IE84033B1 - Cloning and expression of xylanase genes from fungal origin - Google Patents
Cloning and expression of xylanase genes from fungal originInfo
- Publication number
- IE84033B1 IE84033B1 IE1991/2582A IE258291A IE84033B1 IE 84033 B1 IE84033 B1 IE 84033B1 IE 1991/2582 A IE1991/2582 A IE 1991/2582A IE 258291 A IE258291 A IE 258291A IE 84033 B1 IE84033 B1 IE 84033B1
- Authority
- IE
- Ireland
- Prior art keywords
- xylanase
- dna
- expression
- gene
- sequence
- Prior art date
Links
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- A—HUMAN NECESSITIES
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- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/14—Pretreatment of feeding-stuffs with enzymes
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
- A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
-
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
Description
PATENTS ACT 1964
COMPLETE SPECIFICATION
“CLONING AND EXPRESSION OF XYLANASE GENES FROM
FUNGAL ORIGIN"
GIST—BROCADES N.V., a Dutch Body Corporate, of wateringseweg I,
P.O. Box 1, 2600 MA DeTft, The Netherlands
Gist—brocades N.V. ]A Series 2566
Cloning and Expression of Xylanase Genes From Fungal Origin
The present invention relates to the field of
molecular biology. In particular, invention
the present
relates to the cloning and overexpression of a fungal DNA
sequence encoding a protein having the activity of a
xylanase. The present invention also provides methods for
the production and use of a
single xylanase which is
obtainable in a form which is free of other xylanases, and
indeed from other enzymes in general.
Background of the Invention
The composition of a plant cell wall is complex and
variable. Polysaccharides are mainly found in the form of
long chains of cellulose (the main structural component of
the plant cell wall), hemicellulose (comprising various B-
xylan chains) and pectin. The occurrence, distribution and
structural features of plant cell wall polysaccharides are
determined. by (1) plant species; (2) variety; (3) tissue
type, (4) growth conditions; (5) ageing and (6) processing
of plant material prior to feeding.
Basic differences exist between monocotyledons (e.g.
cereals and grasses) and dicotyledons (e.g. clover, rapeseed
and soybean) and between the seed and vegetative parts of
the plant (Chesson, 1987; 1986).
Carré and Brillouet,
Monocotyledons are characterized by the presence of an
arabinoxylan complex as the major hemicellulose backbone.
The main structure of hemicellulose in dicotyledons is a
xyloglucan complex. Moreover, higher pectin concentrations
are found in dicotyledons than in monocotyledons. Seeds are
generally very high in pectic substances but relatively low
in cellulosic material.
A cross-sectional diagram of a plant cell is depicted
in Figure 1. Three more or less interacting polysaccharide
structures can be distinguished in the cell wall:
(1) The middle lamella forms the exterior cell wall.
It also serves as the point of attachment for the
individual cells to one another within the plant
middle
primarily «of calcium salts «of highly esterified
tissue matrix. The
lamella consists
pectins;
(2) The primary wall is situated just inside the
middle lamella. It is a well—organized structure
embedded in an
pectin,
of cellulose microfibrils
amorphous matrix of
hemicellulose,
phenolic esters and proteins;
(3) The secondary wall is
formed as the plant
matures. During" the plant's growth and ageing
phase, cellulose microfibrils, hemicellulose and
lignin are deposited.
The primary cell wall of mature, metabolically active
cells (e.g.
susceptible to enzymatic hydrolysis than the secondary cell
plant mesophyll and epidermis) is more
wall, which by this stage, has become highly lignified.
There is a high degree of interaction between
cellulose, hemicellulose and pectin in the cell wall. The
enzymatic degradation of these rather intensively cross-
linked polysaccharide structures is not a simple process. At
least five different enzymes are needed to completely break
down an arabinoxylan, for example. The endo—cleavage is
effected by the use of an endo—B(l+4)~D—xylanase. Exo~(l»4)~
D—xylanase liberates xylose units at the non—reducing end of
the polysaccharide. Three other enzymes (a—glucuronidase, a-
L-arabinofuranosidase and acetyl esterase) are used to
attack substituents on the xylan backbone. The choice of the
specific enzymes is of course dependent on the specific
hemicellulose to be degraded (McCleary and Matheson, 1986).
For certain applications, however, complete
degradation of the entire hemicellulose into monomers is not
In the
arabinoxylan, for example, one needs simply to cleave the
necessary or is not desirable. liquefaction of
main xylan backbone into shorter units. This may be achieved
by the action of an endo—xylanase, which ultimately results
in a mixture of xylose monomer units and oligomers such as
xylobiose and xylotriose. These shorter subunits are then
sufficiently soluble for the desired use.
Filamentous fungi are widely known for their capacity
to secrete large amounts of a variety of hydrolytic enzymes
such as a—amylases,
proteases and amyloglucosidases and
various plant cell wall degrading enzymes such as
cellulases, hemicellulases, and pectinases. Among these,
multiple xylan-degrading enzymes have been recognized, which
have been shown to possess a ‘variety’ of biochemical and
physical properties. This heterogeneity in xylanase function
allows for the selection of a xylanase of interest which is
best suited for’ a desired application (see Wong gt al.
(1988), Woodward (1984) and Dekker and Richards (1977)).
Multiple Xylanases of ‘various ‘molecular' weights are
known to be produced by micro-organisms such as Aspergillus
niqer, Clostridium Trichoderma
thermocellum, reesei,
Penicillium ianthinellum, as well as species of Bacillus and
Streptomyces.
On the contrary, in yeast no xylanase multiplicity has
Trichosporon,
Cryptococcus and Aureobasidium, only a single xylanase could
be detected.
been observed. In three
east enera
I
In nature, microbial Xylanases are always produced
together with other enzymes having polysaccharide-degrading
activities, such. as exo—arabinanase, acetyl esterase and
cellulases. For some applications, these enzyme activities
are not needed or are unwanted.
It is known that fermentation conditions may be varied
to favor the production of an enzyme of interest. It is also
known that the cloning’ of the gene encoding" the desired
enzyme and overexpressing it in its natural host, or other
compatible expression host will specifically enhance the
production of the enzyme of interest. This latter method is
}.a
U]
particularly useful if the enzyme of interest is to be
obtained in a form which is free of undesired enzyme
activity.
The expression of recombinant bacterial xylanase has
been previously described in European Patent Application
.138. The gene
encoding the bacterial
xylanase was
isolated from Bacillus chromosomal DNA and brought to
expression in an E. coli host. However, E. coli expression
hosts are, in some instances, considered to be unsafe for
the production of proteins by recombinant DNA methods due to
their production of unacceptable by—products such as toxins.
Since bacterial genes contain no
introns, one is
confronted with few problems in cloning and expressing such
hosts. On the hand, the
eukaryotic always so
straightforward. It is well known that genes isolated from
eukaryotic
genes in prokaryotic other
expression of genes is not
strains contain introns. This
inherently
introduces complications in the cloning and expression of
these genes, should a prokaryotic host be preferred.
Furthermore, certain differences exist, in general,
between the physical characteristics of xylanases of fungal
origin and those from bacteria. In general, fungal xylanases
have a pH optimum in the range of between pH 3.5 - 5.5 as
compared to bacterial xylanases which generally have a pH
optimum in the range of pH 5.0 - 7.0. Fungal xylanases also
generally have a broader pH stability range (pH 3 — 10) than
(pH 5.0 — 7.5).
xylanases generally have a temperature optimum of about
°C.
do their bacterial counterparts Fungal
Bacterial xylanases generally have a temperature
optimum between 50°C and 70°C. For a further discussion of
the physical characteristics of xylanases see Wong et gl.
(1988), Woodward (1984) and Dekker and Richards (1977).
Thus, it is clear that bacterial xylanases are less
suitable for use in, for example, processes requiring lower
pH conditions. In other instances, bacterial xylanases are
too thermostable for certain applications such as the
lagering of beer (see European Patent No. 227.159).
Accordingly, it would be of great importance to obtain
genes encoding xylan-degrading enzymes of fungal origin
which may be brought to expression in other, high—producing
microbial expression hosts.
Summary of the Invention
The present invention provides purified and isolated DNA sequences of fungal
ongnrasdefinedincknniL\Nmchencodepnndnshavmg
xylan-degrading activity. These DNA sequences include the
xylanase encoding sequence and preferably the adjacent 5'
and 3' regulatory sequences as well.
It is also an object of the present invention to
provide constructs for the microbial overexpression of the
xylanase—encoding sequences using either their native
regulatory sequences or, in an alternative embodiment, the
xylanase—encoding sequence operably linked to selected
regulatory regions such as promoter, secretion leader and
terminator signals which are
of the
capable of directing the
overexpression xylanase protein in a suitable
expression host.
It is a further object of the present invention to
provide microbial expression hosts, transformed with the
expression constructs of the present invention, which are
capable of the overexpression and, if desired, the secretion
of a xylanase of fungal origin.
It is yet a further object of the present invention to
provide methods for the production of a xylanase of interest
which may, in turn, advantageously be used in an industrial
process. Typically, such an industrial process requires
xylanase activity at a lower pH than that at which xylanases
of bacterial origin optimally function.
Brief Description of the Figures
Figure 1: A cross—sectional diagram of a plant cell.
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
HPLC elution profile of a culture filtrate
obtained from Asperqillus nicer DS16813 (CBS
323.90). This strain was later reclassified as
more likely belonging to the species Aspergillug
tubigensis.
Oligonucleotide probes AB80l - AB806, designed
from the N—terminal amino acid sequence of the
Aspergillus tubiqensis XYL A protein (Formula 1).
Oligonucleotide probe AB1255, designed from the
N—terminal amino acid sequence of an internal 19
kDa fragment of the asperqilius tubigensis XYL A
protein, digested with the
peptidase (Formula 2).
S. aureus V8 endo~
Restriction map of the genomic region containing
the xln A gene, as derived from Southern blot
analysis
of bacteriophage lambdaflfl. Indicated
are the hybridizing fragments and their
corresponding lengths.
Strategy employed to sequence the gspergillus
tupigensis xln A gene. The arrows indicate the
direction and number of bp sequenced.
Restriction map of pIMlOO containing the 6.9 kb
Sall
fragment containing the gspergillus
tubigensis xlg A gene. In addition to the two
HinDIII sites indicated, two further HinDIII
sites are present in the plasmid insert.
Nucleotide sequence of the Aspergillus tubigensis
gin A gene. The positions of the intron and the
propeptide are putative.
Representation of a zymogram exhibiting the XYL A
protein expressed by_transformants TrX2 and TrX9.
SDS—polyacrylamide gel electrophoresis showing
the expression of the XYL A protein in A. niger
CBS 513.88 (A) and A. niger N593 (B).
: Native gradient PAGE exhibiting the XYL A protein
expressed. by
A. niqer CBS 513.88 transformants
Figure 12:
Figure 13:
Figure 14:
Figure 15:
Figure 16:
Figure 17:
Figure 18:
Figure 19:
numbers 10,
with an RBB-xylan overlay (B).
Physical map of pAB 6—1. The 14.5 kbp fiigdIII DNA
on a fragment in
[Abbreviations:
insert -in pUC19 contains the entire
amyloglucosidase (AG) locus from A} niger.
A schematic View of the generation of AG
promoter/xylanase gene fusions performed by the
polymerase chain reaction.
Construction pathway of the intermediate plasmid
pXYL2AG.
Construction pathway of the intermediate
pXYL2.
Construction pathway of the intermediate
pXYL3AG.
[Abbreviationsz see Figure 12]
plasmid
[Abbreviations: see Figure 12]
plasmid
[Abbreviations: see Figure 12]
Construction pathway of the intermediate
pXYL3.
plasmid
[Abbreviations: see Figure 12]
Schematic representation of the constructs made
in the
the gpal site used in
of the A. niger pyr
by creating deletions Xln A promoter
region. For orientation,
the cloning A gene is
indicated.
Detailed Description of the Invention
The present invention describes purified and isolated
DNA sequences of fungal origin which encode xylanases and
genetic variants thereof, as defined in claim 1. The DNA sequence preferably
includes the xylanase—encoding sequence and adjacent 5' and
' regulatory sequences. Genetic variants include hybrid DNA
sequences containing the xylanase-encoding sequence coupled
such as secretion and
to regulatory regions, promoter,
terminator signals, originating from homologous or
heterologous organisms. Genetic variants also include DNA
sequences encoding mutant xylanase proteins and degenerate
DNA sequences wherein the xylan—degrading activity of the
enzyme is retained. The present invention also includes DNA
sequences which are capable of hybridizing to the xylanase-
encoding DNA sequences and genetic ‘variants thereof, as
described above, but which may differ in codon sequence due
to the degeneracy of the genetic code or cross—species
wnmUonTHmsemeab0spedfiednidamiL
The present invention also provides DNA constructs vectors in the claims for .
in a desired
the expression of a xylanase of interest
expression host. These include hybrid
DNA sequences containing the xylanase—encoding region
operably linked to regulatory regions, such as promoter,
secretion and terminator signals originating from homologous
or heterologous organisms, these regulatory regions being
capable of directing the overexpression of the enzyme
encoded by the xylanase—encoding DNA sequence in an
appropriate host. Preferably, the expression construct will
be integrated into the genome of the selected expression
host.
The present invention further
provides vectors,
preferably plasmids, for the cloning and/or transformation
of microbial hosts via the introduction into the microbial
host of the DNA constructs for the expression of the xylanese of interest. Vectors within
theinvennonaredefinedinthe<flanns,asarenncnfinaihogs
In addition, the present invention concerns homologous
hosts transformed by DNA
or heterologous constructs
described above. Microbial expression hosts may be selected
from bacteria, yeasts or fungi.
Within the context of the present invention, the term
"homologous" is Aunderstood to intend all that which is
native to the DNA sequence
encoding the xylanase of
interest, including its regulatory regions. A homologous
host is defined as the species from which such DNA sequence
may be isolated.
The term "heterologous" is thus defined as all that
which is not native to the DNA sequence encoding the
xylanase of interest itself, including regulatory regions. A
"heterologous" host is defined as any microbial species
other than that from which the Xylanase-encoding gene has
been isolated.
Within the scope of the present invention, a xylanase
of interest is ‘understood. to include a xylan-degrading
enzyme which is naturally produced by a filamentous fungus.
interest are those which are
Xylanases of particular
naturally produced by filamentous
Aspergillus.
fungi of the gums
Especially preferred Xylanases are
those originating from
Aspergillus tubiqensis.
An endo—xylanase of interest. may be identified Via
assay methods not critical to the present invention, such as
a spot test assay. .According to this method, a filtrate
obtained from the culturing of a microorganism induced (e.g.
with oat spelts xylan) to produce an endo—xylanase may be
tested for the presence of endo—xylanase activity. Drops of
the elution fractions are placed individually onto an agar
film containing a citrate—phosphate buffer (see Example 1.1,
below) and oat spelt xylan. The film is then incubated. If
.._'LO..
endo~xy1anase activity is present, the location of the
individual drops on the agar film are visibly clear.
Once a xylanase of interest has been identified, the
DNA sequence encoding such xylanase may be obtained from the
filamentous fungus which naturally produces it by culturing
the fungus
in a xylan-containing medium, isolating the
desired xylanase using known methods such as column
chromatography (e.g. HPLC — see Figure 2) and determining at
least a portion of the amino acid sequence of the purified
protein. A,
DNA probes may thereafter be designed by synthesizing
oligonucleotide sequences based (N1 the partial amino acid
sequence. Amino acid sequences may be determined from the N-
terminus of the complete protein and/or from the N—termini
of internal peptide fragments obtained via proteolytic or
chemical digestion of the complete protein. Once obtained,
the DNA probe(s) are then used to screen a genomic or CDNA
library.
If this method is unsuccessful, the genomic library
may’ be differentially‘ screened. with CDNA. probes obtained
from mRNA from non~induced and induced cells. Induced mRNA
is prepared from cells grown on media containing xylan as a
carbon source, while non-induced mRNA must be isolated from
cells grown on a carbon source other than xylan, e.g.
glucose. Among the clones which only hybridize with the
induced cDNA probe, a clone containing the desired Xylanase
gene may be recovered. Alternatively, a xylanase gene may be
identified by cross—hybridization with a related xylanase
sequence .
A genomic library may be prepared by partially
digesting the fungal chromosomal DNA with a suitable
may be screened with a suitable DNA probe.
Alternatively, a cDNA library may be prepared by
synthesized. frtmi mRNA. isolated from fungal
induced for the
cloning CDNA,
cells synthesis of xylanase, into an
appropriate phage vector, e.g. lambda gt 10 or lambda gt 11.
The CDNA library may then be screened with a DNA probe, or
alternatively using
immunological means or via
a plate
assay.
In 21 preferred embodiment of the present invention,
oligonucleotide probes are designed from the N-terminal
amino acid sequence (see Figure 3, formula 1) of a xylanase
having an apparent molecular weight of 25 kDa purified from
an Asperqillus tubiqensis culture filtrate and/or from the
amino acid sequence of an internal peptide fragment (see
Figure 4, formula 2) obtained by digestion of the xylanase
with Staphylococcus aureus
endoprotease V8. The oligo-
DNA isolated from the four phage clones hybridized
with the N—terminal oligo mixture as well as with the oligo
mixture derived from the amino acid sequence of the internal
fragment (see Figure 4). Restriction enzyme analysis
revealed that all four clones contained DNA from the same
genomic region of A. tubigensis.
A region of approximately 2.1 kb which hybridizes with
both oligo mixtures has
been sequenced. The nucleotide
sequence, as depicted in Figure 8, comprises a xylanase
...l2_
coding sequence of 681 bp (which is interrupted by one small
intron of’ 49 bp from: position 1179 to 1230), as well_ as
sequences of 949 and 423 nucleotides of the 5' and 3'
flanking regions, respectively.
Variants among the purified xylanase proteins have
also been discovered. It has been determined that the
corresponding xylanases have three different N-termini,
possibly as a conditions.
Approximately one—third of these xylanases have serine as
the N—termina1 amino acid (Figure 8, position 1), another
result of fermentation
approximately one—third have alanine as the N—termina1 amino
acid (Figure 8, position 2) and the remaining proteins have
glycine as the N—terminal amino acid (Figure 8, position 3).
The availability of a DNA sequence encoding a xylanase
protein enables the construction of mutant xylanases by
site—directed mutagenesis. If the tertiary structure of the
xylanase is known, and its catalytic and substrate binding
domains are localized, amino acids may be selected for
mutagenesis (for example with the aid of‘computer modelling)
which most likely affect catalytic and/or substrate binding
functions. If the tertiary structure of the protein is not
available, random mutants may be either generated along with
the entire coding sequence, or the tertiary structure of the
protein may be predicted by comparison with similar known
xylanases isolated from another microorganism.
To facilitate the insertion of the DNA fragment
containing the xylanase—encoding sequence into expression
constructs comprising one or more heterologous regulatory
regions, the polymerase chain reaction (PCR) (Ehrlich, H.A.
(editor), 1989) may be used for introduction of appropriate
ends of the
xylanase coding sequence. The choice of restriction sites
restriction enzyme sites in the 5' and 3'
depends on the DNA sequence of the expression vector, i.e.
the presence of other restriction sites within the DNA
molecule.
To obtain overexpression of the xylanase protein in
the original (homologous) production species, or
6.9 kb SalI
fragment (see Figure 5) comprising the complete gene with
its 5'
alternatively in another
fungal strain, a
and. 3' regulatory regions, or alternatively, the
complete gene fused to the regulatory regions of other
genes, is introduced into the selected expression
host to increase the copy number of ‘the gene and,
consequently, protein expression. A
If a heterologous expression host is preferred, and a
yeast or a bacterial strain is selected, an uninterrupted
(intronless) DNA sequence is used for the construction of a
heterologous avoid the
expression vector in order to
possibility that splice signals residing on the genomic
fragment are not recognized by the heterologous host. This
uninterrupted DNA sequence may be obtained from a CDNA
library constructed from mRNA isolated from cells, induced
for the synthesis of xylanases. This library may be screened
with an oligonucleotide or CDNA probe obtained as described
before. Alternatively, an uninterrupted DNA sequence may be
obtained by applying a polymerase chain reaction using
appropriate 5' and 3‘ oligonucleotides on the first strand
CDNA synthesized from the RNA of xylan-induced cells.
Within the
overexpression is defined as the expression of the xylanase
context of the present invention,
of interest at levels above that which are ordinarily
In the
same context, overexpression also intends the expression of
encountered in the homologous wild—type organism.
the xylanase of interest in a heterologous organism which
does not normally produce such xylanase except for the
introduction of the DNA sequence encoding the xylanase of
interest into the heterologous expression host. Progeny of
these expression hosts are, of course, also to be understood
to be embraced by the present invention.
overexpression of the xylanase of interest may also be
achieved by the selection of
heterologous regulatory
regions, e.g promoter, secretion leader and terminator
regions, which serve to increase expression and, if desired,
secretion levels of the protein of interest from the chosen
expression host and/or to provide for the inducible control
of the expression of the xylanase of interest.
Aside from the xylanase of interest's native promoter,
other promoters may be used to direct its expression. The
promoter may be selected for its efficiency in directing the
expression of the xylanase of interest in the desired
expression host.
In another embodiment, a constitutive promoter may be
selected to direct the expression of the desired xylanase,
relatively" free from other xylanases. Such. an expression
construct is furthermore advantageous since it circumvents
the need to culture the expression hosts on a medium
containing solid xylans as an inducing substrate.
Examples of
strong constitutive and/or inducible
promoters which are preferred for use in fungal expression
hosts are the ATP-synthetase, subunit; 9 (oliC), triose
phosphate isomerase (tpi), alcohol dehydrogenase (adhA), a-
amylase (amy), amyloglucosidase (AG), acetamidase (amds)
and glyceraldehyde—3—phosphate dehydrogenase (gpd)
promoters.
Examples of strong yeast promoters are the alcohol
dehydrogenase,
lactase, kinase and
—phosphoglycerate
triosephosphate isomerase promoters.
Examples of strong bacterial promoters are the a-
amylase and gpgz
promoters as well as promoters from
extracellular protease genes.
Hybrid promoters may also advantageously be used to
improve inducible regulation of the expression construct.
Preferred promoters according to the present invention
are those originating from the amyloglucosidase (AG) gene
and native xylanase promoters.
It is often desirable for the xylanase of interest to
be secreted from the expression host into the culture medium
from where the xylanase may be more easily recovered.
According to the present invention, the xylanase of
interest's native secretion leader sequence may be used to
effect the secretion of the expressed xylanase.
However, an increase in the expression of the xylanase
sometimes results in the production of the protein in levels
beyond that which the
processing‘ and secreting,
expression host‘ is capable of
creating" a build—up of protein
product within the cell due to a bottleneck in the transport
of the protein through the cell wall. Accordingly, the
present invention also
provides heterologous leader
sequences to provide for the most efficient secretion of the
xylanase from the chosen expression host.
According to the present invention, the secretion
leader may be selected on the basis of the desired
expression host. A heterologous secretion leader may be
chosen which is homologous to the other regulatory regions
of the expression construct. For example, the leader of the
highly secreted amyloglucosidase protein may be used in
combination with the amyloglucosidase promoter itself, as
well as in combination with other promoters. Hybrid signal
sequences may also advantageously be used within the context
of the present invention.
Examples of preferred heterologous secretion leader
sequences are those originating from the amyloglucosidase
gene (fungi), the a—factor gene (yeasts) or the a-amylase
gene (Bacillus).
Most preferred secretion leader sequences according to
the present invention are the those originating from the
amyloglucosidase (AG) gene and the native xylanase leader
sequence.
considered to be
In general, terminators are not
critical elements for the overexpression of genes. If
desired, a terminator may be selected from the same genes as
.._l6_.
the promoters, or alternatively, the homologous terminator
may be employed.
In addition to the genomic fragment mentioned above,
the transforming DNA may contain a selection marker to
discriminate cells which have incorporated the desired gene
from the bulk of untransformed cells. This selection marker,
provided with the
appropriate 5' and 3' regulatory
sequences, may reside on the same DNA molecule containing
the desired gene or be present on a separate molecule. In
the latter case, a co—transformation must be performed. The
ratio of the expression vector/selection vector must be
adjusted in such a manner that a high percentage of the
selected transformants also have incorporated the vector
containing the expression construct of the xylanase of
interest.
The most suitable selection systems for industrial
micro—organisms are those formed by the group of selection
markers which do not
require a mutation in the host
organism. Examples of fungal selection markers are the genes
for acetamidase (amds), ATP synthetase, subunit 9 (oliC) and
benomyl resistance (benA). Exemplary of non-fungal selection
markers are the G418 resistance gene (yeast), the ampicillin
resistance gene (E. coli) and the neomycin resistance gene
(Bacillus).
Once the desired expression construct has been
assembled, it is transformed into a suitable cloning host
such as E. Coli to propagate the construct. Afterwards, the
expression
construct is introduced into a suitable
expression host wherein the
expression construct is
preferably integrated into the genome. Certain hosts such as
Bacillus species may be used as both cloning and expression
hosts, thus avoiding an extra transformation step.
According to the present invention, a variety of
expression hosts may be used to overexpress the xylanase of
interest. In one embodiment, a homologous expression host
may be used. This involves the introduction of the desired
expression construct back into the strain from which the
xylanase encoding DNA sequence was isolated either in
increased gene copy numbers, or under the control of
heterologous regulatory regions as described above, or both.
In another embodiment, a xylanase of interest may be
overexpressed by introducing and expressing the DNA
construct encoding the
xylanase of interest under the
control of the appropriate regulatory regions in
heterologous hosts such as bacteria, yeasts or fungi. For
that purpose, the DNA sequence encoding’ the xylanase of
interest is preferably expressed under the control of
promoter and terminator sequences originating from the
heterologous host. In addition, it may be necessary to
replace the native secretion leader sequence of the xylanase
of interest with a leader sequence homologous to the
expression host in order to achieve the most efficient
expression and secretion of the product.
Factors such as the size (molecular weight), the
possible need for glycosylation or the desirability of the
extracellular secretion of the xylanase of interest play an
important role in the selection of the expression host.
The gram—negative bacterium E. coli is widely used as
a host for heterologous gene expression, but mostly
accumulates large amounts of heterologous protein inside the
cell. Subsequent purification of the desired protein from
the bulk of E. coli intracellular proteins can sometimes be
difficult.
In contrast to E. coli, bacteria from the genus
Bacillus are very suitable as heterologous hosts because of
their capability to secrete
proteins into the culture
medium.
Alternatively, a heterologous host selected from the
group of yeasts or fungi may be preferred. In general, yeast
cells are preferred over fungal cells because they are
easier to manipulate.
However, some proteins are either
poorly secreted from the yeast cell, or in some cases are
_..l8..
not processed properly (e.g. hyperglycosylation in yeast).
In these instances, a fungal host organism should be
selected.
A heterologous host may also be chosen to express the
xylanase of interest
substantially free from other
polysaccharide-degrading enzymes by choosing 21 host which
does not normally produce such enzymes such as Kluyyeromyces
lactis.
Examples of preferred expression hosts within the
scope of the present invention are fungi such as Aspergillus
in EP 184.438 and EP 284.603) and
Bacillus
species (described
Trichoderma species, bacteria such as species
(described in EP 134.048) and yeasts such as Kluyveromyces
species (described in EP 96.430 and EP 301.670) and
Saccharomyces species.
Particularly" preferred expression hosts may be
selected from Asperqillus niqer, Asperqillus awamori,
Asperqillus aculeatus, Asperqillus oryzae, Asperqillus
tubiqensis, Trichoderma reesei, Bacillus subtilis, Bacillus
licheniformis, Kluyveromyces lactis and
Saccharomyces
cerevisiae.
The overexpression of the xylanase of interest is
effected. by the culturing of the expression. hosts, which
have been transformed with the xylanase expression
construct, in a conventional nutrient fermentation medium.
The fermentation medium consists of an
ordinary
culture medium containing a carbon source (e.g. glucose,
maltose, molasses, etc.), a nitrogen source (e.g. ammonium
sulphate, ammonium nitrate,
ammonium chloride, etc.), an
organic nitrogen source (e.g. yeast extract, malt extract,
peptone, etc.) and inorganic nutrient sources (e.g.
phosphate, magnesium, potassium, zinc, iron, etc.).
Optionally, an inducer (e.g. oat spelts xylan) may be
included.
The selection of the appropriate medium may be based
on the choice of expression hosts and/or based on the
_.. _.
regulatory requirements of the expression construct. Such
media are well—known to those skilled in the art. The medium
may, if desired, contain additional components favoring the
expression
transformed
hosts over other
potentially
contaminating microorganisms.
The fermentation is performed over a period of 0.5—20
days in a batch or fed—batch process at a temperature in the
range of between 0 and 45 °C and a pH between 2 and 10.
Preferred fermentation conditions are a temperature in the
range of between 20 and 37 “C and a pH between 3 and 9. The
appropriate conditions are selected based on the choice of
the expression host.
After fermentation, the cells are removed from the
fermentation broth by means of centrifugation or filtration.
After removal of the cells, The xylanase of interest may
then be recovered and, if desired, purified and isolated by
conventional means.
The product is stably formulated either in liquid or
dry form. For certain applications, immobilization of the
enzyme on a solid matrix may be preferred.
xylanases of interest, produced by means of the
present invention, may be applied either alone, or together
with other selected enzymes in a variety of processes
requiring the action of a xylan-degrading enzyme. Moreover,
the fungal xylanases of the present invention, which
generally have lower pH optima than xylanases of bacterial
origin, are particularly well suited for use in industrial
processes which are performed at low pH.
In accordance with the present invention, it has been
found that the xylanases produced via the present invention
may be used in the baking of breads. The incorporation of
small amounts of xylanase to the flour imparts favorable
characteristics to the dough and thus to the bread itself
such as increased loaf
better
characteristics such as break and shred quality and crumb
quality.
volume and textural
_20__
added to
rich in
When added to feeds
Xylanases may also be animal feed
compositions which are arabinoxylans and
glucoxylans. (including silage) for
monogastric animals (e.g. poultry or swine) which contain
cereals such as barley, wheat, maize, rye or oats or cereal
by—products such as wheat bran or maize bran, the enzyme
significantly improves the break—down of plant cell walls
which leads to better utilization of the plant nutrients by
the animal. As a consequence, growth rate and/or feed
conversion are improved. Moreover, Xylanases may be used to
the reduce the viscosity of feeds containing xylans.
Xylanase may be added beforehand to the feed or silage
if pre—soaking or wet diets are preferred. More
advantageously, however, the Xylanases produced via the
present invention when added to feed continue to hydrolyze
xylans in the feed in
vivo. Fungal Xylanases, which
generally’ have lower pH optima, are capable of releasing
important nutrients in acidic environments as the
such
stomach of the animal ingesting such xylanase—supplemented
feed.
The Xylanases produced via the present invention are
also effective in filtration and
improving removing
dissolved organic substances from the broth in processes
wherein apple
microbial biomass.
distillery waste is bioconverted into
Xylanases originating from filamentous
fungi may be advantageously used in this process.
Also according to the present invention, glucose
syrups having improved filterability and/or lower viscosity
are produced from impure cereal starch by subjecting the
impure starch first to the action of an a-amylase, then to
fungal Xylanases produced via the present invention and
finally" to a hydrolysis. Similarly, the Xylanases of the
present invention may be used in beer brewing to improve the
filterability of the wort.
Xylanases may also be used. to remove lignins from
and thus facilitate bleaching by reducing the amount of
chlorine needed in the preparation of paper products.
kraft pulp
In addition, the xylanases produced via the present
invention may be used in other processes such as to increase
yield in the preparation of fruit or vegetable juices, the
enzymatic hydrolysis of sugar beet pulp, the resulting
hydrolyzed fraction being capable of use in microorganism
culture medium; of agricultural residues such as corn cobs,
wheat—straw and ground nutshell; and of certain recyclable
materials such as waste paper.
The following examples are provided so as to give
those of ordinary skill in the art a complete disclosure and
description of how to make and use the invention and are not
intended to limit the scope of what the inventors regard as
their invention. Efforts have been made to ensure accuracy
with respect to numbers used (e.g., amounts, temperature,
pH, etc.) but some experimental errors and deviation should
be accounted for. Unless indicated otherwise, temperature is
in degrees Celsius and pressure is at or near atmospheric.
EXAMPLE 1
Purification and characterization of
Aspergillus
tubigensis endo—xy1anase XYL A.
Example 1.1
Purification of Asperqillus tubiqensis endo—xylanase
XYL A.
approximately 35 ml which was then was ultrafiltrated on a
Diaflo PM 10 filter in a 50 ml Amicon :module to remove
salts.
The supernatant was then concentrated to a volume of
ml and the retentate was washed twice with 25 ml 25 mM
Tris—HCl buffer (pH 7.0). the retentate
volume was brought to 25 ml.
After washing,
This retentate was injected in 1 ml quantities onto a
Syn Chropak. AX 300 column
eluted in the following HPLC regime:
(dimensions 10 x 250 mm) and
elution rate: 2 ml/min.
elution buffer A: 25 mM Tris-HCl pH 7.0
elution buffer B: 25 mM Tris—HCl pH 7.0 + 1 M NaCl
elution gradient: time
(min) %A %B
O 99 1
12 97 3
80 20
50 50 50
70 O 100
90 O 100
95 99 1
Fractions of 1 ml each were collected. Detection of
the eluted protein was performed by continuous measurement
of the UV absorption at 280 nm. The elution profile is shown
in Figure 2.
The fractions were tested for the presence of endo-
xylanase activity by a spot test. This spot test consists of
adding 12 ml citrate—phosphate buffer (prepared by mixing
900 ml 0.2 M Na2HPO, and 125 ml 0.5 M citric acid, followed
by an adjustment of the pH of the solution to pH 5.6 using
0.5 M citric acid or 0.2 M Na2HPOfl containing 0.5% oat
spelt xylan (Sigma) to 180 mg agar (Difco) and heating the
mixture to 100 “C to dissolve the agar. After cooling to 60
“C, the agar mixture is poured evenly onto Agarose gel—bond
film. Drops of the elution fractions are placed individually
onto the film and incubated for 30 min. at 30 °C. If endo-
xylanase activity is present, the location of the individual
drops on the agar film is clear.
Total Xylanase activity in the collected fractions was
quantitatively determined by amount of
measuring the
reducing sugars produced over a predetermined time period in
the microassay as described by Leathers gt gl. (1984), using
oat spelt xylan in 50 mM sodium acetate at pH 5.0 as a
substrate. Activity units are also as defined by leathers
(supra).
Exo—xylanase activity in the eluted fractions was
determined by the method. described by Poutanen and Puls
(1988), using p-nitro—pheny1—B—D—xylopyranoside (0.3 mM,
Sigma) as a substrate at pH 5.0 and 30 °C.
The spot test revealed that the elution fractions
corresponding to peaks B, F and K (see Figure 2) contain
endo—xylanase activity. The total xylanase assay showed
activity in the elution fractions of peaks B, F, H and K.
The elution fractions of peaks B and H were determined to
contain exo-xylanase activity.
The elution fractions of peaks F (XYL2 protein) and K
(XYL A protein)
exchange
were further purified by repeated ion
chromatography. The endo—xylanases contained
therein were characterized by SDS/PAGE (Moonen gg gl., 1982)
and. Iso Electric Focussing (3.5
according to the manufacturer's instructions. The apparent
molecular weight of endo—xylanase F, as determined by SD3-
PAGE, was approximately 22 kDa: the apparent molecular
weight of endo—xylanase K was approximately 24 kDa. The iso-
electric point (IEP) of endo—xylanase F was approximately pH
4.0, while the IEP of endo—xylanase K was determined to be
lower than pH 3.5.
Example 1.2
Amino acid sequencing of the N—terminus of Aspergillus
tubigensis endo-xylanase XYL A.
Approximately 5 pg of endo-xylanase, purified as
described in Example 1.1, was subjected to electrophoresis
on a 12% SDS-polyacrylamide gel, followed by electroblotting
onto Immobilon—P membrane
(Millipore), according to the
method described by Matsudaira (1987). The membrane fragment
containing the main band having an apparent molecular weight
(SDS-PAGE) of 25 kDa is subjected to sequence analysis in a
gas-phase sequenator (Eurosequence, Groningen). The
following N-terminal sequence has been determined:
1 5 ’ 10
Ala—Gly—I1e—Asn—Tyr—Val—Gln—Asn-Tyr—Asn
(Figure 3, Formula 1)
However, roughly equal amounts of two other variants
were also discovered wherein either a serine (Figure 8,
position 1) or a glycine (Figure 8, position 3) were
determined to be the N-terminal amino acid.
Example 1.3
Amino acid sequence determination of endo-proteinase
Glu—C released peptides of endo—xylanase XYL A.
Approximately 260 pg of endo—xylanase,
described in Example 1.1,
purified as
was dissolved in 110 pl of a
solution containing 50 mM ammonium bicarbonate buffer pH 7.5
and 2 mg/ml SDS.
After heating the solution for three
minutes at 100°C and cooling to room. temperature, endo~
proteinase Glu-C (Staphylococcus aureus protease V8) was
added in an 18—fold molar excess. Protein digestion was
performed for 20 minutes at room temperature, after which
the reaction mixture was heated for three minutes at 100°C.
Approximately one—fifth. of the reaction xnixture was
subjected to electrophoresis on a 15% SDS—polyacrylamide
gel, followed by blotting onto Immobilon—P membrane
(Millipore) according to the method described by Matsudaira
(1987). Three fragments were observed with a molecular mass
of 19, 16 and 4 kDa respectively. The two largest fragments
(19 and 16 kDa) were used in gas—phase sequencing (Applied
470A protein
Groningen). Membrane fragments containing 2-3 nmol of the
Biosystems model
se encer Eurose ence,
I
particular peptide were washed and subjected to sequence
analysis,
(1987).
according to the program described by Amons
The following N-terminal amino acid sequence has been
determined from the 19 kDa fragment:
14
Tyr—Tyr-Ile—Val—Glu~Asp—Tyr~Gly— X -Tyr—Asn-Pro—Cys—(Ser)
(Figure 4, Formula 2)
The identity" of the amino acid at position 9 (X),
could not be determined. At position 14, only a trace of Ser
is found as indicated by brackets.
The following amino acid sequence has been determined
from the N—terminus of the 16 kDa fragment:
14
Tyr—Tyr-I1e—Val-Glu—Asp—Tyr—Gly~(Ser)— X —Asn—Pro—Cys-Ser
(Figure 4, Formula 3)
The identity of amino acid (X) at position 10 could
not be determined. The sequence found for this fragment is
almost identical to the sequence of the 19 kDa fragment.
Both peptides share the same N—terminal sequence, which is
not identical to the amino acid
N—termina1 sequence
determined for the intact protein (Example 1.2, Formula 1).
It has been determined that these two internal fragments
correspond to the sequence beginning with position 79 as
illustrated in Figure 8.
EXAMPLE 2
Construction of a genomic library of Aspergillus niger
DSl6813 (CBS 323.90;
tubigensis).
strain later reclassified as A;
Example 2.1
Isolation of DNA from Aspergillus niger DS16813 (CBS
323.90; later reclassified as A. tubigensis).
Fungal DNA was isolated via the procedure described by
de Graaff et a1. (1988). Mycelium, grown overnight in liquid
minimal medium (per 1000 ml: 6.0 g NaNO3; 1.5 g KH2PO,; 0.5 g
MgSO,*HgO; 0.5 g KC1; 1 ml Visniac solution [Visniac and
Santer, 1957: 10 g EDTA; 4.4 g ZnSO4*NgO; 1.0 g Mnclzwugo;
0.32 g CoC12-6H2O: 0.32 g cuso,-5H2o; 0.22 g (NH,)6Mo,02,-4H20;
1.47 g Caclz-ZHZO; 1.0 g Peso,-7H20; pH 4.0]; pH 6.0)
supplemented with 0.2 % casamino acids and 0.5 % yeast
extract, was harvested, washed with cold saline, frozen in
liquid. nitrogen and stored at —80‘C. Nucleic acids were
isolated by disrupting 0.5 g frozen mycelium using a
microdismembrator (Braun). The mycelial powder obtained was
extracted with freshly prepared extraction buffer.
The extraction buffer was prepared as follows: 1 ml
tri—isopropylnaphtalene sulfonic acid (TNS) (20 mg/ml) was
thoroughly mixed with 1 ml p—aminosalicylic acid (PAS) (120
mg/ml) and 0.5 nd.ES x RNB buffer (per 1000 ml: 121.10 g
Tris; 73.04 g NaCl: 95.10 g EGTA; adjusted to pH 8.5 with
Hcl) was added. After the addition of 1.5 ml phenol, the
extraction buffer was equilibrated for 10 minutes at 55 °C.
The warm buffer was then added to the mycelial powder, and
the suspension was thoroughly mixed for 1 minute using a
vortex mixer. After the addition of 21 ml chloroform, the
suspension was remixed for 1 min. After centrifugation at
104 x g for 10 min. using a Sorvall high speed centrifuge,
the aqueous phase was extracted once more with an equal
volume of phenol/chloroformx (1:1) and was then extracted
twice with chloroform. DNA was isolated from the aqueous
phase using the following procedure; the DNA was immediately
precipitated with 2 volumes ethanol at room temperature and
was subsequently collected by centrifugation using a Sorvall
high speed centrifuge at 104 x g for 10 min., washed twice
by redissolving the DNA in distilled, sterile water and
precipitating it again with ethanol. RNA was removed by
adding RNase A (20 g pg/ml) to the final solution.
Example 2
Partial digestion of Asperqillus tubiqensis DNA with
Sau 3A and isolation of DNA fragments after agarose gel
electrophoresis.
DNA (30 pg), isolated from Aspergillus niger DSl6813
(recently reclassified as A. tubigensis) as described in
Example 2.1, was partially digested by incubation of the DNA
with 0.1 U Sau 3A during 30 minutes at 37°C. The resulting
fragments were size fractionated by electrophoresis on 0.4%
agarose in TAE buffer containing 0.5 pg/ml ethidiumbromide.
Fragments of 14 kb to 22 kb in size, compared to fragments
of bacteriophage lambda DNA. digested with fig; II (22.0,
13.3, 9.7, 2.4, 0.65 and 0.44 kb) as size markers, were
recovered from the gel by cutting" the appropriate. region
from the gel.
These fragments were
recovered from the piece of
agarose by electro—elution using ISCO cups. A dialysis
both the and the small
containers of this cup, the cup was filled with 0.005 x TAE
(diluted from 50 x TAE stock solution (per 1000 ml): 242.0 g
57.1 ml glacial 100 ml 0.5 M EDTA;
adjusted to pH 8.0 with HCl) and the piece of agarose was
placed in the large container of the cup. Subsequently, the
cup was placed in the electro—elution apparatus, with the
large container in the cathode chamber containing TAE and
membrane was mounted on
large
Tris: acetic acid;
the small container at the anode chamber containing TAE/3 M
NaCl. The fragments were electro—eluted at 100 V for a
period of 2 hours. Afterwards, the cup was taken from the
electro—elution apparatus and the buffer was removed from
the large container, while the buffer was removed only from
the upper part of the small container. The remaining buffer
(200 pl) containing the DNA fragments was dialyzed in the
cup against distilled water for a period. of 30 minutes.
Finally, the DNA was precipitated by the addition of 0.1
volume 3 M NaAc, pH 5.6 and 2 volumes cold (—20°C) ethanol.
The DNA was collected by centrifugation (Eppendorf centrifu-
ge) for 30 min. at 14,000 x g. at 4°C. After removal of the
DNA pellet was
Speedvac vacuum centrifuge. Following ethanol precipitation,
supernatant, the dried using a Savant
the DNA was dissolved in 10 pl TE buffer (10 mM Tris—HCl pH
8.0; 1 mM EDTA; pH 8.0) and the concentration was determined
by agarose electrophoresis, using lambda DNA with a known
concentration as a reference and ethidiumbromide staining to
detect the DNA.
Example 2.3
Cloning of Asperqillus tubiqensis DNA fragments into
bacteriophage lambda EMBL 3.
Fragments obtained by partial digestion of genomic
DNA, as Example 2.2 were
bacteriophage lambda EMBL 3 Bam HI arms,
The ligated DNA was packaged in vitro using Gigapack
II Gold packaging extract (Stratagene) and plated on E. coli
LE392 (Murray, 1977) using NZYCM medium (per 1000 ml: 10 g
NZ amine; 5 g Nacl; 5 g yeast extract; 1 g casamino acids; 2
g Mgso,-7H20; pH 7.5;
according to the manufacturer's instructions.
for plates 12 g agar is added),
The complete reaction described above was repeated
once, using 3 pl genomic DNA fragments in a final volume of
pl.
Example 2.4
Titration and amplification of the
Aspergillus
tubigensis genomic library.
Dilutions of the primary genomic library were made in
SM buffer (per 1000 ml: 5.8 g NaCl; 2.0 g MgS04*HgO; 50 ml
Tris—HCl; pH 7.5; 5 ml 20% gelatin) and plated on E.coli
LE392 as a host as described by Maniatis et al. (1982, pp.
64) using NZYCM medium. After incubation overnight at 37°C,
the resulting plaques were counted and the amount of phages
was calculated. The first ligation and packaging resulted in
about '7 x 104 pfu (plaque—forming ‘units), the second in
about 4 X 105 pfu, resulting in a total of about 5 x 105
pfu.
” pfu/ml.
centrifugation at 4,000 g for 10 min.
This phage stock contained approximately
EXAMPLE 3
Screening of the Asperqillus tubiqensis genomic
library for the endo—xylanase A gene (Xln A) and isolation
of the gene.
Example 3.1
RP-labelling of synthetic oligonucleotides.
The amino acid sequence derived in Example 1.2
(Formula 1) was used to synthesize oligonucleotide mixes
corresponding 1x) the Neterminal amino acid sequence. The
oligonucleotides were synthesized. by the phosphoramidite
method,
synthesizer.
using an Applied Biosystems oligonucleotide
The oligonucleotide mixes AB801 to AB806 (Figure 3)
were mixed. in equal amounts, hereinafter referred to as
oligonucleotide mix AB800, to give a final concentration of
37 pmol oligonucleotides per pl. This oligonucleotide
mixture was labelled in a reaction mixture of the following
composition: 37 pmol oligonucleotide mixture, 66 mM Tris-HC1
pH 7.6, 1 mM ATP, 10 mM MgCl2, 15 mM
dithiothreitol, 200 pg/ml BSA, 34 pmol gamma—”P ATP (NEN,
6000 Ci/mMo1) and 30 U T1 polynucleotide kinase (BRL) in a
final volume of 50 ul. The reaction mixture was incubated
mM spermidine,
for 60 min. at 37°C, after which the reaction was terminated
by the addition of 4 pl 0.5 M EDTA: pH 8.0.
Oligonucleotide mixture AB1255, derived from the amino
acid sequence obtained in Example 1.3 (Formulas 2 and 3)
(Figure 4), was labelled via the same procedure as described
above. The
oligonucleotide mixtures were used in the
screening of the genomic library (Example 3.2) and in
Southern blot analysis (Example 3.4 and 3.5) without further
purification.
Example 3.2
Screening of the Asperqillus tubiqensis genomic
library for xln A gene.
To screen for the xln A gene in an Aspergillus
tubigensis genomic library, 3 x 103 pfu per plate were
plated in NZYCM top agarose containing 0.7% agarose (NZYCM
medium plus 7 g agarose) on four 85 mm diameter NZYCM (1.2%
agar) plates as described by Maniatis et gl. (1982, pp. 64).
E. coli LE392 were used as plating bacteria.
After incubation of the plates overnight at 37°C, two
replicas of each plate were made on nitrocellulose filters
(Schleicher and Schull BA85) as described by Maniatis gt gl.
(1982, pp. 32o~321).
After baking the filters for 2 hours at 80°C, the
filters were wetted and washed for 60 minutes at room
temperature in 3 x SSC (diluted from 20 X SSC stock solution
(per 1000 ml): 175.3 g Nacl; 107.1 g sodium citrate~5.5 H53;
pH 7.0). The filters were prehybridized. at 65°C for two
6 X SSC
(diluted from the 20 x SSC stock solution (see above)), 0.5
% SDS, 10 X Denhardt's solution (per 5000 ml: 10 g Ficoll-
hours in a prehybridization buffer containing:
; 10 g polyvinylpyrrolidone; 10 g Bovine Serum Albumin
(Pentax Fraction V)) and 100 pg/ml heat denatured herring
After two
prehybridization, the prehybridization buffer was replaced
buffer identical to the
prehybridization buffer, except that this buffer did not
sperm DNA, but RP-labelled
oligonucleotide mix AB800, prepared as described in Example
sperm DNA (Boerhinger Mannheim). hours
by hybridization which was
contain herring contained
3.1. The filters were hybridized for 18 hours at an final
temperature of 38°C, achieved by slow, controlled cooling
from the initial temperature of 65°C.
After hybridization, the filters were first washed in
2 x SSC, after which the filters were washed in prewarmed
hybridization buffer at 38°C for the same period of time.
Finally, the filters were washed for 30 minutes at 38°C in 6
x SSC, 0.05% sodium pyrophosphate. The air dried filters
were taped onto a sheet of Whatman 3MM paper, keying marks
were made with radioactive ink and the Whatman paper and
filters were covered with Saran Wrap”. Hybridizing plaques
were identified by exposure of Kodak XAR X—ray film for 72
hours at —70°C using an intensifying screen.
Four of the oligonucleotide mixture hybridizing
plaques, appearing in duplicate on the replica filters, were
1 to lambdaflm. Each
positive plaque was removed from the plate using a Pasteur
identified and were designated lambdafln
pipette and the phages were eluted from the agar plug in 1
ml of SM buffer containing 20 ul chloroform, as described by
Maniatis gt; al. (1982, p. 64). The phages obtained. were
purified by repeating the procedure described above using
filter replicas from plates containing 50-100 plaques of the
isolated phages.
After purification, the phages were propagated by
plating 55 X 103 phages on NZYCM medium. After incubation
overnight at 37°C, confluent. plates were obtained, from
which the phages were eluted by adding 5 ml SM buffer and
storing" the plate for 2 hours at 4°C with intermittent
shaking. After removal of the supernatant, the bacteria were
removed from the solution by centrifugation at 4,000 X g for
minutes at 4°C. (0.3%) was added to the
supernatent and the number of pfu. was determined. These
Chloroform
phage stocks contained approximately 10w pfu/ml.
,E,>ia.I_rp_le 3 - 3
Isolation of DNA from bacteriophage lambda.
Each of the isolated phages lambdaflfl to lambdafifl were
propagated as described in Example 3.2 using five plates for
each of the phages. The phages were precipitated from the
thus—obtained supernatant (25 ml) by addition of an equal
volume of a solution containing 20% PEG~6000 (w/v) and 2 M
Nacl, followed by thorough mixing and incubation on ice for
collected by
centrifugation at 14,000 x g at 4°C for 20 minutes. The
supernatant was removed by aspiration, while the last traces
minutes. The precipitated phages were
of liquid were removed using a paper towel. The phages were
carefully resuspended in 4 ml SM buffer and extracted once
with chloroform.
Prior to extracting the DNA from the phage particles,
DNA and RNA originating from the lysed bacteria were removed
by incubation of the phage suspension with DNase I and RNase
A (both 100 pg/ml) for 30 minutes at 37°C. The phage DNA was
subsequently released from the phages by the addition of SDS
and EDTA to a final concentration of 0.1% and
mM
respectively, followed by incubation at 65°C for 10 minutes.
Protein was removed from the solution by extracting twice
alcohol
(25:24:1). After separation of the phases by centrifugation
(14,000 X g, 10 min.), the
extracted once with an equal volume
with an equal volume phenol/chloroform/isoamyl
in an Eppendorf centrifuge
aqueous phase was
chloroform/isoamylalcohol (24:1). The phases were separated
by centrifugation 14,000 X g, 10
after which the DNA was precipitated from the
by the addition 0.1
perchlorate and 0.1 volume isopropanol and incubation on ice
(Eppendorf centrifuge,
minutes),
aqueous phase volume 5 M sodium
for 30 min. The DNA was recovered by centrifugation for 10
minutes at 4°C (14,000 X g). The supernatant was removed by
aspiration, after which the DNA was resuspended in 400 pl TE
buffer. The
ethanol:
DNA was once again precipitated Wlfll
The DNA was
centrifugation for 10 minutes at 4°C
collected by
(14,000 X g). The
supernatant was removed by aspiration, the remaining pellet
after" which the DNA. was
resuspended in 125 pl TE buffer containing 0.1 pg/ml RNase
A. This purification procedure resulted in the isolation of
approximately 40-50 pg DNA from each phage.
was briefly dried under vacuum,
Example 3.4
Restriction analysis of xln A containing phages.
The isolated DNA of phages lambdaxm1 to lambdaxm, was
analyzed by Southern analysis using the following
restriction enzymes; BamHI; BqlII; EcoRI; HinDIII; Kpnl;
following solutions; 3 pl DNA
give a final volume of 50 pl. After digestion, the DNA was
precipitated by the addition of 0.1 volume 3 M NaAc and 2
volumes ethanol. The DNA was collected by centrifugation for
minutes at room temperature (14,000 x g). The supernatant
was removed by aspiration. The remaining pellet was briefly
dried under vacuum and resuspended in sterile distilled
water. After addition of :1 pl DNA loading buffer (0.25 %
(w/V) bromophenol blue; 0.25 % (w/V) xylene cyanol; 15 %
(w/V) Ficoll type 400 in Inc), the samples were incubated
for 10 minutes at 65°C and rapidly cooled on ice. The
samples were then loaded on a 0.6% agarose gel in 1 X TAE
buffer. The DNA fragments were separated by electrophoresis
at 25 V for 15-18 hours.
After electrophoresis, the DNA was denatured and
transferred. to 21 nitrocellulose ‘membrane as described. by
Maniatis et gl. (1982, pp. 383-386), followed by subsequent
prehybridization and labelled
described in
hybridization using the
AB8OO and AB1255 as
Example 3.1 and. hybridization conditions as described in
Example 3.2. The
oligonucleotide mixes
hybridization pattern for each
oligonucleotide mixture was obtained by exposure of Kodak
XAR—5 X-ray film for 18 hours at —70°C using an intensifying
screen.
From the results, it was concluded that the DNA of all
four isolated clones hybridized with the oligonucleotide
mixture derived from the N—terminal amino acid sequence (mix
AB800), as well as with the oligonucleotide mixture derived
from the amino acid sequence obtained from the peptide
isolated after S. aureus V8 digestion (AB1255). In all four
clones, fragments originating from the same genomic region
were found.
The restriction fragment patterns and the
hybridization patterns were used to construct an approximate
restriction map of the genomic region where the gig A gene
is located (Figure 5).
Example 3.5
subcloning of the xln A gene.
isolated from phage
vector pUC9
dephosphorylated with alkaline
minutes. The linearized vector‘ was isolated from a 0.6%
agarose gel as described in Example 2.2.
sterile water. 10 pl of the diluted mixture was used to
transform E. coli JMIO1 (Yanisch-Perron gt gl., 1985)
competent cells, prepared by the CM1, CM2 method as
described in the Pharmacia Manual for the M13
cloning/sequencing system. E. coli JM101 containing plasmid
pIMl00 was deposited at the
Schimmelcultures, Baarn, The Netherlands on July 19, 1990
Centraal Bureau voor
and was assigned the designation CBS 322.90.
A selection of six of the resulting colonies was grown
overnight in LB medium (per 1000 ml: 10 g trypticase peptone
(BBL); 5 g yeast extract (BBL); 10 g Nacl; 0.5 mM Tris—HCl;
pH 7.5) containing 100 pg/ml ampicillin.
Plasmid DNA was isolated from the cultures by the
alkaline lysis method as described by Maniatis et al. (1982,
pp. 368—369).
analysis,
This plasmid DNA was used in restriction
as described in Example 3.4 to select £1 clone
harboring the desired plasmid. Plasmid DNA was isolated on a
large scale from 500 ml cultures E. coli JM101 containing
the plasmid pIM100 grown in LB medium containing 100 pg/ml
A 1982, p.86) The plasmid was
ethanol
The yield was
ampicillin (Maniatis gt_ al.,
purified by CsCl centrifugation, phenolized,
precipitated and dissolved. in 400 pl TE.
approximately 500 pg.
The plasmid pIM100 was further analyzed by restriction
enzymes resulting in the restriction map shown in Figure 7.
The orientation of the gene, as indicated, was determined
from hybridization experiments under conditions described in
Example 3.2 using the oligonucleotide mixes AB8OO and ABl255
as probes.
EXAMPLE 4
Characterization of the Asperqillus tubiqensis xln A
gene.
Example 4.1
Sequence determination of the A. tubigensis xln A gene
The sequence of the Asperqillus tubigensis xln A gene,
which comprises its promoter/regulation region, the
structural gene and the termination region, was determined
by subcloning fragments from pIM100 in Ml3mp18/mp19, in
combination with the use of specific oligonucleotides as
primers in the sequencing reactions.
For nucleotide sequence analysis, restriction
fragments were isolated as described in Example 2.2 and were
then cloned in bacteriophage M13 mp18/19 RF DNA ‘vectors
(Messing, 1983: Norrander gt al., 1983), digested with the
appropriate restriction enzymes. The nucleotide sequences
Example 4.2
The A. tubigensis gin A gene
The sequence obtained comprises 2054 bp, 949 bp in the
' non~coding region and 420 bp in the 3' non-coding region.
In the 5' upstream region, a putative TATA box (TATAAAT) was
found at 854, before the
(position. 950). A triplicate
(5'GTCCATTTAGCCA3') was in the
region 190 to 350 bp from the translation initiation site
(positions; 618 to 632: 636 to 650; 656 to 670).
position 848 to position
translation initiation site
repeating
sequence found
The structural section of the zlg A gene is 681 bp
long and is interrupted by a single putative intron 48 bp
long. The polypeptide derived from the sequence is 211 AA in
length. A 17 AA long hydrophobic signal sequence is found at
the N-terminus of this polypeptide, which is followed by a
propeptide which is 12 residues long. The mature protein is
184 AA in size with an predicted molecular weight of 19 kDa
and has a theoretical IEP of 3.6.
EXAMPLE 5
Expression of the xln A gene in an Aspergillus niger
N593.
Example 5.1
Introduction of the xln A gene into Aspergillus niger
N593 by co—transformation.
The plasmid pIM100, obtained in Example 3.5 was
introduced in Asperqillus niqer by co—transformation of
Aspergillus nigg; N593 (py;' mutant of A. niger N402; Goosen
et al., 1987) using the Aspergillus giggr py; A gene as a
selective marker on the plasmid pGW635 (Goosen gt al., 1989)
and the plasmid pIM100 as the co—transforming plasmid.
Protoplasts were prepared from mycelium by growing
Aspergillus giggr N593 on minimal medium supplemented with
0.5% yeast extract, 0.2% casamino acids, 50 mM glucose and
mM uridine for 20 hours at 30°C. The preparation of
protoplasts of Aspergillus Qiggr N593 and the transformation
procedure was performed as described by Goosen gt al.
(1987). The resulting PYR+ transformants were then analyzed
for the expression of the xln A gene.
Example 5.2
Screening of transformants for the expression of the
xln A gene.
The transformants obtained in
filtrate was analyzed. by SDS—polyacrylamide gel
electrophoresis, using a gel containing 12% acrylamide. The
XYL A after
protein was detected on nitrocellulose
electroblotting and incubation with polyclonal antibodies
raised against the XYI. A protein, which. was purified as
described in Example 1.1. The antibody bound, was detected
after incubation with goat—anti—rabbit antibody conjugated
to alkaline phosphatase, according to the Biorad instruction
manual.
Sixteen of the twenty transformants analyzed produced
the XYL A protein as detected by this procedure. The protein
was secreted into the medium. Of the transformants analyzed,
transformant TrX9 was selected by I.E.F. analysis, using a
pH gradient of pH 3 to 7 and. subsequent staining of a
dilution series of transformants TrX2 and TrX9, using the
method as described by Biely gt al. (1985 a and b).
Figure 9 is a zymogram exhibiting the XYL A protein
expressed by transformants TrX2 and TrX9.
SDS-PAGE analysis was performed using 4 pl supernatant
samples of individual transformants and the A. niger control
strain, adjusted to pH 7 with 3 N NaOH and
subsequently brought to a final volume of 20 pl with 1 X SB
buffer, as described by Laemmli (1970). After heating for 5
100°C, total
polyacrylamide gel
first
minutes at
sos/12.5%
mixtures were subjected to a
electrophoresis and
subsequently stained with coomassie brilliant blue. As shown
in Figure 10 B, a protein band having an apparent molecular
weight of 25 kDa (comparable with purified xylanase (lane 2)
could be detected in transformants TrX2 (lane 4) and TrX9
(lane 5), which is absent from the supernatant of the
control strain (lane 3). Molecular weight markers (lane 1)
represent 92, 68, 46 and 30 kDa.
Example 5.3
Deletion analysis of the xln A promoter region
tubiqensis xln A
Regulatory elements in the A.
promoter were studied by promoter deletion analysis. A
series of five constructs of the xln A gene were made and
cloned in combination with the A. niger py; A gene. The pyr
A gene allows selection in transformations experiments as
described in Example 5.1. In addition, the pyr A gene
permits the selection of transformants having a single copy
of the plasmid integrated at the pyr A locus.
The plasmids pIMl12, pIM113, pIM1l4, pIM116 and pIM117
were used to transform A. niger N593 as described in Example
.1. The resulting PYR* transformants of each plasmid were
cultivated and DNA was isolated as described in Example 2.1.
The resulting DNA was digested with fipal and single copy
integrations were selected by Southern analysis using a y
labeled 3.8 kb XbaI fragment
P
(labelled as described in
Example 7.2), which contained the Q1; A gene as a probe.
Transformants having a hybridizing flpal fragment (the size
of which was increased by a unit plasmid length as compared
to the size of the flpgl fragment in A. niger N593) were
selected as single copy integrations at the pyr A locus.
Single copy transformants of each of the plasmids,
selected as described. above, were grown for 36 hours as
described in Example 5.2. The expression of the xln A gene
was analyzed by IEF analysis as described in Example 5.2 and
of total RNA as
described by de Graaff _e3:_ a_1. (1988), using the 32:» labelled
by Northern analysis after isolation
bp Xhgl/gamfll fragment of the xln A gene as a probe.
In transformants originating from the plasmids pIM1l2,
pIM113 and pIMll4, expression of the gin A gene was found as
detected by IEF and
Northern analysis.
from the
However, the
transformants originating plasmids pIM116 and
pIMl17 did not express the x13 A gene, since neither XYL A
protein nor hybridizing RNA were found. From these results
it was concluded that the 158 bp ggtl/ghgl fragment, the
essential difference between pIM114 and pIM116, contains an
element necessary for the induction of the xln A gene in A;
giggr, which were grown on medium using xylan as a carbon
SOUICB .
EXAMPLE 6
Expression in A. niger of the xln A gene fused to the
promoter and/or signal sequence of the A. niger
amyloglucosidase (AG) gene.
Example 6.1
Xylanase expression vectors
To obtain expression of xylanase in the strain A;
gigg; CBS 513.88, additional expression cassettes (pXYL3 and
pXYL3AG) were created in which the xln A gene is under the
control of the A. niger amyloglucosidase (AG) promoter in
combination with different signal sequences.
In expression cassette pXYL3, the AG-promoter sequence
was fused. to the gin A encoding" sequence including the
xylanase leader.
In the expression cassette pXYL3AG, the AG-promoter
sequence, as well as the 18 amino acid (aa) leader sequence
of the AG—gene were
fused to the xln A gene fragment
encoding solely the mature protein.
Example 6.2
Construction of intermediate plasmids.
a) Subcloninq the X1nA locus.
To reduce the length of the genomic xln A locus, the 2
kb Pstl pIM1OO
comprising the entire xln A gene including the 5'— and 3'
fragment of (described in Example 3.5)
flanking sequences, was subcloned. into the Pstl site of
pTZ18R (Promega). The plasmid containing the xln A gene in
the proper orientation
(indicated in Figure 12) was
designated pXYL1.
b) Basic selection Vector pAmdSH
To serve as a selection marker for the transformation
of Aspergillus, the EcoRI/KpnI DNA fragment of plasmid
pGW325 (Wernars, K. (1986)) containing the homologous
Asperqillus nidulans amds gene, was inserted into the
EcoRI/KpnI sites of pTZ18R (Promega). -In the resulting
vector (pAmds), an additional HindIII restriction site was
introduced by insertion of the synthetic fragment:
' AATTCAAGCTTG 3‘
3‘ GTTCGAACTTAA 5'
into the EcoRI-site. The
designated. pAmdSH.
thus-obtained plasmid was
In this basic vector, the AG/xylanase
fusion DNA fragments will be inserted.
c) Isolation of the qenomic AG locus:
QAB6-l.
Plasmid pAB6-1 contains the entire AG locus from A;
construction of
niger, isolated from an A. niger plasmid library containing
the 13-15 kb HindIII fragments, inserted into pUC19.
For this
oligonucleotides were used:
isolation, the following AG-specific
AG-1: 5'-GACAATGGCTACACCAGCACCGCAACGGACATTGTTTGGCCC-3'
AG-2: 5'-AAGCAGCCATTGCCCGAAGCCGAT-3',
both based on the nucleotide sequence published for A. niger
(Boel, gt gl. (1984a); (1984b)). The
derived from the
Boel, et al.
oligonucleotide probes were sequence
surrounding intron 2: oligo AG—1 is located downstream this
intron and has a polarity identical to the AGmRNA; oligo AG-
is found upstream of intron 2 and is chosen antiparallel
to the AGmRNA. Plasmid pAB6—1 contains the entire AG locus
on a 14.5 kb HindIII fragment (see Figure 13).
d) The intermediate plasmids pXYLAG and pXYL2AG.
Fusion of the AG-promoter and the 18 aa AG—leader se-
quence to the 313 A gene encoding the mature protein
(lacking the serine from position 1) was performed by the
Polymerase Chain Reaction (PCR) method.
In the PCR reactions, two templates were used: pXYL1,
containing the gln A gene and pAB6—1, containing the entire
AG genomic locus.
As primers for the PCR DNA—amplifications, four
synthetic oligo nucleotides were designed having the
following sequence:
Oligo AB 177125'-CTCTGCAGGAATTCAAGCTAG-3'
(an AG specific sequence around the EQQRI site approx;
250 bp upstream from the ATG initiation codon).
Oligo AB 1985:5'-GTAGTTGATACCGGCACTTGCCAACCCTGTGCAGAC-3'
mature xylanase -—-—i~—+- 18 aa AG~leader
Oligo AB 1986:5'-GTCTGCACAGGGTTGGCAAGTGCCGGTATCAACTAC-3'
18 aa AG—leader -—i~—- mature xylanase
Oligo AB 1984:5'-CCGGGATCCGATCATCACACC-3'
(a gln A specific sequence located at the figmhl site
on position 1701 as shown in Figure 8).
The PCR was performed as described by Saiki g; 1.
(1988) and according to the supplier of TAQ~polymerase
(Cetus). Twenty-five amplification cycles (each: 2 minutes
at 55°C; 3 minutes at 72°C; 1 minute at 94°C) were carried
out in a DNA—amplifier (Perkin—Elmer/Cetus).
To fuse the AG sequences to the gin A coding sequence,
two separate polymerase chain reactions were performed: the
pAB6~l as the template and
oligonucleotides AB 1771 and AB 1985 as primers to amplify a
300 bp DNA fragment which contained the 3‘ piece of the AG-
promoter and the 18 aa AG—leader sequence, flanked at the
first reaction with
‘—border by the first 18 nucleotides of the coding sequence
-44..
of gin A and the second reaction with pXYLl as the template
and oligonucleotides AB 1986 and 1984 as primers to amplify
Kim A DNA sequences encoding the mature xylanase protein,
flanked at the 5’—border by the last 18 nucleotides of the
AG-signal peptide. A schematic view of these amplifications
is presented in Figure 14.
The two DNA fragments generated were purified by
agarose gel electrophoresis and ethanol precipitation and
subsequently used as templates in the third PCR with oligo
nucleotides AB 1771 and 1984 as primers to generate the AG-
xylanase fusion. The thus—obtained DNA fragment was digested
with EQQRI and fiamfil and subcloned into the appropriate
sites of pTZl8R. The resultant fusion was sequenced and
designated pXYLAG (see Figures 14 and 15).
The remaining
wherefrom the
thus-obtained plasmid pXYL2AG is shown in
Figure 15.
e) The intermediate plasmids pXYL and pXYL2.
Fusion of the AG—promoter sequence to the gin A gene
including the xylanase leader was performed as described in
part d), above. As primers, two additional oligonucleotides
were designed having the following sequence:
Oligo AB 1982: 5'-AGCCGCAGTGACCTTCATTGCTGAGGTGTAATGATG'3'
Xylanase gene ———-i*——- AG—promoter
01190 AB 1983! 5'-CATCATTACACCTCAGCAATGAAGGTCACTGCGGCT‘3'
AG—promoter ———~L——*- Xylanase gene
-45..
To fuse the AG promoter sequence to the xylanase gene
(including the xylanase
chain
signal sequence), two separate
performed: the first
reaction with pAB6—1 as template and oligonucleotides AB
1771 and AB 1982 as primers to amplify a 282 bp fragment
containing the 3'-part of the AG promoter flanked at the 3'-
border by 18 nucleotides of the xylanase leader and the
second with pXYLl as template and the
oligonucleotides AB 1983 and AB 1984 as primers to amplify a
DNA fragment containing the entire xylanase gene (including
the xylanase leader) and flanked at. the 5'—border by 18
nucleotides of the AG—promoter.
The two
polymerase reactions were
reaction
DNA fragments generated were purified by
agarose gel electrophoresis and ethanol precipitation and
subsequently used as templates in a third PCR with
oligonucleotides AB 1771 and 1984 as primers to generate the
AG~Xy1anase fusion. The thus~obtained DNA fragment was digen
sted with EQQRI and fiamhl and subcloned into the appropriate
sites of pTZl8R. The resultant fusion was sequenced and
designated pXYL (see Figures 14 and 16).
(3.5 kb) upstream region of the AG
promoter was inserted into pXYLl as described in part d),
above. The thus—obtained plasmid was designated pXYL2.
The remaining
Exampleiéii
Construction of the
pXYL3AG and pXYL3.
xylanase expression cassettes
Both expression cassettes were created by insertion of
the AG/xylanase fusions of pXYL2AC or pXYL2 into the basic
A, niger vector pAmdSH. For this final construction, pAmdSH
was digested with gpnl and fiindlll (partially) and pXYL2AG
and pXYL2 with fiindxir with gpgl. All
fragments were isolated and purified by gel electrophoresis
and partially
and ethanol precipitation. To the 6.8 kb Kpnl/HindIII DNA
fragment of pAmdSH, either the 5.3 kb Kpnl/HindIII DNA
of pXYL2AG or pXYL2 was added,
subsequently molecular cloned by transferring both ligation
fragment ligated and
mixtures to E.coli. The thus—derived expression cassettes
were designated pXYL3AG (containing the AG-leader) and pXYL3
(containing the xylanase leader), as shown in Figures 17 and
18, respectively.
Example 6.4
Expression of the xln A gene under the control of the
AG promoter in A. niger.
a) Transformation of A. niger (CBS 513.88).
Before transferring both expression cassettes pXYL3AG
and pXYL3 to A. niger, the E. coli sequences were removed by
HindIII ethanol
digestion, gel electrophoresis and
precipitation. Transformation of the strain A. niger (CBS
513.88, deposited October 10, 1988) was performed with 10 pg
linearized DNA fragment by procedures as
Tilburn, J. gt gl.
described by
(1983) and Kelly and Hynes (1985) with
the following modifications:
— Mycelium was minimal medium
Aspergillus
(Cove, D. (1966)) supplemented with 10 mM arginine and
mM proline for 16 hours at 30%: in a rotary shaker
grown on
at 300 rpm.
— Only Novozym 234, and no helicase, was used for
formation of protoplasts.
~ After 90 minutes of protoplast formation, 1 volume of
STC buffer (1.2 M sorbitol, 10 mM Tris—HCl pH 7.5, 50
mM CaCl2) was added to the protoplast suspension and
centrifuged. at 2500 rpnx at. 4°C for 10 Ininutes in a
swing—out rotor. The protoplasts were washed and
resuspended in STC—buffer at a concentration of 108
cells/ml.
— Plasmid DNA was added. in a volume of 10 pl in TE
buffer (10 mM Tris—HCl pH 7.5, 0.1 mM EDTA) to 100 pl
of the protoplast suspension.
— After incubation of the DNA-protoplast suspension at
0°C for 25 minutes, 200 pl PEG solution was added
dropwise (25% PEG 4000 (Merck), 10 mM Tris—HCL pH 7.5,
mM Caclz). Subsequently, 1 ml of PEG solution ( 60%
PEG 4000 in 10 mM Tris-HCl pH 7.5, 50 mM CaCl?) was
added slowly, with repeated mixing of the tubes. After
incubation at room temperature, the suspensions were
diluted with STC—buffer,
centrifuged at 2000 rpm at 4%} for 10 minutes. The
mixed by inversion and
protoplasts were resuspended. gently in 200 pl STC-
buffer and plated on Aspergillus minimal medium with
mM acetamide as sole nitrogen source, 15 mM Cscl, I
M sucrose, solidified with 0.75% bacteriological agar
#1 (Oxoid). Growth. was performed at 33°C for 6-10
days.
b) Growth of transformants in shake flasks.
Single A. niger transformants from each expression
cassette were isolated, and the spores were streaked on
selective acetamide-agar plates. Spores of each transformant
were collected from cells grown for 3 days at 37°C on 0.4%
(Oxoid,
production was tested in shake flasks under the following
potato—dextrose England) agar plates. Xylanase
growth conditions:
— About 1.108 spores were inoculated in 100 ml pre-
1 g KHZPO4; 30 g
maltose; 5 g yeast—extract; 10 g casein—hydrolysate; 0.5 g
MgSO,JNg0 and 3 g Tween 80. The pH was adjusted to 5.5.
culture medium containing (per litre):
- After growing overnight at 34 °C in a rotary shaker, 1
ml of the growing culture was inoculated in a 100 ml main-
2 g 1
dextrin (Maldex MDO3, Amylum); 12.5 g yeast—extract; 25 g
casein—hydrolysate; 2 g Kgxr; 0.5 g MgSO,.7Hg3; 0.03 g Znclfi
culture containing (per litre): 70 g malto-
.02 g CaCl2: 0.05 g MnSO,.4 I50 and FeSO,. ‘The pH was
adjusted to 5.6. The mycelium was grown for at least 140
hours.
c) Analyses of transformants.
Xylanase analyses of individual transformants were
by sDs—
polyacrylamide gel electrophoresis stained with Coomassie
Brilliant Blue A
Remazolbrilliant blue R.
performed by measuring the Xylanase activity;
and by a zymogram stained with Xylan-
Xylanase activities were determined as described by
Leathers gt al. (1984), with some
substrate concentration was increased from 1% to 5% oat
xylan, dissolved in 100 mM NaAc at pH 3.5 and heated to
modification. The
°C for 10 minutes. In addition, enzyme reactions were
carried out at 39°C instead of 30°C.
Xylanase measured in the
production levels were
supernatant of 6 day-shake flask fermentations of several,
randomly chosen transformants obtained from each expression
cassette. The results are shown in Table 1.
Table l
Xylanase production of several A. niqer CBS 513.88 strains
transformed with plasmids containing the xlnA gene under the
control of the A. niqer AG-promoter in combination with
different leaders.
Expression cassette Transformant # Xylanase activity
(U/ml)
.1 2400
pXYL3 1.2 1700
(AG—promoter/ 10 3600
xln—leader) 29 3500
pXYL3AG
(AG~promoter/ 3.1 2400
AG—leader)
A. niger CBS 513.88
(control strain) — O
sDs—PAGE analysis was performed as follows: after 5
days of growth as described in part b) above, 4 pl
supernatant samples from individual transformants and from
the . er control strain were first adjusted to pH 7 with
3 N NaOH and subsequently brought to a final volume of 20 pl
with 1 x SB buffer, as described by Laemmli (1970). After
heating for 5 l00'C, total mixtures were
subjected to a SDS/12.5% polyacrylamide gel electrophoresis
and subsequently stained with coomassie brilliant blue. As
shown in Figure 10 A, a proteirx band having" an apparent
molecular weight of 25 kDa
(lane 1)
minutes at
(comparable with purified
could. be detected in transformants 10
(lane 4), 29 (lane 5) and 1.1 (lane 6). This protein band
was absent from the supernatant of the control strain (lane
3). Molecular weight markers (lane 2) represent 94, 67, 43
, 20 and 14.5 kDa.
xylanase
Zymogram analysis was performed as follows. The same
samples as used above were also applied two times to a
native 8~25% PAA (BRL). Following
electrophoresis, gel A was stained with Coomassie brillant
blue (see Fioure 11A)
Blue—xylan
phast system gel
and gel R with a Remazol Brillant
overlay at pH 3.5 as described by
(see Figure 11B),
to visualize the
supplied to this RBB—xylan
overlay gel contained 5 times less protein as compared to
the samples provided to the gels as shown in Figures 10 and
11A. The identification of the lanes in Figures 11 A and B
Samples
are the same as Figures 10 A and B.
These analyses clearly show the expression and
secretion of active endo-xylanase in A, niger CBS 513.88
transformed with an expression cassette wherein the xln A
gene is under the control of the A. nigcr amyloglucosidase
promoter. Expression and secretion were also observed with
different 5, njge;
protein lacking the
signal sequences. Furthermore, the
serine residue from
position 1
nevertheless retained xylan-deoradinc activity.
EXAMPLE 7
Application of the XYL A protein in animal feed
compositions.
The efficacy’ of endo-xylanase supplementation to a
diet rich in wheat by—products on nutrient digestibility and
zootechnical performance is demonstrated using the following
experimental protocol.
One—day old female chicks were housed in battery cages
with wire floors and fed on a commercial starter diet until
the start of the experimental period. At day 13, the birds
were allocated at random to equal live—weight treatment
groups. Three different experimental diets were assigned to
18 groups of birds with 8 birds per group. The diets were
pelleted under very mild conditions and the chicks were fed
ad libitum during days 13 to 34 (post—hatching).
Feed consumption and growth were monitored weekly for
each cage. Digestibility was measured by a :3 day excreta
collection period. A semi—quantitative collection of the
excreta was performed. Using a marker (HCL—insoluble ash),
individual digestibility coefficients for protein, fat,
crude fibre and Nitrogen Free Extract were calculated. The
(AME) of
calculated from the following equation:
apparent metabolisable energy each diet was
AME (MJ/kg D.M.) = 17.46 a,-+ 38.81 a2-+ 8.0 as + 16.5 a,
a1 = crude protein (gram per kg D.M.) X d.c.”/
a2 = crude fat (gram per kg D.M.) x d.c.
a3 = crude fibre (gram per kg D;M) x d.c.
a, = Nitrogen Free Extract (gram per kg D.M.) X d.c.
U d.c. = digestibility coefficient
The basal diet was based on wheat bran, maize starch
and protein-rich animal by—products (Table 3). To this diet,
endo—xylanase was supplemented at two different levels,
,000 U/kg and 174,000 U/kg
(specific activity 300,000 U/g).
Table 3
Composition of the basal diet
purified endo-xylanase
Ingredient
[o\°
wheat bran
maize starch
maize 4.4
animal by—products (meat meal,
La)-$5
00
fish meal, feather meal) 9.7
soy isolate (81% cp) 6.0
soy oil 6.0
fat blend 0.7
ground limestone 0.32
mono calcium phosphate 0.13
premix (including DL—methionine
& Lysine - HCL) 1.35
SiO2—marker (Diamole) 1
Calculated content
AME (MJ/kg) 12.66
crude protein, % 18.0
lysine, % 1.2
methionine + cystine, % 0.9
Analyzed content
crude protein, % 18.2
crude fat, % 9.7
crude fibre,% 4.1
sioz - marker, % 1.07
Ca, % 0.8
P, % 0.85
ash, % 5.9
The most important results are summarized in Table 4
and Table 5 (digestibility data). The
performance data refer to the entire experimental period,
days 13 to 34 while vthe digestibility
figures are the average Values derived from analyses in
(performance data)
(post-hatching),
excreta collected during days 21 to 24 and days 28 to 31.
Table 4
The effect of endoxvlanase addition on chick performance
from 13 to 34 days of aqe.
diet 1 diet 2 diet 3
36.000 U/kg 174.000 U/kg
Parameter basal endoxvlanase endoxylanase
—growth (g per day) 50.5 50.80 51.9
—feed consumption
(g/bird/day) 95.6 89.7 92.6
—feed: gain (gzg) 1.89 1.77 1.79
Table 5
The effect of endoxvlanase addition on diqestibility
coefficients (d.c.) of the orqanic nutrients and calculated
enerqy value of the diet.
Digestibility diet 1 diet 2 diet 3
36,000 174,000
coefficient zbasal units/kq XYL A units/kq XYL A
crude protein‘, % 79 82 82
crude fat, % 85 91 82
crude fibre, % O 11 11
Nitrogen Free
Extract, % 73 75 75
AME (MJ/kg D.M.) 13.81 14.28 14.
L/I
The nitrogen measured in the excreta was corrected for
the uric acid content in the urine.
This experiment (demonstrates the efficacy of endo-
xylanase addition to feed compositions for broilers which
Both the
performance of the chicks and the energy value of the diets
contain a large proportion of wheat bran.
were affected positively.
Regarding the performance feed conversion, efficiency
which reflected the
influence of enzyme addition. There was a tendency towards a
was the most sensitive parameter
slightly reduced feed consumption in the enzyme supplemented
groups associated with a similar or better growth.
Consequently, the feed: gain ratio was decreased
substantially.
The improvement in performance can be explained by the
effects from enzyme addition on the digestibility
coefficients. All nutrient digestibility figures were
affected positively, although the increase in fat
which led to ‘a 3.5%
increase in the energy value of the enzyme—supplemented
digestibility was most pronounced,
diets.
No dose—response relationship ‘was noticed at these
levels of enzyme inclusion.
EXAMPLE 8
The use of endo—xylanase in breadmaking
Pup—loaves were baked from 150 g dough pieces obtained
by mixing 200 g wheat flour (100%), 106 ml water (53%), 1.2
g instant dry baker's yeast (0.6%: Gist—brocades N.V.,
Delft, The Netherlands), 4 g Nacl (2%), 400 mg caclzcngo
(0.2%), 10 mg fungal
SKB/kg flour)
xylanase (xyl A) activity. After mixing for 6 minutes and 15
a—amylase Pmo (Gist—brocades, 2250
and a variable number of units of endo-
seconds at 52 r.p.m. in a pin mixer, the dough was divided,
proofed for 45 minutes at 31°C, punched, proofed- for an
additional 25 minutes, molded. and panned. After‘ a final
proof of 70 ndnutes at 31°C, the dough was baked for 20
minutes in an oven at 250°C. Loaf volume was determined by
the rapeseed displacement method. The results are summarized
in Table 6, below.
Table 6
Characteristics of bread prepared with various amounts of
endo-xylanase (xvl A) activity
Endo-
xylanase Loaf
activity volume Break{ Crumb *
(units) (ml) Shred structure
0 546 6 6
32 560 7 6
128 579 7.5 7
320 609 8 6-5
640 621 7.5 6.5
960 624 7.5 7
2560 618 7.5 7.5
= Score from 1 (lowest quality) to 10 (highest quality)
From these results, it is clear" that an increasing
amount of endo—xylanase activity added to the dough leads to
an increase in loaf volume and. an improvement. of bread
quality in terms of break and shred and crumb structure.
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Claims (1)
1. A DNA sequence comprising a sequence encoding a polypeptide having fungal xylanase activity which is: (a) a DNA sequence encoding the xylanase amino acid sequence of
Applications Claiming Priority (2)
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EPEUROPEANPATENTOFFICE(EPO)24/07/1 | |||
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IE20040144A IE20040144A1 (en) | 1990-07-24 | 1991-07-23 | Cloning and expression of xylanase genes from fungal origin |
IE258391A IE68859B1 (en) | 1990-07-24 | 1991-07-23 | Enzymatic treatment of silage |
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- 1991-07-24 DE DE0463706T patent/DE463706T1/en active Pending
- 1991-07-24 AT AT91201943T patent/ATE124844T1/en not_active IP Right Cessation
- 1991-07-24 WO PCT/NL1991/000137 patent/WO1992001793A1/en active IP Right Grant
- 1991-07-24 EP EP91201943A patent/EP0468596B1/en not_active Expired - Lifetime
- 1991-07-24 HU HU9200961A patent/HU215234B/en not_active IP Right Cessation
- 1991-07-24 ES ES91201944T patent/ES2086267T3/en not_active Expired - Lifetime
- 1991-07-24 DK DK91201944T patent/DK0463706T3/en active
- 1991-07-24 CA CA002067329A patent/CA2067329A1/en not_active Abandoned
- 1991-07-24 IL IL9894191A patent/IL98941A/en not_active IP Right Cessation
- 1991-07-24 AT AT91201944T patent/ATE233818T1/en not_active IP Right Cessation
- 1991-07-24 EP EP91201944A patent/EP0463706B2/en not_active Expired - Lifetime
- 1991-07-24 DE DE69133201T patent/DE69133201T2/en not_active Expired - Lifetime
- 1991-07-24 JP JP3513261A patent/JPH05501657A/en active Pending
- 1991-07-24 KR KR1019920700672A patent/KR100212232B1/en not_active IP Right Cessation
- 1991-07-24 HU HU92960A patent/HUT60606A/en unknown
- 1991-07-24 DE DE69111148T patent/DE69111148T2/en not_active Expired - Fee Related
- 1991-07-24 US US07/842,349 patent/US5358864A/en not_active Expired - Lifetime
- 1991-07-24 AU AU83205/91A patent/AU647170B2/en not_active Ceased
- 1991-07-24 JP JP3513262A patent/JPH05500907A/en active Pending
- 1991-07-24 CA CA002066734A patent/CA2066734A1/en not_active Abandoned
- 1991-07-24 WO PCT/NL1991/000136 patent/WO1992001389A1/en active Application Filing
- 1991-07-24 DK DK91201943.7T patent/DK0468596T3/en active
-
1992
- 1992-03-20 FI FI921231A patent/FI108944B/en active IP Right Grant
- 1992-03-20 FI FI921232A patent/FI921232A/en not_active Application Discontinuation
- 1992-03-23 NO NO921133A patent/NO921133D0/en unknown
- 1992-03-23 NO NO921134A patent/NO307347B1/en not_active IP Right Cessation
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