Note: Descriptions are shown in the official language in which they were submitted.
W O 93/11249 21 2 4 9 3 9 PCT/DK92/00361~
A method of clonlng proteins in yeast and cellulase from
Humicola insolens.
FIELD OF INVENTION
The present invention relates to a method of screening for DNA
sequences coding for proteins of interest, as well as to a
process for producing such proteins of interest.
BACKGROUND OF THE INVENTION
, ,
The advent of recombinant DNA techniques has made it possible
to select single protein components with interesting properties
and produce them on a large scale. This represents an
improvement over the previously employed production process
15~ using microorganisms isolated from nature and producing a
mixture of proteins which w~uld either be used as such or
separated âfter the production step. However, the conventional
cloning techniques have the drawback that each protein
component has to be purified and characterized by its (partial)
amino acid sequence before it is possible to prepare synthetic
oligonucleotide probes for hybridization experiments. Since
this is a rather time-consuming process, the cloning of novel -
proteins might be considerably expedited by using a screening
method involving selecting clones expressing a desired protein
activity.
Such a screening method has previously been devised for the
cloning of prokaryotic gene products in Bacillus, cf. US
4,469,791; P. Cornelis et al., Mol. Gen. Genet. 186, 19~2, pp.
507-511; I. Palva, Gene 19, 1982, pp. 81-87; S.A. ortlepp, Gene
23, 1983, pp. 267-276; H. Yamazaki et al., J. Bacteriol. 156,
1983, pp. 327-337; N. Tsukagoshi et al., Mol. Gen._Genet. 193,
1984, pp. 58-63; M. Sibakov and I. Palva, Eur. J. Biochem. 145,
1984, pp. 567-572; and J.R. Mielenz, Proc. Natl. Acad. Sci. US~
80, lg83, pp. 5g75-5979. A screening method based on expression
cloning of eukaryotic genes in mammalian cells has been
described, e.g. in D.P. Gearinq et al., The EMBO J. 8, 198g,
WO93/1l2~9 ~1~ 4 ~ 3 9 PCT/D~92/0036~
pp. 3667-3676; N. Harada et al., Proc. Natl. Acad. sci. USA 87,
1990, pp. 857-861; and R. Fukunaga et al., Cell 61, 1sso, pp.
341-350.
5 SU~ARY OF THE INVENTION
It has now been found possible to screen for yeast clones
expressing protein activities of interest with a view to
isolating DNA coding for single protein components.
Accordingly, the present invention relates to a method of
screening for a DNA sequence coding for a protein of interest,
the method comprising
(a) cloning, in suitable vectors, a DNA library from an
organism suspected of producing one or more proteins of
interest,
(b) transforming suitable yeast host cells with said vectors,
~c) culturing the host cells under suitable conditions to
express any protein of interest encoded by a clone in the DNA
library, and
(d) screening for positive clones by determining any activity
of a protein expressed in step (c).
As indicated above, expression cloning of prokaryotic genes in
Bacillus has previously been described. The prokaryotic systems
devised for expression cloning, however, are not operable for
the cloning of eukaryotic genes which are generally difficult
to express in Bacillus. While expression cloning of eukaryotic
genes in mammalian cells has been described, it is more
advantageous to use yeast as a host organism as it is possible
to obtain a much higher transformation frequency than with
mammalian cells~ and as yeast is far easier to culti~ate.
Furthermore, the yeast clones are stable whereas the mammalian
WO93/l1249 2 1 2 4 ~ 3 ~ PCT/DK92/0036
expression cloning system described in the references cited
above is based on transient expression in CoS cells. Unlike the
mammalian system, the yeast system results in pure clones after
the initial screening and, therefore, they need not be screened
in pools and subpools as in the mammalian system. Apart from
this conventional selection systems may be used to select yeast
transformants.
According to the present invention, it has surprisingly been
found that yeast cells appear to be able to express
heterologous genes extracellularly by means of heterologous
secretion signals in amounts which are sufficient for screening
purposes. Although expression cloning of certain proteins in
yeast has been described previously (G.L. McKnight and B.L.
McConaughy, Proc. Nat. Acad. Sci. USA 80, 1983, pp. 4412-4416),
it has not been generally useful as it is based on
complementation of essential genes and therefore is dependent
on yeast host strains which have been mutated to lack these
essential genes. In the present screening method, no such
requirement is necessary for the yeast host strain to bé used
in the method. Besides, the gene products of the previously
described method are intracellular rather than extracellular
as in the present method.
The advantage presented by the present screening method is
primarily that it requires no prior knowledge of the structure
o~ the protein of interest. This means that the rate at which
novel genes may be isolated and, consequently, novel products
be developed may be greatly increased. Furthermore, the method
permits screening for multiple protein activities and may even
result in the isolation of several different genes coding for
the same type of proteins.
.
In another aspect, the presen~ invention relates to a process
35 for producing a protein of interest in a heterologous host ;~
cell, the process comprising transforming a suitable
heterologous host cell with a DNA sequence coding for a protein
WO93/11249 ~l 2 4 ~ 3 9 PCT/DK92/00361
of interest, which DNA sequence has been isolated by the
screening method of the invention, culturing the transformed
cells under suitable conditions to express the protein, and
recovering the expressed protein from the culture.
In a further aspect, the present invention relates to an enzyme
which exhibits cellulase activity,-and which has the following
characteristics
(a) the DNA sequence encoding the enzyme has been isolated from
a DNA library of Humicola insolens,
(b) said DNA sequence comprises at least one of the following
partial sequences
(i) TGGCAGCAGT GTGGTGGCGT TGGCTTCTCG GGCTCTACGT
CCTGTGTGTC CGGTTACACG TGCGTGTACT TGAACGACTG
GTACAGCCAA TGC
~SEQ ID~l)
(ii) CAGCGCAGCC GACGACGTTA CGGACAACAC AACAACGACC
AGGGCAACAT CGACAACAAG GTCAGCCCCG GCTGCCACTT
CAACCACTCCG G
(SEQ ID#~)
~iii) CCAAGGCGAA GTTCAAGTGG TTGGCATCAA CCAGTCCTGC
GCTGAGTTCG GCAAGGAGAG TATCCGGCTA TGGGCAAGCA
CTTACTTCCT TCGCGACGTC GTCGATTCAA GCGCACATCA
ATCGTGGCTT CA (SEQ ID#3)
(iv) CTGACGTGAA CGTGACCAAC AACAACTTGG CCGTAGCGAC
CGAGAACAAG CTGTGTACCA GATGCATCA (SEQ ID#4)
~v) GGACGGTCCG GCACGAGCAC GGCCTGCGTC AGCACCCAGG
TCGGCCTTCA GCGCGTCATT GGCGCGACCA ACTGGCTCAG
GCAAAACGGC AAGGTTGGAC TGCTCGCGAC TTGCCGCGGC (SEQ
ID#5)
212ll93g '~
WO93/11249 PCT/DK92/00361
(vi) GCCAAGTGGG TTTGCCAGCA GGCCATTGAG GGCATGCTGA
ACCACCTCCA GGAGAATAGC GATGTCTGGA CAGGTGCGCT
CTGGTGGGCG GGAGGCCCGT GGTGGGGTTG ACTATATCTA (SEQ
ID#6)
(c) the enzyme comprises a cellulose-binding domain, and
~ .
(d) the enzyme exhibits endocellulase activity in the presence
of linear alkyl benzene sulfonate.
The enzyme of the invention may be isolated by the method of
the invention.
In the present context, the term "cellulose-binding domain" is
intended to indicate an amino acid sequence capable of
effecting binding of the enzyme to a cellulosic subs~rate.
Cellulose-binding domains have been found to be important for
the endoglucanase activity of cellulytic enzymes on substrates
(cf. the discussion in PCT/DK9l/00124). The term "endocellulase
activity" refers to the ability of the enzyme to degrade
cellulose to glucose, cellobiose, triose and other cello-
oligosaccharides, as determined by the formation of cl~aring
zones in a carboxymethyl cel~ulose (CMC) g~l under the
conditions specified below. Unlike the endocellulase described
in PCT/DK9l/00l23) f the enzyme of the present invention shows
substantially unchanged stability in the presence of li~ear
alkyl benzene sulfonates. This is an important advantage as
linear alkyl benzene sulfonates are commonly used in detergent
compositions.
DETAILED DISCL~SURE OF THE INVENTION
According to the invention, the DNA library is preferably a
cDNA library prepared from the mRNA of an organism suspected
of producing one or more proteins of interest. Although it may
also be possible to screen genomic libraries in this manner,
at least some potential yeast hosts may not be able to splice
W093/11249 2 l 2 4 9 3 9 PCT/DK92/0036~
eukaryotic genomic DNA correctly, and therefore a positive
result of the screening may more often be obtained by using
cDNA instead.
To ensure a more accurate result, it may be an advantage to
subject positive clones isolated in step (d) of the present
method to rescreening, reisolation and recloning.
The organism suspected of producing one or more proteins of
interest is typically a eukaryotic organism, in particular a
fungus since fungi are known to produce a large number of
different proteins which makes the traditional process of
isolating a gene coding for a particular protein product by
initially purifying each protein separately particularly
cumbersome. This makes it particularly advantageous to screen
fungal DNA libraries by the method of the invention because a
large number of different protein activities (and DNAs coding
for them) may be identified within a relatively short time-span
using the same library. In this respect, screening of yeast
colonies for different protein activities is far more efficient
than screening of filamentous fungi as a large number (i.e.
about 500-l000) of yeast colonies may be grown on each plate,
compared to 10-50 filamentous fungi/plate.
One type of industrially useful proteins currently obtained
from fungi is enzymes. Thus, yeast clones may be screened by
the method of the invention for expression of one or more
enzyme activities by means of appropriate assays. Examples of
enzymes which may be identified by this method are
carbohydrases, e.g. cellulytic enzymes such as endocellulases,
cellobiohydrolases, ~-glucanases or ~-glucosidases,
hemicellulases or pectinolytic enzymes such as galactanases,
galactosidases,mannanases,xylanases, pectinases, xylosidases,
arabanases, rhamnogalacturonases or amy~ases; esterases, e.g.
lipolytic enzymes such as lipases; proteases; oxidoreductases,
e,g. peroxidases, oxidases or laccases; or isomerases, e.g.
glucose isomerase.
WO 93/11249 2 1 2 4 9 3 9 PCT/DK92/0036(~
A wide range of indicator systems for t~e different types of
enzymes may be used for the screening of yeast colonies on agar
plates. For instance, endocellulases may be identified by
clearing zones in carboxymethyl cellulose after staining with
Congo Red; similar methods may be used to detect glucanases,
xylanases'and galactanases. Endoarabanases may be identified
by blue zones obtained after dissolution of azurine-crosslinked
araban. This principle is general and may be used to detect,
e.g., mannanases, xylanases and cellulases. Pectinases
(polygalacturonases and pectin lyases) may be identified by
clearing zones in pectin after precipitation with quaternary
ammonium ions. Amylases may be identified by clearing zones in
starch after visualisation with iodine. ~-galactosidases may
be detected by the release of p-nitrophenol (yellow) from p-
nitrophenol-~-galactopyranoside or by coupling released
naphthole or naphthole derivatives from, e.g., l-naphthole-~-
galactopyranoside to azo dyes; similar methods may be used to
detect ~-galactosidases, ~- and ~-glycosidases, ~-xylosidase
and ~-mannosidase. Numerous methods are available for the
detection of proteases, e.g. clearing zones in casein after
precipitation with trichloroacetic acid. Peroxidases and
oxidases may be detected by the reaction of 4-a.ninoantipyrine
with ESBT (N-ethyl-N-sulfobutyl-m-toluidine) in the presence
of hydrogen peroxide (generating a purple colour). Lipases may
be detected by the formation of clearing zones in tributyrine
emulsions.
The yeast strain selected to be the host cell for the DNA
library may be any yeast strain conventionally used for the
cloning of heterologous DNA sequences. Thus, the y~ast strain
may suitably be selected from SaccharomYces sp., such as
Saccharomvcescerevisiae,Saccharomvcesklu w eri, Saccharomvces
uvarum or Schizosa,ccharomvces Pombe, Hansenula sp. Pichia sp.,
Yarrowia sp. such as Yarrowia li~olvtica, or Kluyveromvces sp.
such as Klyyyeromvces lactis.
WO93/11249 ? 1 ~ ~ 9 3 9 PCT/DK92/0~36~
The vector used for cloning the DNA library may be any vector
which may conveniently be subjected to recombinant DNA
procedures. In each vector, the DNA sequence derived fr~m the
library should be operably connected to a suitable promoter
sequence. The promoter may be any DNA sequence which shows
transcriptional activity in the yeast cell. Examples of
suitable promoters for use in yeast host cells include
promoters from yeast glycolytic genes (Hitzeman et al., J.
Biol. ~hem. 255, 1980, pp. 12073-12080; Alber and Kawasaki, J.
Mol. A~l. Gen. 1, 1982, pp. 419-434) or alcohol dehydrogenase
genes (Young et al., in Genetic_Enaineerinq of Microorqanisms
for Chemicals (Hollaender et al, eds.), Plenum Press, New York,
1982), or the TPIl (US 4, 599, 311) or ADH2-4c (Russell et al.,
Nature 304, 1983, pp. 6S2-654) promoters.
Each DNA library sequence may also be operably connected to a
suitable terminator, such as the TPIl (Alber and Kawasaki, op.
cit.) or ADH3 (McKnight et al., oP. cit.) or yeast MF~
terminators.
The vector may further comprise a DNA sequence enabling the
vector to replicate in yeast cell. An example of such a
sequence is the yeast plasmid 2~ replication genes REP 1-3 and
origin of replication. If the v~ctor is a yeast/E. coli shuttle
vector, it will also include an oriyin of replication region
which is functional in E. coli. The vector may also comprise
a sele~table marker, e.g. a gene the product of which
complements a defect in the host cell such as URA3, or one
which confers resistance to a drug, e.g. ampicillin, kanamycin,
chloramphenicol, tetracyclin, etc., or ~he Schizosaccharomvces
pombe TPI gene ~describ~d by P.R. Russell, Gene 40, 1985, pp.
125-130~.
The procedures used to ligate the DNA library sequences, the
promoter and the terminator, respectively, and to introduce
them into suitable vectors cont~ining the information necessary
for replication, are well known to persons skilled in the art
WO93/ll249 212 4 9 3 9 PCT/DK92/0036(~
g
(cf., for instance, Sambrook et al., Molecular Cloninq: A
Laboratorv Manual, Cold Spring Harbor, New York, 1989). The
transformation of yeast cells may for instance be effected by
protoplast formation followed by transformation or by the LiAc
method in a manner known per se.
In the process of the invention of producing a protein of
interest after the DNA coding for the protein has been isolated
by the screening method described above, the heterologous host
cell transformed with the isolated DNA sequence may be a strain
of a filamentous fungus, e.g. fungi belonging to the groups
Phycomycetes, Zygomycetes, Ascomycetes/ Basidiomycetes or Fungi
Imperfecti, including Hyphomycetes such as the genera
As~eraillus, Trichoderma, Penicillium, Fusarium or Mumicola.
The filamentous fungus host organism may conveniently be one
which has previously been used as a host for producing
recombinant proteins, e.g. a strain of AsPeraillus sp., such
as A. niaer, A. n dulans or A. or~zae. The use of A. orvzae in
the production of recombinant proteins is extensively described
in, e.g. EP 238 023.
In particular when the host organism is A. orYzae, a preferred
promoter for use in the process of the present invention is the
A. orvzae TAKA amylase promoter as it exhibits a strong
transcriptional activity in ~ orvzae. The sequence of the TAKA
amylase promoter appears from EP 238 023.
Termination and polyadenylation sequences may suitably be
derived from the same sources as the promoter.
The techniques used to transform a fungal host cell may
suitably be as described in EP 238 023.
The medium used to culture the transformed host cell may be
any conventional medium suitable for growing As~erqillus cells.
The mature protein secreted from the host cells may
WOg3/11249 ~1 2 4 ~ ~ 9 PCT/DK9V0036
conveniently be recovered from the culture medium by well-known
procedures including separating the cells from the medium by
centrifugation or filtration, and precipitating proteinaceous
components of the medium by means of a salt such as ammonium
sulphate, followed by chromatographic procedures such as icn
exchange chromatography, affinity chromatography, or the like.
A pre~erred endocellulase enzyme according to the invention is
an enzyme, a crude extract (15 ~1) of which diluted with one
volume of 0.lS% linear alkyl benzene sulfonate and added to a
2% agarose gel containing 1% carboxymethyl cellulose in 50 mM
~odium phosphate buffer, pH 7, mixed with one volume of 0.15%
linear alkyl sulfonate forms a clearing zone in said agarose
gel after 18 hours of incubation, which clearing zone is equal
to (less 3 mm) the clearing zone formed in a similar
carboxymethyl cellulose gel not containing any linear alkyl
benzene sulfonate, provided that the concentration of enzyme
in the extract is such that a clearing zone of at least 10 mm
is formed in a carboxymethyl cellulose gel (with no linear
alkyl benzene sulfonate) under the conditions specified above.
..~
The ~NA sequence coding for the enzyme may for instance be
isolated by screening a cDNA library of Humicola insolens, e.g
strain DSM 1800, deposited on 1 October 1981 at the Deutsche
Sammlung von Mikroorganismen in accordance with the provisions
o~ the Budapest Treaty and selecting for clones expressing the
appropriate enzyme activity (i.e. endocellulase activity as de-
fined above). The appropriate DNA sequence may then be isolated
~rom the clone by standard procedures, e.g. as described in
Example 1.
In a further aspect, the invention relates to a detergent
additive comprising the enzyme of the invention. The detergent
additive may suitably be in the~form;of a non-dusting granula-
te, stabilized liquid or protected enzyme. Non-dusting
granulates may be produced~e.g. according to US 4,1~06,991 and
4,661,452 (both to Novo Industri A/S~) and may optionally be
~: ,
,
. ~
WO93/11249 212 4 9 3 9 PCT/DK92/0036/~
11
coated by methods known in the art. Liquid enzyme preparations
may, for instance, be stabilized by adding a polyol such as
propylene glycol, a sugar or sugar alcohol, lactic acid or
boric acid according to established methods. Other enzyme
stabilizers are well known in the art. Protected enzymes may
be prepared according to the method disclosed in EP 238 216.
It will be understood that the detergent additive may further
include one or more other enzymes, such as a protease, lipase,
peroxidase or amylase, conventionally included in detergent
additives.
In a still further aspect, the present invention relates to a
detergent composition comprising the enzyme of the invention.
The detergent composition of the invention may ~e in any
convenient form, e.g. as powder, granules or liquid. A liquid
detergent may be aqueous, typically containing up to 90% water
and 0-20% organic solvent.
The detergent composition comprises a surfactant which may be
anionic, non-ionic, cationic, amphoteric or a mixture of these
types. The detergent will usually contain 0-50% anionic
surfactant such as linear alkyl benzene sulphonate (LAS),
alpha-olefin sulphonate (AOS), alkyl sulphate (AS), alcohol
ethoxy sulphate (AES) or soap. It may also contain 0-40% non-
ionic surfactant such as nonyl phenol ethoxylate or alcohol
ethoxylate. Furthermore, it may contain a polyhydroxy fatty
acid amide surfactant (e.g. as described in WO 92/06154).
The detergent composition may additionally comprise on~ or more
other enzymes, such as an amylase, lipase, peroxidase, oxidase
or protease.
The pH (measured in aqueous detergent solution) will usually
be neutral or alkaline, e.g. 7-11. The detergent may contain
1-40% of a detergent builder such as zeolite, phosphate,
phosphonate, citrate, NTA, EDTA or DTPA, alkenyl succinic
W O 93/11249 212 4 9 3 9 PC~r/DK92/0036(~ 12
anhydride, or silicate, or it may be unbuilt ti.e. essentially
free from a detergent builder). It may also contain other
conventional detergent ingredients, e.g. fabric conditioners,
foam boosters, bleaching agents, e.g. perborate, percarbonate,
tetraacetyl ethylene diamine (TAED), or nonanoyloxybenzene
sulfonate (NOBS), anti-corrosion agents, soil-suspending
agents, sequestering agents, anti-soil redeposition agents,
stabilizing agents for the enzyme(s), foam depressors, dyes,
bactericides, optical brighteners or perfumes.
Particular forms of detergent composition within the scope of
the invention include:
a) A detergent composition formulated as a detergent powder
1~ containing phosphate builder, anionic surfactant, nonionic
surfactant, silicate, alkali to adjust to desired pH in use,
and neutral inorganic salt.
b) A detergent composition formulated as a detergent powder
containing zeolite builder, anionic surf~ctant, nonionic
surfactant, acrylic or equivalent polymer, silicate, alkali to ~-
adjust to desired pH in use, and neutral inorganic salt. :.
c) A detergent composition formulated as an aqueous detergent
llquid comprising anionic surfactant, nonionic surfactant,
humectant, organic acid, caustic alkali, with a pH in use
adjusted to a Yalue between 7 and 10.5. .
d) A detergent composition formulated as a nonaqueous deter-
gent liquid comprising a liquid nonionic surfactant consisting
essentially of linear alkoxylated primary alcohol, phosphate
builder, caustic alkali, with a pH in use adjusted to a ~Jalue
between about 7 and 10.5. :.
"
e) A detergent composition formulated as a detergent powder
in the form o~ a granulate having a bulk density of at least
600 g/l, containing anionic surfactant and nonionic surfactant,
WO93/1l24~ ~ 12 4 g 3 g PCT/DK92/003
13
low or substantially zero neutral inorganic salt, phosphate
builder, and sodium silicate.
f) A detergent composition formulated as a detergent powder
in the form of a granulate having a bulk density of at least
600 g/l, containing anionic surfactant and nonionic surfactant,
low or substantially zero neutral inorganic salt, zeolite
builder, and sodium silicate.
g) A detergent composition formulated as a detergent powder
containing anionic surfactant, nonionic surfactant, acrylic
polymer, fatty acid soap, sodium carbonate, sodium sulphate,
clay particles, and sodium silicate.
h) A liquid compact detergent comprising 5-65% by weight of
surfactant, 0-50% by weight of builder and 0-30% by weight of
electrolyte.
Apart from these ingredients, the detergent compositions a)-h)
include the cellulase of the invention and optionally one or
more other enzymes, as indicated above.
The softening, soil removal and colour clarification effects
obtainable by means of the enzyme of the invention generally -~
require a concentration of the enzyme in the washing solution
of 0.0001 - 100, preferably 0.0005 - 60, and most preferably
0.01 - 20 mg of enzyme protein per liter. The detergent
composition o~ the inve~tion is typically employed in
concentrations of 0.5 - 20 g/l in the washing solution. In
general, it is most convenient to add the detergent additive
in amounts of 0.1 - 5% w/w or, preferably, in amounts of 0.2 -
2% of the detergent composition.
'~
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a map o~ plasmid pYHD17, wherein "TPI promoter"
indicates the S. cerevisiae triose phosphate isomerase
W093/1l249 ~ 12 4 9 3 g PCT/DK9~/0036
14
promoter, "Terminator" indicates the S. cerevisiae triose
phosphate isomerase terminator, "Amp" indicates the gene
mediating ampicillin resistance, "2~ ori" indicates the yeast
plasmid 2~ origin of replication, and "URA3" indicates a gene
encoding a selection marker complementing a uracil deficiency
in the host strain; and
Fig. 2 is a map of plasmid pHD414, wherein "AMG Terminator"
indicates the A. niaer glucoamylase terminator, and "TAKA
Promoter" indicates the A. orYzae TAK~ amylase promoter;
The present invention is further illustrated in the following
examples which are not in any way intended to limit the scope
of the invention as claimed.
~XAMP~EB
Mater~ls and Methods
Donor organism: mRNA was isolated from the following organisms:
H. insolens, DSM 1800, grown in a cellulose-rich fermentation
medium with agitation to ensure sufficient aeration.
.
Construction of an expression plasmid: The commercially
available plasmid pYES II (Invi~rogen) was cut with SpeI,
filled in with Klenow DNA polymerase ~ dNTP and cut with ClaI.
The DNA was size fractionated on an agarose gel, and a fragment
of about 2000 bp was purified by electroelution. The same
plasmid was cut with ClaI/PvuII, and a fragment of about 34~0
bp was purified by electroelution. The two fragments were
ligated to a blunt-ended SphI/EcoRI fragment containing the
yeast TPI promoter. This fragment was isolated from a plasmid
in which the TPI promoter from S. cerevisiae (cf. T. Albers and
G. Kawasaki, ~. Mol. ApPl. Genet. 1, 198~, pp. 419 434) was
slightly modified: an internal SphI site was removed by
deleting the four bp constituting the core of this site.
Purthermore, redundant sequences upstream of the promoter were
.:
WO93/11249 15 PCT/~92/0036~)
removed by Ball exonuclease treatment followed by addition of
a SphI linker. Finally, an EcoRI linker was added at position -
l0. After these modifications, the promoter is included in a
SphI-EcoRI fragment. Its effeciency compared to the original
promoter appears to be unaffected by the modifications. The
resulting plasmid pYHDl7 is shown in Fig. l.
Isolation of mRNA: Total RNA was isolated from approximately
7 g of mycelium. The mycelium was frozen in liquid nitrogen and
ground in a mortar with l g of quartz sand to a consistency of
flour. The RNA was extracted with guanidinium thiocyanate and
centrifuged through CsCl essentially as described in Sambrook
et al~, 1989, op. cit.. Poly A RNA was isolated from total RNA
by chromatrography on oligo dT cellulose.
cDNA synthesis: cDNA synthesis was carried out by means of a
cDNA syn~hesis kit from Invitrogen according to the
manufacturer's specifications. The DNA was adapted to the
expression vectors by addition of a BstxI linker (Invitrogen)
and size fractionated on an agarose gel. Only DNA larger than
5-600 bp was used in the library construction. The adapted cDNA
was ligated into an appropriate vector cut with BstxI.
Following test ligations (in order to determine the size of the
library) the library was plated onto 50 agar plates. To each
plate containing from approximately 5Q0 to 5000 individual
clones (dependent on the library size) was added 3 ml medium.
The bacteria were scraped off, l ml glycerol was added, and
stored at ~80^C as 50 pools. The remaining 2 ml were used for
DNA isolation. If the amount of DNA was insufficient to give
the required number of yeast transformants (see below), large
scale DNA was prepared from 500ml medium (TB) inoculated with
50 ~l -80 D C bacterial stock propagated over night.
Construction of Yeast Libraries: DNA from one or more pools was
transformed into yeast as described below. To ensure that all
the bacterial clones were tested in yeast a n~mber of yeast
WO93/lt249 2 ~ 2 ~ 9 3 9 PCT/D~92/0~361
16
transformants 5 x larger than the number of bacteria clones in
the original pools was set as a limit.
Transformation of yeast: The yeast strain used was yNG231. (MAT
alpha, leu2, ura3-5~, his4-539, pep4-delta 1, cir+). One colony
was grown at 3aoc overnight in 10 ml YPD (this culture can be
stored for several days at 5 C).
10, 30, and 60 ~1 of this culture were added to 3 shaker flasks
containing 100 ml YPD, and incubated with shaking overnight at
30-C. The culture with an OD600 closest to 0.3-0.4 was selected.
The cells were harvested in 50 ml tubes in a Beckman centrifuge
(speed 6, 10 minutes), the cells were resuspended in 2 x 5 ml
H2O, centrifuged as described above, resuspended in 5 ml buffer
1~ containing 0.1 M LiAc, 10 mM Tris-Cl, 1 mM EDTA, pH 7.5, and
centrifuged again. The cells were resuspended in S00 ~1 of the
above buffer and incubated for 60 minutes at 30-C. 250 ~g
carrier DNA (sterile salmon-sperm DNA 10 mg/ml) was added and
aliquots of 100 ~1 were prepared. The DNA to be transformed
(approx. 5 ~g) was added to the 100 ~1 aliquot, mixed gently,
2nd incubated for 30 minutes at 30C. 700 ~1 40~ PEG 4000, 0.1
M LiAc, 10 mM Tris-Cl, 1 mM EDTA, pH 7.5 was added, and
incubation was continued for 60 minutes at 30C. The
transformation mixture was subjected to heat shock for 5
minutes at 42~C, spun briefly in a micro centrifuge,
resuspended in 100-200 ~1 H2O, and plated on SC plates without
uracil, followed by incubation for three days at 30'C.
~r~paration of ~arrier DNA: 100 mg salmon-sperm DNA was w~ighed
out and dissol~ed overnight in 10 ml 10 mM Tris-Cl, 1 mM EDTA,
pH 7,5 (TE). The solution was then sonicated in a plastic
container in ice water until it was no longer viscous. The
solution was then phenole extracted and EtOH precipitated, and
the pellet was washed and resuspended in 5 ml TE. The
su~pension was EtOH precipitated, and the pellet was washed and
re~uspend in 5 ml TE. The OD260 was measured, and the suspension
was diluted with TE to 10 mg/ml.
WO93/11249 2 1 2 4 9 3 9 PCT/DK92/0036~
17
Media:
YPD: 10 g yeast extract, 20 g peptone, H2O to B10 ml.
Autoclaved, 9o ml 20% glucose (sterile filtered) added.
10 x Basal salt: 66.8 g yeast nitrogen base, 100 g succinic
acid, 60 g NaOH, H2O ad 1000 ml, sterile filtered.
SC-URA: 90 ml 10 x Basal salt, 22.5 ml 20 % casamino acids,
9 ml 1% tryptophane, H2O ad 806 ml, autoclaved, 3.6 ml 5%
threonine and 90 ml 20% glucose added.
SC-H agar: 7.5 g/l yeast nitrogen base without amino acids,
11.3 g/l succinic acid, 6.8 g/l NaOH, S.6 g/l casamino ac~ds
without vitamins, 0.1 g/l tryptophan and 20 g/l agar (Bacto).
1~ Autoclaved for 20 min. at 121C. After autoclaving, 55 ml of
a 22% galactose solution and 1.8 ml of a 5% threonine solution
were adde~ per 450 ml agar.
SC-H broth: 7.5 g/l yeast nitrogen base without amino acids,
11.3 g/l succinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids
without vitamins, 0.1 g/l tryptophan. Autoclaved for 20 min.
at 121C. After autoclaving, 10 ml of a 30% galactose solutio~,
S ml of a 30% glucose solution and 0.4 ml of a 5% threonine
solution were added per 100 ml medium.
YNB-l agar: 3.3 g/l KH2PO~, 16.7 g/l agar, pH adjusted to 7.
Autoclaved for 20 min. at 121C. After autoclaving, 25 ml of
a 13.6~ yeast nitrogen base without amino acids, 25 ml of a 40%
glucose solu~ion, 1.5 ml o~ a 1% L-leucine solution and 1.5 ml
of a 1% histidine solution were added per 450 ml agar.
YNB-1 broth: Composition as YNB-l agar, but without the agar.
CMC overlayer gel: 1% agarose, 1% carboxymethyl ~ellulose in
Tris-malate buffer, pH 7. The gel was boiled and then cooled
to 55-C before the overlayer was poured on~o agar plates.
W093/ll249 ~ t 2 4 ~ 3 g PCT/DK92/0036(l
18
Oat spelt xylan overlayer gel: 1% agarose, 1% oat spelt xylan
(Sigma Chemical Company) in Tris-malate buffer, pH 7. The gel
was boiled and then cooled to 55C before the overlayer is
poured onto agar plates.
Construction of an A~per~illu~ expression ~ector: The vector
pHD414 is a derivative of the plasmid p775 (described in EP 238
023). In contrast to this plasmid, pHD 414 has a string of
unique restriction sites between the promoter and the
terminator. The plasmid was constructed by removal of an
approximately 200 bp long fragment (containing undesirable RE
sites) at the 3'end of the terminator, and subsequent removal
of an approximately 250 bp long fragment at the 5'end of the
promoter, also containing undesirable sites. The 200 bp region -~-
was removed by cleavage with NarI (positioned in the pUC
vector) and XbaI (just 3' to the terminatorj, subsequent
filling in the generated ends with Klenow DNA polymerase +dNTP,
purification of the vector fragment on gel and religation of
the vector fragment. This plasmid was called pHD413. pHD413 was
2~ cut with StuI (positioned in the 5lend of the promoter) and
PvuII (in the pUC vector), fractio~ated on gel and religated.
The plasmid pHD 414 is shown in Fig. 2.
ExamPle 1 ~'
A library from H.-insolens consisting of approx. 300,000
individual clones in 50 pools was constructed.
DNA was isolated from 20 individual clones from the library and
subjected to analysis for cDNA insertion. The insertion
frequency was found to be >90 % and the average insert size was
approximately 1400bp~
DNA was isolated from 10 pools from the Humicola library (2ml
from the original plate). An aliquot was digested with
restriction enzymes in order to excise the cDNA insert and
analyzed by Southern blot using a 43kD cellulase cDNA probe
WO 93/1 1249 212 4 9 3 9 PCT/DK92/0036~
19
(the 43 kD enzyme is disclosed in PCT/DK91/00123 ) and a CBH 2
cDNA probe (the enzyme is disclosed in PCT/DK91/00124) . Several
bands were found to hybridize with the 43kD cellulase probe
after a low stringency wash ( 2x SSC 65-C) in the ~0 pools from
the Humicola library. At higher stringency (0.1 x SSC, 75 C )
one band corresponding to the expected size for 43kD cellulase
was detected in 5 out of 10 pools. Similar results were
obtained with the CBH 2 probe. Here 10 out of 10 pools were
found to have a band corresponding to the expected size for CBH
2. In addition, 4 pools contained bands with a higher molecular
weight. These bands were seen even under stringent conditions,
demonstrating that the library is of an appropriately high
quality.
..:
DNA from the Humicola library, pools l-10, was transformed into
yeast, and plates containing 20-25,000 colonies were obtained
from each pool. The colonies were scraped off and stored in
glycerol at -80-C. -
Yeast cells from the library were spread onto YNB agar to a
total of about ~00,000 c~olonies. The number of colonies per
plate varied from 50 to 500. After 4 or 5 days of growth, the
agar plates were replica plated-onto two sets of SC-H agar
plates. These plates were then incubated for 2-4 days at 30~C
before the two sets of agar plates were overlayered with a CMC
indicator gel for detection of cellulase activity and oat spelt
xylan indicator gel for the detection of xylanase and
cellulase. After incubation overnight at 40'C, enzyme reactions
were visualised with Congo Red. 10-15 ml of a 0.1% solution of
Congo Red was poured onto the overlayer and removed after 10-20
min. The plates were then washed once or twice by pouring 10-15
ml of 2M NaC1 onto the plates. The NaCl solution was removed
af~er 15-25 min. Cellulase-positive colonies were identified
on the plates with the ~CMC overlayer as colonies with
colourless or pale red clearing zone& on a~red background.
Xylanase-positive colonies identif-iéd on the plates with oat
spelt xylan overlayers as colourless or pale red clearing zones
"
.~: , . .. . .
WO93/11249 2 12 4 ~ 3 g PCT/D~92/003
on a red background. Cellulase-positive colonies were also
identified on plates with oat spelt xylan overlayers as pale
red or blue clearing zones on a red background.
Cells from enzyme-positive colonies were spread for single
colony isolation on agar, and an enzyme-producing single colony
was selected for each of the cellulase- or xylanase-producing
colonies identified.
Each of the 133 cellulase-producing colonies and 147 of the
xylanase-producing colonies were isolated. So~e of these
colonies were inoculated into 20 ml YNB-l ~roth in a 50 ml
glass test tube. The tube was shaken for 2 days at 30C. The
cells were harvested by centrifugation for 10 min. at 3000 rpm.
The cells were resuspended in 1 ml 0.9 M sorbitol, 0.1 M EDTA,
pH 7.5. The pellet was transferred to an Eppendorf tube, and
spun for 30 seconds at full speed. The cells were resuspended
in 0.4 ml O.9 M sorbitol, 0.1 M EDTA, 14 mM ~-mercaptoethanol.
100 ~1 2 mg/ml Zymolase was added, and the suspension was
incubated at 37~C for 30 minutes and spun for 30 seconds. The
pellet ~spheroplasts) was resuspended in 0.4 ml TE. 90 ~1 of
( 1. 5 ml O . 5 M EDTA pH 8 . O, O . 6 ml 2 M Tris-Cl pH 8 . O, O . 6 ml
10% SDS) was added, and the suspension was incubated at 65C
for 30 minutes. 80 ~1 5 M KOAc was added, and the suspension
was incubated on ice for at least 60 minutes and spun for 15
minutes at full speed. The supernatant was transferred to a
fresh tube which was filled with EtOH (room temp.) followed by
thorough but gentle mixing and spinning for 30 seconds. The
pellet was washed with cold 70% ETOH, spun for 30 seconds and
dried at room temperature. The pellet was resuspended in 50 ~1
TE and spun for 15 minutes. The supernatant was transferred to
a fresh tube. 2.5 ~1 10 mg/ml RNase was added, followed by
incubation at 37~C for 30 minutes and addition of 500 ~1
isopropanol with gentle mixing. The mixture was spun for 30
seconds, and the supernatant was removed~ The pellet was rinsed
with cold 96% EtOH and dried at room tempexature. The DNA was
WO93/11249 2 12 4 9 3 g PCT/DK92/00361
21
dissolved in 50 ~1 water to a final concentration of
approximately 100 ~l/ml.
The DNA was transformed into E.coli. by standard procedures.
Two E. coli colonies were isolated from each of the
transformations and analysed with the restriction enzymes
HindIII and XbaI which excised the DNA insert. DNA from one of
these clones was retransformed into 5~ cerevisiae strain JG169
(MATa; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-113;
prcl::HIS3; prbl:: LEU2) and rescreened for enzyme activity.
The DNA sequences of several of the positive clones were
partially determined. The partial DNA sequences are shown in
' Sequence Listings SEQ ID#7-15. Based on the DNA sequence, the
clones were classified as follows:
.
Endocellulases:
CMC 1: C3, 26, 27, XY33, XY46 250 amino acids (SEQ ID#7)
20 GMC 4: C46, 47, 50, 51, 54, ~-
101, 102, 103, 104 -1400 bp (the enzyme of the
invention) (SEQ ID#8)
CMC 5: XY49 -1050 bp (SEQ ID#9)
CMC 6: C49 -1000 bp (SEQ ID#10
25 CMC 38K: C13 (SEQ ID#11)
CMC EGl: C6, 11, 15, 16, 17, 21,
22, 23, 25, XY34, 41, 145 (SEQ ID#12)
Xylanases:
XYL 1: XY30, 31, 40, 42, 101, 102,
110, 117, 119, 123, 125,
136, XY56, 60, 137 22 kD (SEQ ID#13)
XYL 2: XY103, 104, 107~ 108, 109 r
113, 114, 118, 120, 121, 124,
126, 128, 130, 134, 142, 143 ~SEQ ID#14)
XYL 3: XY115, 116, 132, 146 (SEQ ID#15)
,j
W O 93/11249 ?~ 24~3~ 22 PCT/D~92/00361~
In order to express the genes in AsPerqillus the cDNA insert
is isolated from one or more representatives of each family and
cloned into the vector pHD414 which is transformed into A.
orYzae or A. niqer according to the general procedure described
below.
Transformation of AsPeraillus oryzae or A~peraillus niaer
(aeneral ~rocedure)
100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold
Spring Harbor Laboratory, 1981) is inoculated with spores of
A. orvzae or A. niaer and incubated with shaking at 370C for
about 2 days. The mycelium is harvested by filtration through
miracloth and washed with 200 ml of 0.6 M MgS04. The mycelium
is suspended in 15 ml of 1.2 M MgS04. 10 mM NaH2P04, pH = 5.8.
The suspension is cooled on ice and 1 ml of buffer containing
120 mg of Novozym 234, batch 1687 is added. After 5 minutes
1 ml of 12 mg/ml BSA (Sigma type H25) is added and incubation
with gentle agitation continued for 1.5-2.5 hours at 37C until
a large numb~r of protoplasts is visible in a sample inspected
under the microscope.
The suspension is filtered through miracloth, the filtrate
transferred to a sterile tu~e and overlayered with 5 ml of 0.6
M sorbitol, 100 mM Tris-HCl, p~ = 7Ø Centrifugation is
performed for 15 minutes at 100 g and the protoplasts are
collected from the top of the MgS04 cushion. 2 volumes of STC
(1.2 M sorbitol~ 10 mM Tris-HCl, pH = 7.5. 10 mM CaC12) are
added to the protoplast suspension and the mixture is
centrifugated for 5 minutes at 1000 g. The protoplast pellet
is resuspended in 3 ml of STC and repelleted. This is repeated.
Finally the protoplasts are resuspended in 0.2-1 ml of STC.
100 ~1 o~ protoplast suspension i5 mixed with 5-25 ~g of the
appropriate DNA in 10 ~1 of STC. Protoplasts are mixed with
p3SR2 (an A. nidulans amdS gene carrying plasmid). The mixture
WO93/11249 212 4 9 3 9 PCT/DK92/0036
23
is left at room temperature for 25 minutes. 0.2 ml of 60% PEG
4000 (BDH 29576). lO mM CaCl2 and lO mM Tris-HCl, pH = 7.5 is
added and carefully mixed (twice) and finally 0.85 ml of the
same solution is added and carefully mixed. The mixture is left
at room temperature for 25 minutes, spun at 2500 g for 15
minutes and the pellet is resuspended in 2 ml of l.2 M
sorbitol. After one more sedimentation the protoplasts are
spread on the appropr`iate plates. Protoplasts are spread on
minimal plates (Cove Biochem.Biophys.Acta 1l3 (1966~ 51-56)
containing l.0 M sucrose, pH = 7.0, lO mM acetamide as nitrogen
source and 20 mM CsCl to inhibit background growth. After
incubation for 4-7 days at 37-C spores are picked and spread
for single colonies. This procedure is repeated and spores of
a single colony after the second reisolation is stored as a
defined transformant.
Bx~mple 2
Cellulase type 4 clones C46 and C51 and a 43 kD cellulase
control clone (obtained by transforming yeast strain JGl69 with
pYHDl7 carrying a DNA sequence coding for the 43 kD cellulase
tisolated as described in PCT/DK9l/000123]) were inoculated in
lO0 ml test tubes with 15 ml YNB-l broth. The tubes were
agitated at 30~C for 2 days. 5 ml of broth from each tube were
th~n used as seed material for shake ~lasks containing lO0 ml
SC-H broth. The shake flasks were agitated for 4 days at 30'G~
The ~ells from 20 ml of broth were collected by centrifugation
and mixed with 1-2 ml O.l M sodium phosphate buffer, pH 7, and
3.3 g of glass beads (420-500 ~m in diameter) in lO ml glass
test tubes. The crude cell extracts were collected after about
8 minutes of agitation by means of a IKA vibrax VXR (available
from IKA Labortechnik).
The cellulase activity of the crude cell extracts from the
yeast clones C46, C51 and 43 kD were measured under different
conditions by the size of the clearing zones formed in GMC
containing gels.
W093/l1249 ?~ ~49 3 ~ PCT/DK92/00~
24
CMC ael: CMC overlayer gel as described above.
CMC LAS ael: 2~ agarose, 1~ CMC in 50 mM sodium phosphate
buf~er, pH 7, boiled and mixed with one volume of 0.12% LAS.
The cellulase activity was measured by adding 15 ~1 crude cell
extract to 4 mm (diameter) holes in the gel. The crude cell
extracts were diluted with one volume of 0.12% LAS before ~
addition to the CMC LAS gel and with one volume of water before ;
addition to the CMC gel. The clearing zones were then
visualised after 18 hours of incubation at 40 C by staining
with Congo Red as described above.
The results are shown in the following table.
. .
Clone C46 C51 43 kD
CMC 14 14 17 -
CMC LAS 15 13 0
Activities are shown as mm clearing zones.
It appears from the table that the enzyme produced by C46~C51
is L~S resis~ant.
W093/l1249 2 12 ~ 9 3 9 PCT/DK92/003~1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Novo Nordisk A/S
(B) STREET: Novo Alle
(C) CITY: Bagsvaerd
tE) COUNTRY: Denmark
(F) POSTAL CODE (ZIP): DK-2880
(G) TELEPHONE: +45 4444 8888
(H) TELEFAX: +45 4449 3256
(I) TELEX: 37304
(ii) TITLE OF INVENTION: A Method of Cloning Proteins in
Yeast
~ (iii) NUMBER OF SEQUENCES: 15
(iv) COMPUTER READABLE FORM:
.(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY- linear
(ii3 MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) OR~ANISM: Humicola insolens
~xi) S~NOE DESCRIPllON: SEQ ID NO: 1:
45 I3G34~2T GDGe~I~ET T~F~CC G~C~GT OCDen~TC o~ CASG ~0
IGo~ CT T~AOGACTG ÇIACA~X~A TGC 93
~2) IN~a~lON ~OR SEQ ID NO: 2:
(A) L~K~ff: 92 ~ase ~ s
(B) TYYE: nucleic acid
(C) SnV~IEn~S: single
(D) T~PO~X~: l~r
WO 93/11249 ~ 12 ~ 9 3 9 PCr/DK92tO0360
26
(ii) M~IECULE T~: c~NA
(vi) ORIG~L S~:
(A) OR~NI5M: ~nicola ~lens
(xi) SE5~OE DESCRI~~ SEX2 ID NO: 2:
CaG~ G~CG~C~ CGG~C~C~C A~CAACGACC AGGGC~CP~ CG~ AG 60
GCI~CI~ CAACC~CC GG 92
(2) INE~:[ION ~ Sl~ ID NO: 3:
(i) SEX~lENCE CHMI~Ll~KISlICS:
(A) ~ff: 132 base pairs
(B) TYl~ mcle~c acid
(C) SlR~NI~: single
(D) IOPOI~GY: linear
20,
(A) OR~N~: ~nicola insolens
(Xi) SE~ENOE I~IP~ON: SEQ ID NO: 3:
IW~ ~AG~ ~l~ClICCI TC~ t~C = It~ G(~ 120
k~GC!3~ C~ 132
(2~ ~NE~LICN F~R S13Q ID ~: 4:
(i) ~UENOE ~ISrIC~:
(A) LEN~: 69 base
(B~ rNcleic acid
(C) SIRAII~: single
(D) ~OPOIDGY: linear
(ii) ~IEa~ ~: cD~
(vi) CRIGIN~L Sa~CE:
(A) ~N~: B~nicola insolens
=~T~ 69
55 ~2) ItlE~ICN E~ SEQ ID NO: 5:
WO 93/11249 2 1 2 4 9 3 9 PCr/DK92/0036~1
(i) SEQUINCE CHAR~ISlICS:
(A) ~: 120 base pairs
(B) T~: nucleic acid
(C) SrRANI~: single
S (D) qOPO~: linear
(vi) ORIGINI~L Sa~E:
(A) ~Nl:~: H~nicola insolens
~xi) S9~NOE IEæE~ON: SEQ ID N~: 5:
~ A =~aG ~AO~GC Aa~l~ IG~l~aC ~3GC 120
20 (2) ~NF~IION EOR S~3Q ID NO: 6:
~A) IEtX~I: 120 base
(B) TYE~: nucleic acid
(C) S~: single
(D) TOPO~GY: linear
(ii) ~E T~: c~
(vi) ~G~L sa~E:
(A) OR~ M: ~nicola insole2~s
(xi~ SE~ENOE DESCRIPlION: SE;Q ID N~: 6:
(2) ~l~ON E~R ~Q ID N0: 7:
(i) SE~UEN~::E C~ACrERIS~CCS:
(A) IEN(~: 1027 base pairs
(B) TY~: rn~cleic acid
(C) Sli~ANI~: sir~gle
(D) ~OPOLOGY: linear
(ii) ~E T~: c~
(Yi) ~I~L sa~::
(A) a~: ~iaola insslens
(Xi) SE~CE DES~LP~[CN: SEQ :lD NO: 7:
WO93/11249 ~ 3~ PCT/DK92/0036n
28
CA~;CC~ q~a:ACC~GC I~AC~ ACCll~ All~GA ICACOK:~C~ 60
5 GP~ a~A~ AG~l~a G~l~G C~l~AGC I~C~ 180
= ~ q~C TaGAaGGC~C C~aGGI' ~1~ GGZ~AQOGC 300
allYI~ ~G~C CXæ1~C~ACGI' CaAG~C~ a~l~Iq~ G~AG~ 360
~a~;GC 0~aAG~ ~1~ C~GCI'.l~;C A~ ~A~A 420
15 a;~ GA~I~ aw~ I~C ll~C Gl~al~ 480
X~ =X~ ~;P~C~ 600
,,20
~3CII~C AA~ACA 1~1~1~ ~11~ ~,~l~i GC~CA~I~G 660
I~,~al~l~ Aa~ C~ ~AI~ Gaa~aC!C ATGGCrACCC 720
25 ~ ~3a~ T:l~ AGll~ GPGIG~ll~ ~X;GG~I~ 780
a~ I:~G~C /~Ga~aAC MGal~ IY~A~GA~ C~AG~ 900
=a~A ACACX;C~G ~:Kr2G GC = ~G cw~a G~ACAG~G 960
~3ac AX;GA~GA A~ TQc~MAcrc AGa~ A~AToe~ 1020
mP~G 1027
(2) ~ON F~ SEQ ID N0: 8:
(i) SI~ENOE C~MAC~RISIICS:
(A) ~: 872 base pairs
(B) TYl~ cleîc a~id
~C) S_: single
(D) TOPOI~GY: lir~ear
(ii) MDIE0IE TY~
(vi) OR:rG~L sa~:
(A) OR~SM: ~nicola insolens
;
AWX~ C~ ~1~ ~C ~Gl~l~ll~ /~ 60
55 a;cl~ ~ ~G~ ql~ a~cl~ G~l~C 120
WO 93/11249 PCl ~DK92/00360
29
Cl~l~l~CC GGl~C~aGT GOGI~ACIT GA~ ;G TAC~T GC~: 180
CCGACGAaGT q~ AC AC~ACAAt~;C C~GGGC~aC~ TCG~ l~C 240
~;CC:I~CT ~CIC C~;GC G~AGlq~ 1~11~ CAA~C 300
1~1~ ~III;GA GAGI~ C~l~Ch~ GC~ClTACIT (Xrl~OEaC 360
a~= ~ ~ c~c~ c~ AACl'Cl~C 420
,.
~1= AAa~ AC~ACl~ /~Gt~ A~GP~ACA AaCl~l~C 480
~al = A~ml~ CT~GG~a;GT ~;aG Cl~a~l~;C Gl~GC~aCC 540
15 A~ l~lC Alq~ ac~AcmG~$ ~AC ~1~ 600
G~C GY~ OEa:~,AGr a~T~ G~ G.~ 660
llaa= ~ u = ~= =~ Ga;GGPI~ 720
=~G =~C q7~Gl~lT~ AA~Cl~ ~IY A~aCrACTA 780
~=T C~A~ A~=A a~ G~~ CI~CI~AG 840
AGa:lTm~C A13~a~ ~mT~ TT 872
~ 2) ~[I~tJ E~ S13Q ID N0: 9:
(A) ~: 368 base pa~rs
(B) lY~ leic acid
(C) SlR~S: single ..
(D) ~OPO~: linear
(ii) I~IE TY~E: cDNA
(A) ~SM: ~mlioola ~r~o1ens
(Xi.) S~OE ~P~ON: SEQ ID N0: 9:
T~a~aa; Al~A~ C~C~A(~ 120
q~GGC~ ~ ~CC~GCC A~ ~T~ ~AC~C 180
~ .a~X~T ~.A~ ~a~.T a~cc a:,~ ~ 240
A=~ GaD a =(X~ ~ = A?~a~ ~1~ 300
a~a~ G:=~ ~ G~;aAC~ ~1~ 360
368
".
WO 93~11249 2 1 2 4 9 3 9 PCI /DK92/00360
(2) INFORM~lqON FOR SEQ ID NO: lO:
(i) SE~ENCE C}~RA~l~LSII~;:
(A) I=: 720 base pair~;
S (B) TYE~E: nucleic acid
(C) S~ANDECNESS: sin~le
(D) IOPOLDGY: linear
(vi) ORIG~L SalR~E:
(A) OR~N~: ~nicola insolens
lS (xi) SE~OE DESCRIPIION: SEQ ID N~: lO:
M = ~r aG~a;Gccc a~aGG~c OEG~CraGC c~cc~ac GrGc~aG~ru 120
T~ C~aGG~C~rG GGC~C~Ca~G ~;c~cr I~C C~A~C~ 240
25 ~;G~C~; GOOGC~Gr l~C~GGC~A ~I'G ~l~CaA~ lT~aC~GG~ 300
C~G~ ~C.~ c~ac T~C~ q~A~ A~Ca 360
AD~ ~: q~C~ ~C~ ~W ~GC 480
~c ~a~ ~AA~ a~GGOG ~C GCrC~AC~u 540
~GCI~ ~1~ GGGtI~ aC AA~C~COG Aa~GGc~ 1~11~1; , 600
Cl~mGC t~ac GC~C~C AA(X~Ct~C a:GCX~GI G~EI~C~ ~60
Gm~,a~ A~ ~.aG~ q~Ç aa~G ~Gm 720
~2) INF~[IGN EOR SEQ ID N0~
(i) ~ENCE CHP,R~C~ICS:
(A) ~H: 724 base pairs
(B) 1~: nucleic acid
(C) STRANDEINESS: sir~le
(vi) ~L sa~:
(A) aE~SM: ~nioc~la insolens
(xi) SEX~CE DESCRIPrIClN: S13Q ID N0: ll:
WO 93/11249 2 1 2 4 9 3 9 PCr/DK92/00360
- 31
a~I~iC (~I~ GCI~GAGCCC (æ~l~ GC~ACCCCIT CI~C 120
A~lK~lGG TC~C~ CI~Ca~ AAI~CC AGA(X~OGOCA GGCCI'll~ 180
/~a=C GAC~CAGA~A A~CI~A GGIC~Gl'AC ~lCCl~GaGA AGG~C 240
~1~1~ GACIl~AC Al'Cll~CC l~C I~ l~CC Al~GAAIG 300
'~ O ''
a;XtXS;OCa AGGC~CG AGI~ACCCCP~ OEI~C GI~CA A(~l~360
a~I~C A~;CGAa;CGG CAGIACCI~ GGCGAC!GITA A~ A~;GCCIG 420
15 A~C~ AaGp.Gr~ a~I~ACCOG T~a A~ a~ ~C 480
Gll~[l~G ~I~lCCr a:~ GIX~ A~I~C GGGCACC~GC 540
G~n:Cc GC~A~G a:G~!l~ A~A~T cGG~T~r A~rcr~I~ 600
~;GG~ ATAA~a GC~ACIGCC C~;Gi~GI~ CACCP,~CrC CAA~660
=~ AAG~G CrrCI~ A~I~C TACAACC~T TCACG~CA~C 720
CGOG 724
~2) ~N=ION F~R SEQ ID N~: 12:
(i) SE5~ENCE C~RACI~RISlICS:
tA) IEt~rH: 71 base pairs
(B) TYPE: rlucleic acid
(C) ~D = S: single
(D) I~POI~GY: linear
3~ (ii) ~IECl~IE TYÆ: c~
(vi) C~IG~L S0~:
(A) ~N~: ~nic~la insolens
a~I~l~:a GrCI~ ~ll~C CI~rC C~ACAACT c~rc~ 60
~ G 71
(2) ~ N F~R SEQ ID N0: 13:
(i) SE~OE C~RACrERISllCS:
~A) IE~H: 572 base pairs
(B) T~E: ~cleic acid
(C) S=~: single
(D) IOPOLOGY: linear
(ii) M~IECUIE TY~E: c~
WO 93/11249 2 12 4 ~ ~ 9 PCr/DK92/003~11
32
(vi) ORIG~L SOUROE:
(A) ~NI5M: Humicola ~lens
(xi) SE~pENOE DESCRIY1~1ON: S~Q ID NO: 13:
q~ c~ ~acx; ~ c~c ~[Tl'r 120
GA~I~ ~ACI~A Cr~CGGCC ~I~C GC~aC CC~AOGC~ 180
G?~ AC~ACGGCI~ crrclAcIaG 1~1~ Aa~ A~ a~I~ 240
q ~ ACC ~ A ~ ~ AC ~G G ~ G~ACACOGG c~Acrra OEc 300
GGIGGTPaGG GnlGGAACOC GGGAACCGGC CGCACEATCA AcIAoGGcGG CTACTTCAAC 360
CCCCAGGGCA AoGGCI~CCT GGCCGICThC GGCT~GACCC GC~ACCoGCT CrTCGAGr~C 420
TPTGTCAIOG AGTcGlAoGG cAoGrAcAAT ClCGGCaGCC AEGC~C~GTA CAAGGGCACA 480
lqCqAIACCG ACGGoG~TCA GTATGACATC TITGIGACCA ~ ACAA CCAGCCCa5C S40
25 AICaCGGCAC CCGGACGTCC ASCTAGTACT GG 572
(2) INFO ~ ON FOR SEQ ID NO: 14:
(i) SEQpENOE CH~RACTERISIICS:
(A) IENGTH: 173 kase pairs
(B) TYl~ cleic acid
(C) S~aN~: single
(1)) TOPOLOGY: linear
3S (ii~ M~IECULE TYPE: c~NA
(vi) CRIG~L SC~E:
(A) ORGANI5~: Humi~la Lnsolens
(xi) SEQUENCE ~ESCRIPTION: SEQ ID NO: 14:
AAAGTAGa~C GTCCTGCrOC CTAGAAAOC~ GICACTCArT C~CAATGCGT TcLATCGCTC 60
ICrCTCTIGC TGCGGCTCCG GGOC~OCTCG CCCAGTCOCA GCrCIG~GOC AGTGCGGqGG 120
CATCGGC~GG AAOGGOC$AC GACTTGCGTC TCGGGCGCTA CCTGCACC~A GAT 173
(2),.INFORMPIION ~OR SEQ ID ~0: 15:
5~
(i) SE~UENCE CH~R~CTERISTICS:
(A) ~ H: 214 base pairs
(B) TYPE: nucleic acid
(C) ST~ANDE~NESS: s mgle
(D) T~P~LDGY: linear
WO 93/11249 2 12 4 9 3 9 PCl /DK92/00360
33
(vi) C~GIN~L Sa~E:
(xi) SE~OE nF~cRIprIoN: SEQ ID N0: 15:
''
A~l~a~ =P~ I~C~CX~OC~; AGCGCI~COG GC~Cl~ OGGC~CAIC 180
