U.S. patent application number 14/255642 was filed with the patent office on 2014-09-25 for methods for eliminating or reducing the expression of a gene in a filamentous fungal strain.
This patent application is currently assigned to Novozymes A/S. The applicant listed for this patent is Novozymes A/S, Novozymes, Inc.. Invention is credited to Howard Brody, Suchindra Maiyuran, Hiroaki Udagawa.
Application Number | 20140287475 14/255642 |
Document ID | / |
Family ID | 34676840 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140287475 |
Kind Code |
A1 |
Brody; Howard ; et
al. |
September 25, 2014 |
Methods For Eliminating Or Reducing The Expression Of A Gene In A
Filamentous Fungal Strain
Abstract
The present invention relates to methods for reducing or
eliminating the expression of a target gene in a filamentous fungal
strain, comprising: (a) inserting into the genome of the
filamentous fungal strain a double-stranded transcribable nucleic
acid construct comprising a first nucleotide sequence comprising a
promoter operably linked to a homologous coding region of the
target gene and a second nucleotide sequence comprising the
homologous coding region, or a portion thereof, of the target gene,
wherein the first and second nucleotide sequences are complementary
to each other and the second nucleotide sequence is in reverse
orientation relative to the first nucleotide sequence; and (b)
inducing production of an interfering RNA encoded by the
double-stranded transcribable nucleic acid construct by cultivating
the filamentous fungal strain under conditions conducive for
production of the interfering RNA; wherein the interfering RNA
interacts with RNA transcripts of the target gene to reduce or
eliminate expression of the target gene. The present invention also
relates to the filamentous fungal strains and to methods of
producing a biological substance of interest in such filamentous
fungal strains.
Inventors: |
Brody; Howard; (Davis,
CA) ; Maiyuran; Suchindra; (Gold River, CA) ;
Udagawa; Hiroaki; (Yokohama-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes A/S
Novozymes, Inc. |
Bagsvaerd
Davis |
CA |
DK
US |
|
|
Assignee: |
Novozymes A/S
Bagsvaerd
CA
Novozymes, Inc.
Davis
|
Family ID: |
34676840 |
Appl. No.: |
14/255642 |
Filed: |
April 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11009890 |
Dec 9, 2004 |
8716023 |
|
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14255642 |
|
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60528367 |
Dec 9, 2003 |
|
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Current U.S.
Class: |
435/171 |
Current CPC
Class: |
C12N 15/1137 20130101;
C12P 1/02 20130101; C12N 15/113 20130101; C12N 2310/53 20130101;
C12N 2310/14 20130101; C12N 2310/111 20130101 |
Class at
Publication: |
435/171 |
International
Class: |
C12P 1/02 20060101
C12P001/02 |
Claims
1. A method for producing a biological substance, comprising: (a)
cultivating an Aspergillus niger, an Aspergillus oryzae, or a
Trichoderma reesei strain under conditions conducive for the
production of the biological substance of interest, wherein the
Aspergillus niger, the Aspergillus oryzae, or the Trichoderma
reesei strain comprises a double-stranded transcribable nucleic
acid construct comprising a first nucleotide sequence comprising a
promoter operably linked to a first homologous transcribable region
of a target gene encoding an undesirable biological substance and a
second nucleotide sequence comprising a second homologous
transcribable region of the target gene, wherein the first and
second homologous regions are complementary to each other and the
second homologous region is in reverse orientation relative to the
first homologous region, wherein interfering RNA encoded by the
double-stranded transcribable nucleic acid construct interacts with
RNA transcripts of the target gene to reduce or eliminate
expression of the target gene encoding the undesirable biological
substance; and wherein the Aspergillus niger, the Aspergillus
oryzae, or the Trichoderma reesei strain comprises a third
nucleotide sequence encoding the biological substance of interest;
and (b) recovering the biological substance from the cultivation
medium.
2. The method of claim 1, wherein the first homologous region
comprises at least 19 nucleotides of the target gene.
3. The method of claim 1, wherein the second homologous region
comprises at least 19 nucleotides of the first homologous region,
wherein the least 19 nucleotides are in reverse order relative to
the corresponding region of the first homologous region.
4. The method of claim 1, wherein the first and second nucleotide
sequences are separated by a fourth nucleotide sequence.
5. The method of claim 4, wherein the fourth nucleotide sequence
comprises at least 5 nucleotides.
6. The method of claim 1, wherein expression of the target gene is
reduced by at least 20%.
7. The method of claim 1, wherein expression of the target gene is
eliminated.
8. The method of claim 1, wherein the interfering RNA interacts
with RNA transcripts of one or more homologues of the target gene
to reduce or eliminate expression of the one or more homologues of
the target gene.
9. The method of claim 8, wherein expression of the one or more
homologues of the target gene is reduced by at least 20%.
10. The method of claim 9, wherein expression of the one or more
homologues of the target gene is eliminated.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 11/009,890, filed Dec. 9, 2004, which claims
the benefit of U.S. Provisional Application No. 60/528,367, filed
Dec. 9, 2003, which applications are fully incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to eliminating or reducing
expression of a gene in a filamentous fungal strain.
[0004] 2. Description of the Related Art
[0005] Filamentous fungal strains are widely used for the
production of biological substances of commercial value. However,
filamentous fungal strains with the desirable traits of increased
expression and secretion of a biological substance may not
necessarily have the most desirable characteristics for successful
fermentation. The production of the biological substance may be
accompanied by the production of other substances, e.g., enzymes,
that degrade the biological substance or co-purify with the
biological substance, which can complicate recovery and
purification of the biological substance.
[0006] One solution to these problems is to inactivate the gene(s)
involved in the production of the undesirable substance.
Inactivation can be accomplished by deleting or disrupting the
gene(s) using methods well known in the art. However, in some
cases, inactivation of the gene may be difficult because of poor
targeting to homologous regions of the gene. Inactivation can also
be accomplished by random mutagenesis, but random mutagenesis is
not always specific for the intended target gene and other
mutations are often introduced into the host organism. In other
situations, the gene and its product may be required for survival
of the filamentous fungal strain. Where multiple genes are to be
inactivated by deletion or disruption, the task can be very
cumbersome and time-consuming. When highly homologous members of
gene families exist, deletion or disruption of all members can be
extremely tedious and difficult.
[0007] In recent years various forms of epigenetic gene regulation
have been described (Selker, 1997, Trends Genet. 13: 296-301;
Matzke and Matzke, 1998, Cell. Mol. Life. Sc. 54: 94-103). These
processes influence gene expression by modulating the levels of
messenger RNA (Hammond and Baulcombe, 1996, Plant Mol. Biol. 32:
79-88; Xi-song Ke et al, 2003, Current Opinion in Chemical Biology
7: 516-523) via micro RNAs (Morel et al., 2000, Curr. Biol. 10:
1591-4; Bailis and Forsburg, 2002, Genome Biol. 3, Reviews 1035;
Grewal and Moazed, 2003, Science 301: 798-802).
[0008] Based on genetic studies of Drosophila and Caenorhabditis
elegans, RNA interference (RNAi), also known as
post-transcriptional gene silencing (in plants), is understood to
involve silencing the expression of a gene by assembly of a
protein-RNA effector nuclease complex that targets homologous RNAs
for degradation (Hannon, 2002, Nature 418: 244-251). The processing
of double-stranded RNA (dsRNA) into small interfering RNAs is
accomplished by a family of enzymes known as Dicer (Bernstein et
al, 2001, Nature 409: 363). Dicer, a member of the RNase III family
of endonucleases that specifically cleaves dsRNA, is responsible
for digestion of dsRNA into siRNAs ranging from 20-25 nucleotides
(Elbashir et al., 2001, Nature 411: 494). These siRNAs denature
with the anti-sense strand and then associate with the RNA Induced
Silencing Complex (RISC) (Elbashir et al., 2001, Genes and Dev. 15:
188; NyKanen et al., 2001, Cell 197: 300; Hammond et al, 2001,
Science 293: 1146.). Although not well understood, RISC targets the
mRNA from which the anti-sense fragment was derived followed by
endo and exonuclease digestion of the mRNA effectively silencing
expression of that gene. RNAi has been demonstrated in plants,
nematodes, insects, and mammals (Matzke and Matzke, 1998, supra;
Kennerdell et al., 2000, Nat. Biotechnol. 18: 896-8; Bosher et al.,
1999, Genetics 153: 1245-56; Voorhoeve and Agami, 2003, Trends
Biotechnol. 21: 2-4; and McCaffrey et al., 2003, Nat. Biotechnol.
21: 639-44).
[0009] WO 98/53083 discloses constructs and methods for enhancing
the inhibition of a target gene within a plant by inserting a
silencing vector comprising an inverted repeat sequence of all or
part of the target gene into the genome of a plant.
[0010] WO 01/49844 describes an inverted repeat gene construct
encoding an inverted repeat gene, comprising a promoter element
operably linked in a 5' to 3' direction to a first coding sequence
and a second sequence in an antisense orientation for disrupting
gene expression in targeted organisms, such as Caenorhabditis
elegans, yeast, Dictostelium, Drosophila, mice, plants, insects,
human cells, and nematodes.
[0011] WO 03/050288 discloses methods of silencing a target gene in
a plant by providing a recombinant DNA construct comprising a
promoter operably linked to a chimeric nucleotide sequence encoding
all or part of the target gene and a transgene, transforming the
plant with the DNA construct such that the expression cassette is
inserted into the genome, and initiating post-transcriptional gene
silencing of the transgene in the plant, whereby initiation of
post-transcriptional gene silencing of the transgene causes
silencing of the target gene.
[0012] It would be an advantage in the art to have alternative
methods for eliminating or reducing the expression of one or more
genes for strain development and improvement, functional genomics,
and pathway engineering of filamentous fungal strains.
[0013] It is an object of the present invention to provide
alternative methods for eliminating or reducing the expression of
one or more genes in a filamentous fungal strain.
SUMMARY OF THE INVENTION
[0014] The present invention relates to methods for reducing or
eliminating the expression of a target gene encoding a biological
substance in a filamentous fungal strain, comprising:
[0015] (a) inserting into the genome of the filamentous fungal
strain a double-stranded transcribable nucleic acid construct
comprising a first nucleotide sequence comprising a promoter
operably linked to a first homologous transcribable region of the
target gene encoding the biological substance and a second
nucleotide sequence comprising a second homologous transcribable
region of the target gene, wherein the first and second homologous
regions are complementary to each other and the second homologous
region is in reverse orientation relative to the first homologous
region; and
[0016] (b) inducing production of an interfering RNA encoded by the
double-stranded transcribable nucleic acid construct by cultivating
the filamentous fungal strain under conditions conducive for
production of the interfering RNA, wherein the interfering RNA
interacts with RNA transcripts of the target gene to reduce or
eliminate expression of the target gene encoding the biological
substance.
[0017] The present invention also relates to filamentous fungal
strains comprising a nucleic acid construct comprising a
double-stranded transcribable nucleic acid construct comprising a
first nucleotide sequence comprising a promoter operably linked to
a first homologous region of a target gene encoding a biological
substance and a second nucleotide sequence comprising a second
homologous region or a portion thereof of the target gene, wherein
the first and second homologous regions are complementary to each
other and the second homologous region is in reverse orientation
relative to the first homologous region, wherein interfering RNA
encoded by the double-stranded transcribable nucleic acid construct
interacts with RNA transcripts of the target gene to reduce or
eliminate expression of the target gene encoding the biological
substance.
[0018] The present invention further relates to methods for
producing a biological substance, comprising:
[0019] (a) cultivating a filamentous fungal strain under conditions
conducive for production of the biological substance of interest,
wherein the filamentous fungal strain comprises a double-stranded
transcribable nucleic acid construct comprising a first nucleotide
sequence comprising a promoter operably linked to a first
homologous transcribable region of a target gene encoding an
undesirable biological substance and a second nucleotide sequence
comprising a second homologous transcribable region of the target
gene, wherein the first and second homologous regions are
complementary to each other and the second homologous region is in
reverse orientation relative to the first homologous region,
wherein interfering RNA encoded by the double-stranded
transcribable nucleic acid construct interacts with RNA transcripts
of the target gene to reduce or eliminate expression of the target
gene encoding the undesirable biological substance; and wherein the
filamentous fungal strain comprises a third nucleotide sequence
encoding the biological substance of interest; and
[0020] (b) recovering the biological substance from the cultivation
medium.
DESCRIPTION OF THE FIGURES
[0021] FIG. 1 shows a restriction map of pAlLo1.
[0022] FIG. 2 shows a restriction map of pMJ04.
[0023] FIG. 3 shows a restriction map of pMJ06.
[0024] FIG. 4 shows a restriction map of pMJ09.
[0025] FIG. 5 shows a restriction map of a Trichoderma reesei CeI6A
inverted repeat fragment.
[0026] FIG. 6 shows a restriction map of pSMai148.
[0027] FIG. 7 shows the relative expression level of Trichoderma
reesei CeI6A cellobiohydrolase II mRNA in transformant 14, a
positive control strain (Trichoderma reesei SaMe02), and the "Empty
vector control" in shake flasks.
[0028] FIG. 8 shows a restriction map of pSMai163.
[0029] FIG. 9 shows detection of small interfering Trichoderma
reesei CeI6A cellobiohydrolase II RNA by Northern analysis. Total
RNAs (25 mg) were electrophoresed onto a 15% polyacrylamide-7M urea
gel, transferred to a nylon membrane and probed with a DIG-labeled
Trichoderma reesei CeI6A cellobiohydrolase II RNA probe.
Trichoderma reesei SaMe02 was a positive control strain where the
Trichoderma reesei CeI6A cellobiohydrolase II gene was knocked-out
by homologous recombination. Trichoderma reesei RutC30 was the host
strain. The ethidium bromide stained gel, which showed the control
28s-rRNA and 18s-rRNA, indicated equal loading.
[0030] FIG. 10 shows SDS-PAGE analysis of day 7 shake flask samples
(#'s 1, 2, 11, 12, 13, and 14) of Trichoderma reesei RutC30
expressing the Trichoderma reesei CeI6A cellobiohydrolase II
inverted repeat fragments. Trichoderma reesei SaMe02 was a positive
control strain where the Trichoderma reesei CeI6A cellobiohydrolase
II gene was knocked-out by homologous recombination. Trichoderma
reesei RutC30 was the host strain. At 7-days post-inoculation, 500
.mu.l of mycelia were used as an inoculum for shake flasks
containing fresh in cellulase-inducing medium. This passaging of
mycelia was performed for a total of five rounds.
[0031] FIG. 11 shows a restriction map of pBANe10.
[0032] FIG. 12 shows a restriction map of pAlLo2.
[0033] FIG. 13 shows a restriction map of pHB506.
[0034] FIG. 14 shows SDS-PAGE analysis of day 7 shake flask samples
of selected Aspergillus niger transformants containing pHB506
(transformants 1-8), a glucoamylase gene deleted strain Aspergillus
niger HowB112, Aspergillus niger JaL303-10, and Aspergillus niger
JaL303-10 transformed with pAlLo2 (empty vector). Transformants 1-4
produced viable amounts of glucoamylase whereas transformants 5-8
produced no detectable glucoamylase.
[0035] FIG. 15 shows a restriction map of pHUda512.
[0036] FIG. 16 shows SDS-PAGE analysis of day 7 shake flask samples
of selected Aspergillus niger transformants containing pHUda512
(lane 1-8) and Aspergillus niger NN049735 (lane 9).
[0037] FIG. 17 shows a restriction map of pDeMi01.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention relates to methods for reducing or
eliminating the expression of a target gene encoding a biological
substance in a filamentous fungal strain, comprising: (a) inserting
into the genome of the filamentous fungal strain a double-stranded
transcribable nucleic acid construct comprising a first nucleotide
sequence comprising a promoter operably linked to a first
homologous transcribable region of the target gene and a second
nucleotide sequence comprising a second homologous transcribable
region of the target gene encoding the biological substance,
wherein the first and second homologous regions are complementary
to each other and the second homologous region is in reverse
orientation relative to the first homologous region; and (b)
inducing production of an interfering RNA encoded by the
double-stranded transcribable nucleic acid construct by cultivating
the filamentous fungal strain under conditions conducive for
production of the interfering RNA, wherein the interfering RNA
interacts with RNA transcripts of the target gene to reduce or
eliminate expression of the target gene encoding the biological
substance.
[0039] The methods of the present invention provide new
opportunities for strain development and improvement, functional
genomics, and pathway engineering in filamentous fungal strains.
For example, the present methods can be used as a tool for
filamentous fungal host strain development by means of gene
manipulation and pathway engineering or as a replacement for gene
knockouts, a time consuming approach with variable rates of
success. A gene may be resistant to inactivation by standard
methods known in the art such as gene knockout. The methods of the
present invention provide a solution to reducing or eliminating the
expression of such a gene. The methods are also particularly useful
and efficient for reducing or eliminating a highly expressed gene
in a particular filamentous fungal strain, which can be very
important, for example, in developing the organism as a production
host. This ability demonstrates the strength of the methods of the
present invention. The methods are also useful for reducing or
eliminating the expression of a multiple of genes that are highly
homologous to each other, especially genes of the same family or
homologous genes in a biosynthetic or metabolic pathway. The
methods are further useful because they can be manipulated to cause
a variable reduction in the expression of a biological substance.
This variability is especially important where a complete knock-out
of a gene encoding a biological substance would be lethal to a
particular filamentous fungal strain, such as in a secondary
pathway that feeds into a biosynthetic pathway of interest.
[0040] In the methods of the present invention, the first
nucleotide sequence comprises a promoter operably linked to a first
homologous transcribable region of the target gene. The second
nucleotide sequence comprises a second homologous transcribable
region of the target gene, where the first and second homologous
regions are complementary to each other and the second homologous
region is in reverse orientation relative to the first homologous
region. In a preferred aspect, the first and second nucleotide
sequences are separated by a third nucleotide sequence to stabilize
the first and second nucleotide sequences in a filamentous fungal
strain against undesirable recombination during construction, such
as in E. coli The nucleotide sequences of the double-stranded
transcribable nucleic acid construct may be of genomic, cDNA, RNA,
semisynthetic, synthetic origin, or any combinations thereof.
Promoter
[0041] The term "promoter" is defined herein as a DNA sequence that
binds RNA polymerase and directs the polymerase to the correct
downstream transcriptional start site of a nucleic acid sequence
encoding a biological substance to initiate transcription. RNA
polymerase effectively catalyzes the assembly of messenger RNA
complementary to the appropriate DNA strand of a coding region. The
term "promoter" will also be understood to include the 5'
non-coding region (between promoter and translation start) for
translation after transcription into mRNA, cis-acting transcription
control elements such as enhancers, and other nucleotide sequences
capable of interacting with transcription factors. The promoter
sequence may be native or foreign (heterologous) to the first
homologous transcribable region and native or foreign to the
filamentous fungal strain. In the methods of the present invention,
the promoter can be a native promoter, heterologous promoter,
mutant promoter, hybrid promoter, or tandem promoter.
[0042] The term "mutant promoter" is defined herein as a promoter
having a nucleotide sequence comprising a substitution, deletion,
and/or insertion of one or more nucleotides of a parent promoter,
wherein the mutant promoter has more or less promoter activity than
the corresponding parent promoter. The term "mutant promoter" also
encompasses natural mutants and in vitro generated mutants obtained
using methods well known in the art such as classical mutagenesis,
site-directed mutagenesis, and DNA shuffling.
[0043] The term "hybrid promoter" is defined herein as parts of two
more promoters that are fused together to generate a sequence that
is a fusion of the two or more promoters, which when operably
linked to a coding sequence mediates the transcription of the
coding sequence into mRNA.
[0044] The term "tandem promoter" is defined herein as two or more
promoter sequences each of which is operably linked to a coding
sequence and mediates the transcription of the coding sequence into
mRNA.
[0045] The term "operably linked" is defined herein as a
configuration in which a promoter sequence is appropriately placed
at a position relative to the homologous regions of the target gene
such that the promoter sequence directs the transcription of the
two regions.
[0046] Examples of promoters useful in the methods of the present
invention include the promoters obtained from the genes for
Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic
proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus
niger acid stable alpha-amylase, Aspergillus niger or Aspergillus
awamori glucoamylase (g/aA), Rhizomucor miehei lipase, Aspergillus
oryzae alkaline protease, Aspergillus oryzae triose phosphate
isomerase, Aspergillus nidulans acetamidase, Coprinus cinereas
beta-tubulin, Fusarium venenatum amyloglucosidase (WO 00/56900),
Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn
(WO 00/56900), Fusarium oxysporum trypsin-like protease (WO
96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma
reesei endoglucanase IV, Trichoderma reesei endoglucanase V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,
Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter
(a hybrid of the promoters from the genes for Aspergillus niger
neutral alpha-amylase and Aspergillus oryzae triose phosphate
isomerase); and mutant, truncated, and hybrid promoters
thereof.
Homologous Transcribable Region
[0047] The term "homologous transcribable region" is defined herein
as a nucleotide sequence which is homologous to the open reading
frame of a target gene or a portion thereof; that is transcribed
into an RNA, e.g., ncRNA (non-coding RNA), tRNA (transfer RNA),
rRNA (ribosomal RNA), miRNA (micro RNA), or mRNA (messenger RNA),
which may or may not be translated into a biological substance,
e.g., polypeptide, when placed under the control of the appropriate
regulatory sequences. The boundaries of the transcribable region
are generally determined by the transcription start site located
just upstream of the open reading frame at the 5' end of the mRNA
and a transcription terminator sequence located just downstream of
the open reading frame at the 3' end of the mRNA. A homologous
transcribable region can include, but is not limited to, genomic
DNA, cDNA, semisynthetic, synthetic, and recombinant nucleic acid
sequences.
[0048] In the methods of the present invention, the first region
homologous to the target gene may be identical to the corresponding
region of the target gene or may be a homologue thereof.
[0049] The degree of identity between the homologue and the
corresponding region of the target gene required to achieve
inactivation or reduction of the expression of the target gene will
likely depend on the target gene. The smaller the homologue's
nucleotide sequence is relative to the entire target gene, the
degree of identity between the sequences should preferably be very
high or identical. The larger the homologue's nucleotide sequence
is relative to the entire target gene, the degree of identity
between the sequences can likely be lower.
[0050] In the methods of the present invention, the degree of
identity of the homologue's nucleotide sequence to the
corresponding region of the target gene is at least 65%, preferably
at least 70%, more preferably at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
90%, even more preferably at least 95%, and most preferably at
least 97%. For purposes of the present invention, the degree of
identity between two nucleic acid sequences is determined by the
Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the
National Academy of Science USA 80: 726-730) using the
LASERGENE.TM. MEGALIGN.TM. software (DNASTAR, Inc., Madison, Wis.)
with an identity table and the following multiple alignment
parameters: Gap penalty of 10 and gap length penalty of 10.
Pairwise alignment parameters were Ktuple=3, gap penalty=3, and
windows=20.
[0051] Alternatively, the ability of the homologue and the
corresponding region of the target gene to hybridize to each other
under various stringency conditions can also provide an indication
of the degree of relatedness required for inactivation or reduction
of expression of a target gene. However, it should be recognized
that the lower the stringency conditions required, e.g., low
stringency, to achieve hybridization between the homologue and the
corresponding region of the target gene, inactivation or reduction
of the expression of the target gene will likely be less efficient.
In a preferred aspect, the homologue and the corresponding region
of the target gene hybridize under low stringency conditions. In a
more preferred aspect, the homologue and the corresponding region
of the target gene hybridize under medium stringency conditions. In
an even more preferred aspect, the homologue and the corresponding
region of the target gene hybridize under medium-high stringency
conditions. In a most preferred aspect, the homologue and the
corresponding region of the target gene hybridize under high
stringency conditions. In an even most preferred aspect, the
homologue and the corresponding region of the target gene hybridize
under very high stringency conditions.
[0052] For probes of at least 100 nucleotides in length, very low
to very high stringency conditions are defined as prehybridization
and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
.mu.g/ml sheared and denatured salmon sperm DNA, and either 25%
formamide for very low and low stringencies, 35% formamide for
medium and medium-high stringencies, or 50% formamide for high and
very high stringencies, following standard Southern blotting
procedures for 12 to 24 hours optimally.
[0053] For probes of at least 100 nucleotides in length, the
carrier material is finally washed three times each for 15 minutes
using 2.times.SSC, 0.2% SDS preferably at least at 45.degree. C.
(very low stringency), more preferably at least at 50.degree. C.
(low stringency), more preferably at least at 55.degree. C. (medium
stringency), more preferably at least at 60.degree. C. (medium-high
stringency), even more preferably at least at 65.degree. C. (high
stringency), and most preferably at least at 70.degree. C. (very
high stringency).
[0054] For probes which are about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
prehybridization, hybridization, and washing post-hybridization at
about 5.degree. C. to about 10.degree. C. below the calculated
T.sub.m using the calculation according to Bolton and McCarthy
(1962, Proceedings of the National Academy of Sciences USA 48:1390)
in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,
1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium
monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml
following standard Southern blotting procedures for 12 to 24 hours
optimally.
[0055] For probes which are about 15 nucleotides to about 70
nucleotides in length, the carrier material is washed once in
6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m.
[0056] The first homologous region preferably consists of at least
19 nucleotides, more preferably at least 40 nucleotides, more
preferably at least 60 nucleotides, more preferably at least 80
nucleotides, even more preferably at least 100 nucleotides, and
most preferably at least 200 nucleotides. The first homologous
region can also consist of the entire open reading frame of the
gene or a homologue thereof.
Inverted Repeat
[0057] The double-stranded transcribable nucleic acid construct
also comprises a second homologous transcribable region of the
target gene, where the first and second homologous regions are
complementary to each other and the second homologous region is in
reverse orientation relative to the first homologous region, i.e.,
an inverted repeat of the first homologous region.
[0058] In a preferred aspect, the second homologous region is 100%
identical to the first homologous region, but in reverse
orientation relative to the first homologous region. In another
preferred aspect, the second homologous region is a portion of the
first homologous region, wherein the portion is 100% identical to
the corresponding portion of the first homologous region. In
another preferred aspect, the second homologous region is a
homologue of the corresponding portion of the first homologous
region. In another preferred aspect, the second homologous region
is a homologue portion of the corresponding portion of the first
homologous region.
[0059] The homologue or homologue portion is at least 65%,
preferably at least 70%, more preferably at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, even more preferably at least 95%, and
most preferably at least 97% identical to the corresponding
sequence of the first nucleotide sequence. Percent identity is
determined according to the Wilbur-Lipman method described
herein.
[0060] The second homologous region which identifies the gene
targeted for reducing or eliminating expression may be any
homologous transcribable part of the target gene, such as the
5'-untranslated region, the biological substance coding sequence,
or the 3'-untranslated region of the target gene.
[0061] In a preferred aspect, the second homologous region
corresponds to the biological substance coding sequence of the
target gene, or a portion thereof.
[0062] In another preferred aspect, the second homologous region
corresponds to the 5'-untranslated region of the target gene, or a
portion thereof.
[0063] In another preferred aspect, the second homologous region
corresponds to the 3'-untranslated region of the target gene, or a
portion thereof.
[0064] The second homologous region preferably consists of at least
19 nucleotides, more preferably at least 40 nucleotides, more
preferably at least 60 nucleotides, more preferably at least 80
nucleotides, even more preferably at least 100 nucleotides, and
most preferably at least 200 nucleotides. The second homologous
region can also consist of the entire open reading frame of the
gene or a homologue thereof.
Spacer Between First and Second Nucleotide Sequences
[0065] The first and second nucleotide sequences comprising the
homologous regions of the target gene may or may not be separated
by a polynucleotide linker or spacer, which is a nucleotide
sequence which has little or no homology to the first and second
nucleotide sequences in the double-stranded transcribable nucleic
acid construct.
[0066] In a preferred aspect, the first and second nucleotide
sequences are separated by a polynucleotide linker. The spacer
preferably consists of at least 5 nucleotides, more preferably at
least 10 nucleotides, more preferably at least 20 nucleotides, more
preferably at least 30 nucleotides, more preferably at least 40
nucleotides, even more preferably at least 500 nucleotides, and
most preferably at least 100 nucleotides.
[0067] The spacer or linker can be any nucleotide sequence without
homology to the first or second nucleotide sequence and preferably
having little or no homology to sequences in the genome of the
filamentous fungal strain to minimize undesirable
targeting/recombination.
Target Gene
[0068] The target gene may be any gene encoding a biological
substance. The biological substance may be an RNA (e.g., ncRNA,
rRNA, tRNA, miRNA, or mRNA). The biological substance may also be
any biopolymer or metabolite. The biological substance may be
encoded by a single gene or a series of genes composing a
biosynthetic or metabolic pathway or may be the direct result of
the product of a single gene or products of a series of genes. The
biological substance may be native to the filamentous fungal strain
or foreign or heterologous to the strain. The term "heterologous
biological substance" is defined herein as a biological substance
which is not native to the cell; or a native biological substance
in which structural modifications have been made to alter the
native biological substance.
[0069] In the methods of the present invention, the biopolymer may
be any biopolymer. The term "biopolymer" is defined herein as a
chain (or polymer) of identical, similar, or dissimilar subunits
(monomers). The biopolymer may be, but is not limited to, a nucleic
acid, polyamine, polyol, polypeptide (or polyamide), or
polysaccharide.
[0070] In a preferred aspect, the biopolymer is a polypeptide. The
polypeptide may be any polypeptide having a biological activity of
interest. The term "polypeptide" is not meant herein to refer to a
specific length of the encoded product and, therefore, encompasses
peptides, oligopeptides, and proteins. The term "polypeptide" also
encompasses two or more polypeptides combined to form the encoded
product. Polypeptides also include hybrid polypeptides, which
comprise a combination of partial or complete polypeptide sequences
obtained from at least two different polypeptides wherein one or
more may be heterologous to the filamentous fungal cell.
Polypeptides further include naturally occurring allelic and
engineered variations of the above-mentioned polypeptides and
hybrid polypeptides.
[0071] In a preferred aspect, the polypeptide is an antibody,
antigen, antimicrobial peptide, enzyme, growth factor, hormone,
immunodilator, neurotransmitter, receptor, reporter protein,
structural protein, and transcription factor.
[0072] In a more preferred aspect, the polypeptide is an
oxidoreductase, transferase, hydrolase, lyase, isomerase, or
ligase. In a most preferred aspect, the polypeptide is an
alpha-glucosidase, aminopeptidase, amylase, carbohydrase,
carboxypeptidase, catalase, cellulase, chitinase, cutinase,
cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,
alpha-galactosidase, beta-galactosidase, glucoamylase,
glucocerebrosidase, alpha-glucosidase, beta-glucosidase, invertase,
laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic
enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, urokinase, or
xylanase.
[0073] In another preferred aspect, the polypeptide is a collagen
or gelatin, or a variant or hybrid thereof.
[0074] In another preferred aspect, the biopolymer is a
polysaccharide. The polysaccharide may be any polysaccharide,
including, but not limited to, a mucopolysaccharide (e.g., heparin
and hyaluronic acid) and nitrogen-containing polysaccharide (e.g.,
chitin). In a more preferred aspect, the polysaccharide is
hyaluronic acid.
[0075] In the methods of the present invention, the metabolite may
be any metabolite. The metabolite may be encoded by one or more
genes, such as a biosynthetic or metabolic pathway. The term
"metabolite" encompasses both primary and secondary metabolites.
Primary metabolites are products of primary or general metabolism
of a cell, which are concerned with energy metabolism, growth, and
structure. Secondary metabolites are products of secondary
metabolism (see, for example, R. B. Herbert, The Biosynthesis of
Secondary Metabolites, Chapman and Hall, New York, 1981).
[0076] The primary metabolite may be, but is not limited to, an
amino acid, fatty acid, nucleoside, nucleotide, sugar,
triglyceride, or vitamin.
[0077] The secondary metabolite may be, but is not limited to, an
alkaloid, coumarin, flavonoid, polyketide, quinine, steroid,
peptide, or terpene. In a preferred aspect, the secondary
metabolite is an antibiotic, antifeedant, attractant, bacteriocide,
fungicide, hormone, insecticide, or rodenticide.
[0078] The biological substance may also be the product of a
selectable marker. A selectable marker is a gene the product of
which provides for biocide or viral resistance, resistance to heavy
metals, prototrophy to auxotrophs, and the like. Selectable markers
include, but are not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hygB (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), trpC (anthranilate synthase), as
well as equivalents thereof.
[0079] It may be necessary in the practice of the present invention
to isolate the target gene. The techniques used to isolate or clone
a gene are known in the art and include isolation from genomic DNA,
preparation from cDNA, or a combination thereof. The cloning of the
gene from such genomic DNA can be effected, e.g., by using the well
known polymerase chain reaction (PCR). See, for example, Innis et
al., 1990, PCR Protocols: A Guide to Methods and Application,
Academic Press, New York. The cloning procedures may involve
excision and isolation of a desired nucleic acid fragment
comprising the gene encoding a biological substance, insertion of
the fragment into a vector molecule, and incorporation of the
recombinant vector into a filamentous fungal cell where multiple
copies or clones of the nucleic acid sequence will be replicated.
The nucleic acid sequence may be of genomic, cDNA, RNA,
semisynthetic, synthetic origin, or any combinations thereof.
[0080] In a preferred aspect, expression of the target gene is
reduced by at least 20%, preferably at least 30%, more preferably
at least 40%, more preferably at least 50%, more preferably at
least 60%, more preferably at least 70%, even more preferably at
least 80%, and most preferably at least 90%. In another preferred
aspect, expression of the target gene is eliminated.
[0081] Where it is desired to use an inverted repeat sequence
within the 5' untranslated region, the coding sequence, or the 3'
untranslated region, gene silencing vectors constructed with
inverted repeats within any one of these regions may additionally
enable the silencing of genes that are homologous to the coding
sequence present in the silencing vector. When it is, therefore,
desired to silence homologues of a gene within an organism, the
construction of a silencing vector containing an inverted repeat
within the 5' untranslated region, the coding sequence, or the 3'
untranslated region may allow the elimination or reduction of
expression of one or more genes exhibiting sequence homology to the
coding sequence within the construct.
[0082] The term "homology" or "homologous" usually denotes those
sequences which are of some common ancestral structure and exhibit
a high degree of sequence similarity of the active regions.
[0083] In a preferred aspect, the interfering RNA interacts with
RNA transcripts of one or more homologues of the target gene to
reduce or eliminate expression of the one or more homologues of the
target gene.
[0084] In a more preferred aspect, expression of one or more
homologues of the target gene is reduced by at least 20%,
preferably at least 30%, more preferably at least 40%, more
preferably at least 50%, more preferably at least 60%, more
preferably at least 70%, even more preferably at least 80%, and
most preferably at least 90%. In another preferred aspect,
expression of one or more homologues of the target gene is
eliminated.
Filamentous Fungal Strains
[0085] The present invention also relates to filamentous fungal
strains comprising a double-stranded transcribable nucleic acid
construct comprising a first nucleotide sequence comprising a
promoter operably linked to a first homologous transcribable region
of the target gene and a second nucleotide sequence comprising a
second homologous transcribable region of the target gene, wherein
the first and second homologous regions are complementary to each
other and the second homologous region is in reverse orientation
relative to the first homologous region, wherein the first and
second nucleotide sequences form a transcribable duplex
polynucleotide comprising a region homologous to a target gene
which silences the expression of the target gene in a filamentous
fungal strain.
[0086] The filamentous fungal strain may be any filamentous fungal
strain useful in the methods of the present invention. "Filamentous
fungi" include all filamentous forms of the subdivision Eumycota
and Oomycota (as defined by Hawksworth et al., 1995, supra). The
filamentous fungi are characterized by a mycelial wall composed of
chitin, cellulose, glucan, chitosan, mannan, and other complex
polysaccharides. Vegetative growth is by hyphal elongation and
carbon catabolism is obligately aerobic. In contrast, vegetative
growth by yeasts such as Saccharomyces cerevisiae is by budding of
a unicellular thallus and carbon catabolism may be
fermentative.
[0087] In a preferred aspect, the filamentous fungal strain is an
Acremonium, Aspergillus, Aureobasidium, Cryptococcus, filibasidium,
Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecllomyces, Penicillium, Piromyces,
Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,
or Trichoderma strain.
[0088] In a more preferred aspect, the filamentous fungal strain is
an Aspergillus awamori, Aspergillus fumigatus, Aspergillus
foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus
niger or Aspergillus oryzae cell. In another more preferred aspect,
the filamentous fungal strain is a Fusarium bactridioides, Fusarium
cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium
graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium
negundi, Fusarium oxysporum, Fusarium reticulatom, Fusarium roseum,
Fusarium sambucinum, Fusarium sarcochroum, Fusarium
sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium
trichothecloides, or Fusarium venenatum cell. In another more
preferred aspect, the filamentous fungal strain is a Bjerkandera
adusta, Ceriporlopsis aneirina, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis
pannocinta, Ceriporlopsis rivulosa, Ceriporiopsis subrufa,
Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,
Humicola insolens, Humicola lanuginosa, Mucor miehei,
Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
[0089] In a most preferred aspect, the Aspergillus oryzae strain is
Aspergillus oryzae strain deposit no. IFO 4177. In another most
preferred aspect, the Fusarium venenatum strain is Fusarium
venenatum A3/5, which was originally deposited as Fusarium
graminearum ATCC 20334 and recently reclassified as Fusarium
venenatum by Yoder and Christianson, 1998, Fungal Genetics and
Biology 23: 62-80 and O'Donnell et al., 1998, Fungal Genetics and
Biology 23: 57-67; as well as taxonomic equivalents of Fusarium
venenatum regardless of the species name by which they are
currently known. In another preferred aspect, the Fusarium
venenatum strain is a morphological mutant of Fusarium venenatum
A3/5 or Fusarium venenatum ATCC 20334, as disclosed in WO 97/26330.
In another preferred aspect, the Trichoderma reesei strain is
Trichoderma reesei ATCC 56765.
[0090] Filamentous fungal strains may be transformed by a process
involving protoplast formation, transformation of the protoplasts,
and regeneration of the cell wall in a manner known per se.
Suitable procedures for transformation of Aspergillus and
Trichoderma strains are described in EP 238 023 and Yelton et al.,
1984, Proceedings of the National Academy of Sciences USA 81:
1470-1474. Suitable methods for transforming Fusarium species are
described by Malardier et al., 1989, Gene 78: 147-156 and WO
96/00787. Yeast may be transformed using the procedures described
by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,
editors, Guide to Yeast Genetics and Molecular Biology, Methods in
Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York;
Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et
al., 1978, Proceedings of the National Academy of Sciences USA 75:
1920.
[0091] Reduction or elimination of expression of a target gene
encoding a biological substance may be detected using methods known
in the art that are specific for the targeted biological substance.
These detection methods may include use of specific antibodies,
high performance liquid chromatography, capillary electrophoresis,
formation of an enzyme product, disappearance of an enzyme
substrate, or SDS-PAGE. For example, an enzyme assay may be used to
determine the activity of the enzyme. Procedures for determining
enzyme activity are known in the art for many enzymes (see, for
example, D. Schomburg and M. Salzmann (eds.), Enzyme Handbook,
Springer-Verlag, New York, 1990).
Methods of Production
[0092] The present invention also relates to methods for producing
a biological substance of interest, comprising: (a) cultivating a
filamentous fungal strain under conditions conducive for production
of the biological substance of interest, wherein the filamentous
fungal strain comprises a double-stranded transcribable nucleic
acid construct comprising a first nucleotide sequence comprising a
promoter operably linked to a first homologous transcribable region
of a target gene encoding an undesirable biological substance and a
second nucleotide sequence comprising a second homologous
transcribable region of the target gene, wherein the first and
second homologous regions are complementary to each other and the
second homologous region is in reverse orientation relative to the
first homologous region, wherein interfering RNA encoded by the
double-stranded transcribable nucleic acid construct interacts with
RNA transcripts of the target gene to reduce or eliminate
expression of the target gene encoding the undesirable biological
substance; and wherein the filamentous fungal strain comprises a
third nucleotide sequence encoding the biological substance of
interest; and (b) recovering the biological substance of interest
from the cultivation medium.
[0093] The biological substance of interest may any biological
substance as described herein. It may be native or foreign to the
filamentous fungal strain. The reduction or elimination of
expression of the target gene encoding the undesirable biological
substance can lead to increased expression of another biological
substance of interest. The undesirable biological substance could
directly affect production or expression of the biological
substance of interest. For example, the undesirable biological
substance may be a protease that attacks the biological substance
of interest thereby lowering the amount of the biological substance
of interest produced. By reducing or eliminating expression of the
protease, more of the biological substance of interest will be
expressed and produced. Or, the undesirable biological substance
may share a cellular process or processes, e.g., transcription
factor or secretory pathway, with the biological substance of
interest thereby lowering the amount of the biological substance of
interest produced. By reducing or eliminating expression of the
undesirable biological substance, more of the cellular process or
processes will be available to the biological substance of
interest, e.g., expression-limiting transcription elements, thereby
increasing the amount of the biological substance of interest
expressed and produced. Moreover, the undesirable biological
substance may be a toxin that contaminates the biological substance
of interest preventing the use of the biological substance of
interest in a particular application, e.g., an enzyme in a food
process.
[0094] In the production methods of the present invention, the
filamentous fungal strains are cultivated in a nutrient medium
suitable for production of the biological substance of interest
using methods known in the art. For example, the strain may be
cultivated by shake flask cultivation, and small-scale or
large-scale fermentation (including continuous, batch, fed-batch,
or solid state fermentations) in laboratory or industrial
fermentors performed in a suitable medium and under conditions
allowing the biological substance to be expressed and/or isolated.
The cultivation takes place in a suitable nutrient medium
comprising carbon and nitrogen sources and inorganic salts, using
procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the biological substance is secreted into the
nutrient medium, it can be recovered directly from the medium. If
the biological substance is not secreted, it can be recovered from
cell lysates.
[0095] The biological substance of interest may be detected using
methods known in the art that are specific for the biological
substances. These detection methods may include use of specific
antibodies, high performance liquid chromatography, capillary
chromatography, formation of an enzyme product, disappearance of an
enzyme substrate, or SDS-PAGE. For example, an enzyme assay may be
used to determine the activity of the enzyme. Procedures for
determining enzyme activity are known in the art for many enzymes
(see, for example, D. Schomburg and M. Salzmann (eds.), Enzyme
Handbook, Springer-Verlag, New York, 1990).
[0096] The resulting biological substance of interest may be
isolated using methods known in the art. For example, a polypeptide
of interest may be isolated from the cultivation medium by
conventional procedures including, but not limited to,
centrifugation, filtration, extraction, spray-drying, evaporation,
or precipitation. The isolated polypeptide may then be further
purified by a variety of procedures known in the art including, but
not limited to, chromatography (e.g., ion exchange, affinity,
hydrophobic, chromatofocusing, and size exclusion), electrophoretic
procedures (e.g., preparative isoelectric focusing (IEF),
differential solubility (e.g., ammonium sulfate precipitation), or
extraction (see, e.g., Protein Purification, J.-C. Janson and Lars
Ryden, editors, VCH Publishers, New York, 1989). A metabolite of
interest may be isolated from a cultivation medium by, for example,
extraction, precipitation, or differential solubility, or any
method known in the art. The isolated metabolite may then be
further purified using methods suitable for metabolites.
Nucleotide Sequences
[0097] The nucleotide sequence encoding the biological substance of
interest may be obtained from any prokaryotic, eukaryotic, or other
source. For purposes of the present invention, the term "obtained
from" as used herein in connection with a given source shall mean
that the biological substance is produced by the source or by a
cell in which a gene from the source has been inserted.
[0098] The techniques used to isolate or clone a nucleotide
sequence encoding a biological substance of interest are known in
the art and include isolation from genomic DNA, preparation from
cDNA, or a combination thereof. The cloning of the nucleotide
sequence from such genomic DNA can be effected, e.g., by using the
well known polymerase chain reaction (PCR). See, for example, Innis
et al., 1990, PCR Protocols: A Guide to Methods and Application,
Academic Press, New York. The cloning procedures may involve
excision and isolation of a desired nucleic acid fragment
comprising the nucleotide sequence encoding the biological
substance, insertion of the fragment into a vector molecule, and
incorporation of the recombinant vector into the mutant filamentous
fungal cell where multiple copies or clones of the nucleic acid
sequence will be replicated. The nucleic acid sequence may be of
genomic, cDNA, RNA, semisynthetic, synthetic origin, or any
combinations thereof.
Nucleic Acid Constructs
[0099] The nucleotide sequence encoding the biological substance of
interest may be contained in a nucleic acid construct in the
filamentous fungal strain. A nucleic acid construct comprises a
nucleotide sequence encoding the biological substance of interest
operably linked to at least one promoter and one or more control
sequences which direct the expression of the nucleotide sequence in
a filamentous fungal strain under conditions compatible with the
control sequences. Expression will be understood to include any
step involved in the production of the biological substance of
interest including, but not limited to, transcription,
post-transcriptional modification, translation, post-translational
modification, and secretion.
[0100] "Nucleic acid construct" is defined herein as a nucleic acid
molecule, either single- or double-stranded, which is isolated from
a naturally occurring gene or which has been modified to contain
segments of nucleic acid combined and juxtaposed in a manner that
would not otherwise exist in nature. The term nucleic acid
construct is synonymous with the term expression cassette when the
nucleic acid construct contains a coding sequence and all the
control sequences required for expression of the coding
sequence.
[0101] An isolated nucleotide sequence encoding the biological
substance of interest may be further manipulated in a variety of
ways to provide for expression of the biological substance.
Manipulation of the nucleotide sequence prior to its insertion into
a vector may be desirable or necessary depending on the expression
vector. The techniques for modifying nucleotide sequences utilizing
recombinant DNA methods are well known in the art.
[0102] The nucleotide sequence may comprise one or more native
control sequences or one or more of the native control sequences
may be replaced with one or more control sequences foreign to the
nucleotide sequence for improving expression of the coding sequence
in a host cell.
[0103] The term "control sequences" is defined herein to include
all components which are necessary or advantageous for expression
of a biological substance of interest. Each control sequence may be
native or foreign to the nucleotide sequence encoding the
biological substance. Such control sequences include, but are not
limited to, a leader, polyadenylation sequence, propeptide
sequence, promoter, signal peptide sequence, and transcription
terminator. At a minimum, the control sequences include a promoter,
and transcriptional and translational stop signals. The control
sequences may be provided with linkers for the purpose of
introducing specific restriction sites facilitating ligation of the
control sequences with the coding region of the nucleotide sequence
encoding a biological substance of interest.
[0104] The control sequence may be a suitable transcription
terminator sequence, a sequence recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3' terminus of the nucleic acid sequence encoding the
biological substance. Any terminator which is functional in the
filamentous fungal strain of choice may be used in the present
invention.
[0105] Preferred terminators for filamentous fungal strain are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease.
[0106] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA which is important for
translation by the filamentous fungal strain. The leader sequence
is operably linked to the 5' terminus of the nucleic acid sequence
encoding the biological substance. Any leader sequence that is
functional in the filamentous fungal strain of choice may be used
in the present invention.
[0107] Preferred leaders for filamentous fungal strains are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus nidulans triose phosphate isomerase, Fusarium venenatum
trypsin, and Fusarium venenatum glucoamylase.
[0108] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3' terminus of the nucleic acid
sequence and which, when transcribed, is recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA.
Any polyadenylation sequence which is functional in the filamentous
fungal strain of choice may be used in the present invention.
[0109] Preferred polyadenylation sequences for filamentous fungal
strains are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0110] The control sequence may also be a signal peptide coding
region that codes for an amino acid sequence linked to the amino
terminus of a polypeptide and directs the encoded polypeptide into
the cell's secretory pathway. The 5' end of the coding sequence of
the nucleic acid sequence may inherently contain a signal peptide
coding region naturally linked in translation reading frame with
the segment of the coding region which encodes the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding region which is foreign to the
coding sequence. The foreign signal peptide coding region may be
required where the coding sequence does not naturally contain a
signal peptide coding region. Alternatively, the foreign signal
peptide coding region may simply replace the natural signal peptide
coding region in order to enhance secretion of the polypeptide.
However, any signal peptide coding region which directs the
expressed polypeptide into the secretory pathway of a fungal host
cell of choice may be used in the present invention.
[0111] Effective signal peptide coding regions for filamentous
fungal strains are the signal peptide coding regions obtained from
the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger
neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei
aspartic proteinase, Humicola insolens cellulase, and Humicola
lanuginosa lipase.
[0112] The control sequence may also be a propeptide coding region
that codes for an amino acid sequence positioned at the amino
terminus of a polypeptide. The resultant polypeptide is known as a
proenzyme or propolypeptide (or a zymogen in some cases). A
propolypeptide is generally inactive and can be converted to a
mature active polypeptide by catalytic or autocatalytic cleavage of
the propeptide from the propolypeptide. The propeptide coding
region may be obtained from the genes for Saccharomyces cerevisiae
alpha-factor, Rhizomucor miehei aspartic proteinase, and
Myceliophthora thermophila laccase (WO 95/33836).
[0113] Where both signal peptide and propeptide regions are present
at the amino terminus of a polypeptide, the propeptide region is
positioned next to the amino terminus of a polypeptide and the
signal peptide region is positioned next to the amino terminus of
the propeptide region.
[0114] It may also be desirable to add regulatory sequences which
allow the regulation of the expression of the biological substance
relative to the growth of the filamentous fungal strain. Examples
of regulatory systems are those which cause the expression of the
gene to be turned on or off in response to a chemical or physical
stimulus, including the presence of a regulatory compound. In
filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus
niger glucoamylase promoter, Aspergillus oryzae glucoamylase
promoter, and Fusarium venenatum glucoamylase promoter may be used
as regulatory sequences. Other examples of regulatory sequences are
those which allow for gene amplification. In eukaryotic systems,
these include the dihydrofolate reductase gene which is amplified
in the presence of methotrexate, and the metallothionein genes
which are amplified with heavy metals. In these cases, the
nucleotide sequence encoding the biological substance of interest
would be operably linked with the regulatory sequence.
Expression Vectors
[0115] The nucleotide sequence encoding the biological substance of
interest may be contained in a recombinant expression vector
comprising a promoter, the nucleotide sequence encoding the
biological substance, and transcriptional and translational stop
signals. The various nucleic acid and control sequences described
above may be joined together to produce a recombinant expression
vector which may include one or more convenient restriction sites
to allow for insertion or substitution of the promoter and/or
nucleic acid sequence encoding the biological substance at such
sites. Alternatively, the nucleotide sequence may be expressed by
inserting the nucleotide sequence or a nucleic acid construct
comprising a promoter and/or sequence into an appropriate vector
for expression. In creating the expression vector, the coding
sequence is located in the vector so that the coding sequence is
operably linked with a promoter and one or more appropriate control
sequences for expression.
[0116] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) which can be conveniently subjected to
recombinant DNA procedures and can bring about the expression of
the nucleic acid sequence. The choice of the vector will typically
depend on the compatibility of the vector with the host cell into
which the vector is to be introduced. The vectors may be linear or
closed circular plasmids.
[0117] The vector may be an autonomously replicating vector, i.e.,
a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome.
[0118] The vector may contain any means for assuring
self-replication. Alternatively, the vector may be one which, when
introduced into the host cell, is integrated into the genome and
replicated together with the chromosome(s) into which it has been
integrated. Furthermore, a single vector or plasmid or two or more
vectors or plasmids which together contain the total DNA to be
introduced into the genome of the host cell, or a transposon may be
used.
[0119] The vectors preferably contain one or more selectable
markers which permit easy selection of transformed cells. A
selectable marker is a gene the product of which provides for
biocide or viral resistance, resistance to heavy metals,
prototrophy to auxotrophs, and the like. Selectable markers for use
in a filamentous fungal host cell include, but are not limited to,
amdS (acetamidase), argB (ornithine carbamoyltransferase), bar
(phosphinothricin acetyltransferase), hygB (hygromycin
phosphotransferase), niaD (nitrate reductase), pyrG
(orotidine-5'-phosphate decarboxylase), sC (sulfate
adenyltransferase), trpC (anthranilate synthase), as well as
equivalents thereof. Preferred for use in an Aspergillus cell are
the amdS and pyrG genes of Aspergillus nidulans or Aspergillus
oryzae and the bar gene of Streptomyces hygroscopicus. Preferred
for use in a Fusarium cell is the bar, amdS, pyrG, or hygB
gene.
[0120] The vectors preferably contain an element(s) that permits
stable integration of the vector into the host cell's genome or
autonomous replication of the vector in the cell independent of the
genome.
[0121] For integration into the host cell genome of a filamentous
fungal strain, the vector may rely on the nucleotide sequence
encoding the biological substance or any other element of the
vector for stable integration of the vector into the genome by
homologous or nonhomologous recombination. Alternatively, the
vector may contain additional nucleic acids for directing
integration by homologous recombination into the genome of the host
cell. The additional nucleic acid sequences enable the vector to be
integrated into the host's genome at a precise location(s) in the
chromosome(s). To increase the likelihood of integration at a
precise location, the integrational elements should preferably
contain a sufficient number of nucleic acids, such as 100 to 10,000
base pairs, preferably 400 to 10,000 base pairs, and most
preferably 800 to 10,000 base pairs, which have a high degree of
identity with the corresponding target sequence to enhance the
probability of homologous recombination. The integrational elements
may be any sequence that is homologous with the target sequence in
the genome of the host cell. Furthermore, the integrational
elements may be non-encoding or encoding nucleic acid sequences. On
the other hand, the vector may be integrated into the genome of the
host cell by non-homologous recombination.
[0122] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. Examples of a plasmid
replicator useful in a filamentous fungal cell are AMA1 and ANSI.
(Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic
Acids Research 15: 9163-9175; WO 00/24883). Isolation of the AMA1
gene and construction of plasmids or vectors comprising the gene
can be accomplished according to the methods disclosed in WO
00/24883. The origin of replication may be one having a mutation
which makes its functioning temperature-sensitive in the host cell
(see, e.g., Ehrlich, 1978, Proceedings of the National Academy of
Sciences USA 75: 1433).
[0123] More than one copy of a nucleotide sequence encoding a
biological substance of interest may be inserted into the
filamentous fungal strain to increase production of the gene
product. An increase in the copy number of the nucleotide sequence
can be obtained by integrating at least one additional copy of the
sequence into the host cell genome or by including an amplifiable
selectable marker gene with the nucleotide sequence where cells
containing amplified copies of the selectable marker gene, and
thereby additional copies of the nucleic acid sequence, can be
selected for by cultivating the cells in the presence of the
appropriate selectable agent.
[0124] The procedures used to ligate the elements described above
to construct the recombinant expression vectors are well known to
one skilled in the art (see, e.g., Sambrook et al., 1989,
supra).
[0125] The present invention is further described by the following
examples which should not be construed as limiting the scope of the
invention.
EXAMPLES
Media and Buffer Solutions
[0126] COVE selection plates were composed per liter of 342.3 g of
sucrose, 20 ml of COVE salt solution, 10 mM acetamide, 15 mM
CsCl.sub.2, and 25 g or 30 g of Noble agar.
[0127] COVE2 plates were composed per liter of 30 g of sucrose, 20
ml of COVE salt solution, 10 mM acetamide, and 25 g or 30 g of
Noble agar.
[0128] COVE salt solution was composed per liter of 26 g of KCl, 26
g of MgSO.sub.4.7H.sub.2O, 76 g of KH.sub.2PO.sub.4, and 50 ml of
COVE trace metals.
[0129] COVE trace metals was composed per liter of 0.04 g of
NaB.sub.4O.sub.2.10H.sub.2O, 0.4 g of CuSO.sub.4.5H.sub.2O, 1.2 g
of FeSO.sub.4.7H.sub.2O, 0.7 g or 1 g of MnSO.sub.4.H.sub.2O, 0.8 g
of Na2MoO.sub.2.2H.sub.2O, and 10 g of ZnSO.sub.4.7H.sub.2O.
[0130] COVE top agarose was composed per liter of 342.3 g of
sucrose, 20 ml of COVE salt solution, 10 mM acetamide, and 10 g of
low melt agarose.
[0131] Cellulase-inducing medium was composed per liter of 20 g of
Arbocel-natural cellulose fibers (J. Rettenmaier USA LP), 10 g of
corn steep solids (Sigma Chemical Co., St. Louis, Mo.), 1.45 g of
(NH.sub.4).sub.2SO.sub.4, 2.08 g of KH.sub.2PO.sub.4, 0.28 g of
CaCl.sub.2, 0.42 g of MgSO.sub.4, 0.42 ml of Trichoderma reesei
trace metals solution, and 2 drops of pluronic acid. The pH was
adjusted to 6.0 with 10 N NaOH before autoclaving.
[0132] Trichoderma reesei trace metals solution was composed per
liter of 216 g of FeCl.sub.3.6H.sub.2O, 58 g of
ZnSO.sub.4.7H.sub.2O, 27 g of MnSO.sub.4H.sub.2O, 10 g of
CuSO.sub.4.5H.sub.2O, 2.4 g of H.sub.3BO.sub.3, and 336 g of citric
acid.
[0133] YP medium was composed per liter of 10 g of yeast extract
and 20 g of Bacto peptone.
[0134] YPG medium was composed per liter of 4 g of yeast extract, 1
g of K.sub.2HPO.sub.4, 0.5 g of MgSO.sub.4, and 15.0 g of glucose
(pH 6.0).
[0135] PEG Buffer was composed per liter of 500 g of PEG 4000, 10
mM CaCl.sub.2, and 10 mM Tris-HCl pH 7.5, filter sterilized.
[0136] STC was composed per liter of 0.8 M or 1 M sorbitol, 10 mM
or 25 mM CaCl.sub.2, and 10 mM or 25 mM Tris-HCl, pH 7.5 or pH 8,
filter sterilized.
[0137] STPC was composed of 40% PEG4000 in STC.
[0138] M400 medium was composed per liter of 50 g of Maltodextrin,
2 g of MgSO.sub.4.7H.sub.2O, 2 g of KH.sub.2PO.sub.4, 4 g of citric
acid, 8 g of yeast extract, 2 g of urea, 0.5 g of CaCl.sub.2, and
0.5 ml of AMG trace metals solution.
[0139] AMG trace metals solution was composed per liter of 6.8 g of
ZnCl.sub.2.7H.sub.2O, 2.5 g of CuSO.sub.4.5H.sub.2O, 0.24 g of
NiCl.sub.2'6H.sub.2O, 13.9 g of FeSO.sub.4'7H.sub.2O, 13.5 g of
MnSO.sub.4H.sub.2O, and 3 g of citric acid.
[0140] Minimal medium plates were composed per liter of 6 g of
NaNO.sub.3, 0.52 g of KCl, 1.52 g of KH.sub.2PO.sub.4, 1 ml of COVE
trace metals, 1 g of glucose, 500 mg of MgSO.sub.4.7H.sub.2O, 342.3
g of sucrose, and 20 g of Noble agar per liter (pH 6.5).
[0141] MLC was composed per liter of 40 g of glucose, 50 g of
soybean powder, and 4 g of citric acid, pH 5.0.
[0142] MU-1 was composed per liter of 260 g of maltdextrin (MD-11),
5 g of KH.sub.2PO.sub.4, 3 g of MgSO.sub.4.7H.sub.2O, 6 g of
K.sub.2PO.sub.4, 5 ml of AMG trace metals solution, and 2 g of
urea, pH 4.5.
[0143] CM-1 agar plates at pH 6.5 were composed per liter of 0.25 g
of NaCl, 0.5 g of MgSO.sub.4.7H.sub.2O, 1.9 g of K.sub.2HPO.sub.4,
3.6 g of KH.sub.2PO.sub.4, 0.1 ml of trace metals solution, 30 g of
Bacto agar (Difco), pH 6.5, 11 ml of 10% urea, and 67 ml of 30%
maltose.
[0144] The trace metals solution (1000.times.) was composed per
liter of 22 g of ZnSO.sub.4'7H.sub.2O, 11 g of H.sub.3BO.sub.3, 5 g
of MnCl.sub.2'4H.sub.2O, 5 g of FeSO.sub.4'7H.sub.2O, 1.6 g of
CoCl.sub.2-5H.sub.2O, 1.6 g of (NH.sub.4).sub.6MO.sub.7O.sub.24,
and 50 g of Na.sub.4EDTA.
[0145] PDA plates were composed per liter of 39 g of Potato
Dextrose Agar (Difco).
Example 1
Construction of pAlLo1 Expression Vector
[0146] Expression vector pAlLo1 was constructed by modifying pBANe6
(U.S. Pat. No. 6,461,837), which comprises a hybrid of the
promoters from the genes for Aspergillus niger neutral
alpha-amylase and Aspergillus oryzae triose phosphate isomerase
(NA2-tpi promoter), Aspergillus niger amyloglucosidase terminator
sequence (AMG terminator), and Aspergillus nidulans acetamidase
gene (amdS). Modification of pBANe6 was performed by first
eliminating three Nco I restriction sites at positions 2051, 2722,
and 3397 bp from the amdS selection marker by site-directed
mutagenesis. All changes were designed to be "silent" leaving the
actual protein sequence of the amdS gene product unchanged. Removal
of these three sites was performed simultaneously with a GeneEditor
Site-Directed Mutagenesis Kit (Promega, Madison, Wis.) according to
the manufacturer's instructions using the following primers
(underlined nucleotide represents the changed base):
TABLE-US-00001 AMDS3NcoMut (2050): (SEQ ID NO: 1)
5'-GTGCCCCATGATACGCCTCCGG-3' AMDS2NcoMut (2721): (SEQ ID NO: 2)
5'-GAGTCGTATTTCCAAGGCTCCTGACC-3' AMDS1NcoMut (3396): (SEQ ID NO: 3)
5'-GGAGGCCATGAAGTGGACCAACGG-3'
[0147] A plasmid comprising all three expected sequence changes was
then submitted to site-directed mutagenesis, using a QuickChange
Mutagenesis Kit (Stratagene, La Jolla, Calif.), to eliminate the
Nco I restriction site at the end of the AMG terminator at position
1643. The following primers (underlined nucleotide represents the
changed base) were used for mutagenesis:
TABLE-US-00002 Upper Primer to mutagenize the AMG terminator
sequence: (SEQ ID NO: 4)
5'-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACAG- 3' Lower Primer
to mutagenize the AMG terminator sequence: (SEQ ID NO: 5)
5'-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCTG- 3'
[0148] The last step in the modification of pBANe6 was the addition
of a new Nco I restriction site at the beginning of the polylinker
using a QuickChange Mutagenesis Kit and the following primers
(underlined nucleotides represent the changed bases) to yield
pAlLo1 (FIG. 1).
TABLE-US-00003 Upper Primer to mutagenize the NA2-tpi promoter:
(SEQ ID NO: 6) 5'-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3'
Lower Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 7)
5'-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3'
Example 2
Construction of pMJ04 expression vector
[0149] Expression vector pMJ04 was constructed by first PCR
amplifying the Trichoderma reesei CeI7A cellobiohydrolase 1 gene
(cbh1) terminator from Trichoderma reesei RutC30 genomic DNA using
primers 993429 (antisense) and 993428 (sense) shown below. The
antisense primer was engineered to have a Pac I site at the 5'-end
and a Spe I site at the 5'-end of the sense primer. Trichoderma
reesei RutC30 (ATCC 56765; Montenecourt and Eveleigh, 1979, Adv.
Chem. Ser. 181: 289-301) was derived from Trichoderma reesei Qm6A
(ATCC 13631; Mandels and Reese, 1957, J. Bacteriol. 73:
269-278).
TABLE-US-00004 Primer 993429 (antisense): (SEQ ID NO: 8)
5'-AACGTTAATTAAGGAATCGTTTTGTGTTT-3' Primer 993428 (sense): (SEQ ID
NO: 9) 5'-AGTACTAGTAGCTCCGTGGCGAAAGCCTG-3'
[0150] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer (New England Biolabs, Beverly,
Mass.), 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic
DNA (isolated using a DNeasy Plant Maxi Kit, QIAGEN Inc., Valencia,
Calif.), 0.3 .mu.M primer 993429, 0.3 .mu.M primer 993428, and 2
units of Vent polymerase (New England Biolabs, Beverly, Mass.). The
reactions were incubated in an Eppendorf Mastercycler 5333
(Hamburg, Germany) programmed as follows: 30 cycles each for 30
seconds at 94.degree. C., 30 seconds at 55.degree. C., and 30
seconds at 72.degree. C. (15 minute final extension). The reaction
products were isolated on a 1.0% agarose gel using 40 mM Tris
base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer where a
229 bp product band was excised from the gel and purified using a
QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.)
according to the manufacturer's instructions.
[0151] The resulting PCR fragment was digested with Pac I and Spe I
and ligated into pAlLo1 digested with the same restriction enzymes,
using a Rapid Ligation Kit (Roche, Indianapolis, Ind.), to generate
pMJ04 (FIG. 2).
Example 3
Construction of pMJ06 Expression Vector
[0152] Expression vector pMJ06 was constructed by first PCR
amplifying the Trichoderma reesei CeI7A cellobiohydrolase 1 gene
(cbh1) promoter from Trichoderma reesei RutC30 genomic DNA using
primers 993696 (antisense) and 993695 (sense) shown below. The
antisense primer was engineered to have a SalI site at the 5'-end
of the sense primer and an Nco I site at the 5'-end of the
antisense primer.
TABLE-US-00005 Primer 993695 (sense): (SEQ ID NO: 10)
5'-ACTAGTCGACCGAATGTAGGATTGTT-3' Primer 993696 (antisense): (SEQ ID
NO: 11) 5'-TGACCATGGTGCGCAGTCC-3'
[0153] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 100 ng of
Trichoderma reesei RutC30 genomic DNA (prepared using a DNeasy
Plant Maxi Kit), 0.3 .mu.M primer 993696, 0.3 .mu.M primer 993695,
and 2 units of Vent polymerase. The reactions were incubated in an
Eppendorf Mastercycler 5333 programmed as follows: 30 cycles each
for 30 seconds at 94.degree. C., 30 seconds at 55.degree. C., and
60 seconds at 72.degree. C. (15 minute final extension). The
reaction products were isolated on a 1.0% agarose gel using TAE
buffer where a 988 bp product band was excised from the gel and
purified using a QIAquick Gel Extraction Kit according to the
manufacturer's instructions.
[0154] The resulting PCR fragment was digested with Nco I and Sal I
and ligated into pMJ04 digested with the same restriction enzymes,
using a Rapid Ligation Kit, to generate pMJ06 (FIG. 3).
Example 4
Construction of pMJ09 expression vector
[0155] Expression vector pMJ09 was constructed by PCR amplifying
the Trichoderma reesei CeI7A cellobiohydrolase 1 gene (cbh1)
terminator from Trichoderma reesei RutC30 genomic DNA using primers
993843 (antisense) and 99344 (sense) shown below. The antisense
primer was engineered to have a Pac I and a Spe I sites at the
5'-end and a Pvu I site at the 5'-end of the sense primer.
TABLE-US-00006 Primer 993844 (sense): (SEQ ID NO: 12)
5'-CGATCGTCTCCCTATGGGTCATTACC-3' Primer 993843 (antisense): (SEQ ID
NO: 13) 5'-ACTAGTTAATTAAGCTCCGTGGCGAAAG-3'
[0156] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 100 ng of
Trichoderma reesei RutC30 genomic DNA (extracted using DNeasy Plant
Maxi Kit), 0.3 .mu.M primer 993844, 0.3 .mu.M primer 993843, and 2
units of Vent polymerase. The reactions were incubated in an
Eppendorf Mastercycler 5333 programmed as follows: 30 cycles each
for 30 seconds at 94.degree. C., 30 seconds at 55.degree. C., and
60 seconds at 72.degree. C. (15 minute final extension). The
reaction products were isolated on a 1.0% agarose gel using TAE
buffer where a 473 bp product band was excised from the gel and
purified using a QIAquick Gel Extraction Kit according to the
manufacturer's instructions.
[0157] The resulting PCR fragment was digested with Pvu I and Spe I
and ligated into pMJ06 digested with Pad and Spe I, using a Rapid
Ligation Kit, to generate pMJ09 (FIG. 4).
Example 5
Fermentation and Mycelial Tissue
[0158] Trichoderma reesei RutC30 was grown under cellulase inducing
standard conditions as described by Mandels and Weber, 1969, Adv.
Chem. Ser. 95: 391-413). Mycelial samples were harvested by
filtration through Whatman paper and quick-frozen in liquid
nitrogen. The samples were stored at -80.degree. C. until they were
disrupted for RNA extraction.
Example 6
Expressed Sequence Tags (ESTs) cDNA Library Construction
[0159] Total cellular RNA was extracted from the mycelial samples
described in Example 5, according to the method of Timberlake and
Barnard (1981, Cell 26: 29-37), and the RNA samples were analyzed
by Northern hybridization after blotting from 1%
formaldehyde-agarose gels (Davis et al., 1986, Basic Methods in
Molecular Biology, Elsevier Science Publishing Co., Inc., New
York). Polyadenylated mRNA fractions were isolated from total RNA
with an mRNA Separator Kit.TM. (Clontech Laboratories, Inc., Palo
Alto, Calif.) according to the manufacturer's instructions.
[0160] Double-stranded cDNA was synthesized using approximately 5
.mu.g of poly(A)+ mRNA according to the method of Gubler and
Hoffman (1983, Gene 25: 263-269), except a Not I-(dT)18 primer
(Pharmacia Biotech, Inc., Piscataway, N.J.) was used to initiate
first strand synthesis. The cDNA was treated with mung bean
nuclease (Boehringer Mannheim Corporation, Indianapolis, Ind.) and
the ends were made blunt with T4 DNA polymerase (New England
Biolabs, Beverly, Mass.). BamH I/EcoR I adaptors were then ligated
to the blunt ends of the cDNA. After digestion with Not I, the cDNA
was size selected (ca. 0.7-4.5 kb) by 0.7% agarose gel
electrophoresis using TAE buffer, and ligated with pYES2
(Invitrogen, Carlsbad, Calif.) which had been cleaved with Not I
plus BamH I and dephosphorylated with calf-intestine alkaline
phosphatase (Boehringer Mannheim Corporation, Indianapolis,
Ind.).
[0161] The ligation mixture was used to transform competent E. coli
TOP10 cells (Invitrogen, Carlsbad, Calif.) according to the
manufacturer's instructions. Transformants were selected on 2YT
agar plates (Miller, 1992, A Short Course in Bacterial Genetics. A
Laboratory Manual and Handbook for Escherichia coli and Related
Bacteria, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.)
supplemented with ampicillin at a final concentration of 50 .mu.g
per ml.
Example 7
Template Preparation and Nucleotide Sequencing of cDNA Clones
[0162] From the cDNA library described in Example 6, approximately
7000 transformant colonies were picked directly from the 2YT plates
into 96-well microtiter plates which contained 100 .mu.l of 2YT
broth supplemented with 50 .mu.g of ampicillin per ml. The plates
were incubated overnight at 37.degree. C. with shaking at 200 rpm.
After incubation, 100 .mu.l of sterile 50% glycerol were added to
each well. The transformants were replicated into secondary,
deep-dish 96-well microculture plates (Advanced Genetic
Technologies Corporation, Gaithersburg, Md.) containing 1 ml of
Magnificent Broth.TM. (MacConnell Research, San Diego, Calif.)
supplemented with 50 .mu.g of ampicillin per ml in each well. The
primary microtiter plates were stored frozen at -80.degree. C. The
secondary deep-dish plates were incubated at 37.degree. C.
overnight with vigorous agitation (300 rpm) on a rotary shaker. To
prevent spilling and cross-contamination, and to allow sufficient
aeration, each secondary culture plate was covered with a
polypropylene pad (Advanced Genetic Technologies Corporation,
Gaithersburg, Md.) and a plastic microtiter dish cover.
[0163] DNA was isolated from each well using a 96-well Miniprep Kit
protocol of Advanced Genetic Technologies Corporation
(Gaithersburg, Md.) as modified by Utterback et al. (1995, Genome
Sal Technol 1:1-8). Single-pass DNA sequencing was performed with a
Perkin-Elmer Applied Biosystems Model 377 XL Automated DNA
Sequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City,
Calif.) using dye-terminator chemistry (Giesecke et al., 1992,
Journal of Virology Methods 38: 47-60) and the T7 sequencing
primer:
TABLE-US-00007 (SEQ ID NO: 14) T7 primer:
5'-TAATACGACTCACTATAGGG-3'
Example 8
Analysis of DNA Sequence Data of cDNA Clones
[0164] Nucleotide sequence data were scrutinized for quality and
vector sequences and ambiguous base calls at the ends of the DNA
sequences were trimmed, and all sequences were compared to each
other with assistance of PHRED/PHRAP software (University of
Washington, Seattle, Wash.). The resulting contigs and singletons
were translated in six frames and searched against publicly
available protein databases using GeneMatcher.TM. software
(Paracel, Inc., Pasadena, Calif.) with a modified Smith-Waterman
algorithm using the BLOSUM 62 matrix.
Example 9
Identification of cDNA Clones Encoding a Family 6 Cellobiohydrolase
II (CeI6A)
[0165] Putative cDNA clones encoding a Family 6 cellobiohydrolase
(CeI6A) were identified by comparing the deduced amino acid
sequence of the assembled ESTs to protein sequences deposited in
publicly available databases such as Swissprot, Genpept, and PIR.
One clone, Trichoderma reesei EST Tr0749, was selected for
nucleotide sequence analysis which revealed an 1747 bp pYES2 insert
which contained a 1413 bp open reading-frame as shown in SEQ ID NO:
15 and a deduced amino acid sequence as shown in SEQ ID NO: 16. The
plasmid containing Trichoderma reesei CeI6A cellobiohydrolase II
was designated pTr0749.
Example 10
Construction of pSMai148 Expression Vector
[0166] Expression vector pSMai148 was constructed for transcription
of double stranded-RNA (ds-RNA) derived from the Trichoderma reesei
CeI6A cellobiohydrolase II gene and intended for the silencing of
expression of the Trichoderma reesei CeI6A cellobiohydrolase II
gene in Trichoderma reesei RutC30 strain. Plasmid pSMai148 was
generated by PCR amplifying a 210 bp of the Trichoderma reesei
CeI6A cellobiohydrolase II coding region from pTr0749 using primers
994991 (antisense) and 994990 (sense) shown below. The antisense
primer was engineered to have an EcoR I site at the 5'-end and a
Nco I site at the 5'-end of the sense primer.
TABLE-US-00008 Primer 994991 (antisense): (SEQ ID NO: 17)
5'-GGAATTCTAGTTCTTATATTTGGCGACGCCACCATCT-3' Primer 994990 (sense):
(SEQ ID NO: 18) 5'-CATGCCATGGAAAGGTTCCCTCTTTTATGTGGCTAG-3'
[0167] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 10 ng of pTr0749,
0.3 .mu.M primer 994990, 0.3 .mu.M primer 994991, and 2.5 units of
Taq polymerase (New England Biolabs, Beverly, Mass.). The reactions
were incubated in an Eppendorf Mastercycler 5333 programmed as
follows: 30 cycles each for 30 seconds at 94.degree. C., 30 seconds
at 55.degree. C., and 30 seconds at 72.degree. C. (15 minute final
extension). The reaction products were isolated on a 1.0% agarose
gel using TAE buffer where a 227 bp product band was excised from
the gel and purified using a QIAquick Gel Extraction Kit according
to the manufacturer's instructions.
[0168] A separate PCR was performed to amplify a 337 bp fragment of
the Trichoderma reesei CeI6A cellobiohydrolase II coding region
from pTr0749 using primers 994993 (antisense) and 994992 (sense)
shown below. The antisense primer was engineered to have an EcoR I
site at the 5'-end and a Pac I site at the 5'-end of the sense
primer.
TABLE-US-00009 Primer 994993 (antisense): (SEQ ID NO: 19)
5'-GGAATTCTGACTGAGCATTGGCACACTTTGGAGTAC-3' Primer 994992 (sense):
(SEQ ID NO: 20) 5'-CCTTAATTAAAAAGGTTCCCTCTTTTATGTGGCTAG-3'
[0169] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 10 ng of pTr0749,
0.3 .mu.M primer 994992, 0.3 .mu.M primer 994993, and 2.5 units of
Taq polymerase. The reactions were incubated in an Eppendorf
Mastercycler 5333 programmed as follows: 30 cycles each for 30
seconds at 94.degree. C., 30 seconds at 55.degree. C., and 30
seconds at 72.degree. C. (15 minute final extension). The reaction
products were isolated on a 1.0% agarose gel using TAE buffer where
a 354 bp product band was excised from the gel and purified using a
QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0170] The resulting Trichoderma reesei CeI6A cellobiohydrolase II
PCR fragment was digested with Pac I and EcoR I and ligated in an
inverted orientation downstream of the Nco I and EcoR I digested
Trichoderma reesei CeI6A cellobiohydrolase II PCR fragment (from
first PCR). The tail-to-tail repeat (FIG. 5) was ligated into Pac I
and Nco I digested pMJ09 vector, using T4 DNA ligase (Roche,
Indianapolis, Ind.) according to manufacturer's protocol, to
generate pSMai148 (FIG. 6).
Example 11
Transformation Trichoderma reesei with an Expression Construct
Possessing an Inverted Repeat Fragment of the CeI6A
Cellobiohydrolase II Gene
[0171] To demonstrate that double stranded RNA-mediated (ds-RNA)
interference occurs in Trichoderma reesei, the pSMai148 expression
vector, expressing the self-complementary hairpin RNA of the CeI6A
cellobiohydrolase II sequence, was transformed into Trichoderma
reesei RutC30 protoplasts. As a control, pMJ09 plasmid ("Empty
vector") (FIG. 4), the parent vector of pSMai148, was also
included. Both constructs contained the amdS gene providing
selection for growth on acetamide as the sole nitrogen source and
the Trichoderma reesei CeI6A cellobiohydrolase II sequence in the
pSMai148 plasmid was placed under the control of the strong
cellulose-inducible Trichoderma reesei CeI7A cellobiohydrolase 1
promoter.
[0172] Protoplast preparation and transformation was performed
using a modified protocol by Penttila et al., 1987, Gene 61:
155-164. Briefly, Trichoderma reesei RutC30 was cultivated in 25 ml
of YP medium, supplemented with 2% (w/v) glucose and 10 mM uridine,
with gentle agitation (90 rpm) at 27.degree. C. for 17 hours.
Mycelia were collected by filtration using a Millipore Vacuum
Driven Disposable Filtration System (Millipore, Bedford, Mass.) and
washed twice with deionized water and twice with 1.2 M sorbitol.
Protoplasts were generated by suspending the washed mycelia in 20
ml of 1.2 M sorbitol containing 15 mg of Glucanex.RTM. 200 G
(Novozymes Switzerland AG, Neumatt, Switzerland) per ml and 0.36
units of chitinase (Sigma Chemical Co., St. Louis, Mo.) per ml for
15-25 minutes at 34.degree. C. with gentle shaking (90 rpm).
Protoplasts were collected by centrifuging for 7 minutes at
400.times.g and washed twice with cold 1.2 M sorbitol. The
protoplasts were counted using a haemacytometer and re-suspended to
a final concentration of 1.times.10.sup.8 protoplasts/ml in STC.
Excess protoplasts were stored in a Cryo 1.degree. C. Freezing
Container (Nalgene, Rochester, N.Y.) at -80.degree. C.
[0173] Approximately 7 .mu.g of Pme I digested expression plasmid
(pSMai148 or pMJ09) was added to 100 .mu.l of the protoplast
solution and mixed gently. PEG buffer (250 .mu.l) was added, mixed,
and incubated at room temperature for 30 minutes. STC (3 ml) was
then added, mixed, and plated onto COVE plates. The plates were
incubated at 28.degree. C. for 5-7 days. Transformants were
sub-cultured onto COVE2 plates and grown at 28.degree. C.
Example 12
Detection of Trichoderma reesei CeI6A Cellobiohydrolase II Protein
by SDS-polyacrylamide gels
[0174] Twenty transformants (SMA148-01 to SMA148-20) harboring the
Trichoderma reesei
[0175] CeI6A cellobiohydrolase II inverted repeat fragment and 10
transformants (M309-01 and M309-10) containing "Empty vector" were
randomly selected and cultured in 125 ml baffled shake flasks
containing 25 ml of cellulase-inducing medium at pH 6.0 inoculated
with spores of the transformants and incubated at 28.degree. C. and
200 rpm for 7 days. Trichoderma reesei RutC30 was run as a control.
Culture broth samples were removed 7 days post-inoculation,
centrifuged at 15,700.times.g for 5 minutes in a micro-centrifuge,
and the supernatants transferred to new tubes.
[0176] The extent of Trichoderma reesei CeI6A cellobiohydrolase II
silencing was initially evaluated at a protein level by analyzing
total broth on SDS-PAGE gels. SDS-PAGE was carried out using
Criterion.TM. Tris-HCl gels (Bio-Rad Laboratories, Hercules,
Calif.) with The Criterion.TM. Cell (Bio-Rad Laboratories,
Hercules, Calif.). Five .mu.l of day 7 samples were suspended in
2.times. concentration of Laemmli Sample Buffer (Bio-Rad
Laboratories, Hercules, Calif.) and boiled for 3 minutes in the
presence of 5% beta-mercaptoethanol. All the samples were loaded
onto a polyacrylamide gel and subjected to electrophoresis in
1.times. Tris/Glycine/SDS running buffer (Bio-Rad Laboratories,
Hercules, Calif.). The resulting gel was stained with Bio-Safe.TM.
Coomassie Stain (Bio-Rad Laboratories, Hercules, Calif.).
[0177] SDS-PAGE analysis showed that the majority of the
Trichoderma reesei RutC30 transformants carrying the Trichoderma
reesei CeI6A cellobiohydrolase II inverted repeat fragment
(SMA148-01 to SMA148-20) produced significantly lower amounts of
Trichoderma reesei CeI6A cellobiohydrolase II protein compared to
transformants harboring the "Empty vector" (pMJ09-01 to pMJ09-10).
One transformant (transformant 14) exhibited complete lack of the
protein.
[0178] Six SMA148 transformants (transformants 1, 2, 11, 12, 13,
14) showing varying degrees of Trichoderma reesei CeI6A
cellobiohydrolase II protein reduction and two "Empty vector"
transformants (M309-03 and M309-04) were selected for further
analysis. Two rounds of single spore isolation were performed on
these transformants to obtain pure strains. These single spore
isolated transformants were grown in 25 ml of cellulase-inducing
medium (pH 5.0) at 28.degree. C. In addition, the host strain
(Trichoderma reesei RutC30) and a positive control strain
[0179] (Trichoderma reesei SaMe02) where the Trichoderma reesei
CeI6A gene was knocked-out by homologous recombination was also
cultured under the same conditions. Both supernatant and mycelia
were collected from each of these samples at 3 days
post-inoculation and submitted to SDS-PAGE analysis as described
earlier.
[0180] SDS-PAGE showed that single-spore isolation further
decreased the level of Trichoderma reesei CeI6A cellobiohydrolase
II produced, presumably due to the transformants becoming more
homozygous as a result of spore purification.
Example 13
Detection of Trichoderma reesei CeI6A cellobiohydrolase II mRNA by
Northern blots
[0181] Northern hybridization was conducted to determine if
incorporation of Trichoderma reesei CeI6A cellobiohydrolase II
inverted repeat fragment (pSMai148) in a strain resulted in a
reduction in the amount of Trichoderma reesei CeI6A
cellobiohydrolase II mRNA. Total RNA was extracted from frozen
mycelia obtained in Example 11 using Fenozol.TM. (Active Motif,
Carlsbad, Calif.) and following a slightly modified protocol by the
manufacturer. Briefly, frozen mycelia were ground to a fine powder
in an electric coffee grinder with a few chips of dry ice. The
ground mycelia were mixed with 20 ml of Fenozol.TM., vortexed, and
incubated in a 50.degree. C. water-bath for 15 minutes, after which
5 ml of chloroform (Sigma, St. Louis, Mo.) was added, vortexed, and
allowed to stand at room temperature for 10 minutes. The samples
were then centrifuged at 700.times.g at room temperature for 20
minutes and the aqueous phase transferred to a new tube and an
equal volume of phenol-chloroform-isoamylalcohol was added. The
samples were vortexed and centrifuged for 10 minutes at 700.times.g
and the top aqueous layer was added to an equal volume of
chloroform. The samples were further centrifuged for 10 minutes at
700.times.g and the aqueous layer was transferred to a tube
containing 0.5 ml of 3 M sodium acetate pH 5.2 and 6.25 ml of
isopropanol. The samples were mixed, incubated at room temperature
for 15 minutes, and centrifuged at 13400.times.g for 30 minutes to
recover RNA. The RNA was washed with 70% ethanol, centrifuged,
dried, and re-suspended in DEPC-water. The quantity and quality of
the extracted RNA were assessed on an Agilent 2100 Bioanalyzer
(Agilent Technologies, Wilmington, Del.) in conjunction with the
RNA 6000 Nano LabChip.RTM. Kit (Agilent Technologies, Wilmington,
Del.) according to manufacturer's protocol.
[0182] Isolated total RNA was fractionated by electrophoresis for
4-6 hours on a 1% agarose/formaldehyde gel and blotted onto Nytran
SuperCharge membrane (Schleicher & Schuell BioScience, Keene,
N. H.) using a Turboblotter (Schleicher & Schuell BioScience,
Keene, N. H.) for 14-16 hours, following the manufacturer's
recommendations. The membrane was first hybridized with a 505 bp
digoxigenin-labeled Trichoderma reesei CeI6A cellobiohydrolase II
probe, which was synthesized by incorporation of
digoxigenin-11-dUTP during PCR using primers 996118 (antisense) and
996117 (sense) shown below:
TABLE-US-00010 Primer 996118 (antisense): (SEQ ID NO: 21)
5'-AAATCGTGGCGCACTGCTGT-3' Primer 996117 (sense): (SEQ ID NO: 22)
5'-TGAGTGCATCAACTACGCCG-3'
[0183] The amplification reaction (50 .mu.l) was composed of
1.times. ThermoPol Reaction Buffer, 5 .mu.l of PCR DIG Labeling Mix
(Roche Molecular Biochemicals, Indianapolis, Ind., USA), 10 ng of
pTr0749, 0.3 .mu.M primer 996118, 0.3 .mu.M primer 996117, and 2.5
units of Taq polymerase. The reactions were incubated in an
Eppendorf Mastercycler 5333 programmed as follows: 30 cycles each
for 30 seconds at 94.degree. C., 30 seconds at 50.degree. C., and
30 seconds at 72.degree. C. (15 minute final extension). Five
microliters of the PCR product was size-selected on 1.5% agarose
gels using TAE buffer, stained with ethidium bromide, and
visualized under a UV transilluminator. Incorporation of
digoxigenin was indicated by increase in molecular mass.
[0184] Hybridization was performed in DIG Easy Hyb buffer (Roche
Molecular Biochemicals, Indianapolis, Ind., USA) at 50.degree. C.
for 15-17 hours. The membrane was then washed under high stringency
conditions in 2.times.SSC plus 0.1% SDS for 5 minutes at room
temperature followed by two washes in 0.1.times.SSC plus 0.1% SDS
for 15 minutes each at 50.degree. C. The probe-target hybrids were
detected by chemiluminescent assay (Roche Molecular Biochemicals,
Indianapolis, Ind.) following the manufacturer's instructions. The
membrane was then stripped in 50% formamide/5% SDS/50 mM Tris-HCl,
pH 7.5 at 80.degree. C. for 2 hours and re-probed with a 407 bp
digoxigenin-labeled probe encoding the Trichoderma reesei actin 1
gene (Accession Number X75421) (Matheucci et al., 1995, Gene 161:
103-106) prepared using the exact same conditions as before. The
407 bp actin 1 gene probe was synthesized by incorporation of
digoxigenin-11-dUTP during PCR using primers 996120 (antisense) and
996119 (sense) shown below.
TABLE-US-00011 Primer 996120 (antisense): (SEQ ID NO: 23)
5'-GTCAACACGACGAATGGCGT-3' Primer 996119 (sense): (SEQ ID NO: 24)
5'-TGATCGGTATGGGTCAGAAGG-3'
[0185] The amplification reaction (50 .mu.l) was composed of
1.times. ThermoPol Reaction Buffer, 5 .mu.l of PCR DIG Labeling Mix
(Roche, Indianapolis, Ind.), 100 ng Trichoderma reesei RutC30
genomic DNA, (isolated using a DNeasy Plant Maxi Kit), 0.3 .mu.M
primer 996119, 0.3 .mu.M primer 996120, and 2.5 units of Taq
polymerase. The reactions were incubated in an Eppendorf
Mastercycler 5333 programmed as follows: 30 cycles each for 30
seconds at 94.degree. C., 30 seconds at 50.degree. C., and 30
seconds at 72.degree. C. (15 minute final extension).
[0186] Five microliters of the PCR product was size-selected on
1.5% agarose gels using TAE buffer, stained with ethidium bromide,
and visualized under a UV transilluminator. Incorporation of
digoxigenin was indicated by increase in molecular mass.
[0187] The Northern showed that transformants which had the
Trichoderma reesei CeI6A cellobiohydrolase II inverted repeat
sequences produced substantially less Trichoderma reesei CeI6A
cellobiohydrolase II transcripts compared with transformants that
received the "Empty vector" (negative control) and the host strain
(Trichoderma reesei RutC30). This result was particularly
noticeable in transformants 11 and 14 where the Trichoderma reesei
CeI6A cellobiohydrolase II mRNAs were not detectable although actin
1 gene transcripts (actin 1 was used as a control for equivalent
RNA loading) were clearly present. The apparent 100% suppression in
Trichoderma reesei CeI6A cellobiohydrolase II mRNA correlated
strongly with the 100% reduction in the Trichoderma reesei CeI6A
cellobiohydrolase II protein in these transformants, indicating
that RNA interference in Trichoderma resulted from a reduction in
the amount of target mRNA available for translation.
Example 14
Detection of Trichoderma reesei CeI6A Cellobiohydrolase II mRNA by
Real-Time Reverse-Transcription-PCR
[0188] The relative expression levels of Trichoderma reesei CeI6A
cellobiohydrolase II mRNA in the different transformants were
quantitated with real-time Reverse Transcription-PCR(RT-PCR). Total
RNA was extracted from each transformant as described in Example 13
to serve as a template for RT-PCR reactions. The Trichoderma reesei
actin 1 gene was used as an internal control. Primers were designed
using Primer Express.TM. software (Applied Biosystems, Foster City,
Calif.). After primer lists were generated according to
favorability ratings, pairs were chosen by selecting the first set
of primers that had no more than two G+C in the last 5 bases at the
3'-end. The following primers were used:
TABLE-US-00012 Trichoderma reesei Cel6A cellobiohydrolase II
forward primer (996006): (SEQ ID NO: 25)
5'-CTGGTCCAACGCCTTCTTCAT-3' Trichoderma reesei Cel6A
cellobiohydrolase II reverse primer (996007): (SEQ ID NO: 26)
5'-GGAACGTAGTGAGGCTCGCTAA-3' Trichoderma reesei actin 1 forward
primer (996121): (SEQ ID NO: 27) 5'- CATGGCTGGTCGTGATCTTACC-3'
Trichoderma reesei actin 1 reverse primer (996122): (SEQ ID NO: 28)
5'- CCTTGATGTCACGGACGATTTC-3'
[0189] The RT-PCR assay was performed using an ABI Prism.RTM. 7700
System (Applied Biosystems, Foster City, Calif.) and SYBR.RTM.
Green PCR master mix (Applied Biosystems, Foster City, Calif.).
Each reaction mixture contained 12.5 .mu.l of SYBR.RTM. Green PCR
Master Mix, 6 units of SuperscriptII (Invitrogen, Carlsbad,
Calif.), 0.83 .mu.M forward primer, 0.83 .mu.M reverse primer, and
varying concentrations of RNA template in a total volume of 25
.mu.l. Reverse transcription was performed for 30 minutes at
50.degree. C., followed by inactivation of SuperscriptII and
activation of AmpliTaq.RTM. at 95.degree. C. for 10 minutes. Forty
PCR cycles were carried out under the following conditions: 15
seconds of denaturation at 95.degree. C. followed by 1 minute at
60.degree. C. for annealing and extension. Each sample was prepared
in triplicate. A negative control with no SuperscriptII was run on
every plate tested to assess specificity. Furthermore, since
SYBR.RTM. Green binds non-specifically to double stranded-DNA, an
aliquot of the PCR amplification products was electrophoresed on a
2.0% agarose gel using TAE buffer to confirm the absence of
non-specific amplification.
[0190] Data obtained from the ABI PRISM 7700 Sequence Detection
System was analyzed using "Standard Curve Method for Relative
Quantitation" as described in ABI user Bulletin #2 (Applied
Biosystems, Foster City, Calif.). In this method, the quantity of
expression of treated sample was calculated relative to the
untreated control sample. The quantity of the treated sample was
determined from the standard curve and divided by the quantity of
the untreated control sample. Thus, the untreated sample was
designated the 1.times. sample, and all other quantities were
expressed as an n-fold difference relative to the untreated sample.
Then, the treated sample amount was normalized to an endogenous
control, actin 1, to account for differences in the amount of total
RNA added to each reaction.
[0191] FIG. 7 shows that the relative expression level of
Trichoderma reesei CeI6A cellobiohydrolase II mRNA was lowest in
transformant 14 and the positive control strain (SaMe02) when
compared to the "Empty vector control", correlating strongly with
the Northern hybridization data.
Example 15
Construction of a Subtracted and Normalized cDNA Library from
Trichoderma reesei Using Suppression Subtractive Hybridization
(SSH)
[0192] Trichoderma reesei strain RutC30 was grown in two-liter
Applikon laboratory fermentors using a base Celluclast.TM. medium
and fermentation conditions. The carbon sources included glucose,
cellulose, or pre-treated and washed corn stover. All were loaded
based on the carbon equivalent of 52 g of glucose per liter. The
fermentation method employed a temperature of 28.degree. C., pH
4.5, and a growth time of approximately 120 hours. In addition to
the carbon source, all fermentations contained per liter the
following medium components: 5 g of glucose, 10 g of corn steep
solids, 2.08 g of CaCl.sub.2, 3.87 g of (NH.sub.4).sub.2PO.sub.4,
2.8 g of KH.sub.2PO.sub.4, 1.63 g of MgSO.sub.4.7H.sub.2O, 0.75 ml
of trace metals, and 1.8 ml of pluronic acid. The trace metals
solution was composed per liter of 216 g of FeCl.sub.3.6H.sub.2O,
58 g of ZnSO.sub.4.7H.sub.2O, 27 g of MnSO.sub.4.H.sub.2O, 10 g of
CuSO.sub.4.5H.sub.2O, 2.4 g of H.sub.3BO.sub.3, and 336 g of citric
acid. Samples of mycelia were harvested one, two, three, four, and
five days post-inoculum and were quickly separated from the culture
medium by filtration through Miracloth.TM. (Calbiochem, La Jolla,
Calif.), frozen in liquid nitrogen, and stored at -80.degree.
C.
[0193] Total cellular RNA was isolated from frozen cells grown on
glucose, cellulose, or pre-treated corn stover (Example 1) using
slight modifications to the method of Timberlake and Barnard, 1981,
supra. RNA extraction buffer was prepared by adding a freshly
prepared solution of p-aminosalicylic acid (9.6 g in 80 ml of
DEPC-treated water) to a solution of triisopropylnaphthalene
sulfonic acid (1.6 g in 80 ml of DEPC-treated water). This mixture
was added to 40 ml of 5.times.RNB solution (1 M Tris-HCl, pH 8.5,
1.25 M NaCl, 0.25 M EGTA) with stirring. Frozen mycelia were ground
to a fine powder in an electric coffee grinder with a few chips of
dry ice. The ground mycelia were poured directly into 20 ml of RNA
extraction buffer on ice, and an equal volume of TE-saturated
phenol was added. After vigorous agitation, the samples were
centrifuged at 2500 rpm (Sorvall RT7 centrifuge equipped with a
H1000B rotor) for 10 minutes to separate phases. The aqueous phase
was transferred to a new tube that contained 10 ml of phenol and 10
ml of chloroform-isoamyl alcohol (24:1), while an additional 5 ml
of extraction buffer was added to the phenol phase. The latter
mixture was incubated at 68.degree. C. for 5 minutes to liberate
RNA trapped in polysomes and in the interface material. Following
the incubation, the tubes were centrifuged at 2500 rpm (Sorvall RT7
centrifuge equipped with a H1000B rotor) for 10 minutes and the
aqueous phase was combined with that obtained from the first
extraction. These mixtures were subjected to repeated extraction
with phenol-chloroform until there was no longer protein at the
interface (usually five or six times).
[0194] The RNA was recovered by centrifugation (30 minutes at
12,000.times.g) following precipitation with 0.3 M sodium acetate
pH 5.2 and 50% isopropanol. From each sample consisting of
approximately 1-2 grams of frozen mycelia generated in
laboratory-scale fermentors, 0.4-1.8 mg of total cellular RNA was
obtained.
[0195] The quality of RNA from cultures grown on cellulose and PCS
was appraised by formaldehyde-agarose gel electrophoresis followed
by Northern blotting and hybridization (Thomas, 1980, Proc. Nat.
Acad. Sc. USA 77: 5201-5205) with a Trichoderma reesei cbh1
specific probe. The cbh1 probe fragment was amplified by standard
PCR methods based on the published nucleotide sequence information
available from the EMBL database (accession number E00389). The
probes were labeled with horseradish peroxidase (HRP) and
hybridized at 55.degree. C. using the buffers and protocols
provided in a North2South Direct HRP Labeling and Detection Kit
(Pierce, Rockford, Ill.). The blots were washed three times in
2.times.SSC with 0.1% SDS at 55.degree. C. for five minutes each,
followed by three additional washes in 2.times.SSC (no SDS) for
five minutes each. Following exposure of the blot to X-ray film, it
was clear that virtually all of the hybridization signal in each
lane was contained in a 1.8 kb cbh1 mRNA species that migrated to a
position just slightly above the 18S ribosomal RNA band. There was
no evidence of significant mRNA degradation on either the
autoradiogram or on the ethidium bromide stained gel.
Polyadenylated (polyA.sup.+) mRNA fractions were purified using an
Oligotex.TM. mRNA Isolation Kit according to the manufacturer's
instructions (QIAGEN, Valencia, Calif.). Yields of polyA+ mRNA from
each of these samples ranged from 2 .mu.g to 25 .mu.g. Each of the
mRNA fractions was subsequently analyzed by Northern blot
hybridization using HRP-labeled probes derived from the Trichoderma
reesei .gamma.-actin and cbh1 genes. The .gamma.-actin probe
fragment was amplified by standard PCR methods and the following
gene-specific primers.
TABLE-US-00013 (SEQ ID NO: 29) 5'-CCAGACATGACAATGTTGCCGTAG-3' (SEQ
ID NO: 30) 5'-TTTCGCTCTTCCTCACGCCATTG-3'
As expected, the hybridization signals were localized in bands that
corresponded to the .gamma.-actin and cbh1 mRNAs (ca. 1.2 kb and
1.8 kb, respectively) in each lane, indicating that the mRNA
samples were of high quality and suitable for cDNA synthesis.
[0196] The suppression subtractive hybridization (SSH) method
described by Diatchenko et al, 1996, supra, was used to generate a
cDNA pool from Trichoderma reesei RutC30 that was both enriched for
cellulose- and PCS-induced sequences and normalized to aid in
recovery of rare transcripts. Table 1 below lists the combinations
of driver and tester cDNAs used for these experiments.
TABLE-US-00014 TABLE 1 Driver and tester cDNA pools used for SSH.
SSH Reaction Driver cDNA source Tester cDNA source 1 Glucose-grown
cells PCS-grown cells 2 Glucose-grown cells Cellulose-grown cells 3
Cellulose-grown cells PCS-grown cells
[0197] The resulting cDNA pools from the SSH reactions in Table 1
were used to generate subtractive libraries of cellulose- and
PCS-induced sequences. For synthesis of cDNA, 400 ng of
polyA.sup.+mRNA derived from each time point (1-5 days) was
combined for a total of 2 .mu.g of template. Synthesis and
subtraction of cDNA was done using a PCR-Select.TM. Kit (Clontech,
Palo Alto, Calif.). The methods are based on the procedure of
suppression subtractive hybridization (SSH) as outlined by
Diatchenko et al, 1996, supra. The overall scheme is shown in FIG.
1. First, mRNA was converted from three separate fermentations of
Trichoderma reesei RutC30 grown on glucose, cellulose, and PCS into
double-stranded cDNA using reagents supplied with the
PCR-Select.TM. Kit (Clontech, Palo Alto, Calif.). The
differentially expressed cDNAs were present in both the "tester"
cDNA pool (i.e., from cells grown on cellulose or corn stover) and
the "driver" cDNA, but were present at much lower levels in the
"driver" pool (Table 1). Both of these cDNA pools were digested
with the restriction enzyme Rsa I which recognizes a four-base pair
palindrome and yields blunt-end fragments (GTIAC). The tester cDNA
pool was then divided into two samples and ligated with two
different adaptor oligonucleotides (provided with the Clontech
PCR-Select.TM. Kit) resulting in two populations of tester cDNA.
The adaptors were designed without 5'-phosphate groups such that
only the longer strand of each adaptor could be covalently linked
to the 5'-ends of the cDNA.
[0198] In the first of two hybridizations using conditions
specified in the Clontech PCR-Select.TM. Kit an excess of driver
cDNA was added to each portion of tester cDNA. The mixtures were
denatured by heating to 95.degree. C. then allowed to anneal. Four
types of molecules were generated by this annealing (designated as
a, b, c, and d molecules). Type a molecules included equal
concentrations of high- and low-abundance cDNAs, because the
second-order kinetics of hybridization were faster for more
abundant molecules in the pool which preferentially formed b type
molecules. At the same time, type a molecules were significantly
enriched for differentially expressed (e.g., cellulose- or
PCS-induced) sequences, since common non-target cDNAs formed type c
molecules with the driver. In a second hybridization, the two pools
of primary hybridized products were combined so that the type a
molecules from each tester sample could associate and form new type
e hybrids. These were double-stranded tester molecules with
different adaptor sequences on each end. Fresh denatured driver
cDNA was also added to further enrich the pool of e molecules for
differentially expressed sequences.
[0199] In the final step of the SSH procedure, the differentially
expressed cDNAs were selectively amplified by PCR (conditions
specified in the PCR-Select.TM. Kit) Only type e molecules that
have two different primer annealing sites were amplified
exponentially.
[0200] As a quality check, the cDNA clones from approximately 360
randomly picked colonies were purified by rolling circle
amplification using an Amersham TempliPhi Kit and analyzed by
[0201] DNA sequencing (70 from Reaction 1, 96 from Reaction 2, and
192 from Reaction 3). Clustering of the sequences using Transcript
Assembler.TM. software (Paracel, Inc., Pasadena, Calif.) showed
that each pool contained a high percentage of non-redundant
clones--76% for Reaction 1, 90% for Reaction 2, and 67% for
Reaction 3. In addition, the contigs (overlapping sequences of the
same cDNA) identified in this analysis contained on average only
two sequences. Collectively, these observations suggested that
efficient normalization of the libraries was achieved during the
SSH reactions, yielding a low level of redundancy in the
corresponding cDNA libraries. These differentially expressed
sequences were greatly enriched in the final subtracted cDNA pool,
and useful as a hybridization probe or to create a subtractive
library.
[0202] Subtracted and normalized cDNA fractions generated by the
SSH procedure were ligated with pCRII-TOPO (Invitrogen, Carlsbad,
Calif.) and the ligation mixtures were used to transform
electrocompetent E. coli TOP10 cells (Invitrogen, Carlsbad,
Calif.). Transformants were selected on LB agar plates (Miller, J.
H. 1992. A short course in bacterial genetics. A laboratory manual
and handbook for Escherichia coli and related bacteria. Cold Spring
Harbor Press, Cold Spring Harbor, N.Y.) that contained 250 .mu.g/ml
X-Gal (no IPTG) and ampicillin at a final concentration of 100
.mu.g/ml.
[0203] In order to evaluate the efficiency of subtraction and
normalization in SSH cDNA libraries, two approaches were used:
colony hybridization and sequencing of random clones from each SSH
library. The procedure for colony hybridization is detailed in
Birren et al 1998. Genome Analysis, A Laboratory Manual, Vol. 2,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).
Colony-hybridization analysis included approximately 700
independent clones from each subtracted [PCS minus glucose (SG),
cellulose minus glucose (CG), PCS minus cellulose (SC)] and
un-subtracted (cellulose, PCS) cDNA libraries with DIG-labeled cbh1
probe (abundant transcript), and .gamma.-actin probe, a moderately
abundant transcript representing a house-keeping gene (Table
2).
TABLE-US-00015 TABLE 2 Colony hybridization of SSH libraries probed
with cbh1 and .gamma.-actin cDNA fragments. Library Frequency cbh1
Frequency .gamma.-actin Cellulose (no SSH) 3.3% <0.17% PCS (no
SSH) 3.7% 0.13% PCS minus glucose 0.4% ND Cellulose minus glucose
0.5% ND PCS minus cellulose 0.1% ND ND, not detected.
[0204] While the cbh1 was a rather abundant in the non-subtracted
cellulose and PCS libraries (3.3% and 3.6% correspondingly), the
subtracted SG and CG libraries contained almost 10 times less cbh1
clones, which indicated that the abundant transcript was
successfully normalized. Colony-hybridization of the SC library
showed very low occurrence of cbh1 (only 0.1% of cbh1 clones)
indicating an efficient subtraction of this abundant transcript
when performing SSH with cell populations both expressing high
levels of cbh1.
[0205] The cDNA clones from approximately 360 randomly picked
colonies were purified by rolling circle amplification (RCA) (Dean
et al, 2001, Genome Res. 11: 1095-1099) and analyzed by DNA
sequencing (70 from Reaction 1, 96 from Reaction 2, and 192 from
Reaction 3). Clustering of the sequences using Transcript
Assembler.TM. software showed that each pool contained a high
percentage of non-redundant clones: 76% for reaction 1, 90% for
reaction 2, and 67% for reaction 3. In addition, the contigs
(overlapping sequences of the same cDNA) identified in this
analysis contained on the average only two sequences. Collectively,
these observations suggested that efficient normalization of the
libraries was achieved during the SSH reactions, yielding a low
level of redundancy in the corresponding cDNA libraries.
Example 16
Fabrication and Use of DNA Microarrays
[0206] A total of 3,608 white and light blue colonies were picked
from the subtracted and normalized cDNA library described in
Example 15. These were grown overnight in LB medium supplemented
with 100 .mu.g of ampicillin per ml in 96-well plates, and frozen
at -80.degree. C. Rolling circle amplification (Dean et al., 2001,
Genome Res. 11: 1095-1099) of plasmid DNA from frozen cells was
done using TempiPhi.TM. reagents from Amersham (Arlington Heights,
Ill.). High-throughput processing of these reactions was performed
using a Beckman Biomek.RTM. Fx robot (Beckman Coulter, Inc.,
Fullerton, Calif.) according to the manufacturer's instructions.
The amplified and diluted genomic clones were then spotted from
384-well plates onto poly-L-lysine coated glass microscope slides
using the equipment and methods described by Eisen and Brown, 1999,
Methods EnzymoL 303: 179-205.
Example 17
Analysis of Trichoderma reesei Strains that Contain Gene Silencing
Vectors
[0207] The RNA profiles of several Trichoderma reesei strains were
compared as shown in Table 3 below. Fluorescent probes were
prepared by reverse transcription of total RNA, incorporating
aminoallyl-dUTP into first strand cDNA (total RNA was prepared
using Fenozol reagents that are available commercially from Active
Motif, Carlsbad, Calif.). The amino-cDNA products were subsequently
labeled by direct coupling to either Cy3 or Cy5 monofunctional
reactive dyes (Amersham, Arlington Heights, Ill.) and purified as
described previously (Berka et al, 2003, Proc. Nat. Acad. Sc. USA
100: 5682-5687). Cy3 and Cy5 labeled probes were combined, purified
and dried under a vacuum, resuspended in 15.5 .mu.l of water, and
combined with the following: 3.6 .mu.l of 20.times.SSC, 2.5 .mu.l
of 250 mM HEPES (pH 7.0), 1.8 .mu.l of poly-dA (500 .mu.g/ml), and
0.54 .mu.l of 10% SDS. Before hybridization, the solution was
filtered with a 0.22 .mu.m filter, heated to 95.degree. C. for 2
minutes, and cooled to room temperature. The probe was applied to
the microarray under a cover glass, placed in a humidified chamber,
and incubated at 63.degree. C. overnight (15-16 hours). Before
scanning, the arrays were washed consecutively in 1.times.SSC with
0.03% SDS, 0.2.times.SSC, and 0.05.times.SSC and centrifuged for 2
minutes at approximately 100.times.g to remove excess liquid.
[0208] Microarray slides were imaged using an Axon 4000B scanner
(Molecular Devices Corp., Sunnyvale, Calif.) according to the
manufacturer's instructions. Fluorescence intensity values for
microarray spots were measured, and the ratio of Cy5 to Cy3 (650
nm:532 nm) intensity for each spot was calculated following
background subtraction. Those spots for which the intensity ratio
varied by more than two-fold from unity were deemed to be
differentially expressed.
TABLE-US-00016 TABLE 3 Trichoderma reesei RNA pools compared using
DNA microarrays. Exper- iment Number Cy3-label Comment Cy5-label
Comment 1 SMA148- Total RNA isolated MJ09-4 Total RNA 11-1-3 from a
strain (Empty isolated from a harboring a gene vector) control
strain silencing vector to harboring an inhibit cbh2 mRNA empty
vector 2 SaMe02 Total RNA isolated MJ09-4 Total RNA from a cbh2-
(Empty isolated from a deleted strain vector) control strain
harboring an empty vector 3 SaMe02 Total RNA solated SMA148- Total
RNA from a cbh2- 11-1-3 isolated from a deleted strain strain
harboring a gene silencing vector to inhibit cbh2 mRNA
[0209] In experiments 1 and 3, a total of 72 spots were identified
that satisfied the criterion of two-fold change in their
fluorescence intensity ratios, and were therefore classified as
differentially expressed sequences. DNA sequence analysis revealed
that 63/72 (88%) corresponded to cbh2-specific transcripts. The
remaining 9 spots corresponded to unknown or hypothetical gene
sequences. It was possible that these may contain nucleotide
sequences that are closely related to cbh2 and may encode conserved
modules such as cellulose binding domains. In contrast to what was
expected, the cbh2-mRNA levels in strains harboring the gene
silencing vector appeared to be elevated compared to the
corresponding mRNA levels in the control strain. This observation
reflected the fact that the gene silenced transcript itself can
hybridize to the cbh2 targets on the arrays, thereby giving the
illusion that cbh2-specific mRNA levels were increased in the gene
silencing strains. Nevertheless, the effect of gene silencing in
Trichoderma reesei appeared to be highly specific in that there
were few (if any) transcripts/genes other than cbh2 that were
affected.
[0210] In experiment 2, none of the sequences on the microarrays
appeared to be differentially expressed indicating that
cbh2-specific RNA sequences were still produced by the cbh2-deleted
strain at levels comparable to the control strain. It should be
noted, however, that this presumably reflected the fact that the
deletion/disruption event in Trichoderma reesei SaMe02 did not
remove the entire coding region for cbh2. Thus, it was possible for
the strain to make truncated transcripts that still hybridized to
cbh2 targets on the microarrays.
Example 18
RNAi During Small-Scale Fermentation
[0211] Two-liter fermentations were performed as described in
Example 5 on transformants 11 and 14, which showed almost 100%
reduction for cbh2 mRNA, to determine whether gene silencing is
successfully maintained throughout small-scale fermentations.
Fermentations were also performed on the host strain Trichoderma
reesei RutC30 and a positive control strain Trichoderma reesei
SaMe02 where the cbh2 gene was knocked-out by homologous
recombination. The level of cellobiohydrolase II protein was
measured by SDS-PAGE analysis as described in Example 12.
[0212] The level of cellobiohydrolase II protein was substantially
reduced in transformants 11 and 14, and Trichoderma reesei SaMe02,
when compared to the host strain Trichoderma reesei RutC30.
Example 19
Detection of Small Interfering RNA of Trichoderma reesei CeI6A
Cellobiohydrolase II by Northern Blot Hybridization
[0213] Northern analysis was performed to examine transformants 1,
2, 11, 12, 13, and 14 for the presence or absence of Trichoderma
reesei CeI6A cellobiohydrolase II-specific small interfering RNA
(siRNA). The same total RNA that was used in Example 13 was used
for this experiment. Isolated total RNA (25 .mu.g) was mixed with 1
part TBE-Urea sample buffer (Bio-Rad Laboratories, Hercules,
Calif.), heated for 15 minutes at 65.degree. C., and fractionated
by electrophoresis on a Criterion.TM. 15% polyacrylamide-7M urea
gel (Bio-Rad Laboratories, Hercules, Calif.) with a Criterion.TM.
Cell in 1.times. TBE running buffer (Bio-Rad Laboratories,
Hercules, Calif.). RNA was then electroblotted to a Zeta-Probe.RTM.
GT blotting membrane (Bio-Rad Laboratories, Hercules, Calif.) using
a Criterion.TM. Blotter (Bio-Rad Laboratories, Hercules, Calif.)
for an hour at 50V in 0.5.times.TBE transfer buffer (Bio-Rad
Laboratories, Hercules, Calif.). After the transfer, RNA was fixed
onto the membrane in a Stratalinker.RTM. 1800 UV Crosslinker
(Stratagene, La Jolla, Calif.) using Auto-Crosslink setting. The
membrane was hybridized with a 363 bp digoxigenin-labeled
Trichoderma reesei CeI6A cellobiohydrolase II single stranded RNA
probes, which was synthesized by in vitro transcription of
Trichoderma reesei CeI6A cellobiohydrolase II template DNA in the
presence of digoxigenin-11-dUTP using a DIG RNA Labeling Kit
(Roche, Indianapolis, Ind.), following a protocol supplied by the
manufacturer. The template DNA was generated by PCR amplifying a
363 bp fragment of the Trichoderma reesei CeI6A cellobiohydrolase
II coding region, representing the entire double stranded RNA
sequence from pTr0749 using primers 998305 (sense) and 998306
(antisense) shown below. The sense primer was engineered to have an
Xba I site at the 5'-end and a Hind III site at the 5'-end of the
antisense primer.
TABLE-US-00017 Primer 998305 (sense): (SEQ ID NO: 31)
5'-CTAGTCTAGAGTCGCAAAGGTTCCC-3' Primer 998306 (antisense): (SEQ ID
NO: 32) 5'-GGGGGAAGCTTTGACTGAGCATT-3'
[0214] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 10 ng pTr0749,
0.3 .mu.M primer 998305, 0.3 .mu.M primer 998306, and 2.5 units of
Taq polymerase. The reactions were incubated in an Eppendorf
Mastercycler 5333 programmed as follows: 30 cycles each for 30
seconds at 94.degree. C., 30 seconds at 56.degree. C., and 30
seconds at 72.degree. C. (15 minute final extension). The reaction
products were isolated on a 1.0% agarose gel using TAE buffer where
a 383 bp product band was excised from the gel and purified using a
QIAquick Gel Extraction Kit according to the manufacturer's
instructions. The resulting Trichoderma reesei Cel6A
cellobiohydrolase II PCR fragment was digested with Xba I and Hind
III and ligated into Xba I and Hind III digested pSPT19 vector
(Roche Molecular Biochemicals, Indianapolis, Ind., USA), using T4
DNA ligase according to manufacturer's protocol, to generate
pSMai163 (FIG. 8).
[0215] Hybridization was performed in DIG Easy Hyb buffer (Roche
Molecular Biochemicals, Indianapolis, Ind., USA) at 42.degree. C.
for 15-17 hours. The membrane was then washed under high stringency
conditions in 2.times.SSC plus 0.1% SDS for 5 minutes at room
temperature followed by two washes in 0.1.times.SSC plus 0.1% SDS
for 15 minutes each at 42.degree. C. The probe-target hybrids were
detected by chemiluminescent assay (Roche Molecular Biochemicals,
Indianapolis, Ind.), following the manufacturer's instructions.
[0216] Northern blot analysis revealed that transformants
expressing the Trichoderma reesei CeI6A cellobiohydrolase II
inverted repeat fragments produced small interfering Trichoderma
reesei CeI6A cellobiohydrolase II RNA of approximately 21 bp (FIG.
9). The same RNAs were not detected in transformants containing
vector controls, i.e., Trichoderma reesei RutC30) or Trichoderma
reesei SaMe02 (where the Trichoderma reesei CeI6A cellobiohydrolase
II gene was knocked-out by homologous recombination). These results
indicated that double stranded RNA-mediated regulation of
Trichoderma reesei Ce/6A cellobiohydrolase II gene expression in
Trichoderma reesei involved the production of small RNAs of the
size range expected for a mechanism involved in RNA
interference.
Example 20
Long Term Stability of the Trichoderma reesei Silenced CeI6A
Cellobiohydrolase II Transformants
[0217] To evaluate the stability of the silenced Trichoderma reesei
CeI6A cellobiohydrolase II transformants, spores from six
transformants (#1, 2, 11, 12, 13 and 14) exhibiting varying degrees
of Trichoderma reesei CeI6A cellobiohydrolase II protein reduction,
the host strain Trichoderma reesei RutC30, and a positive control
strain Trichoderma reesei SaMe02 were grown under inducing
conditions in cellulase-inducing medium at pH 6.0. At 7-days
post-inoculation, 500 .mu.l of mycelia were used as an inoculum for
shake flasks containing fresh in cellulase-inducing medium. This
passaging of mycelia was performed for a total of five rounds.
Supernatant from each round of passaging for each transformant was
collected and analyzed by SDS-PAGE as described in Example 12. As
FIG. 10 shows, silencing was maintained throughout the five
generations for all the transformants.
Example 21
Construction of pAlLo2 Expression Vector
[0218] The amdS gene of pAlLo1 (Example 1) was swapped with the
Aspergillus nidulans pyrG gene. Plasmid pBANe10 (FIG. 11) was used
as a source for the pyrG gene as a selection marker. Analysis of
the sequence of pBANe10 showed that the pyrG marker was contained
within an Nsi I restriction fragment and does not contain either
Nco I or Pac I restriction sites. Since the amdS is also flanked by
Nsi I restriction sites the strategy to switch the selection marker
was a simple swap of Nsi I restriction fragments. Plasmid DNA from
pAlLo1 and pBANe10 were digested with the Nsi I and the products
purified by agarose gel electrophoresis. The Nsi I fragment from
pBANe10 containing the pyrG gene was ligated to the backbone of
pAlLo1 to replace the original Nsi I DNA fragment containing the
amdS gene. Recombinant clones were analyzed by restriction digest
to determine that they had the correct insert and also its
orientation. A clone with the pyrG gene transcribed in the
counterclockwise direction was selected. The new plasmid was
designated pAlLo2 (FIG. 12).
Example 22
Construction of pHB506
[0219] Two fragments, one composed of a 184 bp Inverted Repeat (IR)
and the other composed of 290 bp containing the 184 bp sequence in
the opposite orientation along with a 106 linker region designated
Inverted Repeat-Linker (IR-L) were PCR amplified from exon 4 of an
amyloglucosidase gene of Aspergillus niger JaL303-10 (Aspergillus
niger NN049453 is a strain originally generated from Aspergillus
niger C40, which was isolated from the soil in 1960's, of which
amyloglucosidase activity has been enhanced by mutagenesis. The
Aspergillus niger strain JaL303 was constructed by site-directed
gene disruption to cause the interruption of the resident
tripeptidyl aminopeptidase gene in Aspergillus niger NN049453.
Aspergillus niger JaL303-10 is a pyrG minus mutant strain of JaL303
spontaneously isolated on agar containing 5-FOA.)
[0220] Using 200 ng of genomic DNA as template, the primers below
were employed. The sense primer was engineered to have an Nco I
site at the 5'-end and a Not I site at the 5'-end of the antisense
primer.
TABLE-US-00018 Primer 997491 (sense): (SEQ ID NO: 33) Sense
5'-GGGGCCATGGTCCTGGTGTGATTCTCAGGCACCCGAAATTC TCTGC-3' Primer 997492
(antisense): (SEQ ID NO: 34)
5'-GGGGGCGGCCGCAGCAGGGCTGGAAGGTGGAGTCGTCGCATG-3'
[0221] Four hundred .mu.l of Aspergillus niger JaL303-10 spores
were grown in 50 ml of YP medium in a baffled shake flask at
34.degree. C. and 150 rpm for 18 hours. Genomic DNA was then
extracted from the mycelia using a DNeasy Plant Mini Kit (QIAGEN
Inc., Valencia, Calif.) according to manufacturer's
instructions.
[0222] The amplification reactions (50 .mu.l) were composed of
1.times. Pfx Reaction Buffer (Invitrogen, Carlsbad, Calif.), 100 ng
of Aspergillus niger JaL303-10 genomic DNA, 0.3 .mu.M sense primer,
0.3 .mu.M antisense primer, and 2.5 units of Pfx polymerase
(Invitrogen, Carlsbad, Calif.). The reactions were incubated in an
Eppendorf Thermocycler 5333 programmed as follows: 30 cycles each
for 30 seconds at 94.degree. C., 30 seconds at 56.degree. C., and
30 seconds at 72.degree. C. (15 minute final extension). The
reaction product was isolated on a 1.0% agarose gel using TAE
buffer where a 184 bp product band was excised from the gel and
purified using a QIAquick Gel Extraction Kit according to the
manufacturer's instructions.
[0223] For amplification of the inverted repeat in the reverse
orientation plus the linker sequence, the sense primer was
engineered to have a Pac I site at the 5' end and a Not I site at
the 5' end of the antisense primer.
TABLE-US-00019 Primer 997493 (sense): (SEQ ID NO: 35)
5'-GGGGTTAATTAATCCTGGTGTGATTCTCAGGCACCCGAAATTC TC-3' Primer 997494
(antisense): (SEQ ID NO: 36)
5'-GGGGGCGGCCGCTACCGACCCACCGCAACAGCCTCGCTGTCA-3'
[0224] The amplification reactions (50 .mu.l) were performed as
described above. The reaction product was isolated on a 1.0%
agarose gel using TAE buffer where a 290 bp product band was
excised from the gel and purified using a QIAquick Gel Extraction
Kit according to the manufacturer's instructions.
[0225] The resulting PCR fragment of 290 bp was then inserted into
pTOPO Blunt (Invitogen Carlsbad, Calif.) according to the
manufacturer's instructions to produce pTOPO-IR-L.
[0226] Plasmid pTOPO-IR-L was digested with Pac I and Not I,
purified using 1.0% agarose gel electrophoresis and a QIAquick Gel
Extraction Kit as described above, and inserted into the
corresponding restriction sites of pAlLo2 resulting in pAILo2IR-L.
The 184 bp fragment was isolated after digestion of pTOPO-IR and
inserted into Nco I/Not I digested pAlLo2IR-L resulting in pHB506
(FIG. 13).
Example 23
Transformation of pHB506 into Aspergillus niger JaL303-10
[0227] Protoplasts of Aspergillus niger JaL303-10 were prepared
using the modified protocol described in Example 11. The
protoplasts were counted using a haemacytometer and re-suspended to
a final concentration of 1.times.10' protoplasts per ml of STC.
Excess protoplasts were stored in a Cryo 1.degree. C. Freezing
Container (Nalgene, Rochester, N.Y.) at -80.degree. C.
[0228] Transformation of the Aspergillus niger JaL303-10
protoplasts with pHB506 and pAlLo2 (as a control) was performed as
described in Example 11 with the following modifications.
[0229] Approximately 7 .mu.g of pHB506 or pAlLo2 was added to 100
.mu.l of the protoplast solution and mixed gently. PEG buffer (250
.mu.l) was added, mixed, and incubated at room temperature for 30
minutes. STC (3 ml) was then added, mixed, and plated onto Minimal
Medium plates. The plates were incubated at 34.degree. C. for 5-7
days.
[0230] Approximately 70 transformants were obtained. Spores from
the 70 transformants were streaked onto Minimal Medium plates and
incubated at 34.degree. C. for 6 days to obtain primary
transformants.
[0231] The primary transformants, along with Aspergillus niger
JaL303-10 transformed with pAlLo2, were then submitted to a plate
assay to measure glucoamylase activity using maltose as a
substrate. Release of glucose was determined using a coupled
glucose oxidase/horseradish peroxidase/ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay which
produces a violet color. All reagents were from Sigma Chemical
Company, St. Louis, Mo. M400 medium containing 20% Noble agar was
autoclaved, cooled to 50.degree. C. at which time 50 ml of 20%
maltose (sterile filtered), 3 ml of 2.65% w/v ABTS stock solution,
and 2 ml of HRP stock solution (100 U of horseradish peroxidase per
ml of 0.1 M sodium acetate pH 5.0) were added. Two ml of the medium
were added to each well of a 12 well cell culture plate. The plates
were either wrapped in foil or placed in a light-proof box and
stored at 4.degree. C. Inoculating loops were used to transfer
spores of each primary transformant to the assay wells. The plates
were wrapped in foil and incubated at 34.degree. C. for 1-2 days.
Glucose oxidase solution (1.5 U of glucose oxidase per ml of 0.1 M
sodium acetate pH 5.0) was then sprayed onto the plates which were
then wrapped in foil and incubated at 34.degree. C. for 1-2 hours.
Glucoamylase activity was determined by the oxidation of ABTS
indicated by color intensity of the colonial halo.
[0232] After incubation, the results showed that the color
intensity of each transformant varied in its shade of violet with
that of Aspergillus niger JaL303-10 transformed with pAlLo2 being
the darkest and several of the transformants displaying little or
no glucoamylase activity.
Example 24
Shake Flask Analysis of Transformants
[0233] Based on the colorimetric screen described in Example 23,
spores from eight transformant colonies were streaked onto Minimal
Medium plates. Four transformants displaying little or no
glucoamylase activity and 4 randomly chosen transformants producing
variable amyloglucosidase activity based on colorometric plate
assay were selected for growth in shake flasks. The above
transformants along with Aspergillus niger HowB112 having
glucoamylase and neutral amylase deletions, prepared as described
in WO 04/090155, Aspergillus niger JaL303-10, and Aspergillus niger
JaL303-10 transformed with pAlLo2 were grown in shake flasks
containing M400 medium.
[0234] Shake flasks (125 ml, baffled) containing 25 ml of M400
medium were inoculated with spores of each Aspergillus niger
transformant that grew on Minimal Medium plates and the controls
described above and incubated at 34.degree. C. and 200 rpm for 3
days. Culture broth samples were removed 3 days post-inoculation
and centrifuged at 15,700.times.g for 5 minutes in a
micro-centrifuge. The supernatants were transferred to new tubes
and stored at 4.degree. C. until SDS-PAGE analysis.
[0235] SDS-PAGE was carried out using Criterion.TM. Tris-HCl gels
with The Criterion.TM. Cell. Five .mu.l of day 3 samples were
suspended in 2.times. concentration of Laemmli Sample Buffer
(Bio-Rad Laboratories, Hercules, Calif.) and boiled for 3 minutes
in the presence of 5% .beta.-mercaptoethanol. Samples were loaded
onto a SDS-PAGE gel and subjected to electrophoresis in lx
Tris/Glycine/SDS running buffer (Bio-Rad Laboratories, Hercules,
Calif.). The resulting gel was stained with Bio-Safe.TM. Coomassie
Stain (Bio-Rad Laboratories, Hercules, Calif.).
[0236] The SDS-PAGE results (FIG. 14) showed the presence of two
proteins, a predominant band at approximately 125 kDa, thought to
be a precursor and a more diffuse band, or bands, at 75 kDa known
to be the processed enzyme. Both of these protein bands were
present in Aspergillus niger JaL303-10 transformed with pAlLo2. In
contrast, the glucoamylase gene deleted strain Aspergillus niger
HowB112 lacked either band. The selected transformants produced
lesser amounts of these two proteins. Transformants 1-4
representing randomly picked strains produced viable amounts of
glucoamylase whereas transformants 5-8, lacking glucoamylase as
measured in the plate screen, produced no detectable glucoamylase.
These results suggested that expression of the Aspergillus niger
glucoamylase was suppressed as a result of RNAi.
Example 25
Construction of pHUda512 expression vector
[0237] Expression vector pHUda512 was constructed for transcription
of double stranded-RNA derived from the amyloglucosidase gene of
Aspergillus niger NN049735. The production of the cDNA sequence of
the Aspergillus niger amyloglucosidase gene and the cDNA clone of
the Aspergillus niger amyloglucosidase gene are described in WO
00/004136. A PCR reaction with the cDNA clone of the Aspergillus
niger amyloglucosidase gene as template was performed with an
Expand.TM. PCR system (Roche Diagnostics, Japan) using primers
HU704 to introduce Bgl II, Kpn I, and Xho I sites and primer HU705
to introduce a BamH I site, as shown below.
TABLE-US-00020 HU704: (SEQ ID NO: 37)
5'-TTTAGATCTCTCGAGGTACCAAATGTGATTTCCAAGCGCGCG-3' HU705: (SEQ ID NO:
38) 5'-TTTGGATCCAAGAGATCGACGAGGGTCTTG-3'
[0238] The amplification reactions (50 .mu.l) were composed of 1 ng
of template DNA per .mu.l, 250 mM dNTP each, 250 nM primer HU704,
250 nM primer HU705, 0.1 U of Taq polymerase per .mu.l in 1.times.
buffer (Roche Diagnostics, Japan). The reactions were incubated in
a DNA Engine PTC-200 (MJ-Research, Japan) programmed as follows: 1
cycle at 94.degree. C. for 2 minutes; 30 cycles each at 92.degree.
C. for 1 minute, 55.degree. C. for 1 minute, and 1 cycle at
72.degree. C. for 1 minute; 1 cycle at 72.degree. C. for 10
minutes; and a hold at 4.degree. C.
[0239] The reaction products were isolated on a 1.0% agarose gel
using TAE buffer where a 213 bp product band was excised from the
gel and purified using a QIAquick.TM. Gel Extraction Kit (QIAGEN
Inc., Valencia, Calif.) according to the manufacturer's
instructions.
[0240] The 213 bp amplified DNA fragment was digested with Bgl II
and BamH I and ligated into pMT2188 (WO 03/089648) digested with
BamH I. The ligation mixture was transformed into E. coli DB6507
(ATCC 35673) using the Saccharomyces cerevisiae URA 3 gene as
selective marker to create the expression plasmid pHUda508. The
amplified plasmid was recovered using a QIAprep.RTM. Spin Miniprep
kit (QIAGEN Inc., Valencia, Calif.) according to the manufacturer's
instructions.
[0241] Plasmid pMT2188 comprised an expression cassette based on
the Aspergillus niger neutral amylase II promoter fused to the
Aspergillus nidulans triose phosphate isomerase non translated
leader sequence (Na2/tpi promoter) and the Aspergillus niger
amyloglucosidase terminator (AMG terminator), the selective marker
amdS from Aspergillus nidulans enabling growth on acetamide as sole
nitrogen source, and the URA3 marker from Saccharomyces cerevisiae
enabling growth of the pyrF defective Escherichia coli strain
DB6507.
[0242] A separate PCR was performed with the cDNA clone of the
Aspergillus niger amyloglucosidase gene as template with an
Expand.TM. PCR system using primer HU704 to introduce Bg/II, Kpn I
and Xho I sites and primer HU706 to introduce a BamH I site, as
shown below.
TABLE-US-00021 HU704: (SEQ ID NO: 39)
5'-TTTAGATCTCTCGAGGTACCAAATGTGATTTCCAAGCGCGCG-3' HU706: (SEQ ID NO:
40) 5'-TTTGGATCCTAGGCAGTCTCATCGACATTG-3'
[0243] The amplification reactions (50 .mu.l) were composed of 1 ng
of template DNA per .mu.l, 250 mM dNTPs each, 250 nM primer HU704,
250 nM primer HU706, 0.1 U of Taq polymerase per .mu.l in 1.times.
buffer (Roche Diagnostics, Japan). The reactions were incubated in
a DNA Engine PTC-200 programmed as follows: 1 cycle at 94.degree.
C. for 2 minutes; 30 cycles each at 92.degree. C. for 1 minute,
55.degree. C. for 1 minute, and 1 cycle at 72.degree. C. for 1
minute; 1 cycle at 72.degree. C. for 10 minutes; and a hold at
4.degree. C.
[0244] The reaction products were isolated on a 1.0% agarose gel
using TAE buffer where a 366 bp product band was excised from the
gel and purified using a QIAquick.TM. Gel Extraction Kit (QIAGEN
Inc., Valencia, Calif.) according to the manufacturer's
instructions.
[0245] The 366 bp amplified DNA fragment was digested with Xho I
and BamH I and ligated into pHUda508 digested with BamH I and Xho
I. The ligation mixture was transformed into E. coli DB6507 (ATCC
35673) to create the expression plasmid pHUda512 (FIG. 15)
containing a 213 bp inverted repeat derived from the Aspergillus
niger amyloglucosidase coding region. The amplified plasmid was
recovered using a QIAprep.RTM. Spin Miniprep Kit according to the
manufacturer's instructions.
Example 26
Expression of Double Strand-RNA Derived from an Aspergillus Niger
Amyloglucosidase Gene
[0246] Aspergillus niger strain NN049735 produces both Aspergillus
niger a mylog I u cosidase and hybrid enzyme between Aspergillus
niger acid stable alpha-amylase and a carbohydrate-binding module
derived from Aspergillus kawachii acid stable alpha-amylase.
[0247] Aspergillus niger NN049735 was cultivated in 100 ml of
non-selective YPG medium at 32.degree. C. for 16 hours on a rotary
shaker at 120 rpm. Cells were collected by filtering, washed with
0.6 M KCl, and resuspended in 20 ml of 0.6 M KCl containing
beta-glucanase (GLUCANEX.TM., Novozymes A/S, Bagsvrd, Denmark) at a
final concentration of 600 .mu.l per ml. The suspension was
incubated at 32.degree. C. and 80 rpm until protoplasts were
formed, and then washed twice with STC buffer. The protoplasts were
counted with a haemacytometer and resuspended and adjusted in an
8:2:0.1 solution of STC:STPC:DMSO to a final concentration of
2.5.times.10' protoplasts/ml. Approximately 3 .mu.g of pHUda512 was
added to 100 .mu.l of the protoplast suspension, mixed gently, and
incubated on ice for 20 minutes. One ml of SPTC was added and the
protoplast suspension was incubated for 30 minutes at 37.degree. C.
After the addition of 10 ml of 50.degree. C. COVE top agarose, the
reaction was poured onto COVE agar plates and the plates were
incubated at 32.degree. C. After 5 days transformants were selected
from the COVE medium.
[0248] Eight randomly selected transformants were inoculated into
100 ml of MLC medium and cultivated at 30.degree. C. for 2 days.
Ten ml of MLC medium was inoculated into 100 ml of MU-1 medium and
cultivated at 30.degree. C. for 7 days. Supernatants were obtained
by centrifugation at 3,000.times.g for 10 minutes.
[0249] Glucoamylase activity in the supernatant samples was
determined as an increase in NADH production by glucose
dehydrogenase and mutarotase reaction with generating glucose and
measured the absorbance at 340 nm. Six .mu.l of enzyme samples
dissolved in 100 mM sodium acetate pH 4.3 buffer was mixed with 31
.mu.l of 23.2 mM of maltose in 100 mM sodium acetate pH 4.3 buffer
and incubated at 37.degree. C. for 5 minutes. Then, 313 .mu.l of
color reagent (430 U of glucose dehydrogenase per liter, 9 U
mutarotase per liter, 0.21 mM NAD, and 0.15 M NaCl in 0.12 M
phosphate pH 7.6 buffer) was added to the reaction mixture and
incubated at 37.degree. C. for 5 minutes. Activity was measured at
340 nm on a spectrophotometer. Six .mu.l of distilled water was
used in place of the enzyme samples as controls.
[0250] Glucoamylase activity was measured in AmyloGlucosidase Units
(AGU), which was determined relative to an enzyme standard obtained
from Novozymes A/S, Bagsvrd, Denmark. One AGU is defined as the
amount of enzyme that hydrolyzes 1 micromole of maltose per minute
at 37.degree. C. in 23.2 mM maltose in 0.1 M sodium acetate pH 4.3
buffer.
[0251] Acid stable alpha-amylase activity was determined in the
supernatant samples as a decrease of blue color of starch-iodine
complex measured at 590 nm. Twenty-five .mu.l of enzyme samples
dissolved in 51.4 mM calcium chloride in 2 mM citrate pH 2.5 buffer
were mixed with 135 .mu.l of 0.6 g of soluble starch (Merck 1253,
Germany) and 12 g of sodium acetate per liter of 100 mM sodium
citrate pH 2.5 buffer, and incubated at 37.degree. C. for 325 sec.
After 325 sec, 90 .mu.l of iodine solution (1.2 g of potassium
iodine and 0.12 g of iodine per liter) was added to the reaction
mixture and incubated at 37.degree. C. for 25 seconds. Activity was
measured at 590 nm on a spectrophotometer. Twenty-five .mu.l of
distilled water was used in place of the enzyme samples as
controls.
[0252] Acid stable alpha-amylase activity was measured in AFAU
(Acid Fungal Alpha-amylase Units), which was determined relative to
an enzyme standard obtained from Novozymes A/S, Bagsyrd, Denmark.
One FAU is defined as the amount of enzyme which degrades 5.260 mg
starch dry matter per hour under the conditions described
above.
[0253] Table 2 shows amyloglucosidase activity and acid stable
alpha-amylase activity of the selected transformants, relative to
the activity of the host strain, Aspergillus niger NN049735, which
was normalized to 1.0. The results demonstrated the decrease of
amyloglucosidase activity and increase of acid stable alpha-amylase
activity simultaneously compared to the host strain Aspergillus
niger NN049735.
TABLE-US-00022 TABLE 2 Shake flask results of the selected
transformants Acid stable alpha- A. niger AMG amylase (AGU/ml)
(AFAU/ml) Strains Relative activities Relative activities # 5 0.01
1.43 # 19 0.03 1.57 # 20 0.01 1.23 # 50 0.02 1.37 # 64 0.05 1.34 #
65 0.01 0.97 # 77 0.04 1.46 # 97 0.01 1.23 NN049735 1.0 1.00
[0254] SDS-PAGE analysis was carried out using e-PAGEL gels E-T 7.5
L (ATTO Corporation, Japan). Ten .mu.l of day 7 samples were
suspended in 2.times. concentration of Sample Buffer (1% SDS, 1%
.beta.-mercaptoethanol, 1 mg/ml bromophenolblue, 10% glycerol, and
50 mM Tris-HCl pH 6.8), and boiled for 5 minutes. The samples were
loaded onto an e-PAGEL gel and subjected to electrophoresis in
1.times. Tris/Glycine/SDS running buffer (25 mM Tris-HCl, 0.1% SDS,
192 mM glycine) at 20 mA for 90 minutes. The resulting gel (FIG.
16) was stained with stain solution (1 g/L Coomassie brilliant
blue, 10% acetic acid, 30% methanol) for 30 minutes and then
destained with destaining solution (10% acetic acid, 30% methanol)
until the bands were visible.
[0255] Dominant bands at approximately 100 kDa and 80 kDa
corresponding to Aspergillus niger amyloglucosidase and fusion
enzyme between Aspergillus niger acid stable alpha-amylase fusion
protein, respectively, were present in Aspergillus niger NN049735.
In contrast, the selected transformants produced lower amounts of
the Aspergillus niger amyloglucosidase band and higher amounts of
the Aspergillus niger acid stable alpha-amylase fusion protein
band.
Example 27
Screening for Morphological Mutants
[0256] The Ae@ pools and Ab@ pools described in WO 98/11203
(Example 5) were screened for colonies having altered morphology by
plating on CM-1 agar and incubating at 34EC for 4 days. Colonies
having altered plate morphology within the pool were transferred to
a fresh CM-1 agar plate and incubated 5 days at 34EC for single
colony isolation. Each morphological mutant on a plate was
transferred from a single colony to the center of a CM-1 plate and
a PDA plate, and incubated 6-8 days at 34EC before the morphology
was evaluated, i.e., the diameter and the appearance. A total of
218 morphological mutants was transferred to COVE2 plates and
incubated at 34EC for 1-2 weeks to generate spores. A mutant was
identified which produced white spores and designated Aspergillus
oryzae P2-5.1.
Example 28
Rescue and Characterization of Plasmid DNA and Flanking DNA from
Morphological Mutant Aspergillus Oryzae P2-5.1
[0257] The plasmid DNA and genomic flanking loci were isolated from
mutant Aspergillus oryzae P2-5.1 using the procedure described in
WO 98/11203 (Example 9) except for the restriction endonuclease
used. An E. coli HB101 transformant containing a Bg/II rescued
locus from mutant Aspergillus oryzae P2-5.1 was isolated.
[0258] The Aspergillus oryzae P2-5.1 rescued locus containing 535
and 750 bp regions on either side of the integration event was
sequenced with an Applied Biosystems Model 373A Automated DNA
Sequencer (Applied Biosystems, Inc., Foster City, Calif.) on both
strands using the primer walking technique with dye-terminator
chemistry (Giesecke et al., 1992, Journal of Virol. Methods 38:
47-60) using the M13 reverse (-48) and M13 forward (-20) primers
(New England Biolabs, Beverly, Mass.) and primers unique to the DNA
being sequenced. A 400 bp nucleic acid sequence (SEQ ID NO: 41)
representing one flank of the rescued wA gene was chosen as the
template of construction of the inverted repeat. The nucleic acid
sequence suggested that the integration event occurred within an
open reading frame for a homolog of the Aspergillus fumigatus wA
gene (accession number Y17317). Nucleic acid homology of 74% was
found and the deduced amino acid sequence (SEQ ID NO: 42) of the
400 bp fragment of the rescued locus shared 71% identity when
compared to the deduced amino acid sequence of the Aspergillus
fumigatus wA gene. The wA gene encodes a polytketide synthetase
involved in the synthesis of the green pigment in wild-type spores.
Mutants of this gene produce spores which lack pigment and appear
white. Disruption of the wA gene in Aspergillus nidulans is known
to change the spore color to white (Mayorga and Timberlak, 1992,
Mol. Gen. Genet. 235: 205-212), which is the observed phenotype of
Aspergillus oryzae P2-5.1.
Example 29
Construction of pDeMi01
[0259] To express dsRNA derived from the Aspergillus oryzae wA
gene, all of one half of the inverted repeat of 175 base pairs from
within the open reading frame of the wA gene was PCR amplified
using a sense strand primer possessing a Not I restriction site and
an antisense primer possessing a 5' Nhe I restriction site shown
below.
TABLE-US-00023 Sense: (SEQ ID NO: 43)
5'-GGGGGCGGCCGCAGCACTTCGATTGCATTAGTCAAAA-3' Antisense: (SEQ ID NO:
44) 5'-GGGGGCTAGCAGAACGAACGCAGGTTTTAT-3'
[0260] The amplification reactions (50 .mu.l) were composed of
1.times. Pfx Reaction Buffer, 100 ng of Aspergillus oryzae P2-5.1
genomic DNA (which was isolated using a DNeasy Plant Maxi Kit), 0.3
mM dNTPs, 0.3 .mu.M sense primer, 0.3 .mu.M antisense primer, and
2.5 units of Pfx polymerase. The reactions were incubated in an
Eppendorf Thermocycler 5333 programmed as follows: 30 cycles each
for 30 seconds at 94.degree. C., 60 seconds at 58.degree. C., and 1
minute at 72.degree. C. (15 minute final extension). The reaction
product was isolated on a 1.0% agarose gel using TAE buffer where a
175 bp product band was excised from the gel and purified using a
QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0261] The other half of the inverted repeat including a 103 base
pair spacer was amplified using the sense primer above and the
antisense primer below having a Nhe I restriction site at the
primer's 5' end.
TABLE-US-00024 Antisense: (SEQ ID NO: 45)
5'-GGGGGCTAGCGGGTAGCCCGACGGCCACAAAGG-3'
[0262] The amplification reactions (50 .mu.l) were performed in
1.times. Pfx Reaction Buffer, 0.3 mM dNTPs, 100 ng of Aspergillus
oryzae P2-5.1 genomic DNA, 0.3 .mu.M sense primer, 0.3 .mu.M
antisense primer, and 2.5 units of Pfx polymerase. The reactions
were incubated in an Eppendorf Thermocycler 5333 programmed as
follows: 30 cycles each for 30 seconds at 94.degree. C., 45 seconds
at 58.degree. C., and 1 minute at 72.degree. C. and a 15 minute
final extension. The reaction product was isolated on a 1.0%
agarose gel using TAE buffer where a 273 bp product was excised
from the gel and purified using a QIAquick Gel Extraction Kit
according to the manufacturer's instructions.
[0263] The two PCR products were digested with Nhe I. The 273 bp
repeat plus spacer and the 175 bp repeat were mixed and ligated
with T4 DNA ligase using conditions specified by the manufacturer.
The ligation products were resolved on a 1.5% agarose gel. The 448
bp fragment corresponding to the inverted repeated was gel-isolated
using a Qiaex II DNA Purification Kit (QIAGEN Inc., Valencia,
Calif.) followed by Not I digestion. To drive the transcription of
the inverted repeat, plasmid pBANe6 (U.S. Pat. No. 6,461,837) which
contains the NA2-tpi promoter directly upstream of the Not I site
was used. The plasmid was digested with Not I and dephosphorylated
using shrimp alkaline phosphatase (Roche, Indianapolis, Ind.).
De-phosphorylated linear plasmid and the 448 bp inverted repeat
fragment were mixed and ligated using T4 DNA Ligase at 14.degree.
C. for 16 hours. Two .mu.l of the ligation mix were used to
transform competent E. coli SURE Cells (Stratagene, La Jolla,
Calif.) according to the manufacturer's instructions.
[0264] Plasmid DNA from several transformants was recovered,
digested with Not I, and resolved on a 1.5% agarose gel as above.
Transformants containing a single fragment of approximately 500 bp
was confirmed by DNA sequence analysis to consist of both the 175
bp repeat fragment the 175 bp fragment in the reverse orientation
separated by the 103 bp spacer. One of the isolated plasmids was
designated pDeMi0 (FIG. 17).
Example 24
Transformation of Aspergillus oryzae and Analysis of
Transformants
[0265] Five .mu.g of pDeMi01 or pBANe6 were used to transform
protoplasts prepared from Aspergillus oryzae strain Ja1250 (WO
98/11203) using growth on acetamide for selection. Growth on
acetamide requires expression of the amdS gene present on pDeMi01.
The transformation was performed as described in WO 98/11203
(Example 2). After incubation at 34.degree. C. for 30 minutes, the
protoplast/DNA mix was brought to 3 ml with STC and spread on COVE
plates supplemented with 10 mM uridine. The plates where then
incubated at 34.degree. C. for 6 days.
[0266] Approximately 50 transformants were obtained using pDeMi01
or pBANe6. All the pBANe6 transformants possessed the dark green
spores characteristic of wild-type Aspergillus oryzae. In contrast,
the pDeMi01 transformants produced colors ranging from light yellow
to dark green. The results suggested that the inverted repeat, when
transcribed, formed a hairpin dsRNA inducing genes specific RNAi.
Variation in spore color could be the result of differential
transcription of the inverted repeat, possibly due to the
chromosome integration site of the plasmid.
[0267] Eight primary transformants obtained using pDeMi01 and 1
transformant obtained with pBANe6 were streaked on COVE2 plates
supplemented with 10 mM uridine. All colonies derived from pBANe6
were uniformly dark green. In contrast, the colonies obtained from
the pDeMi01 transformants varied in spore color. Subsequent spore
purifications yielded uniformity of spore color. Spores from plates
transformed with pDeMi01 or pBANe8 were suspended in water, placed
on slides and observed by light microscopy. Spores from the control
pBANe8 strain remained green, whereas spores from pDeMi01
transformants appeared clear, the expected result of inactivation
by RNA interference.
[0268] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed,
since these embodiments are intended as illustrations of several
aspects of the invention. Any equivalent embodiments are intended
to be within the scope of this invention. Indeed, various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims. In the
case of conflict, the present disclosure including definitions will
control.
[0269] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
45122DNAAspergillus nidulens 1gtgccccatg atacgcctcc gg
22226DNAAspergillus nidulens 2gagtcgtatt tccaaggctc ctgacc
26324DNAAspergillus nidulens 3ggaggccatg aagtggacca acgg
24445DNAAspergillus niger 4caccgtgaaa gccatgctct ttccttcgtg
tagaagacca gacag 45545DNAAspergillus niger 5ctggtcttct acacgaagga
aagagcatgg ctttcacggt gtctg 45644DNAAspergillus niger 6ctatatacac
aactggattt accatgggcc cgcggccgca gatc 44744DNAAspergillus niger
7gatctgcggc cgcgggccca tggtaaatcc agttgtgtat atag
44829DNATrichoderma reesei 8aacgttaatt aaggaatcgt tttgtgttt
29929DNATrichoderma reesei 9agtactagta gctccgtggc gaaagcctg
291026DNATrichoderma reesei 10actagtcgac cgaatgtagg attgtt
261119DNATrichoderma reesei 11tgaccatggt gcgcagtcc
191226DNATrichoderma reesei 12cgatcgtctc cctatgggtc attacc
261328DNATrichoderma reesei 13actagttaat taagctccgt ggcgaaag
281420DNAEscherichia coli 14taatacgact cactataggg
20151416DNATrichoderma reesei 15atgattgtcg gcattctcac cacgctggct
acgctggcca cactcgcagc tagtgtgcct 60ctagaggagc ggcaagcttg ctcaagcgtc
tggggccaat gtggtggcca gaattggtcg 120ggtccgactt gctgtgcttc
cggaagcaca tgcgtctact ccaacgacta ttactcccag 180tgtcttcccg
gcgctgcaag ctcaagctcg tccacgcgcg ccgcgtcgac gacttctcga
240gtatccccca caacatcccg gtcgagctcc gcgacgcctc cacctggttc
tactactacc 300agagtacctc cagtcggatc gggaaccgct acgtattcag
gcaacccttt tgttggggtc 360actccttggg ccaatgcata ttacgcctct
gaagttagca gcctcgctat tcctagcttg 420actggagcca tggccactgc
tgcagcagct gtcgcaaagg ttccctcttt tatgtggcta 480gatactcttg
acaagacccc tctcatggag caaaccttgg ccgacatccg caccgccaac
540aagaatggcg gtaactatgc cggacagttt gtggtgtatg acttgccgga
tcgcgattgc 600gctgcccttg cctcgaatgg cgaatactct attgccgatg
gtggcgtcgc caaatataag 660aactatatcg acaccattcg tcaaattgtc
gtggaatatt ccgatatccg gaccctcctg 720gttattgagc ctgactctct
tgccaacctg gtgaccaacc tcggtactcc aaagtgtgcc 780aatgctcagt
cagcctacct tgagtgcatc aactacgccg tcacacagct gaaccttcca
840aatgttgcga tgtatttgga cgctggccat gcaggatggc ttggctggcc
ggcaaaccaa 900gacccggccg ctcagctatt tgcaaatgtt tacaagaatg
catcgtctcc gagagctctt 960cgcggattgg caaccaatgt cgccaactac
aacgggtgga acattaccag ccccccatcg 1020tacacgcaag gcaacgctgt
ctacaacgag aagctgtaca tccacgctat tggacctctt 1080cttgccaatc
acggctggtc caacgccttc ttcatcactg atcaaggtcg atcgggaaag
1140cagcctaccg gacagcaaca gtggggagac tggtgcaatg tgatcggcac
cggatttggt 1200attcgcccat ccgcaaacac tggggactcg ttgctggatt
cgtttgtctg ggtcaagcca 1260ggcggcgagt gtgacggcac cagcgacagc
agtgcgccac gatttgactc ccactgtgcg 1320ctcccagatg ccttgcaacc
ggcgcctcaa gctggtgctt ggttccaagc ctactttgtg 1380cagcttctca
caaacgcaaa cccatcgttc ctgtaa 141616471PRTTrichoderma reesei 16Met
Ile Val Gly Ile Leu Thr Thr Leu Ala Thr Leu Ala Thr Leu Ala 1 5 10
15 Ala Ser Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly
20 25 30 Gln Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala
Ser Gly 35 40 45 Ser Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln
Cys Leu Pro Gly 50 55 60 Ala Ala Ser Ser Ser Ser Ser Thr Arg Ala
Ala Ser Thr Thr Ser Arg 65 70 75 80 Val Ser Pro Thr Thr Ser Arg Ser
Ser Ser Ala Thr Pro Pro Pro Gly 85 90 95 Ser Thr Thr Thr Arg Val
Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr 100 105 110 Ser Gly Asn Pro
Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr 115 120 125 Ala Ser
Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met 130 135 140
Ala Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu 145
150 155 160 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala
Asp Ile 165 170 175 Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly
Gln Phe Val Val 180 185 190 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala
Leu Ala Ser Asn Gly Glu 195 200 205 Tyr Ser Ile Ala Asp Gly Gly Val
Ala Lys Tyr Lys Asn Tyr Ile Asp 210 215 220 Thr Ile Arg Gln Ile Val
Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu 225 230 235 240 Val Ile Glu
Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr 245 250 255 Pro
Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr 260 265
270 Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala
275 280 285 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro
Ala Ala 290 295 300 Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser
Pro Arg Ala Leu 305 310 315 320 Arg Gly Leu Ala Thr Asn Val Ala Asn
Tyr Asn Gly Trp Asn Ile Thr 325 330 335 Ser Pro Pro Ser Tyr Thr Gln
Gly Asn Ala Val Tyr Asn Glu Lys Leu 340 345 350 Tyr Ile His Ala Ile
Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn 355 360 365 Ala Phe Phe
Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly 370 375 380 Gln
Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly 385 390
395 400 Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe
Val 405 410 415 Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp
Ser Ser Ala 420 425 430 Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp
Ala Leu Gln Pro Ala 435 440 445 Pro Gln Ala Gly Ala Trp Phe Gln Ala
Tyr Phe Val Gln Leu Leu Thr 450 455 460 Asn Ala Asn Pro Ser Phe Leu
465 470 1737DNATrichoderma reesei 17ggaattctag ttcttatatt
tggcgacgcc accatct 371836DNATrichoderma reesei 18catgccatgg
aaaggttccc tcttttatgt ggctag 361936DNATrichoderma reesei
19ggaattctga ctgagcattg gcacactttg gagtac 362036DNATrichoderma
reesei 20ccttaattaa aaaggttccc tcttttatgt ggctag
362120DNATrichoderma reesei 21aaatcgtggc gcactgctgt
202220DNATrichoderma reesei 22tgagtgcatc aactacgccg
202320DNATrichoderma reesei 23gtcaacacga cgaatggcgt
202421DNATrichoderma reesei 24tgatcggtat gggtcagaag g
212521DNATrichoderma reesei 25ctggtccaac gccttcttca t
212622DNATrichoderma reesei 26ggaacgtagt gaggctcgct aa
222722DNATrichoderma reesei 27catggctggt cgtgatctta cc
222822DNATrichoderma reesei 28ccttgatgtc acggacgatt tc
222924DNATrichoderma reesei 29ccagacatga caatgttgcc gtag
243023DNATrichoderma reesei 30tttcgctctt cctcacgcca ttg
233125DNATrichoderma reesei 31ctagtctaga gtcgcaaagg ttccc
253223DNATrichoderma reesei 32gggggaagct ttgactgagc att
233346DNAAspergillus niger 33ggggccatgg tcctggtgtg attctcaggc
acccgaaatt ctctgc 463442DNAAspergillus niger 34gggggcggcc
gcagcagggc tggaaggtgg agtcgtcgca tg 423545DNAAspergillus niger
35ggggttaatt aatcctggtg tgattctcag gcacccgaaa ttctc
453642DNAAspergillus niger 36gggggcggcc gctaccgacc caccgcaaca
gcctcgctgt ca 423742DNAAspergillus niger 37tttagatctc tcgaggtacc
aaatgtgatt tccaagcgcg cg 423830DNAAspergillus niger 38tttggatcca
agagatcgac gagggtcttg 303942DNAAspergillus niger 39tttagatctc
tcgaggtacc aaatgtgatt tccaagcgcg cg 424030DNAAspergillus niger
40tttggatcct aggcagtctc atcgacattg 3041400DNAAspergillus
oryzaemisc_feature(265)..(265)N=A, C, G, OR T 41ggaaggcaac
tatacgaaaa atgctctcaa ttccgggcaa caatgcagca cttcgattgc 60attagtcaaa
accaagggtt tccttcgatc cttcccttgg ttgacggaag cgtgcccgtg
120gaggagctgg gccctatcgt gacacagctc ggcaccacat gtcttcagat
ggctttggtc 180aactattggg gttcactagg tataaaacct gcgttcgttc
ttgggcatag tctcggggag 240tttgctgctt tgaataccgc aggantatta
tcgacttccg ataccatcta cctttgtggc 300cgtcgggcta ccctccttac
agaatactgc caggttggga cacacgccat gctggctgtc 360aaggcttcct
acccccaggt caagcagtta ctgaaagaaa 40042133PRTAspergillus
oryzaeMISC_FEATURE(89)..(89)XAA=ANY AMINO ACID 42Gly Arg Gln Leu
Tyr Glu Lys Cys Ser Gln Phe Arg Ala Thr Met Gln 1 5 10 15 His Phe
Asp Cys Ile Ser Gln Asn Gln Gly Phe Pro Ser Ile Leu Pro 20 25 30
Leu Val Asp Gly Ser Val Pro Val Glu Glu Leu Gly Pro Ile Val Thr 35
40 45 Gln Leu Gly Thr Thr Cys Leu Gln Met Ala Leu Val Asn Tyr Trp
Gly 50 55 60 Ser Leu Gly Ile Lys Pro Ala Phe Val Leu Gly His Ser
Leu Gly Glu 65 70 75 80 Phe Ala Ala Leu Asn Thr Ala Gly Xaa Leu Ser
Thr Ser Asp Thr Ile 85 90 95 Tyr Leu Cys Gly Arg Arg Ala Thr Leu
Leu Thr Glu Tyr Cys Gln Val 100 105 110 Gly Thr His Ala Met Leu Ala
Val Lys Ala Ser Tyr Pro Gln Val Lys 115 120 125 Gln Leu Leu Lys Glu
130 4337DNAAspergillus oryzae 43gggggcggcc gcagcacttc gattgcatta
gtcaaaa 374430DNAAspergillus oryzae 44gggggctagc agaacgaacg
caggttttat 304533DNAAspergillus oryzae 45gggggctagc gggtagcccg
acggccacaa agg 33
* * * * *