 1mM of ATP, 1 mM Phe and
1.25 uM 14C-labeled Asp in a buffer (50 mM HEPES, 20 mM
MgClz, pH 8) for 6 hours at 37 °C. Control experiments
carried out were done omitting one of ATP, Phe or
°.n zyme .
In a further, analogous, set of experiments
the same amount of said enzyme was incubated under

CA 02365594 2001-09-27
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- 40 -
identical conditions with 1 mM Asp and 1.25 ~M 1''C-
labeled Phe (instead of Phe and 1''C-labeled Asp). Also
the appropriate blanks were carried out.
To finish the reactions, 100 ~.1 of n-butanol
S were added to precipitate the enzyme which then was
removed. The remaining clear solutions were evaporated
and replenished to a volume of 20 ~.l with 10 vol% of
methanol in water. An appropriate volume of each of
these samples was applied to a silica TLC plate, which
l0 then was developed using a solution of butanol/acetic
acid/ethyl acetate/water 1:1:1:1 (v/v/v/v) as the
eluent. After elution the plate was dried and an X-ray
film was put on top of it for 2 to 5 days of exposure.
Finally the film was processed to show spots of
15 radioactively labeled compounds.
Only if Asp, Phe, ATP and enzyme were present
in the reaction mixture a spot could be detected that
had the same retention factor (Rf) as Asp-Phe.
20 In a second set of experiments (method to
determine dipeptide synthetase bound Asp-Phe) 1 nmol of
Asp-Phe-His6 were incubated in a total volume of 600 ~.l
with 1mM of ATP, 1 mM Asp and 1 ~.M 14C-labeled Phe in a
buffer (50 mM HEPES, 20 mM MgCl2, pH 8) at 37 °C.
25 Reactions were stopped after 10, 20 and 30
minutes, respectively, by the addition of 300 ~.1 of 200
(weight) aqueous trichloroacetic acid (TCA) and the
mixtures were cooled to 4 °C. All further steps were
carried out at 4 °C.
30 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. Next the pellet was dried for 15 minutes at

CA 02365594 2001-09-27
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_ :~ 1 _
37 °C, before being resuspended in 200 ~,1 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. To separate the released dipeptide
form the protein, 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 ~C1 of loo aqueous
methanol.
Analysis then was done by TLC with radio-
active detection as described in the previous set of
experiments. A clear spot of the dipeptide Asp-Phe
could be observed in each of the three samples. The
intensity of these spots increased significantly for
the samples taken at 10, 20 and 30 minutes,
respectively. This shows that formation of the
dipeptide Asp-Phe really takes place on the dipeptide
synthetase.
In a final experiment formation of Asp-Phe was
also confirmed by comparing the HPLC retention times
and mass-spectra of the dipeptide formed. In a total
volume of 200 ~C1 SO pmol of Asp-Phe-TE-Hi s6 was
incubated with 1mM of ATP, 1 mM of Asp and 0.5 mM of
Phe in a buffer (50 mM HEPES, 20 mM MgCl2, pH 8) for 6
hours at 37 °C. To finish the reactions, 100 ~l of n-
butanol were added to precipitate the enzyme which then
was removed. The remaining clear solution was
evaporated in vacuo for 3 hours and replenished to a
volume of 20 ~.l with 10 vole of methanol in water.
For further analyses this volume was diluted
lOx (again with 10 vol% methanol in water) and portions
thereof then were subjected to HPLC followed by

CA 02365594 2001-09-27
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- 42 -
photometric detection (as shown below) and to
Electrospray Ionisation LC-MS analysis. Reference
samples of Asp-Phe synt~:esised chemically were used for
comparison.
For the HPLC with photometric detection a 50 ~.1
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. H3BO3 buffer pH = 3.0)
and B (acetonitrile; Merck, HPLC-grade) were used at a
gradient (t = 0 min: 98% A, 2% B; t = 35 mi.n: loo A, 90%
B) and a flow of 1.2 ml/min, at 40 °C.
Detection was done photometrically (at 210 nm
and 257 nm, with quantification at 210 nm).
In the resulting HPLC chromatogram for the
experimental sample a peak was found at 6.59 minutes
which was at exactly the same retention time as for the
Asp-Phe reference compound. Moreover, the UV-spectrum
recorded for the Asp-Phe peak from the sample was shown
to be identical (as to extinction versus wavelength in
the region from 200 to 380 nm) to the one recorded for
the Asp-Phe reference compound. The amount of Asp-Phe in
the sample was calculated to be 16.1 mg/1.
For the Electrospray Ionisation LC-MS
analysis a 4 ~.1 portion of the lOx diluted experimental
sample (see above) was injected onto the column. As a
column a Nucleosil 120-3 C18 reversed-phase (Macherey &
Nagel, 250 x 4 mm) was used. The eluents were eluents A
(demi-water containing 0.05% of formic acid) and B
(HPLC-grade methanol containing 0.05% of formic acid).
As a gradient eluents A and B were used as follows: t =
0: 10%B; t = 25 min: 60°s B; t = 30 min: 100% B; t = 34
min: 100% B at a flow of 0.4 ml/min at ambient
temperature. Detection was done by total ion current
(TIC) using electrospray ionisation in the positive ion

CA 02365594 2001-09-27
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_ 43 _
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 in the
sample was calculated to be about 20 mg/1.

CA 02365594 2001-09-27
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SEQUENCE LISTING
<110> Holland Sweetener Company V.O.F.
<120> Microbiological Production Method For
Alpha-L-Aspartyl-L-Phenylalanine
<130> 4024ep
<140> 99203518.8
<141> 1999-10-27
<160> 47
<170> PatentIn Ver. 2.1
<210> 1
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 1
Leu Thr Tyr Xaa Glu Leu
1 S
<210> 2
<211> 'o
<2.12> PRT
<213> Artificial Seauence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 2
Leu Ser Tyr Xaa Glu Leu
1 5
<210> 3
<211> '_2
<212> PRT
<213> Artificial Sequence

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 3
Leu Lys Ala Gly Xaa Ala Tyr Leu Val Pro Leu Asp
1 5 10
<210> 4
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 4
Leu Lys Ala Gly Xaa Ala Tyr Leu Leu Pro Leu Asp
1 5 10
<210> 5
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 5
Leu Lys Ala Gly Xaa Ala Tyr Leu Val Pro Ile Asp
1 5 10
<210> 6
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Seauence:Consensus
Sequence
<400> 6
2

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
Leu Lys Ala Gly Xaa Ala Tyr .eu Leu Pro Ile Asp
1 5 10
<210> 7
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
seauence
<400> 7
Leu Ala Tyr Xaa Xaa Tyr Thr Ser Gly Ser Thr Gly Xaa Pro Lys Gly
1 5 10 15
<210> 8
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 8
Leu A1a Tyr Xaa Xaa Tyr Thr Ser Gly Thr Thr G1y Xaa Pro Lys Gly
1 5 10 15
<210> 9
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Secruence
<400> 9
Phe Asp Xaa Ser
1
<210> 10
<211> i

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 10
Asn Xaa Tyr Gly Pro Thr Glu
1 5
<210> 11
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 11
G1y G1u Leu Xaa Ile Xaa Gly Xaa Gly Val Ala Arg Gly Tyr Leu
1 5 10 15
<210> 12
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 12
Gly G1u Leu Xaa Ile Xaa Gly Xaa Gly Leu Ala Arg Gly Tyr Leu
1 5 10 15
<210> 13
<211> 6
<212> PRT
<213> Artificial Sequence
<22O>
<223> Description of Artificial Sequence: Consensus
seQUence
4

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
<400> 13
Tyr Arg Thr Gly Asp Leu
i 5
<210> 14
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 14
Tyr Lys Thr Gly Asp Leu
1 5
<210> 15
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 15
Gly Arg Xaa Asp Xaa G1n Val Lys I1e Arg Gly Xaa Arg Ile Glu Leu
1 5 10 15
Gly Glu I1e Glu
<210> 16
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Cc.~.ser.sus
Sequence
<400> 16
Leu Pro Xaa Tyr Met T_~.~e Pro
5

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
1 5
<210> 17
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 17
Leu Pro Xaa Tyr Met Val Pro
1 5
<210> 18
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 18
Asn Gly Lys Val Asp Arg
1 5
<210> 19
<211> 6
<212> PRT
<213> Artificial Seauence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 19
Asn Gly Lys Leu Asp Arg
1 S
<210> 20
<211> 12
<212> PRT
0

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
<213> Artificial Sequence
<220>
<223> Description of Artificia':. Sequence: Consensus
Sequence
<400> 20
Asp Xaa Phe Phe Xaa Xaa Leu Gly Gly His Ser Leu
1 5 10
<210> 21
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 21
Asp Xaa Phe Phe Xaa Xaa Leu Gly Gly Asp Ser Leu
1 5 10
<210> 22
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 22
Asp Xaa Phe Phe Xaa Xaa Leu Gly Gly His Ser Ile
1 S 10
<210> 23
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificia'~ Sequence: Consensus
Sequence

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
<400> 23
Asp Xaa Phe Phe Xaa Xaa Leu Gly Gly Asp Ser Ile
1 5 10
<210> 24
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 24
Ser Xaa Ala Gln Xaa Arg Leu Trp Xaa Leu
1 5 10
<210> 25
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 25
Ser Xaa Ala Gln Xaa Arg Met Trp Xaa Leu
1 5 10
<210> 26
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 26
Ser Xaa A1a Gln Xaa Arg Leu Tyr Xaa Leu
1 5
8

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
<210> 27
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:COnsensus
Sequence
<400> 27
Ser Xaa Ala Gln Xaa Arg Met Tyr Xaa Leu
1 5 10
<210> 28
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 28
Arg His Glu Xaa Leu Arg Thr Xaa Phe
1 5
<210> 29
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<900> 29
Met His His Xaa Ile Ser Asp Gly Trp Ser
1 5 10
<210> 30
<211> 10
<212> PRT
<213> Artificial Secuence
<220>
9

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 30
Met His His Xaa Ile Ser Asp Gly Val Ser
1 5 10
<210> 31
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 31
Tyr Xaa Asp Phe Ala Val Trp
1 5
<210> 32
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:COnsensus
Sequence
<400> 32
Tyr Xaa Asp Tyr Ala Val Trp
1 5
<210> 33
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 33
Ile Gly Xaa Phe Val Asn Thr Gln Cys Xaa Arg
1 5 10

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
<210> 34
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
sequence
<400> 34
Val Gly Xaa Phe Val Asn Thr Gln Cys Xaa Arg
1 5 10
<210> 35
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 35
Ile Gly Xaa Phe Val Asn Thr Leu Cys Xaa Arg
1 5 10
<210> 36
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<900> 3'0
Val Gly Xaa Phe Val Asn Thr Leu Cys Xaa Arg
1 5 10
<210> 37
<211> "~1
<212> PRT
<213> Artificial Sequence
11

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 37
Ile Gly Xaa Phe Val Asn Thr Gln Ala Xaa Arg
1 5 10
<210> 38
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 38
Val Gly Xaa Phe Val Asn Thr Gln Ala Xaa Arg
1 5 10
<210> 39
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 39
Ile Gly Xaa Phe Va1 Asn Thr Leu Ala Xaa Arg
1 5 10
<210> 40
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 40
12

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
Val Gly Xaa Phe Val Asn Thr Leu Ala Xaa Arg
1 S 10
<210> 41
<211> 7
<212> PRT
<213> Artificial Seouence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 41
His Gln Asp Tyr Pro Phe Glu
1 5
<210> 42
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
sequence
<400> 42
Asn Gln Asp Tyr Pro Phe Glu
1 5
<210> 43
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 43
His Gln Asp Val Pro Phe Giu
1 5
<210> 44
13

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 44
Asn Gln Asp Val Pro Phe Glu
1 5
<210> 45
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 45
Arg Asp Xaa Ser Arg Asn Pro Leu
1 5
<210> 46
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
Sequence
<400> 46
Gly His Ser Xaa Gly
1 5
<210> 47
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Consensus
14

CA 02365594 2001-09-27
WO 00/58478 PCT/NL00/00206
Sequence
<400> 47
Gly Tyr Ser Xaa Gly

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Administrative Status

2024-08-01:As part of the Next Generation Patents (NGP) transition, the Canadian Patents Database (CPD) now contains a more detailed Event History, which replicates the Event Log of our new back-office solution.

Please note that "Inactive:" events refers to events no longer in use in our new back-office solution.

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Event History

Description Date
Inactive: IPC from MCD 2006-03-12
Inactive: IPC from MCD 2006-03-12
Inactive: IPC from MCD 2006-03-12
Application Not Reinstated by Deadline 2003-03-31
Inactive: Dead - Application incomplete 2003-03-31
Inactive: Status info is complete as of Log entry date 2003-02-13
Inactive: Abandoned - No reply to Office letter 2002-12-30
Deemed Abandoned - Failure to Respond to Notice Requiring a Translation 2002-04-02
Deemed Abandoned - Failure to Respond to Maintenance Fee Notice 2002-03-28
Inactive: Incomplete PCT application letter 2002-03-12
Inactive: Cover page published 2002-02-12
Inactive: Courtesy letter - Evidence 2002-02-12
Inactive: First IPC assigned 2002-02-10
Inactive: Notice - National entry - No RFE 2002-02-08
Application Received - PCT 2002-01-23
Application Published (Open to Public Inspection) 2000-10-05

Abandonment History

Abandonment Date Reason Reinstatement Date
2002-04-02
2002-03-28

Fee History

Fee Type Anniversary Year Due Date Paid Date
Basic national fee - standard 2001-09-27
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
HOLLAND SWEETENER COMPANY V.O.F.
Past Owners on Record
MOHAMED ABDALLA MARAHIEL
PETER JAN LEONARD MARIO QUAEDFLIEG
SASHA DOEKEL
THEODORUS SONKE
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Description 2001-09-27 58 1,934
Claims 2001-09-27 5 211
Abstract 2001-09-27 1 63
Cover Page 2002-02-12 1 41
Reminder of maintenance fee due 2002-02-11 1 111
Notice of National Entry 2002-02-08 1 194
Courtesy - Abandonment Letter (Maintenance Fee) 2002-04-25 1 183
Courtesy - Abandonment Letter (incomplete) 2002-04-23 1 173
Request for evidence or missing transfer 2002-09-30 1 108
Courtesy - Abandonment Letter (Office letter) 2003-02-03 1 167
PCT 2001-09-27 15 519
Correspondence 2002-02-08 1 25
Correspondence 2002-03-08 1 31