Note: Descriptions are shown in the official language in which they were submitted.
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Promoter and constructions for expression of recombinant
g~oteins in filamentous funai
This invention relates to improvements in the expression of
proteins, particularly of fusion proteins, by recombinant
DNA technology; using filamentous fungi as the host. These
improvements refer mainly to the use of a new promoter and
new DNA constructions containing it.
DESCRIPTION OF THE PRIOR ART
Filamentous fungi are known to produce in nature a wide range
of homologous proteins in large amounts. For this reason,
filamentous fungi have been regarded as attractive hosts for
the expression of recombinant proteins. For instance,
Asperaillus awamori has been used for the production of
recanbinant proteins such as bovine chymosin and human
lactof errin.
Some recombinant proteins, however, have proved to be very
difficult to express in filamentous fungi. This is the case
for exan~le of interleukin-6 and thaumatin. The thaumatins are
proteins with a very sweet taste and the ability to increase
the palatability of food. In industry they are currently
extracted from the arils of the fruit of the plant
Thaiunatoccocus~ daniellii Benth (M. Witty, J.D. Higginbotham,
Thaumatin ,1994, CRC Press, Boca Rat6n, Florida). Thaumatins
can be isolated from these arils in at least five different
forms ( I , I I , III , b and c ) , thaiuna,tins I and I I being the
most abundant types in the arils. Despite its advantages,
industrial use of thaumatins of plant origin is ver~J limited
because of the extreme difficulty involved in obtaining the
fruit from which it is extracted. Attgnpts have been made to
produce thaumatins by genetic engineering in different hosts
such as bacteria, yeasts and transgenic plants, but until now
the results have been considered disheartening and thus the
thaumatin available to industry is very scarce and expensive.
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European patent EP 684312 describes a process for preparing
recombinant thaumatin in filamentous fungi. One problem of
this process is that the yields obtained are low in comparison
with those needed for industrial protection of thaumatins.
It is known in the. art that yields of recombinant proteins can
be improved when the recombinant protein of interest is
expressed as a fusion with another protein, and when
expression of this cassette ~is driven by a strong fungal
promoter. This other protein, named "carrier protein", is
usually a~ highly expressed protein of fungal origin. Up to
now, the most frequently used expression system involves the
glucoamylase promoter and gene from. p,,.p~'aillus awamori as the
promoter and the carrier protein, respectively (P.P. Ward et
al., Biotechnolocr~ 1995, vol. 13, pp. 498-502). However, in
some cases the use of this expression system does not lead to
high levels of the desired recombinant protein. One of these
specially problematic cases is the expression of recombinant
thau<natin in filamentous fungi.
~1 i~~r~ pd~e ~.a. ~"
In view of the above, it is clear that there is the need to
provide new and more efficient expression systems that allow
the production of higher concentrations of those proteins that
are difficult to express in filamentvus fungi, such as
i. _.
-~ 25 thaumatins. This goal is achieved with the new promoter and
DNA constructions provided in the present invention, as
explained below.
DESCRIPTION OF THE INVENTION
3 0 -.
The present invention provides a .new expression system that
makes use of the promoter from the. glutamate dehydrogenase
(gdh) gene from filamentous fungi of the genus As~ercrillus,
particularly, from Asperaillus awamori.
One of the objects of the present invention is a new
promoter for the expression of recombinant proteins - in
AMENDED SHEET
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Insertion for page 2
Gene (1983) , vol. 26, pp. 253-260 discloses the complete nucleotide sequence
of the
Neurospora crassa NADP-specific glutamate dehydrogenase gene.
Appl. Microbiol. Biotechnol. (1997), Vol. 47, pp 1-11 discloses the efficient
production of
secreted proteins by Aspergillus. Particular focus is laid on the gene fusion
strategies.
EP 0 684 312 A2 relates to a preparation process of a natural protein
sweetener, thaumatin.
Said document discloses a new nucleotide sequence encoding thaumatin with
optimised codon
usage for expression in filamentous fungi.
AMENDED SHEET
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filamentous fungi that comprises a nucleotide sequence - or
a complementary strand thereof - selected from the group
consisting of: (a) the nucleotide sequence numbered 1-740 in
the enclosed SEQ ID No. 1; and (b) a nucleotide sequence that hy-
bridizes under stringent conditions:-to that defined in (a) with the
proviso that the sequence is not the promoter of the gdh gene from
Aspergillus nidnlans. Particularly preferred is the promoter
comprising the sequence defined in (a), i.e. the nucleotide
sequence numbered as 1-740 in SEQ ID No. 1, which
corresponds to the gdhA promoter of the glutamate
dehydrogenase A gene from Asnergillus ~wamori.
Although glutamate dehydrogenase A disclosed herein is the
_ first glutamate dehydrogenase identified and described in
the filamentous fungus Asperaillus awamori, there may exist
other glutamate dehydrogenases in AsQeraillua awamori. The
novel nucleotide sequence of the AsnercLillus awamori gdhA:
promoter and/or gene shown in SEQ ID No. 1 or a portion _-
thereof can be used as a probe for the identification and
isolation of other homologous promoters/genes of glutamate
dehydrogenases in As~eraillus awamori as well as in other
organisms, preferably in filamentous fungi, more preferably
in fungi of the genus Asperaillus, still more preferably in
Asperaillus awamori and Asperaillus nig~er, and specially in
Asneraillus awamori, following the teachings of the present
invention. Consequently, the present invention is not
limited to .the specific gdhA promoter from AsDerail_1-us
awamori disclosed herein but also relates to the promoter of
any glutamate dehydrogenase gene from a fungus of the genus
As~eraillus with the proviso that it is not from A~peraillus
nidulans. Examples of said Asperailli include Asneraillus
awamori, Asne_raillus niger, As~eraillus oryzae and
As~eraillus o'a ,In a preferred embodiment, the invention
relates to a promoter of a glutamate dehydrogenase gene from
As~eraillus awamori or Asr~eraillus niQer In a more
preferred embodiment, the invention relates to a promoter of
a glutamate dehydrogenase gene from Asperqillus awamori. The
AMENDED SHEET
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use of the novel nucleotide sequence shown in SEQ ID No. 1
or a portion thereof as probe is also a object oz the
present invention. The term "a portion thereof" denotes
any part of the nucleotide sequence of SEQ ID No.l that is
functional as a probe.
Another object of the present invention is a new DNA
sequence, purified and isolated, that encodes a glutamate
dehydrogenase protein and that comprises a nucleotide
sequence - or a complementary strand thereof - selected trom
the group consisting of: (a) the nucleotide sequence
numbered 741-2245 in the enclosed SEQ ID No. 1; and (b,~
_ a nucleotide sequence that hybridizes under stringent conditions to
that defined in (a) with the proviso that the sequence is not the gdh
_ 15 'I gene from Aspergi l lus nidulans . In a pre f erred
embodiment, the nucleotide sequence encoding a glutamate
dehydrogenase is the sequence defined in (a), i.e. the
nucleotide sequence numbered as 741-2245 in SEQ ID No. 1. _
The present invention is not limited, however, to the _ __
specific gdhA gene from AsDergillus awamori disclosed herein
but also relates to any glutamate dehydrogenase gene from a
fungus of the genus Ast~erc~.~illus with the proviso that it is
not from As~eraillus ~idulans. In a preferred embodiment,
the invention relates to the DNA sequences encoding
25~ glutamate dehydrogenase from Aspergillus awamori or
y Asperaillus niQer. In a more preferred embodiment, the
invention relates to the DNA sequences encoding glutamate
dehydrogenase from Asneraillus awamori.
30! Another object of the invention are the novel proteins
encoded by any of the DNA sequences defined above. In a
preferred embodiment, this protein has the amino acid
sequence shown in the enclosed SEQ ID No. 2. But are also
included in the present invention any glutamate
35 dehydrogenase from a fungus of the genus Asperctillus with
the proviso that it is not from Asgergillus nidulans, more
preferably a glutamate dehydrogenase from Asperaillus
awamori or As~erQillus nicer, and still more preferably a
glutamate dehydrogenase from As~eraillus ~wamori.
AMENDED SHEET
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The invention further relates to the use of the glutamate
dehydrogenase promoters above described for the expression
of recombinant proteins in . filamentous fungi. Certain
5 glutamate dehydrogenases from several microorganisms are
already known and their genes have been disclosed, in
particular the glutamate dehydrogenase A (gdhA) gene from
Asberaillus nidulans (A. R. Hawkins et al., Mol. Gen. Genet.
1989, 218(1), pp. 105-111): However, to the best of our
knowledge, there has been no disclosure up to now of the
expression of a recombinant protein making use° of the gdhA
promoter from A. nidulans nor has it ever been mentioned
that it might be useful for improving the expression of
recombinant proteins in filamentous fungi. As shown in the
examples below, the glutamate dehydrogenase promoter from
Asperaillus awamori has proven to be very strong in
promoting transcription of heterologous genes. Therefore,
this promoter as well as related ghd promoters from
Asperailli are expected to drive high-level transcription of
genes and thus are expected to be of use in the expression
of recombinant proteins in filamentous fungi. It is thus a
further object of the present invention the use of a
promoter from a glutamate dehydrogenase gene from a fungus
of the genus ~_peraillus for the expression of recombinant
proteins in filamentous fungi. Preferably, the gdh promoter
is from a fungus of the genus Asperctillus with the proviso
that it is not from A.~percLllus nidulans, more preferably it
is from Aspercrillus awamori or Aspercrillus ni er, still more
preferably it is from Aspergillus-awam~i, and particularly
preferably it is one of. the novel gdh promoters described
above.
There is in principle no limitation on the desired
recombinant protein to be expressed. Examples of such
desired proteins (which term, as used herein, includes
proteins and smaller polypeptides) include, but are not
limited to, enzymes, hormones, cytokines, growth factors,
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structural proteins, plasma proteins and others. A non-
limiting list of examples of proteins that can be expressed
includes human proteins such as interferons, interleukins,
tissue plasminogen activator, serum albumin, growth hormone,
and growth factors. Other proteins can be of non-human
origin such as lipases of both fungal and non-fungal origin,
proteases, thaumatins, bovine chymosin, etc. Polypeptides,
which can be of human and non-human origin, include
calcitonin, glucagon, insulin, nerve growth factor,
epidermal growth factor, the anticoagulant Hirudin and
analogs such as R3-hirulog.
A further object of the present invention are the DNA
constructions that comprise: a) a promoter from a glutamate
dehydrogenase gene from a fungus of the genus Asperaillus;
b) a DNA sequence encoding a protein normally expressed from
a filamentous fungus or a portion thereof; c) a DNA sequence
encoding a cleavable linker peptide; and d) a DNA sequence
encoding a desired protein. In a preferred embodiment, the
promoter under a) comprises a gdh promoter from a fungus of
the genus Aspergillus with the proviso that it is not from
~speraillus nidulans, more preferably it is from As~ergillus
awamori or Ast~eraillus nigger, still more preferably it is
from Ast~ercrillus awamori, yet more preferably it comprises
any of the new promoters described above, and more
particularly it comprises the nucleotide sequence 1-740 in
SEQ ID No. 1. The DNA sequence under b) encodes a protein
normally expressed from a filamentous fungus or a portion
thereof that is functional, i.e. that is capable of
producing increased secretion of the desired protein.
Examples of such protein under b) include glucoamylase, a-
amylase and aspartyl proteases from Aspergillus awamori,
Asgergillus nicer, Aspergillus oryzae and Asperaillus so'ae,
cellobiohydrolase I, cellobiohydrolase II, endoglucanase I
and endoglucanase III from Trichoderma species, glucoamylase
from Neurospora and Humicola species, the protein B2 from
Acremonium chrvsocrenum and a glutamate dehydrogenase from a
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filamentous fungi. In a preferred embodiment, the DNA
sequence under b) encodes a protein or portion thereof
selected from the group consisting of: i) glucoamylase from
Asperaillus awamori, Asperaillus nigger, Asneraillus orvzae
or Asperaillus solae; ii) B2 from Acremonium chrvsoaenum;
and iii) a glutamate dehydrogenase from a filamentous fungi;
more preferably, the DNA sequence under b) encodes a protein
or portion thereof selected from the group consisting of: i)
glucoamylase from Asperaillus awamori, Asperaillus ni r,
As~eraillus oryzae or Asperaillus so' e; ii) B2 from
Acremonium chryso e~; and iii) a glutamate dehydrogenase
from Asperyillus .awamori or Asperaillus nicer. The DNA
sequence under c) encodes a cleavable linker peptide; as
used herein, cleavable linker peptide means a peptide
sequence which under certain circumstances allows the
separation of the sequences bordering the cleavable linker,
for example sequences that are recognized and cleaved by a
protease or cleaved after exposure to certain chemicals. In
a preferred embodiment, the DNA sequence under c) contains a
KEX2 processing sequence. As mentioned above, the desired
protein under d) can be in principle any recombinant
protein. In a preferred embodiment, the DNA sequence under
d) encodes thaumatin; particularly preferred constructions
for the preparation of thaumatin include those wherein the
DNA sequence encoding thaumatin under d) is the synthetic
gene encoding ahaumatin II coming from plasmid pThIX, which
is disclosed in EP 684312.
Although in the context of the present invention it is
preferred, when expressing.a desired protein, to use the gdh
promoters in fusion constructions,~it is also possible 'to
use a gdh promoter to express directly a desired protein.
Therefore, it is a further object of the present invention
the new DNA constructions that comprise a gdh promoter from
a fungus of the genus Asperaillus operatively linked to a
DNA sequence encoding the protein that it is desired to
express. In a preferred embodiment, the gdh promoter is from
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a fungus of the genus Aspe~c~illus with the proviso that it
is not from Asberaillus nidulans, more preferably it is from
Asberaillus awamori or Asperaillus niger, still more
preferably it is from Asperqillus awamori, yet more
preferably it is one of the new promoters described above,
and more particularly it comprises the nucleotide sequence
1-740 in SEQ ID No. 1.
As will be obvious to those skilled in the art of
recombinant DNA technology, all the above DNA constuctions
may additionally contain other elements which include, but
are not limited to, signal sequences, termination sequences,
polyadenylation sequences, selection sequences, sequences
that allow the replication of the DNA; etc. There is no
limitation on the number and nature of these additional
sequences and any of the known sequences for exerting these
functions can in principle be used in the constructions
according to the present invention. For example, as a signal
sequence functional as a secretory sequence we can mention
the signal sequences from glucoamylase, oc-amylase and
aspartyl proteases from Asperaillus awamori, Asperaillus
~aer, Asperaillus o~yzae and Asperaillus s_olae, signal
sequences from cellobiohydrolase I, cellobiohydrolase II,
endoglucanase I and endoglucanase III from Trichoderma
species, signal sequences from glucoamylase from Neurosnora
and Humicola ,species and the signal sequence from the
protein B2 from Acremonium chrysoaenum. In general it is
preferred to use as signal sequence those derived from
proteins secreted by the filamentous fungus used as
expression host to express and secrete the recombinant
protein or, in case fusion constrLictions are used, also
those derived from the protein used as carrier. protein. A
termination sequence is a nucleotide sequence which is
recognized~~ by the expression host to terminate
transcription. Examples include the terminators from the A.
nidulans trpC gene, the A. awamori, A. nicer, A. oryzae or
A. s_osae glucoamylase gene, the A. awamori, A. ni er, A.
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orvzae or A. 'ae oc-amylase genes and the Saccharomyces
cerevisiae cycl gene. A selection sequence is a sequence
useful as selection marker to allow the selection of
transformed host cells. In principle any known selection
marker for the filamentous fungus that is intended to be
used as host can~be employed. Examples of such selection
markers include genes confering resistance to a drug such as
an antibiotic (e.g. hygromycin or phleomycin) as well as
auxotrophic markers such as argB, trpC, niaD and pyre. A
polyadenylation sequence is a nucleotide sequence which
when transcribed is recognized by the expression host to add
polyadenosine residues to transcribed mRNA. Examples include
the polyadenylation sequences from the A. nidulans trpC
gene, the A. awamori, ~ ni_ecL~r, ~ orvzae or A. s_oiae
glucoamylase genes and the Mucor miehei carboxyl protease
gene.
The present invention also relates to the filamentous fungus
cultures capable of producing a recombinant protein that
have been transformed with plasmids that contain any of the
DNA constructions mentioned above. Examples of species of
filamentous fungi that may be used as expression hosts
include the following genera: Aspercrillus, Trichoderma,
Neurospora, Penicillium, Acremonium, ~e~halosporium, Achlya,
Phanerochaete, Podosnora, Endothia, uc , Fusarium,
Humicola, ~ochliobolus, Rhizo~us and Pvricularia.
Particularly preferred are those cultures wherein the
filamentous fungus is selected from a fungus of the genus
Aspergillus, and more preferably it is selected from
Asperaillus awamori, Aspergillus nicer, Asneraillus oryzae,
Asperaillus nidulans or Aspergillils .s_Qjae. In another
preferred embodiment, the recombinant protein produced is
thaumatin.
A further object of the present invention is to provide a
process for producing a recombinant protein in a filamentous
fungus that comprises the following steps: a) preparation of
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an expression plasmid that contains a DNA construction as
defined above; b) transformation of a strain of filamentous
fungus with said expression plasmid; c) culture of the
transformed strain under appropriate nutrient conditions to
5 produce the desired protein, either intracellularly,
extracellularly or in both ways simultaneously; and d)
depending on each case, separation and purification of the
desired protein from the fermentation broth. Preferred is
the process wherein the recombinant protein produced is
10 thaumat~n..
The accompanying examples describe the identification and
isolation of the glutamate dehydrogenase A gene and its
promoter region from Asperaillus awamori. This was achieved
using a probe from ~leurospora crassa. The selection of a
suitable DNA fragment from the glutamate dehydrogenase gene
in Neurospora crassa to be used as a probe to get the
homologous gene in Asperg~illus awamori is not, however,
straightforward. In this case, there were no clear homology
sequences that could be detected, and therefore what was
used was a 2.6 kb BamFiI fragment that contained the
Neuros~ora crassa gdh gene. This is a large fragment of DNA,
and is certainly not the optimal size fragment. Ideally, one
wants to use as a probe a highly homologous fragment of DNA,
no more than 200-300 by long. Here a much larger fragment
(2600 bp) with, undefined homology was used. Yet the present
inventors managed ~to clone a sequence that was later on
proven to be the gdh from Aspercrillus awamori.
The accompanying examples also describe the application of
the above described novel promoters~and DNA constructions to
the expression of the recombinant protein thaumatin in the
filamentous fungus Asperaillus awamori. As shown in these
examples, ~anc1 as illustrated graphically in Figure 12, the
expression system of the present invention offers several
advantages over the prior art systems. On the one hand, it
allows to reach concentrations of expressed protein of about
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100 mg/1, which are one order of magnitude higher than the
best described (for example, using the process described in
EP 684312, concentrations of about 5-10 mg/1 are attained;
see I. Faus et al., Appl. Microbiol. Biotechnol., 1998, vol.
49, pp. 393-398). On the other hand, for a same carrier
protein and a same fermentation time, the use of the
promoter of the present invention . leads to higher
concentrations of expressed protein. And last but not least,
with the constructions of the present invention it is
possible to use a more economical nitrogen source (ammonium
sulfate) than the one that is commonly used (asparagine).
DEFINITIONS
The term "promoter" means a DNA sequence operative in a
filamentous fungus capable of promoting transcription of a
coding region when operatively associated therewith.
The term "recombinant protein" means a protein that is not
expressed under standard normal conditions by the host, and
that is only expressed by the host as a result of the
introduction into said host of a DNA sequence that allows
for the expression of said recombinant protein. This
recombinant protein can be fungal or non-fungal, and it can
even be found in the'non-recombinant host.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1, parts A, B and C. Schematic representation of the
steps involved in the construction of the B2KF~ expression
cassette.
Figure 2. Restriction map of a 28.7 kb region of A. awamori
DNA including the gdhA gene. Map of phages FAN1 and FAN2.
Thick lines indicate the overlapping zone between the two
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phages containing the gdhA gene. pBlO, pB5.5 and PB2.7
indicate the DNA fragments subcloned in the corresponding
plasmids . B = BamFiC, S = Sal I .
Fiaure 3. Restriction map of the 2.1 kb XbaI-BamHI fra~nent
from pB5.5 plasmid~that was sequenced. The 3' end of the gdhA
gene was contained in the left region of the insert in pBl.7.
B = BamHI, E = EcoRI, EV = EcoRV, P - PstI, S - SalI, X -
Xbal .
Fiaure 4, parts A and B. Alignment of the deduced amino acid
sequences of NADP-specific glutamate dehydrogenases of A.
awamori, A. nidulans (Genebank accession number P18819), N.
crassa (P00369), S. cerevisiae (P07262), S. occidentalis
(P29507), ~ bisporus (P54387), ~ typhimurium (P15111), E.
coli (P00370) and ~ g~lutamicum (P31026). Identical amino
acids ~ are shadowed. Motifs a-i with several consecutive
conserved residues are overlined.
Fiaure 5. Complementation of the gdhA mutation in two strains
of A. nidulans with the gdhA gene of A. awamori. Part A: 1,
nidulans A686 mutant; 2, transformant A686-4; 3,
transformant A686-6; 4, transformant A686-7. Part B. 1, A.
nidulans A699 mutant; 2, transfomant A699-2; 3, transformant
A699-3; and 4, transformant A699-4.
Fiaure 6. Primer extension identification of the 5' end of
the gdhA gene transcript. One protected band (arrow) is
observed in the lane corresponding to the extension reaction
(lane Pe). G, A, T, C lanes correspond to the sequencing
reactions of M13 phage from the -40 primer.
Figure 7. Northern blot analysis of the transcripts of the
gdhA and .!~-'actin genes . A: hybridization with a probe
3 5 internal to the gdhA gene ( 0 . 694 kb PvuII f ragrnent ) . B
hybridization with the f5-actin gene of A. nidulans as
control.
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Figure 8. Slot Blot analysis of the trancript of the A.
awamori gdhA gene, during the course of a fermentation in
1~FA medium with 1~ glucose and 10 mM ammonium sulfate (part
A). For comparative purposes, the transcript of the f3-actin
gene in the same RNA sample was also studied. Part B:
relative level of the expression of the gdhA to the i3-actin
gene. Part C: NADP-dependent glutamate dehydrogenase activity
in the same cultures from where the mRNAs were extracted.
Ficrure 9. Slot Blot analysis of the transcript ~of the A.
awamori gdhA gene during the course of a fermentation in I~mFA
medium with different nitrogen sources (part A). The medium
contained ammonium sulfate 10 mM as a control and glutamic
acid, glutamine, sodium nitrite, sodium nitrate and
asparagine as nitrogen source, all of them at a concentration
of 10 mM. The transcript of the iS-actin gene was also studied
for comparative purposes. Part B: Relative level of
expression of the gdhA to the Q-actin gene.
Ficture 10, parts A, B and C. Schematic representation of the
steps involved in the construction of the GDH expression
cassette.
Figure 11, parts A and B. Schanatic representation of the
steps involved in the construction of the GPD expression
cassette.
Figure 12. Production (expressed as concentratin CT of
secreted protein in mg/1) of thaumatin from ~ awamori
strains TB2b1-44 and TGDTh-4 in fermentor studies. The medium
used was NmFA supplemented with the components described
below. Empty squares: Strain TB2b1-44; 6.0~ sucrose, pH 6.2,
fedbatch with asparagine. Empty circles: TB2b1-44, 6.0~
sucrose, pH 6.2, fedbatch with ammonium sulfate. Filled
triangles: Strain TGDTh-4; 6.0 ~ sucrose, pH 6.2, fed-batch
with ammonium sulfate.
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DETAILED DESCRIPTION OF ONE MODE OF CARRYING OUT THE
INVENTION
This section describes the application of the new promoter
and constructions described in the present invention to the
preparation of recombinant thaumatin. The teachings of the
examples below can be applied to the expression and
production of any other recombinant protein and thus these
examples should not be construed as limiting the scope of the
present ir~vention in any way.
A: CONSTRUCTS:
The starting point for all of the constructs that have been
prepared in the present patent application is plasmid pThIX,
which is described in European patent application EP 684312.
This plasrnid contains: (i) a sulfanilamide resistance marker;
(ii) a DNA sequence which encodes a fusion protein comprising
in.his turn (a) the synthetic gene encoding thaumatin II, (b)
a spacer sequence which in turn contains a KEX2 processing
sequence,--and (c) the complete glucoamylase gene (genomic) of
~speraillus nicer; (iii) the signal sequence ("pre") and the
"pro" sequence of the glucoamylase gene (glaA) of Aspercxillus
n~, and finally (iv) the promoter region sequence of the
glucoamylase gene (glaA) of ~.spergillus niQer.
In the context of the present invention three new expression
cassettes were prepared, which contained: (i) a drug
resistance marker (most of the times .it was a phleomycin
resistance marker); (ii) a DNA sequence which encodes a
fusion protein comprising in his turn (a) the synthetic gene
of thamnatin II, (b) a spacer sequence which in turn contains
a KEX2 processing sequence, and (c) a cDNA sequence that
encodes most of the B2 protein (except sequences in the COON
end) from Acremonium chrvsoaenum; (iii) the signal sequence
of the B2 gene of Acr~nonium chrysocrenum and (iv) three
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1~
different promoter regions.
In all the cloning and sub-cloning manipulati~s described in
this patent application, Escherichia coli DFi5a served as the
recipient strain for high-frequency plasmid transformation.
E. coli WK6 was used as host for obtaining single-stranded
DNA from pBluescript plasmids for sequencing purposes.
A1. Construction of the expression cassette B2KEX
Protein B2 is an extracellular protease produced by the
filamentous fungus Acrgnonium chrvso eq num. This protein is
expressed and secreted in the late stages of growth of
Acrgnonium_ chrvsoaenum (between 120 and 144 hours after the
start of growth).
Plasmid pJElA (Laboratory of Prof. Juan-Francisco Martin,
Universidad de Leon, Leon, Spain) contains the promoter
region, leader peptide (including the signal sequence) and
coding region of the B2 gene from Acremonium chrysoaenum. The
gene itself has 1298 base pairs and two introns. These two
introns are not present in the sequence that has been
subcloned in pJElA, since these subcloned sequences were
obtained from a cDNA. Upstream from the ATG start point of
translation there is a prompter region of 477 base pairs.
When Acranonium chrys~ oaen,~m is grown in a defined medium
which contains sucrose and glucose as carbon sources and
asparagine as nitrogen source, the gene is expressed at its
highest levels between 72 and 96 hours of growth.
The steps involved in the construction of the B2KEX cassette
are detailed in Figure 1 (parts A=C). Plasmid pJElA was
digested sequentially with BamHI arid NcoI, releasing a 560 by
fra~nent that was purified from a 0.8~ agarose gel. This
fragment contains most of the coding region of the B2 gene,
but excludes the active center of the protein. Similarly,
plasmid pJL43b (J.L. Barredo, Ph.D. Thesis, Universidad de
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Leon, Leon, Spain) was also digested with BamHI and NcoI,
releasing a large fragment (3740 bp), which was purified from
a 0.8~ agarose gel. This fragment was ligated with the 560 by
BamHI-NcoI fra~nent from pJElA, yielding plasmid p43)aB2CT
(4300 bp).
Plasmid p43bB2CT was digested with NcoI, treated with the
Klenow fragment of DNA polymerase I (in order to obtain
blunt ends) and then digested with Stul, yielding a fragment
of 3874, by that was also purified from a 0.8~ agarose gel.
The single-stranded oligonucleotides ThS1 and ThS2 (sequences
shown below) where used, using plasmid pThIX as a template,
to amplify by polymerase chain reaction (PCR) the KEX2-like
and thatmnatin sequences present in pThIX. The first 18
nucleotides present in ThSl correspond to the KEX2-like
sequence.
ThSl: 5 ' - ~ 'A ~ AAA AC' A~1A A~ ATGGCCACCT'ICGAG - 3 '
Arg Met Lys Arg Lys Arg
ThS2: 5'- TTA TTA GGC GGT GGG GCA - 3'
A 655 by DNA fragment was obtained by PCR using plasmid pThIX
as the template and ThSl and ThS2 oligonucleotides as
primers. This DNA fragment was ligated with the previously
obtained fragment from p43bB2CT, yielding plasmid p43bB2CTTh.
This plasmid (aprox. 4530 bp) contains part of the B2 protein
gene fused to a KEX-2 sequence and to the synthetic gene
encoding thaumatin II. The transcription termination signal
present in this construct is the terminator sequence from the
cycl gene of Saccharornyces cerevisiae.
Plasmid p43bB2CTTh was digested with BamI~, treated with calf
intestinal- alkaline phosphatase (CIP) and purified from a
0.8~ agarose gel. A 900 by BamKt-BamHI fragment from pJElA
was also isolated. Subsequent ligation of these two DNA
fragments generated plasmid pB2KEX (5430 bp). The 900 by
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BamHI-BamHI fragment from pJElA contains the B2 gene promoter
sequence (477 bp), the leader peptide sequence (318 bp) and
107 by of the amino terminal sequence of the B2 gene.
Plasmid pB2KEX was then digested with XbaI, treated with the
Klenow fragment of DNA polymerase I (in order to obtain
blunt ends) and then digested with Sall, yielding a fragment
of 2400 by that was purified in a 0.8~ agarose gel. Plasmid
pJL43b was digested with HindIII, also treated with the
Klenow fragment of DNA polymerase I, and then digested with
XhoI. A fragment of 4500 by was purified as before. Finally,
the two gel purified fragments described above were ligated,
generating plasmid pB2KTh (6900 bp; Fig. 1C).
On the final sub-cloning step, both plasmids pB2KTh and
pJL43b1 were digested with SacI and StuI, yielding fragments
of 5714 and 1305 bp, respectively, which were purified in a
0.8~ agarose gel. These two fragments were then ligated, thus
obtaining plasmid pB2KThb1 (7020 bp; Fig. IC). Plasmid
pJL43b1 is a derivative of plasmid pJL43b, where the promoter
that drives expression of the phleomycin resistance gene
(PpcbC from Penicillium chr~ocrenum) was substituted by the
glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter from
Asperaillus ~nidulans (P. Punt et al., gene 1990, vol. 93,
pp.101-109).
This plasmid contains a cassette to express thaumatin that
comprises: (i) a phleomycin resistance marker; (ii) a DNA
sequence which encodes a fusion protein comprising in his
turn (a) the synthetic gene of thaumatin II, (b) a spacer
sequence which in turn contains a KEX2 processing sequence,
and (c) a cDNA sequence that encodes most of the B2 protein
(except sequences in the COOH end) from Acremonium
chrysoc~enuiin; ( iii) the signal sequence of the B2 gene of
Acremonium chrvso eq num and (iv) the promoter region of the B2
gene of Acremonium chryso e~. In this particular
construct, expression of the phleomycin resistance gene
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(phleo) is driven by the promoter of the glyceraldehyde-3-
phosphate dehydrogenase gene from Asperaillus nidulans.
A2 Construction of the expression cassette GDHTh
A 2.1. Cloning of a DNA fracanent of Asperaillus awamori
containing the qdhA gene.
~ awamori ATCC 22342 was used as the source of DNA and
RNA. A. nidulans mutants A686 (gdhAl, yA2, methH2, galA1)
and ~ nidulans A699 (gdhAl, biA1) (J. R. Kinghorn, J.A.
Pateman, J. Gen. Microbiol. 1973, vol. 78, pp. 39-46) were
obtained from the Fungal Genetics Stock Center, and were used
for complementation studies with the gdhA gene from A.
awamori. The partial glutamate ~ auxotrophy of these two
strains was confirmed by growth on media with glutamic acid
or high ammonium sulfate concentrations (100 mM) as nitrogen
source. Both gdhA mutants grow very poorly under high
ammonium sulfate concentrations but show normal growth when
glutamic acid is used as nitrogen source. E. coli NM539
served as host for Lambda GEM12 (Prcgnega Co., Wis) phage
derivatives.
Filamentous fungi were routinely maintained on solid Power
sporulation medium (F. Fierro et al. , Appl. Microbiol . .
Biotechnol. 1996, vol-. 43, pp. 597-604) at 30pC for 3 days.
A. awamori and A. nidulans seed cultures in CM medium
(containing 20 g/1 malt extract; 5 g/1 yeast extract; 5 g/1
glucose) were inoculated with 106 spores/ml and grown at 28gC
in a rotary G10 incubator (New Brunswick Scientific, New
Brunswick, N.J.) for 48 h. For gdhA transcript isolation and
characterization studies, A. awamori cultures in MDFA medium
(Y. Q. Shen et al., J. Antibiot. 1984, vol. 37, pp. 503-511)
were incubated with a 15 ~ seed culture and grown at 30gC for
48-72 h in a rotary shaker, as described above.
A.2.1.1. Asperaillus awamori aen~nic library
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A genomic library of total DNA of A. awamori ATCC 22342 was
constructed in a Lambda GEM12 phage vector. Total DNA was
extracted and partially digested with Sau3AI to obtain DNA
fragments of between 17 and 23 kb. This DNA was purified by
sucrose-gradient centrifugation, ligated to Lambda GEM12
phage arms, and packaged in vitro using a Gigapack III Gold
packaging system (Stratagene) resulting in a total of 8x104
reccenbinant phages .
In the next step, and using as probe a 2.6 kb BamHI fragment
containing the gdhA gene of Neurosgora crassa (J. H. Kinnaird,
J.R.S. Fincham, Gene 1983, vol. 26, pp. 253-260), two phages,
FANl and FAN2, that gave a clear hybridization signal were
isolated and purified by three rounds of infection.
Restriction mapping of these two phages showed that they
overlap in 7.2 kb. The total DNA region cloned in the two
phages extended for 28.7 kb.
BamHI fragments of 1.7, 5.5 and 10 kb were subcloned in
pBluescript KS+ plasnid, giving rise to plasmids pBl.7, pB5.5
and pBlO,-as shown in Figure 2. They were then sequenced by
generating ordered sets of deletions with the Erase-a-base
system ( Prcanega Co . , Wis . ) by digestion with exonuclease III
from appropriate ends, followed by removal of single-stranded
DNA with S1 exonuclease. Sequencing of fragments of the gdhA
gene was performed by the dideoxynucleotide chain termination
method. For sequencing the cDNA clones containing the intron-
exon junctions, reactions were performed with 90 ng of dsDNA
using the GeneAmp PCR 2400 system coupled to the ABI-PRISM
310 autcenatic sequencer (Perkin Elmer). Computer analysis of
nucleotide and amino-acid sequences were made with the
DNASTAR software (DNASTAR, Inc., UK).
Initial sequencing showed that an open reading frame (ORF1)
occurred in the right end of the 5.5 kb insert of pB5.5
extending into the left region of the 1.7 kb BamHI fragment
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of pB1.7 , as shown in Figure 3 . The 5 . 5 kb and 1. 7 kb BamFiI
fragments were mapped in detail.
A 2.1 kb Xbal-Xbal fragment corresponding to the right end of
5 plasmid pB5.5 was subcloned in pBluescript SK+ plasmid,
creating plasmid pBSGh. More specifically, this 2.1 Kb XbaI-
XbaI fragment was generated by digesting pB5.5 at an internal
Xbal site and at a second XbaI site in the polylinker of
pBSKS+ (and close to the BamHI site shown in Fig. 3).
A region.~.of 2570 nt was sequenced in both strands by the
dideoxynucleotide chain termination method. This region
contained ORF1 (1380 bp), which started at an ATG located 740
by downstream from the left end of the insert in pBSGh and
extended until the end of the 5.5 kb BamHI fragment, with 60
additional by into the adjacent 1.7 kb fragment. ORF1 was
preceeded by a 740 nucleotide region that contained the
necessary signals required for transcription initiation and
regulation (see SEQ. ID No. 1).
ORF1 contained two putative introns at positions 785-850 and
1414-1471 (following the numbering in SEQ ID No. 1) that
showed lariat and 5' and 3' splicing sequences similar to
those of other fungal introns (D. J. Ballance, Yeast 1986,
vol. 2, pp. 229-236). The presence of the two introns was
confirmed by sequencing the DNA regions corresponding to
introns I and II obtained by PCR from a A. awamori cDNA
library using as primers oligonucleotides IA and I$ for intron
I, and IIA and II$ for intron II (sequences shown below).
cDNA for these experiments was obtained from total RNA
extracted as described above, from mycelia grown for 48 h in
MDFA medium. The first and second cDNA strands were
synthetized.using a cDNA synthesis kit from Stratagene (La
Jolla, Ca) . This cDNA was used for PCR amplification of the
fragments containing the exon-exon junctions by the following
program: 1 cycle at 94~C for 5 min, 50~C for 1 min, 72pC for
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1 min followed by 30 cycles at 94~C for 1 min, 504C for 1
min, 724C for 1 min and finally one cycle at 72qC for 8 min.
Oligonucleotides:
10
IA 5 ' TCT AAC CTT CCT CAC 3 '
ATG
IB 5' CTT ACC ACC ACC CAT 3'
ACC
IIA5' TTC TGT GTT TCC TTC 3'
CGC
IIB5 ' CTT GAA CTT GTT GGC 3 '
GTA
A RF1 encodes a puta tive NADP-dependent glutamate
2.1.2.
~b
dehydrocrenas a
ORF1 encoded a protein of 460 amino acids (see SEQ ID No. 2)
with a deduced molecular mass of 49.4 kDa and a pI value of
5.62. Comparison of the protein encoded by ORF1 with other
proteins in the SWISS-PROT data base showed that the encoded
protein has a high homology with NADP-dependent glutamate
dehydrogenases of A. nidulans (84.7 of identical amino
acids), N. crassa (74.4 identity), Saccharomyces cerevisiae
(66.5 identity) and Schwanniomvces occidentalis (66.9
identity); as shown in Figure 4. The homology is extensive
throughout the entire protein. All these proteins are NADP-
dependent glutamate dehydrogenases that catalyze the
reductive amination of a-ketoglutarate, in the presence of
ATP, to form. L-glutamate. The protein encoded by ORF1
contains nine conserved motifs when compared with other
fungal and yeast glutamate dehydrogenases. One of the
conserved domains (amino acids 108-121) corresponds to a
region implicated in the catalytic mechanism of the enzyme.
The consensus sequence of this region is ~ [LIV] -X ( 2 ) --G-G-
[SAG]-K-X-[GV]-X(3)-[DNS]-[PL] (PROSITE PS00074). The lysine
residue K11° located in the glycine-rich region GGGK11°GG
corresponds to the lysine in the active center of
Glu/Leu/Phe/Val (GLFV) dehydrogenases. Therefore, following
standard fungal gene nomenclature, the gene encoded by ORF1
was named gdhA.
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1 3 The cloned gene complements A nidulans adhA mutants
A. nidulans A686 and A699 strains were transformed by a known
method (M.M. Yelton et al . Proc. Natl. Acad. Sci . USA 1984,
vol. 81, pp. 1470-4) with plasmid pGDHaw (7.1 kb), which
contains the ~ awamori gdhA gene in a 2570 by Xbal-XbaI
fragment. This fragment contains also an upstream promoter
region of 740 by and a 322 by region downstream from ORF1
(gdhA gene). The 2570 by XbaI-XBaI fragment was inserted into
the XbaI ;site of the fungal vector p43gdh, which contains
the phleomycin resistance marker under control of the A.
awamori gdhA prompter as shown later in this patent
application.
Seven transformants of A. nidulans A686 with the ~ awamori
gdhA gene and 15 transformants of A. nidulans A699 were
analyzed on minimal medium supplemented with different
concentrations (10, 50 and 100 mM) of ammanium sulfate as
nitrogen source, and their growth was compared with that of
wild type A. nidulans. As a control, growth was also tested
on medium -containing 10 mM glutamic acid. As shown in Figure
5, the untransformed ~ nidulans mutants A686 and A699 grow
very poorly in plates with 100 mM ammanium sulfate, whereas
three randomly selected transformants grow very well in this
medium. The residual growth of A. nidulans gdhA mutants A686
and A699 in ammanium sulfate as nitrogen source is known
(J.R. Kinghorn, J.A. Pateman, Heredity 1973, vol. 31, pp.
427) and is due to the presence of a sec and glutamate
dehydrogenase activity that allows partial growth of these
mutants .
A 2 1 4 Glutamate dehydrog~enase activity in the
transformants
Nicotinamide adenine dinucleotide phosphate (NADP)-specific
glutamate dehydrogenase (NADP-GDH) activity was assayed by
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following the reductive amination of a-ketoglutarate in the
presence of ammonium and NADPH and expressed as units of
enzyme activity per mg protein. The initial reaction velocity
was estimated from the change in optical density at 340 nm in
a Hitachi U-2001 spectrophotometer. One unit of glutamate
dehydrogenase was defined as the activity that catalyzes the
oxydation of one nancmol of NADPH per minute.
To confirm the complementation results, the NADP-dependent
glutamate. dehydrogenase activity was measured in the
nidulans ~dhA mutants A686 and A699, and in three randomly
selected transformants complemented with the ~ awamori gdhA
gene. Results are shown in Table 1 and they clearly indicated
that while the glutamate dehydrogenase activity in strains
A686 and A699 was clearly belay the detection levels,
significant levels of glutamate dehydrogenase activity were
obtained in the transformants with the A. awamori gdhA gene,
particularly at 24 and 48 h of growth. Some of the
transformants, like A699-4, sho~n~ed relatively high levels of
glutamate dehydrogenase activity, perhaps due to integration
of more than one copy of the gdhA gene in the gencene of this
transformant.
Table 1: NADP-dependent glutamate dehydrogenase
activity (U/mg of protein), in the A. nidulans gdhA mutants
A686 and A699, and in three transformants of each of these
mutants with the ~ awamori gdhA gene.
strain t = 24 h t = 48 h t = 72 h
A. Awamori 550 0' 0
A686 0 0' 0
A686-4 350 280 100
A686-6 340 200 80
A686-7 310 160 90
A699 0 0 0
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A699-2 270 240 100
A699-3 410 420 400
A699-4 500 670 580
A 2 1 5 Characterization of the x~romoter region of the crdhA
gene
Analysis of the nucleotide sequence upstream from the ATG
translation initiation codon revealed the presence of GTATA,
CTATA and:. TCAATC sequences at positions -316, -61 and -17,
respectively, with respect to the translation initiation
codon, which may correspond to putative TATA and CAAT boxes
involved in regulation of gene expression (see SEQ ID No. 1).
Identification of the transcription start point was performed
by "primer extension" with 2 ~..t,g of mRNA obtained from mycelia
gro~nm in 1~FA for 48 h, as shown in Figure 6.
Primer extension analysis using as primer the oligonucleotide
"Pe" 5'-GGGGTTCTTCTGGAAGAGGGT-3' (corresponding to the
nucleotide sequence 70 by downstream from the ATG) revealed a
single band in the extension reaction (Fig. 8). The 5'-end of
the mRNA corresponds to a thymine (T) located 86 by upstream
of the ATG initiation codon.
A 2 1 6 The crdhA gene is transcribed as a monocistroni~
transcript of 1 7 kb and its e~p~ession is regulated by
nitrocren .
In order to perform expression studies, total RNA of A.
awamori was obtained by the phenol-~SDS method from mycelia
grown for 12, 24, 48, 60 or 72 h iii 1~FA medium with 55.5 mM
glucose and 10 mM ammonium sulfate as carbon and nitrogen
sources, respectively. For nitrogen regulation studies, the
NmFA base medium (without ammonium sulfate) was supplemented
with glutamic acid, L-glutamine, sodium nitrite, sodium
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nitrate and L-asparagine at 10 mM final concentrations.
For Northern analysis, total RNA (5 l.i.g) was run on a 1.2~
agarose-formaldehyde gel. The gel was blotted onto a nylon
5 filter (NYTRAN 0.45; Schleicher and Schuell) by standard
methods. The RNA was fixed by W irradiation using an W-
Stratalinker 2400 lamp (Stratagene, La Jolla, Calif.).
For slot blotting, the RNA ~(5 ~,.t,g) was loaded on a filter
10 (NYTRAN~ 0.45) by vacuum in a Bio-Dot SF Microfiltration
apparatus:(Slot Blotting, Bio-Rad). The RNA was fixed by W
irradiation as above. The filters were pre-hybridized for 3 h
at 42 gC in 50~ form~nicle, 5 x Denhardt' s solution, 5 x SSPE,
0.1~ SDS, 500 ~Lg of denatured salmon-sperm DNA per ml, and
15 hybridized in the same buffer containing 100 ~.g of denatured
salmon-sperm DNA per ml at 42~C for 18 h, using as probe an
internal DNA fra~nent (0.694 kb PvuII) of the A. awamori gdhA
gene. The filters were washed once in 2 x SSC, 0.1~ SDS at
42~C for 15 min, once in 0.1 x SSC, 0.1~ SDS at 42~C for 15
20 min, and once more in 0.1 x SSC, 0.1~ SDS at 55qC for 20 min
and then autoradiographed with Amersham X-ray film. mRNA was
purified from total RNA by using the Poly(A) Quick mRNA
isolation kit (Stratagene, La Jolla, Calif.).
25 Northern analysis of the transcription of the gdhA gene
revealed that it is strongly expressed as a 1.7 kb transcript
(mRNA) with a size slightly larger than that of the i~-actin
gene mRNA, as shown in Figure 7. Since ORF1 contains 1380 nt,
this size of the transcript indicates that the gdhA gene is
expressed as a monocistronic transcript.
Since the same amount of total RNA was used in all lanes of
Fig. 7, it was concluded that the gdhA steady state
transcript'levels in the cell are higher than those of the f~-
actin gene (arrows) indicating that the glutamate
dehydrogenase A is expressed from a very efficient promoter.
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To determine the pattern of expression of the gdhA gene
during the time-course of growth of A. ~wamori, gdhA
hybridizing RNA was compared to f3-actin hybridizing RNA in
NmFA medium with ammonium sulfate (Figure 8A) and expressed
as the ratio of counts in the gdhA-hybridizing band to the i3-
actin hybridizing counts (Figure 8B). Results indicate that
expression of both genes (gdhA and f5-actin) is associated
with the growth of A awamori but whereas low steady state
levels of i3-actin mRNA remained in the cells until 96 hours
of growth, the levels of glutamate dehydrogenase mRNA
decreaseddrastically after 48 hours.
The glutamate dehydrogenase enzymatic activity detected when
~ awamori is groHm in NmFA medium with ammonium sulfate (10
mM) as nitrogen source at different times of the culture is
shown in Fig. 8C. There is a sharp decrease in glutamate
dehydrogenase activity between 24 and 48 h after start of
growth, which is in good agreement with the decrease in
transcript levels at this time of the culture, as shown in
Fig. 8B.
Since glutamate dehydrogenase plays a central role in
nitrogen utilization by ~ awamori, it was also of interest
to study if expression of gdhA was regulated by different
nitrogen sources. As shown in Figure 9, very high gdhA
transcript (mRNA) levels were obtained in media containing
NH4~, or asparagine as sole nitrogen sources . Glutamic acid
repressed transcription of the gdhA gene, whereas
intermediate levels of expression (normalized with respect to
the f3-actin gene) were observed in media that contained
nitrate, glutamine or nitrite as nitrogen source. These
results show that the NADP-dependentglutamate dehydrogenase
is subject to a strong nitrogen regulation at the
transcriptional level.
The glutamate dehydrogenase activity in 24-hour cultures
grown in I~FA medium containing different nitrogen sources,
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all at a concentration of 10 mM, is shown in Table 2. The
highest activity (per ml of culture) was observed in cultures
with NH4+ or asparagine as nitrogen sources. Moreover, these
two nitrogen sources favoured a strong growth of A. awamori.
When the results were expressed per mg of protein in the cell
extracts, the highest specific activity was observed in MDFA
medium with nitrate as the sole nitrogen source. This is due
to the fact that in the presence of nitrate, A. awarnori grows
very slowly. The lowest activity was observed in MDFA medium
with glutamate as nitrogen source, confirming the results
observed previously at the transcription level.
Table 2: NADP-dependent glutamate dehydrogenase activity in
~ awamori cultures grown for 24 h in MDFA medium
supplemented with different nitrogen sources.
Nitrogen source Total Activity Specific Activity
(lOmM) (U/ml) (U/mg protein)
_______________ ______________ _________________
ammonium 1450 800
glutamic acid 330 280
glutamine 1100 600
nitrite 990 660
nitrate 1150 ~ 1680
asparagine 1300 720
A.2.2. Construction of the expression cassette GDHTh
Once the promoter region of the gdhA gene was located, a
thaiunatin expression cassette similar to 'the one described
previously was constructed. Plasmid pBSGh was used as a
template to obtain a 750 by DNA fragment corresponding to, the
promoter region of the gdhA gene. This fragment was obtained
by DNA amplification using the oligonucleotides gdhl and gdh2
and the Pfu enzyme (Stratagene) .
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28'
gdhl : 5 ' - TTTT GTC TTG CGA CGG CGT ATT GCT - 3 '
Sal I
gdh2 : 5 ' - TTTT CCA~ TCT GAA GGG GAG GAT TGA - 3 '
NCO I
This amplified DNA fragment was digested with SalI and NcoI
and purified in a 0.8~ agarose gel.
Plasmid pJL43 (a derivative of pJL43b, Dr. Jose Luis Barredo,
Ph.D. Thesis, Universidad de Lebn, Leon, Spain) was digested
with Sall and NcoI and a large fragment (3740 bp) was
purified in a 0.8~ agarose gel. This DNA fragment was then
ligated with the SalI-NcoI fragment previously amplified,
yielding plasmid p43gdh (4500 bp), where the pcbC promoter
from Penicillium chrysoaenum has been replaced by the gdhA
promoter from As~aillus awamori.
In the next step, plasmid p43gdh was digested with NcoI,
treated first with the Klenow fragment of DNA polymerase I
and then with calf-intestinal phosphatase (CIP). In paralell,
a fragment of 1140 by containing the B2 protein gene was
amplified via the PCR technique, using plasmid pJElA as the
template and oligonucleotides NTB2b and CTB2b as primers
(sequences given belcaa) . This 1140 by fragment was digested
with BamHI and then treated with the Klenow fragment of DNA
polymerase I. From this reaction mix a 425 by DNA fragment
containing the amino terminal sequences of the B2 gene was
purified from a 1.0 g agarose gel. This fragment of DNA was
ligated by blunt-end ligation to the fragment of DNA from
p43gdh previously described, resulting in plasmid p43gdhB2,
where the BamHI site that is shown in Figure 10 has been
regenerated. This plasmid is 4925 by long and contains the
gdhA promoter fused "in frame" to the amino terminal portion
of the B2 gene.
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The next step in the construction of the complete expression
cassette was the addition of the second portion of the B2
gene, the KEX2 sequence and the synthetic thatunatin II gene.
For this part of the work, plasanid pB2KEX was used.
pB2KEX was sequentially digested with XbaI, treated with the
Klenow fragment from DNA polymerase I and finally digested
with BamHI . A fragment of 4637 by was purif ied from a 0 . 8~
agarose gel. In paralell, plasmid p43gdhB2 was sequentially
digested with SalI, treated with the Klenow fragment from DNA
polymerase I and finally digested with BamHI. A fragment of
1173 by was purified from a 0.8~ agarose gel. The ligation of
these two fragments yielded plasmid pGDHTh (5810 bp), where a
new SalI site was created. This allows for the excision of
the complete GDHTh cassette as a 2670 by SalI-SalI fragment.
Starting with plas~ntid pGDHTh, two new plasmids were
constructed. The first one was p43GDTh, constructed as
follows. Plasmid pJL43 was linearized by digestion with SalI
and ligated to a 2170 by SalI-DraI fragment from pGDHTh (see
Fig. 10, part B) .
Similarly, plasmid pGD71 was constructed as follows: plasmid
pAN7-1 (P.J. Punt et al., J. Biotecnol. 1990, vol. 17, pp.
19-34) was sequentially digested with Xbal, treated with the
Klenow fragment from~DNA polymerase I, and finally digested
with HindIII, and purified from a 0.8~ agarose gel. In
paralell, plasmid pGDHTh was digested with Ec1136II (or
SacI*, a variant of SacI from Fermentas that recognizes the
standard SacI restriction site but leaves a blunt end),
HindIII and DraI. A fra~nent of 2175 by was purified from an
agarose gel. Ligation of these two fra~nents yielded plasmid
pGD71 (see Fig. 10, part C).
Plasmids p43GDTh and pGD71 contain a cassette to express
thatnnatin that comprises: (i) a DNA sequence which encodes a
fusion protein comprising in his turn (a) the synthetic gene
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of thaumatin II, (b) a spacer sequence which in turn contains
a KEX2 processing sequence, and (c) a cDNA sequence that
encodes most of the B2 protein (except sequences in the COOH
end) from Acremonium chrvsogenum; (ii) the signal sequence of
5 the B2 gene of Acremonium chrysoaenum, (iii) the promoter
region from the Aspercrillus awamori glutamate dehydrogenase A
gene, and (iv) a drug resistance gene that can be used as a
transformation marker. Plasmid p43GDTh has the phleomycin
resistance gene (phleo) driven by the the pcbC promoter from
10 Pgnicillium chrysog~enum. Plasmid pGD71 contains the
hygromyciri B resistance gene driven by the glyceraldehyde-3-
phosphate dehydrogenase prompter from Asperaillus nidulans.
A 3. Construction of the expression cassette GPDTh
The expression cassette GPDTh is similar to the expression
cassette B2K~, exc~t that the B2 promoter from Acr~nonium
chrvsogenum has been replaced by the promoter from the
glyceraldehyde-3 phosphate dehydrogenase (named "gpd" from
now on) gene from As~g~illus nidulans.
The complete promoter region of the gpd gene is present in
plasmid pAN52-1 (P.J. Punt et al. , J. Biotecnol. 1990, vol.
17, pp. 19-34). A SacI-NcoI fragment (880 bp) from pAN52-1
has been subcloned, generating pJL43b1.~
Plasmid pJL43b1 was digested with NcoI and treated first
with the Klenow fra~nent of DNA polymerase I and then with
calf-intestinal phosphatase (CIP), as shown in Figure 11. In
parallel, a 1140 by fragment of DNA was obtained by DNA
amplification using the PCR technique, using pJE2A as
template and oligonucleotides NTB2b and CTB2b as primers.
This fragment of DNA was digested with BamHI and treated with
the Klenow fragment from DNA polymerase I, yielding a
fragment of 425 by that was purified from a 0.8~ agarose gel.
The final ligation reaction yielded plasmid pblB2 (see Fig.
11) .
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NTB2b: 5' - ATG CGT GCT GCT ACT CTC - 3'
CTB2b: 5' - CTG GCC GTT GTT GAT GAG - 3'
As with the GDHTh cassette, the next step in the construction
of a complete expression cassette was the addition of the
second portion of the B2 gene, the KEX2 sequence and the
synthetic thatmnatin II gene. For this part of the work,
plasmid pB2KEX was once again used.
pB2KEX was sequentially digested with XbaI, treated with the
Klenow fragment from DNA polymerase I and finally digested
with BamHI. A fra~nent of 4637 by was purified from a 0.8~
agarose gel. In paralell, plasmid pblB2 was sequentially
digested with BamHI and Ec1136II (or SacI*) (leaves blunt
ends), and a 1300 by fragment was purified from a 0.8~
agarose gel. The ligation of these two fra~nents yielded
plasmid pGPDTh (5800 bp).
In the next step, the GPDTh cassette was isolated from pGPIyfh
by digestion with Ec1136II (or SacI*), HindIII and DraI,
yielding a DNA fragment 2800 by long. In parallel, plasmid
pB2KThb1 was sequentially digested with BamHI, treated with
the Klenow fra~nent from DNA polymerase I and finally
digested with HindIII. A 4500 by fragment was isolated from a
0.8~ agarose gel. The plasmid resulting from the ligation of
these two fragments was named pGPThbl.
This plasmid contains a cassette for the expression of
thaumatin that is identical to the expression cassette, B2KEX
except that the prompter from the~B2 gene of Acrgnonium
ch~ysocrenum has been replaced by ~ the promoter from the gpd
gene from Aspercrillus nidulans .
B. Strains used and transformation protocol
Asperaillus awamori strain NRRL312 was obtained from the
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American Type Culture Collection (ATCC). Using standard
mutagenesis tectmiques with nitrosoguanidine (NTG), a
derivative of this strain was obtained, and was named LpR66.
This mutant strain secretes into the growth medium an
inactive exoprotease aspergillopepsin A (named "pepA" from
now on). In all of the transformation experiments that are
described below the strain that was used was Asperaillus
awamori strain LpR66.
The three expression cassettes that have been described
previously were used to transform Asperg'h~lus awam~ri strain
LpR66. _
In all single transformation experiments, the antibiotic
phleomycin was used as the selection marker. Strain LpR66 can
grow in plates that contain 20 Ei.g/ml of phleomycin.
Therefore, all transformants were selected in plates with 25
~.g/ml of the antibiotic. The regeneration medium that was
used is TSAS, which contains 30 g/1 of Triptone-Soja (Difco),
103 g/1 of sucrose and 1.5~ agar (Difco).
The transformation protocol was similar to the one described
by Melton (see above) with some modifications. A plate
containing Power medium was inoculated with 10' spores. This
plate was incubated for 72 hours at 30~C, at which point the
spores were scraped from the plate and were inoculated in 100
ml of CM medium (500 ml shake flask). Incubation was for 16-
18 hours at 250 rpm and 28QC. The mycelium obtained from this
growth was filtered through a 30 ~.m nylon filter (Nytal) and
washed with 10 mM sodium phosphate buffer (pH 5.8) which also
contained 0.6 M magnesium sulfate. One gram of mycelium was
re-suspended in "protoplast buffer" (10 mM sodium phosphate
buffer (pH 5.8) which also contained 1.2 M magnesium
sulfate). An equal volume of buffer containing the enzyme
"Lysing" (Sigma) was added, yielding a final concentration of
3 mg/ml of the enzyme. The mycelium solution was left to
incubate for 3-4 hours at 100 rpm and 30QC until protoplasts
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were formed. Protoplast formation was monitored by visual
inspection using a light microscope. Protoplasts were
filtered, washed and finally resuspended in STC solution, to
a final concentration of 108 protoplasts/ml.
100 ~.l of protoplast solution was mixed with 10-20 ~,g of DNA
and left in ice for 20 minutes. After this time interval, 500
~.1 of PTC were added, and left at room temperature for
another 20 minutes . Then, 600 ~,l of STC medium were added and
the transformation mix was aliquoted in different test tubes.
Finally, the phleomycin antibiotic solution and TSAS medium
that contained agar were added. The contents of the tubes
were gently homogenized and added to TSAS plates that
contained phleomycin. Plates were incubated at 30$C until the
transformants were visualized as individual colonies. TnThen
hygromycin B was used as selection marker, a similar protocol
was used.
The linearization of all the plasmids that have been
described in this work gave a 4-fold increase in the
efficiency of transformation as compared to transformations
performed with plasmids that had not been linearized.
Therefore, in most transformation experiments the plasmids
were used linearized.
Several transformants were obtained and analyzed. Initial
screens were performed in plates containing 25 ~.~.g/ml of
phleomycin. Confirmation screens were then performed using
phleomycin concentrations as high as 200 Ei.g/ml.
Transformants were analyzed by PCR to detect whether the
thaumatin II gene had been incorporated into their gencene
essentially as described (cf. EP 684312). Those transformants
that were positive were then further analyzed for expression
of thaumatin by immunoblot analysis and ELISA (enzyme-linked
immunoassay) also as described (cf. EP 684312).
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,~ Recombinant strains that produce thaumatin
C.1. Materials and methods
C.1_.1. Culture media
CM medium: malt extract, 5 g/1; yeast extract, 5 g/1;
glucose, 5 g/1.
SMM medium: 8~ sodium citrate; 1.5~ (NHa)ZSO4; 0.13
NaHaP04.2Hz0; 0.2~ MgS04.7HZ0; 0.1~ Tween 80; 0.1~ uridine,
0.1~ antifoam AF and 7~ soya milk. The carbon source
(glucose, sucrose, maltose, etc.) is present at a final
concentration of 15~. The pH of the medium is adjusted to 6.2
with HZS04.
MDFA medium: 1.2~ L-asparagine; 0.8~ of salt solution I [2~
Fe (I~i4) Z (S04) 2. 6HZ0] ; and 14 .4~ of salt solution II [10.4
KZHP04; 10.2 KH2POq; 1.15 NaZCuS04.5Hz0; 0.2~MgS04.7H20; 0.02
ZnS04 . 7H20; 0 . 005 CuS04 . 5Hz0; 0 . 05~ CaCla . 2H20] . The carbon
source used was either maltose (usually 6.5~) or a mix of
sucrose (3.6~) and glucose (2.7~). Other amounts of carbon
source are indicated in each experiment that is described.
The initial pH of this medium is 6.5.
C.1.2. Fermentation anal~rsis
Growth and expression studies were conducted in SMM and MDFA
media, first in shake flasks, and later in several fermentors
equipped with measurement and control systems for the
following variables: stirring, dissolved oxygen, pH, antifoam
and culture level.
Experiments were conducted in 1-liter shake flasks with a
working volume of 150 ml. Inoculation was to a final
concentration of 3 x 105 spores/ml. Stirring was at 150 rpm,
and the incubation temperature was 30°C. The media used was
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either SMM or MDFA.
The experiments conducted in the fermentor were analogous to
the ones in shake flasks, exc~t that the pH of the medium
5 was maintained constant at a pre-set value, and adjusted by
the automatic addition of either 30~ NaOH or 0.5N HZS04.
C 1 3 Analytical methods
10 2-10 ml .samples were taken at different times from the
fermentation culture and processed to determine the dry
weight, thaumatin, maltose and glucose concentrations that
were present.
15 Dry weight was determined by passing a sample through a pre-
filter (Nucleopore, Cat.No. 211114). The biological material
retained in the pre-filter was washed with 40 ml of pure
ethanol and 50 ml of distilled water. It was then incubated
at 90°C until a constant weight could be recorded. The
20 filtrate was aliquoted and frozen for further analysis.
Thau<natin concentration in the culture broth was determined
by an enzyme-linked immunoassay (ELISA) and by immunoblotting
(Western blot) analysis, essentially as described (cf . EP
25 684312), using an anti-thaumatin polyclonal antibody. For
immunoblotting, samples were sometimes concentrated as
follows : 500 ).~,1 of filtrate were mixed with an equal volume
of 10~ trichloroacetic acid (TCA), and frozen for 12 h. The
sample was then allowed to regain room temperature and
30 centrifuged in a table-top centrifuge (15,000 rpm; 20 min.
4°C ). The pellet that is recovered contains all the proteins
that were present in this .sample. The pellet was then
resuspended in protein loading buffer, boiled for 5 minutes,
and subjected to SDS-PAGE as described (cf EP 684312).
Approximately 1 ml of filtrate was used for glucose/maltose
determination. Glucose levels were determined using a SIGMA
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DIAG~10STICS kit (Procedure number 510).
Maltose concentration in the culture broth was determined as
follows: 250 ~.l of sample filtrate were placed in a test-tube
that had been previously chilled; 1.250 ml of anthrone
solution (prepared by dissolving 2 g anthrone in 50 ml
absolute ethanol and then adding 950 ml of 75~ HZSOq) were
then added, and the sample was kept chilled for five minutes.
The sample was then transferred to a boiling water bath, and
incubated. for 10 minutes. Finally the samples were once again
chilled end the absorbance read at 625 nm. Maltose
concentrations were determined by comparison to a calibration
curve generated by measuring the absorbance of maltose
solutions of known concentrations (range: 0 - 0.2 g/1).
c' 2 . Thatunat in nroduc incr s trains
r.2.1. Strain TB2b1-44
This strain is a derivative of Lpr66 that was obtained by
transformation of the aforementioned LpR66 strain with the
expression plasmid pB2KTh-b1. This expression cassette
contains the synthetic thaimnatin II gene under the control of
the promoter of the B2 protein from Acremonium chrysoaenum.
In shake-flask cultures with MDFA medium this strain secretes
6-8 mg thaumatin/1.
Further optimization studies were performed in a 5-liter New
Brunswick fermentor. The inoculum was obtained by growing the
strain for 40 hours at 30pC in CM medium. 450 ml of this
inoculum were then used to seed the 5-liter fermentor
(working volume of 4.5 liters). RPMs were between 250 and
500, and varied according to the oxygen status of the system,
which was always set at 30~.
Different parameters were tested, such as the pH of the
medium and the carbon and nitrogen sources. Representative
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experiments are described in Figure 12:
1. Growth in MDFA medium with 6.0~ sucrose and L-asparagine
as the nitrogen source. The set-point for the pH was set at
6.2, and a fed-batch system was installed. Feedings were done
at 36, 48, 60 and 72 hours after the beginning of the
fermentation. In each feeding, 45 ml of a 0.5 g/ml sucrose
solution were added.
2: The conditions were identical to those described under 1
above, but=i L-asparagi.ne was replaced by ammonium sulfate ( the
molar amounts were the same in both experiments) as the
nitrogen source .
The best productivity was obtained with the conditions
described under 1 above, with asparagine as nitrogen source,
and with 6~ sucrose as the carbon source, with four
"feedings" of sucrose every 12 h after 36 h of fermentation.
Under these conditions, yields of 100 mg thaumatin/1 were
obtained.
C . 2 .2 . Strain TGDTh-4
This strain was deposited according to the Budapest Treaty
with Access No. CECT20241 on March 25, 1998 (25.03.98) in the
following institution:
Coleccibn Espar~ola de Cultivos Tipo (CELT)
Edificio de Investigacibn, planta baja, no. 34
Universidad de Valencia
Campus de Burjasot
46100 Valencia, Spain
It is a derivative of Lpr66 which was obtained by
transformation of the aforementioned LpR66 strain with the
expression cassette p43GDTh. This expression cassette
contains the synthetic thaumatin II gene under the control of
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the prompter of the gdhA gene from Aspercrillus awamori. In
shake-flask cultures with NmFA medium (with 6.0~ sucrose)
this strain secretes 6-8 mg thaumatin/1.
Experiments were also conducted in the controlled
environment of a 5-liter New Brunswick fermentor, as
described before for strain TB2b1-44. Ammonium sulfate was
used in place of asparagine as nitrogen source, at the same
molar levels. In this experiment, also shown in Figure 12,
the following conditions were tested: strain TGDTh-4 was
grown Vin. 1~9.~FA medium supplemented with 6~ sucrose and
ammonium sulfate as nitrogen source. The pH set-point was
6.2. and a fed batch system was also installed. Feedings
were done at 36, 48, 60 and 72 h after the beginning of the
fermentation. In each feeding, 45 ml of a 0.5 g/ml sucrose
solution were added.
The results (Fig. 12) indicate that the production of
thaumatin is also in the order of 100 mg/1, but with the
added advantage of having an earlier production and the use
of a more economical nitrogen source. Therefore, it is
concluded that the glutamate dehydrogenase promoter from
Asperaillus awam~ri is more efficient than the B2 protein
promoter from Acranonium ~hrvsoQern~m.
C.2.3. Strain TGP-3
This strain is a derivative of Lpr66 which was obtained by
transformation of the aforementioned LpR66 strain with the
expression cassette pGPThbl. This expression cassette
contains the synthetic thaumatin II gene under the control of
the promoter of the gpd gene from AsperQll~.~is nidulans . In
shake-flask cultures with NB7FA medium this strain secretes 9-
10 mg thaumatin/liter.
x.2.4. Double transformants
Strains TB2b1-44 and TGP-3 were re-transfornned with
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expression plasmid pGD7l, which contains the thaumatin gene
under control,of the glutamate dehydrogenase promoter from A.
awamori and a hygromycin B resistance gene as a selection
marker for transformation experiments. A battery of different
transformants (see Table 3) was analyzed in shake flask
experiments. It was shov~m that re-transformation of strain
TGP-3 did not result in better producing strains. However,
re-transfornnation of TB2b1-44 did result in better producing
strains when cultured in shake-flasks under the standard
conditions mentioned before.
Table 3: Production of thawnatin in shake flasks by
retransformed strains grown in NmFA medium for 96 h.
Quantification by ELISA. All strains were retransformed using
hygromicin B resistance as selection marker.
Transformant Production (mg/1) Original strain
TGP3-GD1 2.08 TGP3
TGP3-GD2 0.40 TGP3
TGP3-GD3 9.44 TGP3
TGP3-GD4 8.25 TGP3
TGP3-GD5 0.40 TGP3
TGP3-GD6 9.71 TGP3
TB2b1-44-GD1 3.84 TB2b1-44
TB2b1-44-GD2 0. 00 TB2b1-44
TB2b1-44-GD3 9.85 TB2b1-44
TB2b1-44-GD4 11.10 TB2b1-44
TB2b1-44-GD5 11.82 TB2b1-44
TB2b1-44-GD6 10.75 TB2b1-44
TB2b1-44-GD7 10 . 52 TB2b1-44
TB2b1-44-GD8 8.09 TB2b1-44
TB2b1-44-GD9 7 .13 TB2b1-44
______________ _____________________________--___-____
D ~ Purification of recc~nbinant thaimnatin
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Two procedures were employed for the purification of
recanbinant thaumatin. In the first one the fermentation
broth was simply clarified, concentrated and diafiltered,
5 yielding a concentrated and cleaner extract that was used for
sensory experiments to ascertain the sweet profile of the
recanbinant thaumatin. The second procedure involved a
classic purification protocol that yielded pure thaumatin.
10 D 1 Clarification concentration and diafiltration of the
fermentation broth
Biomass was removed by filtration through filter paper. The
filtrate was collected in a filtering flask that was
15 submerged in ice. The clarified broth was then centrifugated
at 6000 rpm for 15 minutes at 4qC.
The clarified fermentation broth was further concentrated by
ultrafiltration using a ProFluxTM M12 Tangential Filtration
20 System. The system configuration was: base unit, level
switch, 2.5 1 reservoir, cooling coil, inlet and oulet
pressure transducers, secondary pump, one Spiral-wound
membrane cartridges S1Y3 (Molecular weight cut-off 3,000
Daltons).
The system was operated as follows: (1) Calibration of the
pressure sensors. (2) Adjustment of alarm set points: low
inlet pressure 3.0 Bars, high inlet pressure 3.5 Bars,
differential pressure 0.3 Bars. (3) Washing of the system
and the cartridges with deoinized, distilled water (4) Fill-
up of the reservoir with process solution; the solution is
kept at 8-10~C by recirculating cold water (HAAKE, DC1-K20
refrigerated circulator) through the cooling coil. (5)
Setting of the level switch at the desired concentration
volume (1/4 to 1/5 of the initial volume) . (6) Operation of
the recirculation pump at 75 ~ . ( 7 ) Adjustment of the Back
Pressure Valve to obtain a 3.0 Bar inlet pressure. If
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necessary, back pressure was reduced during operation.
Once the fermentatian broth was concentrated to the desired
volume, the solution was diafi.ltered in order to remove low
molecular weight solutes (Salts; sugars, etc.).
The system configuration allows the operation in the "pined
diafiltration with autcanatic safety stop" mode. The dialysate
(five volumes of deionized . water) was transferred by the
secondary. pump in steps as directed by the level switch. Once
the dial~sate supply is exhausted, the system and the
secondary pump will shut off autcanatically.
The diafiltered solution is drained from the system,
sterilized by filtration (Stericup, 0.22 Eun, Millipore) and
stored at 4QC.
D 2 Purification of recanbinant ~haumatin to homogeneitv
Reccanbinant thaumatin was purified to homogeneity using a
four step purification scheme that is detailed in Table 4.
The starting point for the particular purification protocol
that is described here are 500 ml of fermentation broth
obtained from the growth of strain TGDTh-4, with thaumatin
present at a concentration of 50 mg/1.
Proteins from this broth were precipitated with ammonium
sulfate (20-50~ range). The precipitate was then re-suspended
in 25 mM phosphate buffer, pH 7Ø
This mix was then passed through a Sephadex G-25 column (for
desalting purposes) and eluted with the same buffer. Fir_ally
the sample was loaded onto a CM-Sepharose column at a flux of
0.5 ml/minute. The column was washed with 25 mM phosphate
buffer, pH 7.0 in order to eliminate proteins in the flow-
through fraction. Thaumatin was eluted with a NaCl linear
gradient ( 0-400 mM) . Thaumatin is eluted from this column in
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almost pure form as detected by Coomassie Blue staining.
Table 4:Purification of thaumatin from the fermentation broth
for growing strain TGDTh-4 in MDFA medium
SAMPLE VOLLaHE CONC. TOTAL YIELD
(ml) (mg/1) (mg) (~)
___________________________________________________________
Broth 500 50 25 100
Ammonium sulfate 11 1745 19.2 76.8
Sephadex G-25 30 596 17.9 71.6
CM-Sepharose 24 704 16.9 67.6
While the foregoing illustrative examples are directed to
the production of recombinant thaumatin, the production of
any other recombinant protein by means of the new
methodology provided in the present invention, particularly
the new promoter and DNA constructions disclosed herein, is
also encompassed by the present invention.
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1
SEQUENCE LISTING
SEO ID 1. Nucleotide sequence of 2570 by of a DNA fragment
present in plasmid~pBSGh, as well as the 5' end from plasnid
pBl.7, which contains the gdhA gene of gi . The
. numbers to the right indicate the numbering of the sequence.
The promoter region of the gdhA gene of $," precedes
the initiating ATG codon at position 742 in the sequence. The
initiation of transcription is at T of position -86 with
respect to the translation initiation triplet (position 655
in the sequence). The part of the gene encoding the protein
begins at position 741. The numbers underneath the amino
acids refer to their numbering. There are a total of 460
amine acids. The two introns present are also showil.
TCTAGATTGC GACGGCGTAT TGCTTATCCT TAGfAGGACT 60
CCGTAA1GGA TTOCGAOCAA
GAAAAGACTG T?1GGCGTG? ACCAATOGCT CATAGTACCA 120
GCAAGAQ1AG AATTTTCTCT
CTCGCTZCGA GAAAGCAATC ARAAAAAAAT CCTATCC1'AC 180
CCTACCCTAC CCTAATACTT
2 CCATTGOCAC CCGATTCCTC CCGATAGfAG AGOGGGOGAC 240
5 TGOCATTTGG CGOGCGGGCC
AGOGGATTCC CGOCGATAGA TAACGGGCAG ATTCTGTGAC 300
CTCAAACTAT CGACTAACAG
CCQ'sAAG2TC GGOGGCCACC GCCAAAOCCG CCOCGGAAGC 360
CGGLCTCATT TGOCGTTTGG
GCGTGCCAGG AAATGCOGCC TGCAGC(A',AG ACTCCCTAG?420
GTOGTC1GTG TTC4rCTGTGT
CG1GTGTGTA GTATACTAGT TACTAGTCTA CTACTGTACA 480
GTGGATGGCC TGAGGGOGGG
3 ACTTTA1GTC CGACTCCGGC TGTTCTOCTC CCTCTATCCA 540
S CTCTACLCTC TTC~CTCTCT
TCTGTCTPTC TCOCCGCTCT CGCCCCTCCC CTOCTCGAAA 600
ACATAAATCG GCCTTTCCCC
CTOGCCATCT TCTTCTTCTT CTOCCTCTCC TTTCTCTTTC 660
40 TTCTTCAGAC TACTTCTCTT
TCTTTCATCT TTTCTCTATA TTOCTGTTTT CCTAGATACC 720
CCAGTTAAAA AAGTTCTCTC
AATCAATCCT CCOCTTCAGA ATG TCT AAC CTT CC? 770
CAC GAG CCC GAG TTC
Met Ser nen Leu Pro His Giu Pro G1u Phe
45
1 s . . io ,
GAG CAG GCC TAC AAG GGTATGTTCC ATTGCCOCTC 825
CGAAATTGAT GAT~GAAAAA
Glu Gin Ala Tyr Lys '-
15
50
AAATTCTAAC AACATCGTCT TACA GAG CTT GCC TCG 876
ACC CTT GAG AAC TCC
Glu Leu Ala Ser Thr Leu Glu Asn 8er
20
55 ACC CTC TTC CAG AAG AAC CCC GAA TAC CGC AAG 924
GCC CTT GCT GTC GTC
Thr Leu Phe Gln Lys Asn Pro Glu Tyr Arg Lys
Ala Leu Ala Val Val
30 35 40
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TCC GTC CCC GAG CGT GTC ATC CAG TTC CGT GTC 972
GTC TGG GAG GAT GAT
Ser Val Pro Glu Arg Val Ile Gln Phe Arg Val
Val Trp Glu Asp Asp
45 50 55
S GCC GGC AAC GTC CAG GTC AAC CGC GGT TTC CGT 1020
GTC CAG TTC AAG AGC
S
er
Ala Gly Aan Val Gln Va1 Asn Arg GIy Phe Arg
Val GIn Phe Asn
60 65 ' 70
GCC CTC GGT CCC TAC AAG GGT GGT CTT CGT TTC 1068
CAC CCC TCC GTC AAC
ZO Ala Leu Gly Pro Tyr Lys G1y Gly Leu Arg Phe
His Pro Ser Vai Asn
75 80 85
TTG TCC ATC CTC AAG TTC CTT GGT TTC GAG CAG 1116
ATC TTC AAG AAT GCT
Leu Ser I1e Leu Lys Phe Leu Gly Phe G1u Gin
Ile Phe Lys Aan Ala
15 90 95 100
CTC ACT GGC CTG AAC ATG GGT GGT GGT AAG GGT 1169
GGT TCC GAC TTC GAC
Leu Ths Gly Leu Asn Met Gly Gly Gly Lys Gly
Gly Ser Asp Phe Asp
' 110 115 120
105 '
CCC AAG GGC ~JIiAG TCC GAC AAC GAG ATC CGT 1212
CGC TTC TGT GTT TCC TTC
Pro Lya G1y Lys Set Asp Asn G1u Ile Arg Arg
Phe Cya Val Ser Phe
125 , 130 135
25 ATG ACC GAG CTC TGC AAG CAC ATC' GGT GCC GAC 1260
ACT GAT GTT CCC GCT
Met Thr Glu Leu Cys Lys His Ile Gly Ala Asp
Thr Asp Val Pro A1a
140 145 150
CGT GAC ATC GGT GTC ACC GGT CGT GAG GTC GGT 1308
TTC CTC TTC GGC CAG
3O Gly Asp Ile Gly Val Thr Gly Arg Glu Val Gly
Phe Leu Phe Gly Gln
155 160 165
TAC CGC AAG ATC CGC AAC CAG TGG GAG GGT GTT 1356
CTC ACC GGT AAG GGT
Tyr Arg Lya Ile Arg Asn Gln Trp Giu Gly vai
Leu Thr Gly Lya Gly
35 170 175 180
GGC AGC TGG GGT GGT TCC CTC ATC CGC CCT GAG 1404
GCC ACC GGT TAC GGT
G1y Sex Trp Gly Gly Ser Leu Ile Arg Pro Glu
Ala Thr Gly Tyr Gly
185 190 195 200
GTT GTC TRC GTATGTCAAT TCCTCT'iCTT ATC~1TTATCT1453
ATGrATNICA
Val Val Tyr
4 GCGACTAACG CGTAACAG TAC GTC GAG CAC ATG ATT 1504
GCT CAC GCC ACC AAC
Tyr Val Glu His Met Ile Ala His Ana Thr Asn
205 210
GGC CAG GAG TCC T?C AAG GGC RAG CGC GTT GCC 1552
ATC TCC GGT TCC GGT
5O Giy G1n Giu Ser Phe Lya Gly Lys Arg Val Aie
iie Ser G1y Ser Giy
215 . 220 _, 225 230
AAC GTT GCC CAG TAC GCC GCC CTC AAG GTC ATT 1600
GAG CTC GGC GGT TCC
Asn Vai Aia G1n Tyr Ala Ala Leu Lys Val I1e
Glu Leu Gly Gly Ser
55 235 240 245
GTC GTC TCC CTG AGC GAC ACG CAG GGC TCC CTC 1648
ATC ATC AAC GGC GAG
Yal Val Ser Leu Ser Aap Thr GIn Gly Ser Leu
Ile ile Asn Gly Giu
250 255 260
E GGT AGC TTC RCC CCC GAG GAG ATC GAG CTC ATC 1696
O GCT CAG'ACC AAG GTC
Gly Ser Phe Thr Pro Glu Glu Ile Giu Leu Ile
Aia Gln Thr Lya Val:,
265 270 275
GAG CGC AAC GAG CTC GCC AGC ATC GTC GGT GCT 1744
GCT CCC .TTC AGC GAC
Glu Arg Asn Glu Leu A1a Ser Ile Va1 Gly Ala
Ala Pro Phe Ser Asp
280 285 290
GCC AAC AAG 1'TC,AAG TAC ATT GCT GGT GCC CGC 1792
CCC TGG GTT CAC GTC
7O Ala Asn Lys Phe Lys Tyr Ile Ale Gly Ala Arg
Pro Trp Yal His Val
295 300 305 310
CA 02325571 2000-09-29
WO 99/51756 PCT/EP99/02243
3
GGC AAG GTC GAC GTC GCT CTC CCC ?CC GCT ACC 1840
CAG AAC GAA GTT TCC
Gly Lya Vnl Asp Yal Ala Leu Pro Ser Ala Thr
Gln Asn Glu Val Ser
315 320 325
GGC GAG GAG GCC CAG GTC CTC ATC AAC GCT GGC 1888
TGC AAG TTC ATC GCC
Gly Glu GIu Ale Gln Val Leu Ile Asn Ala Gly
Cys Lys Phe Ile Ala
330 335 ~ ~ 340
GAG GGT TCC AAC ATG GGT TGC ACC CAG GAG GCC 1936
ATC GAC ACC TTC GAG
Glu G1y Ser Asn Met Gly Cys Thr Gln Glu Ala
Ile Asp Thr phe Glu
345 ~ 350 355
GCC CAC CGT ACC GCC AAC GCT GGC GCG GCT GCC 1984
ATC TGG TAC GCC CCC
Ala His Arg Thr Ala Asn Ala Gly Ala Ala Ala
ile Trp Tyr Ala Pro
360 365 370
GGT AAG GCC CCC AAC GCC GGT GGT GTC GCT GTC 2032
TCC GGT CTG GAG ATG
Gly Lys Ala Ala Aan Ala Gly Gly Val Ala Val
Sar Gly Leu Glu Met
2 375 . . 380 385 390
O
GCT CAG AAC TCT GCC CGC CTC AGC TGG ACT TCT 2080
GAG GAG GTT GAT GCC
Aln Gln Asn Ser Ala Arg Leu Ser Trp Thr Ser
Glu G1u Yal Asp Ale
395 400 405
2 CGT CTT AAG GAC ATC ATG CGC GAC ; TGC TTC 2128
S AAG AAC GGT C?T GAG ACT
Arg Leu Lya Asp Ile Met Arg Asp Cys Phe Lys
Asn Gly Leu Glu Thr
410 115 420
GCT CAG GAG TAC GCC ACC CCC GCT GAG GGT GTC 2176
30 CTG CC? TCC CTG GTG
Ala Gin Glu Tyr Ala Thr Pro Ala Glu Gly Vel
Leu Pro Ser Leu Val
425 430 435
ACC GGA TCC AAC ATT GCC GGT TTC ACC AAG GTG 2224
GCT GCC GCC ATG AAG
Thr Gly Ser Asn Ile Ala Gly Phe Thr Lys Val
35 Ala Ala Ala Met Lys
140 445 450
GAC CAG GGT GAC TGG TGG TAAATGC~GA AAOCCGCAAA2272
CCOCCGOGGC
Gln Gly Asp Trp Trp
40 155
4so .
T?ATGTCATG ACGP1TTATGT AGTTTGATGT TCCCTT'I~CAG2332
CGCGGA1GGA TAGi,GGCGCC
GGTGTTT1'CT TGQ'AG1TTA GA1GGA1GCA TAATGATATC 2392
CTT1'TCTTAA TCCTCAAATT
45 CTTGTA7~fTT GTTGTATCAA TAGI'AGATAA TAUAC1GTA 2452
GT(91ACTACC CT1GCATCTT
CAGTATTTGC AGATGCATTC ATCfCTATTC CGiIGCAf9ITG2512
CACAAAOCCA TCOGACOGCA
GTTCACTAGT AC1TAGCCTG TTATCTTCCC TCTATCOCAT 2570
CT~1AAC~1AC TATCTAGA
50
SEO ID 2. Amino acid sequence of the glutamate dehydrogensse
A tgdh A) protein from Asnerro;lly~ ~,as deduced from
the nucleotide sequence in SEQ ID 1.
Met Ser Asn Leu Pro His Glu Pro Glu Phe Glu Gln Ala Tyr !Lys GIu
1 5 10 . 15
60. Leu Ala Ser Thr Leu Glu Asn Ser Thr Leu Phe Gln Lys Asn Pro Glu
20 25 30
Tyr Arg Lys Ala ~Leu Ala Val Val Ser Val Pro Glu Arg Val Ile Gln
35 90 45
ss
CA 02325571 2000-09-29
WO 99/51756 4 PCT/EP99/02243
Phe Arg Val Val Trp Glu Asp Asp Ala Gly Asn Val Gln Val Asn Arg
50 55 ~ 60
Gly Phe Arg Val Gln Phe Asn Ser Ala Leu Gly Pro Tyr Lys Gly Gly
65 70 ; 75 80
Leu Arg Phe His Pro Ser Val Asn Leu Ser Ile Leu Lys Phe Leu Gly
85 - 90 95
Phe Glu Gln Ile Phe Lys Asn Ala Leu Thr Gly Leu Asn Met Gly Gly
100 105 110
Gly Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Asn Glu
115 120 125
Ile Arg krg Phe Cys Val Ser Phe Met Thr Glu Leu Cys Lys His Ile
130 ; 135 190
Gly Ala Asp Thr Asp Val Pro Ala Gly Asp Ile Gly Val Thr Gly Arg
145 150 . 155 160
Glu Val Gly Phe Leu Phe Gly Gln Tyr Arg Lys Ile Arg Asn Gln Trp
. 165 170 175
Glu Gly Val Leu Thr Gly Lys Gly Gly Ser Trp Gly Gly Ser Leu Ile
_. 180 1B5 190
Arg Pro Glu Ala Thr Gly Tyr Gly Val Val Tyr Tyr Val Glu His Met
195 200 205
Ile Ala His Ala Thr Asn Gly Gln Glu Ser Phe Lys Gly Lys Arg Val
210 215 ~ 220
Ala Ile Ser Gly Ser Gly Asn Val Ala Gln Tyr Ala Ala Leu Lys Val
225 230 235 240
Ile Glu Leu Gly Gly Ser Val Val Ser Leu Ser Asp Thr Gln~ Gly Ser
245 250 255
Leu Ile Ile Asn Gly Glu Gly Ser Phe Thr Pro Glu Glu Ile Glu Leu
260 265 270
Ile Ala Gln Thr Lys Val Glu Axg Asn Glu Leu Ala Ser Ile Val Gly
275 280 285
Ala Ala Pro Phe Ser Asp Ala Asn Lys Phe Lys Tyr Ile Ala Gly Ala
290 295 300
Arg Pro Trp Val His Val Gly Lys Val Asp Val Ala Leu Pro Ser Ala
305 310 . 315 320
Thr Gln Asn Glu Val Ser Gly Glu Glu Ala Gln Val Leu Ile Asn Ala
325 ~ .'330 ~ 335
Gly Cys Lys Phe Ile Ala GIu Gly Ser Asn Met Gly Cys Thr Gln Glu
390' 345 350
Ala Ile Asp. Thr Phe Glu Ala His Arg Thr Ala Asn Ala Gly Ala Ala
355 360 365
CA 02325571 2000-09-29
WO 99/51756 PCT/EP99/02243 ,
Ala Ile Trp Tyr Ala Pro Gly Lys Ala Ala Asn Ala Gly Gly Val Ala
370 375 380
Val Ser Gly Leu Glu Met Ala Gln Asn Ser Ala Arg Leu Ser Trp Thr
5 385 390 395 400
Ser Glu Glu Val Asp Ala Arg Leu Lys Asp Ile Met Arg Asp Cys Phe
405 410 415
Lys Asn Gly Leu Glu Thr Ala Gln Glu Tyr Ala Thr Pro Ala Glu Gly
420 925 430
Val Leu Pro Ser Leu Val Thr Gly Ser Asn Ile Ala Gly Phe Thr Lys
935 440 495
Val Ala Ala Ala Met Lys Asp Gln Gly Asp Trp Trp
450 955 960
