U.S. patent application number 14/167292 was filed with the patent office on 2017-02-09 for expression and high-throughput screening of complex expressed dna libraries in filamentous fungi.
This patent application is currently assigned to Dyadic International, Inc.. The applicant listed for this patent is Dyadic International, Inc.. Invention is credited to Richard P. BURLINGAME, Mark A. EMALFARB, Peter J. PUNT, Cornelius VAN DEN HONDEL, Cornelia VAN ZEIJL, Jan Cornelis VERDOES.
Application Number | 20170037417 14/167292 |
Document ID | / |
Family ID | 53678454 |
Filed Date | 2017-02-09 |
United States Patent
Application |
20170037417 |
Kind Code |
A9 |
EMALFARB; Mark A. ; et
al. |
February 9, 2017 |
EXPRESSION AND HIGH-THROUGHPUT SCREENING OF COMPLEX EXPRESSED DNA
LIBRARIES IN FILAMENTOUS FUNGI
Abstract
The invention is generally directed to modified filamentous
fungal host cells comprising one or more nucleic acids encoding one
or more polypeptides under the control of one or more promoters
that are functional in said cells. Methods of using the modified
cells to express one or more polypeptides are also disclosed,
including methods of screening cells transformed with one or more
expression vectors comprising nucleic acids derived from synthetic
or genomic nucleic acids including, cDNAs. Methods of purifying one
or more polypeptides or complexes comprising one or more
polypeptides expressed in the modified cells, intended for use as
substrates in structure/function studies, as therapeutic agents, as
diagnostic reagents, or as human or animal vaccines, are also
disclosed.
Inventors: |
EMALFARB; Mark A.; (Jupiter,
FL) ; PUNT; Peter J.; (Houten, NL) ; VAN
ZEIJL; Cornelia; (Vleuten-de-Meern, NL) ; VAN DEN
HONDEL; Cornelius; (Gouda, NL) ; VERDOES; Jan
Cornelis; (Bennekom, NL) ; BURLINGAME; Richard
P.; (Jupiter, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dyadic International, Inc. |
Jupiter |
FL |
US |
|
|
Assignee: |
Dyadic International, Inc.
Jupiter
FL
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
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US 20150211013 A1 |
July 30, 2015 |
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Family ID: |
53678454 |
Appl. No.: |
14/167292 |
Filed: |
January 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12518595 |
Mar 1, 2010 |
8680252 |
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PCT/US2007/087020 |
Dec 10, 2007 |
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14167292 |
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60869341 |
Dec 10, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/561 20130101;
C12N 15/80 20130101; C12P 21/00 20130101; C40B 40/06 20130101 |
International
Class: |
C12N 15/80 20060101
C12N015/80; G01N 33/561 20060101 G01N033/561; C12P 21/00 20060101
C12P021/00 |
Claims
1. A modified cell from a filamentous fungus comprising one or more
nucleic acids, each nucleic acid comprising at least one coding
region encoding at least one polypeptide, wherein at least one
coding region is operably-linked to at least one regulatory region
comprising at least one promoter active in said cell; wherein said
fungus is in a genus selected from the group consisting of
Aspergillus, Fusarium, Chrysosporium, Myceliophthora, and
Trichoderma; wherein said fungus has been modified to have less
than 50% of the protease activity of a parental or non-modified
fungus; wherein said fungus has a phenotype characterized by a
culture viscosity of less than 200 cP at the end of fermentation,
when cultured in suspension, in the presence of adequate nutrients,
under optimal or near-optimal conditions.
2. The fungus of claim 1, wherein the genus and species is
Chrysosporium lucknowense.
3. The fungus of claim 2, wherein the fungus is the strain
designated Chrysosporium lucknowense C1, deposited as accession
number VKM F-3500-D, now reclassified as the strain designated
Myceliophthora thermophila C1, or a derivative or mutant
thereof.
4. The fungus of claim 3, wherein said fungus is a derivative or
mutant of the C1 strain selected from the group consisting of:
Chrysosporium strain deposited as ATCC 44006, Chrysosporium strain
deposited as CBS 251.72, Chrysosporium strain deposited as
CBS143.77, Chrysosporium strain deposited as CBS 272.77,
Chrysosporium strain UV13-6 deposited as VKM F-3632 D,
Chrysosporium strain NG7 C-19 deposited as VKM F-3633 D), and
Chrysosporium strain UV18-25 having accession number VKM F-3631 D,
or a derivative or mutant thereof.
5. The fungus of claim 1, wherein the fungus is selected from the
group consisting of: Trichoderma longibrachiatum strain X-252,
Aspergillus sojae strain pclA, and Aspergillus niger strain pclA,
or a derivative or mutant thereof.
6. The fungus of claim 1, wherein said fungus has at least one
protease gene modified by insertion, deletion, and/or substitution
of one or more nucleotide residues.
7. The fungus of claim 6, wherein at least one protease gene is
selected from the group consisting of alp1, alp2 and pep4.
8. The fungus of claim 6, wherein the fungus is a derivative of the
strain designated UV18#100f selected from the group consisting of:
UV18#100f .DELTA.alp1, UV18#100f .DELTA.pyr5 .DELTA.alp1,
UV18#100.f .DELTA.alp1.DELTA.pep4.DELTA.alp2, UV18#100.f
.DELTA.pyr5 .DELTA.alp1.DELTA.pep4 .DELTA.alp2, and UV18#100.f
.DELTA.pyr4 .DELTA.pyr5 .DELTA.alp1.DELTA.pep4 .DELTA.alp2.
9. The fungus of claim 1, wherein said fungus has been modified to
have less than 10% of the protease activity of a parental or
non-modified fungus.
10. The fungus of claim 1, wherein said fungus has been modified to
have less than 1% of the protease activity of a parental or
non-modified fungus.
11. The fungus of claim 1, wherein said fungus has a phenotype
characterized by a culture viscosity of less than 100 cP at the end
of fermentation, when cultured in suspension, in the presence of
adequate nutrients, under optimal or near-optimal conditions.
12. The fungus of claim 1, wherein said fungus has a phenotype
characterized by a culture viscosity of less than 60 cP at the end
of fermentation, when cultured in suspension, in the presence of
adequate nutrients, under optimal or near-optimal conditions.
13. The fungus of claim 1, wherein said fungus has a phenotype
characterized by a culture viscosity of less than 10 cP at the end
of fermentation, when cultured in suspension, in the presence of
adequate nutrients, under optimal or near-optimal conditions.
14. A method of producing the modified fungal cell of claim 1,
comprising introduction of at least one expression vector into a
parental cell, wherein each expression vector comprises a nucleic
acid comprising at least one coding region encoding at least one
polypeptide, wherein at least one coding region is operably-linked
to at least one regulatory region comprising at least one promoter
active in said cell.
15. The method of claim 14, wherein said expression vector is a
circular expression vector capable of autonomous replication
followed by spontaneous linearization and integration into the
genome of a fungal host cell transformed with said vector,
comprising in order, (a) a first region comprising a telomeric
sequence and a first selection marker; (b) a second region
comprising a promoter operable in a fungal host cell; (c) a third
region that promotes autonomous replication in a bacterial host
cell; (d) a fourth region comprising a second selection marker and
a telomeric sequence; and (e) a fifth intervening region linking
the first and fourth regions in the circular form of the expression
vector which comprises one or more sites for linearization between
the telomeric sequences of said first and fourth regions, but does
not comprise a nucleotide sequence encoding a selectable marker
operable in a bacterial host cell; wherein said telomeric sequences
promote autonomous replication and enhance transformation in said
fungal host cell; wherein said linearization occurs in vivo in the
intervening region in the circular form of said expression vector
introduced into said fungal host cell; wherein said second and
third regions may be in any order between said first and fourth
regions.
16. The method of claim 15, wherein the first selection marker in
said first region is pyrE and the second selection marker in said
fourth region is pyrG.
17. The method of claim 15, wherein said second region further
comprises a promoter operably linked to a sequence encoding a
fungal signal peptide fused in frame to at least one coding region
encoding at least one polypeptide.
18. The method of claim 15, wherein said second region further
comprises comprising a at least one terminator sequence
operably-linked in a 3' direction downstream from at least one of
said promoters operable in said modified fungal cell.
19. The method of claim 15, wherein at least one of said promoters
operable in said modified fungal cell is an inducible promoter.
20. The method of claim 15, wherein the telomeric sequences in said
first and fourth regions are human or fungal telomeric sequences,
or homologues thereof.
21. A method of producing one or more polypeptides from a modified
fungal cell claim 1, by a process comprising the steps of: (a)
culturing the modified fungal cell in media under conditions
conducive to the expression of one or more polypeptides encoded by
at least one nucleic acid comprising at least one coding region
operably-linked to at least one regulatory region comprising at
least one promoter active in said cell; and (b) purifying at least
one of said polypeptides or a complex comprising at least one of
said polypeptides from the cells or cell-free media obtained from
culture media.
22. A method of producing one or more polypeptides from a modified
fungal cell of claim 1, by a process comprising the steps of: (a)
culturing said cell under conditions conducive to formation of a
plurality of transferable reproductive elements in suspension; (b)
separating the suspension of transferable reproductive elements
into liquid or solid media suitable for the growth of monoclonal
cultures or monoclonal colonies; (c) culturing the monoclonal
cultures or monoclonal colonies in media under conditions conducive
to the expression of one or more polypeptides encoded by at least
one nucleic acid comprising at least one coding region
operably-linked to at least one regulatory region comprising at
least one promoter active in said cell; and (d) purifying at least
one of said polypeptides or a complex comprising at least one of
said polypeptides from the cells or cell-free media obtained from
culture media.
23. A method of screening a plurality of polypeptides encoded by a
combinatorial library of vectors for a functional activity or
structural property of interest from the modified fungal cell of
claim 1, by a process comprising the steps of: (a) culturing said
cell under conditions conducive to formation of a plurality of
transferable reproductive elements in suspension; (b) separating
the suspension of transferable reproductive elements into liquid or
solid media suitable for the growth of monoclonal cultures or
monoclonal colonies; and (c) culturing the monoclonal cultures or
monoclonal colonies in media under conditions conducive to the
expression of one or more polypeptides encoded by at least one
nucleic acid comprising at least one coding region operably-linked
to at least one regulatory region comprising at least one promoter
active in said cell; and (d) identifying the presence or amount of
at least one of said polypeptides or a complex comprising at least
one of said polypeptides obtained from the cells or cell-free media
of said cultured cells.
24. The method of claim 23, wherein the step (d) is carried out by
high-throughput screening.
25. The method of claim 23, wherein the step (d) comprises an assay
selected from the group consisting of: Western blot, immunoblot,
enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA),
immunoprecipitation, surface plasmon resonance, chemiluminescence,
fluorescent polarization, phosphorescence, immunohistochemical
analysis, matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry, microcytometry,
microarray, microscopy, fluorescence activated cell sorting (FACS),
flow cytometry, or protein microchip, microarray and cell-based
bioassays.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 12/518,595, filed Mar. 1, 2010, now allowed,
which is a national stage entry under 35 USC .sctn.371 of
PCT/US2007/087020, filed Dec. 10, 2007, published as WO
2008/073914, which claims the benefit of priority under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Application No. 60/869,341, filed
on Dec. 10, 2006, the entire disclosures of which are incorporated
herein by reference.
[0002] Each of the following applications and patents is
incorporated herein by reference in its entirety: U.S. patent
application Ser. No. 09/548,938, now U.S. Pat. No. 6,573,086; U.S.
patent application Ser. No. 09/834,434, now U.S. Pat. No.
7,122,330; PCT Publication No. WO 01/25468; PCT Publication No. WO
01/79558; and PCT Publication No. NL/99/00618.
INCORPORATION-BY-REFERENCE of a SEQUENCE LISTING
[0003] This application contains a Sequence Listing submitted as an
electronic text file named "124702_0279_US_ST25.txt", having a size
in bytes of 94,401 bytes, and created on Dec. 26, 2013. This file
was based information in the Sequence Listing for U.S. Ser. No.
12/518,595, downloadable as a file named "12518595.raw", version
1.1, containing 17 sequences, item size 94.12. The information
contained in this electronic file is hereby incorporated by
reference in its entirety pursuant to 37 CFR .sctn.1.52(e)(5).
FIELD OF THE INVENTION
[0004] The invention provides a method for the expression and
subsequent screening of DNA libraries, particularly synthetic,
genomic, and cDNA libraries, in filamentous fungal hosts. In
particular, the invention provides vectors, host strains, and a
method for the expression and screening of complex DNA libraries,
including, but not limited to, combinatory (combinatorial)
libraries expressing one, two or more variable constituents and/or
prepared from two or more sublibraries (e.g., for the expression
and screening of immunoglobulin (including fragments and
derivatives of whole immunoglobulin proteins) and other receptor or
complex DNA libraries or libraries of libraries). The invention is
useful for the expression and screening for a large variety of
proteins and protein complexes, including human proteins. The
system employs transformed or transfected filamentous fungal
strains that generate transferable reproductive elements, for
example by efficient sporulation, in submerged culture. The fungi
preferably exhibit a morphology that minimizes or eliminates the
formation of entangled mycelia. Particularly preferred fungal
strains are also capable of expressing isolatable quantities of
exogenous proteins, including, but not limited to, large proteins
having two or more heterogeneous domains, subunits, or constituents
as well as protein complexes, for evaluation. The mutant fungal
strains of the invention are particularly well-suited for
high-throughput screening techniques, due to their production of
transferable reproductive elements, high levels of expression, low
protease activity, and very low culture viscosity and improved
recombination characteristics for both integrative and replicating
vector molecules. In addition, the mutant fungal strains and the
methods of the invention are well suited for the efficient
selection of proteins expressed by large libraries or libraries of
libraries. They are also suitable for the large-scale, economical
production of such proteins, providing a platform in which
expression, screening and production of the proteins all take place
in a single host species.
BACKGROUND OF THE INVENTION
[0005] There is a need in the art for improved systems for the
expression and screening of a variety of peptides and proteins,
including proteins and protein complexes that are heterologous to
the host in which they are expressed, and particularly including
proteins with two or more heterogeneous or heteromeric domains,
subunits and/or constituents. There is a need in the art for
improved systems for the expression and screening of mammalian
proteins and even more particularly, human proteins, in order to
elucidate protein-protein interactions or interactions of proteins
with other molecules, for directed molecular evolution strategies,
and/or for the production and selection of recombinant complex
proteins and/or engineered proteins for research, diagnostic and/or
therapeutic applications. To name just a few non-limiting examples,
there is a continued need in the art for improved systems for the
expression and screening of complex proteins including, but not
limited to, immunoglobulins (antibodies, including fragments and/or
domains and/or derivatives thereof), other receptors, enzymes,
hormones, lymphokines and DNA binding proteins. For example, the
ability to engineer and rapidly identify useful therapeutic and/or
diagnostic antibodies (or fragments and/or domains and/or
derivatives thereof) or to provide an affinity-based screen for the
selection of a variety of receptors and/or ligands is highly
desirable. Proteins having two or more heterogeneous or variable
domains, subunits, or constituents are particularly challenging to
engineer and efficiently screen. Having a rapid method to identify
proteins, including complex proteins, that are useful as
therapeutic, diagnostic and/or research tools for use in mammalian,
and particularly, human, applications, is therefore invaluable.
[0006] Moreover, in order to effectively screen for certain
proteins, especially those with highly variable domains (e.g.,
immunoglobulins, T cell receptors, MHC-peptide complexes), the
expression and screening of very large libraries, including
libraries of libraries, may be desirable, if not necessary, in
order to be able to select the best candidates for further
development or to cover all of the possible permutations of protein
structures encompassed by the variability in the proteins. With
large libraries, it can be especially difficult and/or
prohibitively time-consuming and/or costly to produce sufficient
quantities of proteins to effectively screen and select candidates
from the large pool of candidates, and then perform additional
evaluation as needed to identify the best candidates, and/or
perform further screening and selection to ensure that all of the
best candidates are identified from the original pool. Accordingly,
being able to efficiently and effectively express and select a
desired protein from a large pool of proteins (and further evolve
such proteins to a preferred candidate, if desired) in a system
that is economical (cost-effective) and provides results in a
relatively short time frame, and then readily produce such proteins
on an economical, large-scale production basis, is highly
desirable. If most or all of these goals could all be achieved in a
single host organism or cell, the advantages would be great.
Accordingly, there remains a pressing need for new approaches to
the characterization of proteins and polypeptides (the term
"protein" as used hereinafter should be understood to encompass
peptides and polypeptides as well), and to the design,
identification, and/or modification, and isolation of the genes
encoding these proteins, so as to enable the modification and/or
production of the proteins.
[0007] One approach to the problem of expressing proteins or
polypeptides is through the expression of a genomic DNA library in
a bacterium such as E. coli, where the expressed proteins are
screened for a property or activity of interest. This approach
suffers from several serious disadvantages, one of which is that
bacteria typically do not effectively express genes having introns.
Eukaryotic genomes of higher organisms are generally too complex
for comprehensive expression of DNA libraries in bacteria. When all
eukaryotic species are considered, bacteria represent only about
0.3% of all known species (E. O. Wilson, "The Current State of
Biological Diversity", in Biodiversity, National Academy Press,
Washington D.C., 1988, Chapter 1); thus the fraction of the world's
genetic diversity accessible to bacterial expression systems is
extremely limited.
[0008] To avoid problems with introns, it is possible to prepare a
cDNA library and express it in bacteria. However, this approach
relies upon the presence of RNA transcripts, and any genes not
actively being transcribed will not be represented in the library.
Many desirable proteins are expressed only under specific
conditions (e.g., virulence factors in pathogenic fungi) and these
conditions may not exist at the time the mRNA is harvested. In
order to obtain sufficient RNA to prepare a cDNA library, it is
necessary to culture suitable quantities of the organism or host
cell of interest. In contrast, sufficient genomic DNA can be
obtained from a very small number of individual cells by PCR
amplification, using either random primers or primers designed to
favor certain classes of genes. Finally, genes that are highly
expressed in an organism or host cell will tend to be
over-represented in the mRNA, and thus over-represented at the
expense of minimally-expressed genes, which are often some of the
more interesting genes, in a cDNA library. In order to have a high
level of coverage of the mRNA species present, a much larger number
of clones must be screened if a cDNA library is employed instead of
a genomic library, since the latter will have a more nearly equal
representation of the variety of genes present. Clearly it is more
desirable to screen a genomic DNA library if at all possible.
[0009] Also, most bacteria, including E. coli, are incapable of
secretion of many proteins, and thus are undesirable as a host cell
for screening purposes where the screening relies upon secretion of
the gene product. An additional disadvantage for E. coli, and for
bacterial hosts in general, is that prokaryotes cannot provide many
of the post-translational modifications required for the activity
of numerous eukaryotic proteins. Moreover, expression of complex
multi-domain or multi-subunit proteins (e.g., immunoglobulin) is
not readily feasible in E. coli. In addition to glycosylation,
subunit cleavage, disulfide bond formation, and proper folding of
proteins are examples of the post-translational processing often
required to produce an active protein.
[0010] To ensure such processing one can sometimes use mammalian
cells, but mammalian cells are difficult to maintain, require
expensive media, and are not generally transformed with high
efficiency, and development of stable production cell lines
requires long timeframes. Such transformation systems are therefore
not convenient for high-throughput screening of proteins, although
efforts have been made to employ mammalian cells as hosts for cDNA
library screening (Schouten et al., WO 99/64582). An approach
involving fusion of transformed protoplasts with mammalian cells
prior to library screening has been described (U.S. Pat. No.
5,989,814), but expression of the protein library occurs in
bacteria or yeast prior to cell fusion. There have been efforts to
modify glycosylation patterns enzymatically after expression in
host cells (Meynial-SalIes and Combes, J. Biotechnol., 46:1-14
(1996)), but such methods must be tailored for specific products
and are not suitable for expression of proteins from a DNA library.
More recently, Maras et al., Eur. J. Biochem., 249:701-707 (1997)
(see also U.S. Pat. No. 5,834,251) have described a strain of
Trichoderma reesei engineered to express human GlcNAc transferase
I. The enzyme transfers N-acetylglucosamine to mannose residues on
other expressed exogenous proteins, a first step toward more
closely approximating natural mammalian products.
[0011] The use of yeast as host cells solves some of the above
problems, but introduces others. Yeast tend to hyper-glycosylate
exogenous proteins (Bretthauer and Castellino, 1999, Biotechnol.
Appl. Biochem. 30:193-200), and the altered glycosylation patterns
often render expressed mammalian proteins highly antigenic (C.
Ballou, in Molecular Biology of the Yeast Saccharomyces, J.
Strathern et al., eds., Cold Spring Harbor Laboratory Press, NY,
1982, 335-360). Although yeast are capable of coping with a limited
number of introns, they are not generally capable of handling
complex genes from higher species such as vertebrates. Even genes
from filamentous fungi are usually too complex for yeast to
transcribe efficiently, and this problem is compounded by
differences in expression and splicing sequences between yeast and
filamentous fungi (see e.g., M. Innis et al., Science 1985
228:21-26). Despite these drawbacks, transformation and expression
systems for yeast have been extensively developed, generally for
use with cDNA libraries. Yeast expression systems have been
developed which are used to screen for naturally secreted and
membrane proteins of mammalian origin (Klein, et al., Proc. Natl.
Acad. Sci. USA 1996 93:7108-7113; Treco, U.S. Pat. No. 5,783,385),
and for heterologous fungal proteins (Dalboge and Heldt-Hansen,
Mol. Gen. Genet. 243:253-260 (1994)) and mammalian proteins
(Tekamp-Olson and Meryweather, U.S. Pat. No. 6,017,731).
[0012] Proper intron splicing, and glycosylation, folding, and
other post-translational modifications of fungal gene products
would be most efficiently handled by a fungal host species, making
filamentous fungi superior hosts for screening genomic DNA from
soil and other samples. It also makes them excellent hosts for the
production of fungal enzymes of commercial interest, such as
proteases, cellulases, and amylases. It has also been found that
filamentous fungi are capable of transcribing, translating,
processing, and secreting the products of other eukaryotic genes,
including mammalian genes. The latter property makes filamentous
fungi attractive hosts for the production of proteins of biomedical
interest (e.g., antibodies, other receptors, hormones, etc.).
Glycosylation patterns introduced by filamentous fungi more closely
resemble those of mammalian proteins than do the patterns
introduced by yeast. For these reasons, a great deal of effort has
been expended on the development of fungal host systems for
expression of heterologous proteins, and a number of fungal
expression systems have been developed. For reviews of work in this
area, see Maras et al., Glycoconjugate J., 16:99-107 (1999);
Peberdy, Acta Microbiol. Immunol. Hung. 46:165-174 (1999);
Kruszewsa, Acta Biochim. Pol. 46:181-195 (1999); Archer et al.,
Crit. Rev. Biotechnol. 17:273-306 (1997); and Jeenes et al.,
Biotech. Genet. Eng. Rev. 9:327-367 (1991).
[0013] High-throughput expression and assaying of DNA libraries
derived from fungal genomes would also be of use in assigning
functions to the many mammalian genes that are currently of unknown
function. For example, once a fungal protein having a property of
activity of interest is identified, the sequence of the encoding
gene may be compared to the human genome sequence to look for
homologous genes.
[0014] Yelton et al., U.S. Pat. No. 4,816,405, discloses the
modification of filamentous Ascomycetes to produce and secrete
heterologous proteins. Buxton et al., in U.S. Pat. No. 4,885,249,
and in Buxton and Radford, Mol. Gen. Genet. 196:339-344 (1984),
discloses the transformation of Aspergillus niger by a DNA vector
that contains a selectable marker capable of being incorporated
into the host cells. McKnight et al., U.S. Pat. No. 4,935,349, and
Boel, in U.S. Pat. No. 5,536,661, disclose methods for expressing
eukaryotic genes in Aspergillus involving promoters capable of
directing the expression of heterologous genes in Aspergillus and
other filamentous fungi. Royer et al., in U.S. Pat. No. 5,837,847,
and Berka et al., in WO 00/56900, disclose expression systems for
use in Fusarium venenatum employing natural and mutant Fusarium
spp. promoters. Conneely et al., in U.S. Pat. No. 5,955,316,
disclose plasmid constructs suitable for the expression and
production of lactoferrin in Aspergillus. Cladosporium glucose
oxidase had been expressed in Aspergillus (U.S. Pat. No.
5,879,921).
[0015] Similar techniques have been used in Neurospora. Lambowitz,
in U.S. Pat. No. 4,486,533, discloses an autonomously replicating
DNA vector for filamentous fungi and its use for the introduction
and expression of heterologous genes in Neurospora. Stuart et al.,
describe co-transformation of Neurospora crassa spheroplasts with
mammalian genes and endogenous transcriptional regulatory elements
in U.S. Pat. No. 5,695,965, and an improved strain of Neurospora
having reduced levels of extracellular protease in U.S. Pat. No.
5,776,730. Vectors for transformation of Neurospora are disclosed
in U.S. Pat. No. 5,834,191. Takagi et al., describe a
transformation system for Rhizopus in U.S. Pat. No. 5,436,158.
Sisniega-Barroso et al., describe a transformation system for
filamentous fungi in WO 99/51756, which employs promoters of the
glutamate dehydrogenase genes from Aspergillus awamori.
Dantas-Barbosa et al., FEMS Microbiol. Lett. 1998 169:185-190,
describe transformation of Humicola grisea var. thermoidea to
hygromycin B resistance, using either the lithium acetate method or
electroporation.
[0016] Fungal expression systems in Aspergillus and Trichoderma,
for example, are disclosed by Berka et al., in U.S. Pat. No.
5,578,463; see also Devchand and Gwynne, J. Biotechnol. 17:3-9
(1991) and Gouka et al., Appl. Microbiol. Biotechnol. 47:1-11
(1997). Examples of transformed strains of Myceliophthora
thermophila, Acremonium alabamense, Thielavia terrestris and
Sporotrichum cellulophilum are presented in WO 96/02563 and U.S.
Pat. Nos. 5,602,004, 5,604,129 and 5,695,985, which describe
certain drawbacks of the Aspergillus and Trichoderma systems. In
addition, the fungal expression system described in U.S. Pat. No.
6,573,086 and PCT Publication No. WO 00/20555 describe a
transformation system using filamentous fungal hosts that
particularly describe an expression system using Chrysosporium
hosts as well as other filamentous fungi.
[0017] Methods for the transformation of phyla other than
Ascomycetes are known in the art; see for example Munoz-Rivas et
al., Mol. Gen. Genet. 1986 205:103-106 (Schizophyllum commune); van
de Rhee et al., Mol. Gen. Genet. 1996 250:252-258 (Agaricus
bisporus); Arnau et al., Mol. Gen. Genet. 1991 225:193-198 (Mucor
circinelloides); Liou et al., Biosci. Biotechnol. Biochem. 1992
56:1503-1504 (Rhizopus niveus); Judelson et al., Mol. Plant Microbe
Interact. 1991 4:602-607 (Phytophthora infestans); and de Groot et
al., Nature Biotechnol. 1998 16:839-842 (Agaricus bisporus).
[0018] In addition to the usual methods of transformation of
filamentous fungi, such as for example protoplast fusion,
Chakraborty and Kapoor, Nucleic Acids Res. 18:6737 (1990) describe
the transformation of filamentous fungi by electroporation. De
Groot et al., in Nature Biotechnol. 16: 839-842 (1998), describe
Agrobacterium tumefaciens-mediated transformation of several
filamentous fungi. Biolistic introduction of DNA into fungi has
been carried out; see for example Christiansen et al., Curr. Genet.
29:100-102 (1995); Durand et al., Curr. Genet. 31:158-161 (1997);
and Barcellos et al., Can. J. Microbiol. 44:1137-1141 (1998). The
use of magnetic particles for "magneto-biolistic" transfection of
cells is described in U.S. Pat. Nos. 5,516,670 and 5,753,477, and
is expected to be applicable to filamentous fungi.
[0019] Most prior efforts in the field of filamentous fungal
expression systems have been directed to the identification of
strains suitable for industrial production of enzymes, and
therefore attention has been focused on culture viscosity,
stability of transformation, yield of heterologous protein per unit
volume, and yield as a percentage of biomass. DNA libraries have
been expressed in fungi; see for example Gems and Clutterbuck,
Curr. Genet. 1993 24:520-524, where an Aspergillus nidulans library
was expressed in A. nidulans, and Gems et al., Mol. Gen. Genet.
1994 242:467-471, where a genomic library from Penicillium was
expressed in Aspergillus. The cloning of an Aspergillus niger
invertase gene by expression in Trichoderma reesei was described by
Berges et al., Curr. Genet. 1993 24:53-59.
[0020] U.S. patent application Ser. No. 09/548,938, now U.S. Pat.
No. 6,573,086; U.S. patent application Ser. No. 09/834,434, now
U.S. Pat. No. 7,122,330; PCT Publication No. WO/0125468; PCT
Publication No. WO/0179558; and PCT Publication No. NL/99/00618,
described a system for expression of heterologous proteins in
fungal host cells, and methods for expressing the gene products of
a DNA library, including genomic and/or eukaryotic genomic DNA
libraries. These applications also disclose mutant fungal strains
that have partially lost their filamentous phenotype and thus
provide low-viscosity cultures.
[0021] The present invention fulfills a continued need in the art
for improved fungal host cell strains, vectors, and methods for the
expression and screening of complex DNA libraries, including
combinatorial libraries expressing proteins having one, two or more
domains, subunits, or constituents, such as immunoglobulins and
other receptors or protein complexes.
SUMMARY OF THE INVENTION
[0022] The present invention provides expression vectors comprising
telomeric sequences and selection markers pyrE and pyrG.
[0023] The present invention also provides expression vectors
comprising at least one sequence that promotes autonomous
replication and enhances transformation in a fungal host, and two
selection markers that flank an expression cassette in the vector,
wherein the entire expression cassette integrates into the genome
of a fungal host transformed with the vector.
[0024] In some embodiments, one of the selection markers is pyrE or
pyrG. In others, the two selection markers are pyrE and pyrG.
[0025] In some embodiments, the selection markers pyrE and pyrG
flank the expression cassette in the vector, and the entire
expression cassette integrates into the genome of a fungal host
transformed with the vector.
[0026] In some embodiments, the sequence that promotes autonomous
replication and enhances transformation in a fungal host comprises
telomeric sequences. In some embodiments, the telomeric sequences
are human telomeric sequences, fungal telomeric sequences, or
homologues thereof.
[0027] In some embodiments, the expression vector further comprises
a fungal signal sequence. In some embodiments, the fungal signal
sequence is the signal sequence of a fungal gene encoding a protein
selected from the group consisting of cellulase,
.beta.-galactosidase, xylanase, pectinase, esterase, protease,
amylase, chitinase, chitosanase, polygalacturonase and
hydrophobin.
[0028] In some embodiments, the expression vector further comprises
a terminator sequence. In some embodiments, the terminator sequence
is TtrpC or Tcbh1. In some embodiments, the terminator sequence
comprises SEQ ID NO:11.
[0029] In some embodiments, the vector comprises one or more copies
of the C1 Repetitive Sequence (CRS). In some embodiments, the
vector comprises from one to ten copies of the CRS. In some
embodiments, the CRS is located in an upstream region of a promoter
within the vector. In some embodiments, the CRS comprises the
nucleic acid sequence of SEQ ID NO:12.
[0030] In some embodiments, the expression vector further comprises
a promoter sequence. In some embodiments, the promoter sequence is
Pcbh1 or PgpdA.
[0031] In some embodiments, the expression vector further comprises
at least one autonomously replicating sequence.
[0032] In some embodiments, the expression vector further comprises
at least one nucleic acid sequence for replication in a non-fungal
host cell.
[0033] In some embodiments, the vector is a self-replicating
vector.
[0034] In some embodiments, the vector is an integrating
vector.
[0035] In some embodiments, the vector is an autonomous vector when
initially transformed into the filamentous fungus, and integrates
into the fungus upon extended culture.
[0036] In some embodiments, the vector comprises the following
elements: Pcbh1, Tcbh1, pyrE, tel, and pyrG.
[0037] In some embodiments, the vector comprises a nucleic acid
sequence encoding a protein between the elements Pcbh1 and
Tcbh1.
[0038] In some embodiments, the protein is a fusion protein.
[0039] In some embodiments, the protein is an immunoglobulin light
chain or a fragment thereof, or an immunoglobulin heavy chain or a
fragment thereof.
[0040] In some embodiments, the protein comprises both an
immunoglobulin light chain or a fragment thereof, and an
immunoglobulin heavy chain or a fragment thereof.
[0041] In some embodiments, the vector is pPcbh1 glaA(II) heavy(88)
Tcbh1 Pcbh1 glaA(II) light(90) Tcbh1 pyrE tel pyrG. In some
embodiments, the vector comprises the nucleic acid sequence of SEQ
ID NO:10.
[0042] In some embodiments, the selection markers flank at least
one nucleic acid sequence encoding a protein.
[0043] In some embodiments, the selection markers flank nucleic
acid sequences encoding components of a heterogeneous or
heteromultimeric protein.
[0044] In some embodiments, the expression vector further comprises
an expression-regulating region operably linked to the nucleic acid
sequence encoding the protein or components of the heterogeneous or
heteromultimeric protein. In some embodiments, the expression
regulating region comprises an inducible promoter.
[0045] In some embodiments, the nucleic acid sequence encoding each
component of the heterogeneous or heteromultimeric protein is
operably linked to a different promoter sequence.
[0046] In some embodiments, the nucleic acid sequence encoding each
component of the heterogeneous or heteromultimeric protein is
operably linked to a different terminator sequence.
[0047] In some embodiments, the vector comprises nucleic acid
sequences for transfer of the nucleic acid sequences encoding the
components of the heterogeneous or heteromultimeric protein to or
from a non-fungal host cell.
[0048] In some embodiments, the non-fungal cell is selected from
the group consisting of a bacterium, a yeast, and a mammalian
cell.
[0049] In some embodiments, the vector comprises nucleic acid
sequences for transfer of the nucleic acid sequences encoding the
components of the heterogeneous or heteromultimeric protein to or
from a bacteriophage. In some embodiments, the nucleic acid
sequences for transfer comprise nucleic acid sequences for in vitro
homologous recombination.
[0050] In some embodiments, the nucleic acid sequences encoding the
components of the heterogeneous or heteromultimeric protein are
linked to each other.
[0051] In some embodiments, the nucleic acid sequences encoding the
components of the heterogeneous or heteromultimeric protein are
separately linked to different expression-regulating regions.
[0052] In some embodiments, the vector comprises at least one
nucleic acid sequence encoding a fusion partner, wherein the
nucleic acid sequence encoding the fusion partner is operatively
linked to a nucleic acid sequence encoding a component of the
heterogeneous or heteromultimeric protein.
[0053] In some embodiments, the fusion partner is linked to the
component with a protein-processing site. In some embodiments, the
protein-processing site is a kex2 cleavage site.
[0054] In some embodiments, each of the components of the
heterogeneous or heteromultimeric protein is linked to a different
fusion partner.
[0055] In some embodiments, each of the components of the
heterogeneous or heteromultimeric fusion protein are linked to each
other and to a fusion partner.
[0056] In some embodiments, the fusion partner and each of the
components is linked by a protease processing site.
[0057] In some embodiments, the fusion partner enables secretion of
the heterogeneous or heteromultimeric protein from the filamentous
fungus.
[0058] In some embodiments, each of the nucleic acid sequences
encoding a component of the multimeric protein is flanked by
restriction enzyme sites.
[0059] In some embodiments, the nucleic acid sequence comprising
all of the nucleic acid sequences encoding a component of the
heterogeneous or heteromultimeric protein is flanked by restriction
enzyme sites.
[0060] In some embodiments, any one or more of the nucleic acid
sequences encoding a component of the heterogeneous or
heteromultimeric protein is mutated to reduce the presence of
protease processing sites recognized by the fungal proteases, or
wherein any one or more of the nucleic acid sequences encoding a
component of the heterogeneous or heteromultimeric protein is
selected to have reduced presence of protease processing sites
recognized by the fungal proteases.
[0061] In some embodiments, the nucleic acid sequences encoding the
components of the heterogeneous or heteromultimeric protein are
optimized to allow efficient transcription, translation, and
protein folding in the fungal host.
[0062] The present invention also provides an isolated fungus that
has been mutated or selected to have low protease activity, wherein
the fungus has less than 50% of the protease activity as compared
to a non-mutated fungus.
[0063] In some embodiments, the fungus has less than 10% of the
protease activity of the non-mutated fungus. In some embodiments,
the fungus has less than 1% of the protease activity of the
non-mutated fungus.
[0064] In some embodiments, the fungus is of a genus selected from
the group consisting of Aspergillus, Fusarium, Chrysosporium,
Myceliophthora, and Trichoderma.
[0065] In some embodiments, the fungus is a strain of C.
lucknowense. In some embodiments, the fungus is a strain of C.
lucknowense C1 (VKM F-3500-D).
[0066] In some embodiments, the fungus is UV18#100.f having
accession number CBS 122188, or a derivative or mutant thereof.
[0067] In some embodiments, at least one protease gene selected
from alp1, alp2 or pep4 has been disrupted. In some embodiments,
each of the protease genes alp1, alp2 or pep4 has been disrupted.
In some embodiments, the protease gene comprises a nucleic acid
sequence described in this application.
[0068] In some embodiments, the fungus is selected from the group
consisting of UV18#100f .DELTA..alp1, UV18#100f .DELTA.pyr5
.DELTA.alp1, UV18#100.f .DELTA.alp1 .DELTA.pep4 .DELTA.alp2,
UV18#100.f .DELTA.pyr5 .DELTA.alp1 .DELTA.pep4 .DELTA.alp2 and
UV18#100.f .DELTA.pyr4 .DELTA.pyr5 .DELTA.alp1 .DELTA.pep4
.DELTA.alp2.
[0069] In some embodiments, the fungus has a phenotype
characterized by a culture viscosity, when cultured in suspension,
of less than 200 cP at the end of fermentation when grown with
adequate nutrients under optimal or near-optimal conditions.
[0070] In some embodiments, the fungus has a phenotype
characterized by a culture viscosity, when cultured in suspension,
of less than 100 cP at the end of fermentation when grown with
adequate nutrients under optimal or near-optimal conditions.
[0071] In some embodiments, the fungus has a phenotype
characterized by culture viscosity, when cultured in suspension, of
less than 60 cP at the end of fermentation when grown with adequate
nutrients under optimal or near-optimal conditions.
[0072] In some embodiments, the fungus has a phenotype
characterized by a culture viscosity, when cultured in suspension,
of less than 10 cP at the end of fermentation when grown with
adequate nutrients under optimal or near-optimal conditions.
[0073] In some embodiments, the fungus has or has been mutated to
have improved homologous recombination of integrated nucleic acid
sequences.
[0074] In some embodiments, the fungus has or has been mutated to
have reduced non-homologous recombination of integrated nucleic
acid sequences.
[0075] In some embodiments, the fungus is of the class
Euascomycetes.
[0076] In some embodiments, the fungus is of the order
Onygenales.
[0077] In some embodiments, the fungus is of the order
Eurotiales.
[0078] In some embodiments, the fungus is of the division
Ascomycota, with the proviso that it is not of the order
Saccharomycetales.
[0079] In some embodiments, the fungus is of a genus selected from
the group consisting of: Aspergillus, Trichoderma, Chrysosporium,
Myceliophthora, Neurospora, Rhizomucor, Hansenula, Humicola, Mucor,
Tolypocladium, Fusarium, Penicillium, Talaromyces, Emericella,
Hypocrea, Thielavia, Aureobasidium, Filibasidium, Piromyces,
Cryplococcus, Acremonium, Tolypocladium, Scytalidium,
Schizophyllum, Sporotrichum, Gibberella, Mucor, Fusarium, and
anamorphs and teleomorphs thereof.
[0080] In some embodiments, the fungus is of a genus selected from
the group consisting of Aspergillus, Fusarium, Chrysosporium,
Myceliophthora, and Trichoderma.
[0081] In some embodiments, the fungus is selected from the group
consisting of: Chrysosporium ATCC 44006 or a derivative or mutant
thereof, Chrysosporium CBS 251.72 or a derivative or mutant
thereof, Chrysosporium CBS 143.77 or a derivative or mutant
thereof, Chrysosporium CBS 272.77 or a derivative or mutant
thereof, Chrysosporium VKM F-3500D (C1) or a derivative or mutant
thereof, Chrysosporium UV13-6 (Accession No. VKM F-3632 D) or a
derivative or mutant thereof, and Chrysosporium NG7 C-19 (Accession
No. VKM F-3633 D) or a derivative or mutant thereof.
[0082] In some embodiments, the fungus is Chrysosporium strain
UV18-25 having accession number VKM F-3631 D, or a derivative or
mutant thereof.
[0083] In some embodiments, the fungus is Trichoderma
longibrachiatum strain X-252, or a derivative or mutant
thereof.
[0084] In some embodiments, the fungus is Aspergillus sojae strain
pclA, or a derivative or mutant thereof.
[0085] In some embodiments, the fungus is Aspergillus niger strain
pclA, or a derivative or mutant thereof.
[0086] The present invention also provides a method of expressing a
plurality of proteins encoded by a combinatorial library of DNA
vectors, wherein the combinatorial library of vectors comprises a
plurality of different vectors of present invention, each different
vector comprising a different protein-encoding nucleic acid
sequence, said nucleic acid sequences being operably linked to an
expression-regulating region and optionally a secretion signal
encoding sequence or a fusion partner, the method comprising the
steps of:
[0087] stably transforming a filamentous fungus with said library
of DNA vectors in order to introduce into each of a plurality of
said filamentous fungus at least one of said DNA vectors, wherein
said filamentous fungus has a phenotype characterized by growth in
suspension and characterized by the production of transferable
reproductive elements in suspension;
[0088] culturing the transformed filamentous fungus under
conditions conducive to formation of transferable reproductive
elements in suspension;
[0089] separating from one another a plurality of transferable
reproductive elements; and
[0090] culturing into monoclonal cultures or monoclonal colonies
the individual transferable reproductive elements, under conditions
conducive to expression of the encoded proteins.
[0091] The present invention also provides a method of screening a
plurality of proteins encoded by a combinatorial library of DNA
vectors for an activity or property of interest, comprising the
steps of:
[0092] expressing the plurality of proteins in monoclonal
filamentous fungal cultures or monoclonal filamentous fungal
colonies, by a method of the present invention; and
[0093] screening individual clonal cultures or clonal colonies for
the activity or property of interest.
[0094] The present invention also provides a method of producing a
DNA molecule encoding a protein having an activity or property of
interest, comprising the steps of: [0095] expressing a plurality of
proteins in monoclonal filamentous fungal cultures or monoclonal
filamentous fungal colonies, by a method of the present
invention;
[0096] screening individual clonal cultures or clonal colonies for
the activity or property of interest; and
[0097] isolating DNA from a clonal culture or clonal colony
exhibiting the activity or property of interest.
[0098] The present invention also provides a method of producing
the nucleotide sequence of a DNA molecule encoding a protein having
an activity or property of interest, comprising the steps of:
[0099] isolating DNA from a clonal culture or clonal colony
exhibiting the activity or property of interest, by a method of the
present invention; and
[0100] sequencing said DNA.
[0101] The present invention also provides a method of producing
the amino acid sequence of a protein having an activity or property
of interest, comprising the steps of:
[0102] producing the DNA sequence of the protein having an activity
or property of interest, by a method of the present invention;
and
[0103] translating said DNA sequence into an amino acid
sequence.
[0104] The present invention also provides a method of optimizing a
protein's activity or property of interest, comprising the steps
of:
[0105] (a) stably transforming a filamentous fungus with a
combinatorial library of DNA vectors of the present invention
comprising mutant forms of the protein, in order to introduce into
each of a plurality of said filamentous fungus at least one of said
DNA vectors; wherein said filamentous fungus has a phenotype
characterized by growth in suspension and by the production of
transferable reproductive elements in suspension;
[0106] (b) culturing the transformed filamentous fungi under
conditions conducive to the formation of transferable reproductive
elements;
[0107] (c) separating from one another a plurality of transferable
reproductive elements;
[0108] (d) culturing into clonal cultures or clonal colonies the
individual transferable reproductive elements, under conditions
conducive to expression of the proteins encoded by the nucleic acid
sequences;
[0109] (e) screening each individual organism, clonal culture, or
clonal colony for an expressed protein having the activity or
property of interest;
[0110] (f) isolating one or more individual organisms, clonal
cultures, or clonal colonies that express said protein exhibiting
the activity or property of interest;
[0111] (g) isolating nucleic acid sequences from the isolated
individual organisms, clonal cultures, or clonal colonies that
encode the protein exhibiting the activity or property of
interest;
[0112] (h) mutating nucleic acid sequences from the isolated
individual organisms, clonal cultures, or clonal colonies that
encode the protein exhibiting the activity or property of
interest;
[0113] (i) providing a library of vectors which comprise the
mutated nucleic acid sequences obtained in step (h); and
[0114] repeating steps (b) through (h), until the property or
activity of interest either reaches a desirable level or no longer
improves.
[0115] In some embodiments, the method further comprises, between
steps (f) and (g), the steps of: culturing one or more of the
individual organisms, clonal cultures, or clonal colonies isolated
in step (f); isolating the expressed protein exhibiting the
activity or property of interest; and evaluating the isolated
protein for the property of interest.
[0116] In some embodiments, the filamentous fungus is a fungus
according to the present invention.
[0117] The present invention also provides a method of expressing a
plurality of proteins encoded by a combinatorial library of DNA
vectors, wherein the combinatorial library of vectors comprises a
plurality of different vectors, each different vector comprising a
different protein-encoding nucleic acid sequence, said nucleic acid
sequences being operably linked to an expression-regulating region
and optionally a secretion signal encoding sequence or a fusion
partner, the method comprising the steps of:
[0118] stably transforming a filamentous fungus of the present
invention with said library of DNA vectors in order to introduce
into each of a plurality of said filamentous fungus at least one of
said DNA vectors, wherein said filamentous fungus has a phenotype
characterized by growth in suspension and characterized by the
production of transferable reproductive elements in suspension;
[0119] (a) culturing the transformed filamentous fungus under
conditions conducive to formation of transferable reproductive
elements in suspension;
[0120] (b) separating from one another a plurality of transferable
reproductive elements; and
[0121] (c) culturing into monoclonal cultures or monoclonal
colonies the individual transferable reproductive elements, under
conditions conducive to expression of the encoded proteins.
[0122] The present invention also provides a method of screening a
plurality of proteins encoded by a combinatorial library of DNA
vectors for an activity or property of interest, comprising the
steps of:
[0123] (a) expressing the plurality of proteins in monoclonal
filamentous fungal cultures or monoclonal filamentous fungal
colonies, by a method of the present invention; and
[0124] (b) screening individual clonal cultures or clonal colonies
for the activity or property of interest.
[0125] The present invention also provides a method of producing a
DNA molecule encoding a protein having an activity or property of
interest, comprising the steps of:
[0126] (a) expressing a plurality of proteins in monoclonal
filamentous fungal cultures or monoclonal filamentous fungal
colonies, by a method of the present invention;
[0127] (b) screening individual clonal cultures or clonal colonies
for the activity or property of interest; and
[0128] (c) isolating DNA from a clonal culture or clonal colony
exhibiting the activity or property of interest.
[0129] The present invention also provides a method of producing
the nucleotide sequence of a DNA molecule encoding a protein having
an activity or property of interest, comprising the steps of:
[0130] isolating DNA from a clonal culture or clonal colony
exhibiting the activity or property of interest, by a method of the
present invention; and
[0131] sequencing said DNA.
[0132] The present invention also provides a method of producing
the amino acid sequence of a protein having an activity or property
of interest, comprising the steps of:
[0133] producing the DNA sequence of the protein having an activity
or property of interest, by a method of the present invention;
and
[0134] translating said DNA sequence into an amino acid
sequence.
[0135] The present invention also provides a method of optimizing a
protein's activity or property of interest, comprising the steps
of:
[0136] stably transforming a filamentous fungus of the present
invention with a combinatorial library of DNA vectors comprising
mutant forms of the protein, in order to introduce into each of a
plurality of said filamentous fungus at least one of said DNA
vectors; wherein said filamentous fungus has a phenotype
characterized by growth in suspension and by the production of
transferable reproductive elements in suspension;
[0137] (a) culturing the transformed filamentous fungi under
conditions conducive to the formation of transferable reproductive
elements;
[0138] (b) separating from one another a plurality of transferable
reproductive elements;
[0139] (c) culturing into clonal cultures or clonal colonies the
individual transferable reproductive elements, under conditions
conducive to expression of the proteins encoded by the nucleic acid
sequences;
[0140] (d) screening each individual organism, clonal culture, or
clonal colony for an expressed protein having the activity or
property of interest;
[0141] (e) isolating one or more individual organisms, clonal
cultures, or clonal colonies that express said protein exhibiting
the activity or property of interest;
[0142] (f) isolating nucleic acid sequences from the isolated
individual organisms, clonal cultures, or clonal colonies that
encode the protein exhibiting the activity or property of
interest;
[0143] (g) mutating nucleic acid sequences from the isolated
individual organisms, clonal cultures, or clonal colonies that
encode the protein exhibiting the activity or property of
interest;
[0144] (h) providing a library of vectors which comprise the
mutated nucleic acid sequences obtained in step (h); and
[0145] repeating steps (b) through (h), until the property or
activity of interest either reaches a desirable level or no longer
improves.
[0146] In some embodiments, the method further comprises, between
steps (f) and (g), the steps of: culturing one or more of the
individual organisms, clonal cultures, or clonal colonies isolated
in step (f); isolating the expressed protein exhibiting the
activity or property of interest; and evaluating the isolated
protein for the property of interest.
[0147] In some embodiments, the vectors are expression vectors
according to the present invention.
[0148] In some embodiments, the screening step is carried out by
high-throughput screening.
[0149] In some embodiments, the screening step comprises an assay
selected from the group consisting of: Western blot, immunoblot,
enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA),
immunoprecipitation, surface plasmon resonance, chemiluminescence,
fluorescent polarization, phosphorescence, immunohistochemical
analysis, matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry, microcytometry,
microarray, microscopy, fluorescence activated cell sorting (FACS),
flow cytometry, or protein microchip, microarray and cell-based
bioassays.
[0150] In some embodiments, the protein is a heterogeneous or
heteromultimeric protein.
[0151] In some embodiments, the heterogeneous or heteromultimeric
protein is selected from the group consisting of: a member of the
immunoglobulin supergene family, a hormone, a hormone receptor, a
cytokine, a cytokine receptor, a growth factor, and a growth factor
receptor.
[0152] In some embodiments, the member of the immunoglobulin
supergene family is selected from the group consisting of: an
immunoglobulin, a major histocompatibility complex protein, a major
histocompatibility complex linked to a peptide, a T cell receptor,
a CD3, and an adhesion molecule.
[0153] In some embodiments, the member of the immunoglobulin
supergene family is an immunoglobulin.
[0154] In some embodiments, the immunoglobulin is selected from the
group consisting of: a whole antibody, a fragment of a whole
antibody, a single chain antibody, a heavy chain antibody, a
bi-specific or multi-specific antibody, and a catalytic
antibody.
[0155] In some embodiments, the fragment of a whole antibody is
selected from the group consisting of: Fv, Fab, Fab', F(ab').sup.2,
CH, and Fc.
[0156] In some embodiments, the immunoglobulin is selected from the
group consisting of: a genetically-engineered antibody or fragment
thereof, a humanized antibody or fragment thereof, a catalytic
antibody and a neutralizing antibody.
[0157] In some embodiments, the expressed protein to biomass ratio
is at least 1:1.
[0158] In some embodiments, the expressed protein to biomass ratio
is at least 2:1.
[0159] In some embodiments, the expressed protein to biomass ratio
is at least 6:1.
[0160] In some embodiments, the expressed protein to biomass ratio
is at least 8:1.
[0161] In some embodiments, the transferable reproductive elements
are individual fungal cells.
[0162] In some embodiments, the transferable reproductive elements
are spores.
[0163] In some embodiments, the transferable reproductive elements
are hyphal fragments.
[0164] In some embodiments, the transferable reproductive elements
are micropellets.
[0165] In some embodiments, the transferable reproductive elements
are protoplasts.
[0166] The present invention also provides a method for obtaining a
heterogeneous or heteromultimeric protein having an activity or
property of interest, comprising the steps of:
[0167] screening a plurality of heterogeneous or heteromultimeric
proteins encoded by a combinatorial library of DNA vectors for an
activity or property of interest, by a method of the present
invention;
[0168] culturing on appropriate scale the monoclonal culture or
monoclonal colony expressing the activity or property of interest,
under conditions conducive to expression of the heterogeneous or
heteromultimeric proteins; and
[0169] isolating the expressed heterogeneous or heteromultimeric
protein.
[0170] The present invention also provides a method for obtaining a
heterogeneous or heteromultimeric protein having an activity or
property of interest, comprising optimizing the activity or
property of interest by a method of the present invention,
culturing on an appropriate scale an individual organism, clonal
culture, or clonal colony isolated in the final step (h), and
isolating the expressed protein from the culture.
[0171] The present invention also provides a method of making a
library of transformed filamentous fungi, comprising the steps
of:
[0172] providing a filamentous fungus having a phenotype
characterized by growth in suspension and characterized by the
production of transferable reproductive elements in suspension;
and
[0173] stably transforming said filamentous fungus with a
combinatorial library comprising a plurality of different vectors
of the present invention, each different vector comprising two or
more different nucleic acid sequences each encoding a component of
the heterogeneous or heteromultimeric protein, said nucleic acid
sequences being operably linked to an expression-regulating region
and optionally a secretion signal encoding sequence or a fusion
partner, in order to introduce into each of a plurality of said
filamentous fungus at least one of said DNA vectors.
[0174] In some embodiments, the filamentous fungus is a fungus of
the present invention.
[0175] The present invention also provides a library of transformed
filamentous fungi, prepared by a method of the present
invention.
[0176] In some embodiments, the transformed filamentous fungi
express substantially the same level of each of the components of
the heteromultimeric protein.
[0177] The present invention also provides a method for obtaining a
transformed filamentous fungal host expressing a protein having an
activity or property of interest, comprising the steps of:
[0178] screening a plurality of proteins encoded by a library of
DNA vectors for an activity or property of interest, by a method of
the present invention; and
[0179] isolating the monoclonal culture or monoclonal colony
expressing the activity or property of interest.
[0180] The present invention also provides a method for expression
and/or screening for heterogeneous or heteromultimeric proteins,
comprising any of the methods as substantially described
herein.
[0181] The present invention also provides a library of transformed
filamentous fungi for the expression and/or screening of
heterogeneous or heteromultimeric proteins, as substantially
described herein.
[0182] The present invention also provides a combinatorial library
of vectors comprising a vector of the present invention.
[0183] The present invention also provides a vaccine produced from
proteins expressed, produced, obtained or optimized by a method of
the present invention.
[0184] The present invention also provides a method for producing a
vaccine, comprising the steps of:
[0185] screening by a method of the present invention, a plurality
of antigens, or a plurality of immunoglobulins or fragments
thereof, for an immunogenic antigen or immunoglobulin or fragment
thereof, wherein the plurality is encoded by a library of DNA
vectors;
[0186] isolating the monoclonal culture or monoclonal colony
expressing the immunogenic antigen or immunoglobulin or fragment
thereof;
[0187] further cultivating the monoclonal culture to express the
immunogenic antigen or immunoglobulin or fragment thereof;
[0188] isolating the immunogenic antigen or immunoglobulin or
fragment thereof for use in a vaccine.
[0189] The present invention also provides isolated nucleic acid
molecules comprising a nucleic acid sequence selected from the
group consisting of:
[0190] a nucleic acid sequence encoding a protein comprising an
amino acid sequence selected from the group consisting of: SEQ ID
NO:2, SEQ ID NO:5, and SEQ ID NO:8;
[0191] a nucleic acid sequence encoding a fragment of the protein
of (a), wherein the fragment has a biological activity of the
protein of (a); and
[0192] a nucleic acid sequence encoding an amino acid sequence that
is at least 70% identical to an amino acid sequence of (a) and has
a biological activity of the protein comprising the amino acid
sequence.
[0193] In some embodiments, the nucleic acid sequence encodes an
amino acid sequence that is at least 90% identical to the amino
acid sequence of (a) and has a biological activity of the protein
comprising the amino acid sequence.
[0194] In some embodiments, the nucleic acid sequence encodes an
amino acid sequence that is at least 95% identical to the amino
acid sequence of (a) and has a biological activity of the protein
comprising the amino acid sequence.
[0195] In some embodiments, the nucleic acid sequence encodes an
amino acid sequence that is at least 97% identical to the amino
acid sequence of (a) and has a biological activity of the protein
comprising the amino acid sequence.
[0196] In some embodiments, the nucleic acid sequence encodes an
amino acid sequence that is at least 99% identical to the amino
acid sequence of (a) and has a biological activity of the protein
comprising the amino acid sequence.
[0197] In some embodiments, the nucleic acid sequence encodes a
protein comprising an amino acid sequence selected from the group
consisting of: SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:8.
[0198] In some embodiments, the nucleic acid sequence consists of a
nucleic acid sequence selected from the group consisting of: SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, and SEQ
ID NO:9.
[0199] The present invention also provides isolated nucleic acid
molecules comprising a nucleic acid sequence that is fully
complementary to a nucleic acid sequence of the present
invention.
[0200] The present invention also provides isolated proteins
comprising an amino acid sequence encoded by the nucleic acid
molecule of the present invention.
[0201] The present invention also provides isolated fusion proteins
comprising an isolated protein of the present invention fused to a
protein comprising an amino acid sequence that is heterologous to
the isolated protein.
[0202] The present invention also provides isolated antibodies or
antigen binding fragments thereof that selectively bind to a
protein of the present invention.
[0203] The present invention also provides a kit for degrading a
polypeptide or proteinaceous material comprising at least one
isolated protein of the present invention.
[0204] The present invention also provides compositions for the
degradation of a polypeptide or proteinaceous material comprising
at least one isolated protein of the present invention.
[0205] The present invention also provides recombinant nucleic acid
molecules comprising an isolated nucleic acid molecule of the
present invention operatively linked to at least one expression
control sequence.
[0206] In some embodiments, the recombinant nucleic acid molecule
comprises an expression vector.
[0207] In some embodiments, the recombinant nucleic acid molecule
comprises a targeting vector.
[0208] The present invention also provides an isolated host cell
transfected with a nucleic acid molecule of the present
invention.
[0209] In some embodiments, the host cell is a fungus.
[0210] In some embodiments, the filamentous fungus is from a genus
selected from the group consisting of: Chrysosporium, Thielavia,
Neurospora, Aureobasidium, Filibasidium, Piromyces, Corynascus,
Cryplococcus, Acremonium, Tolypocladium, Scytalidium,
Schizophyllum, Sporotrichum, Penicillium, Gibberella,
Myceliophthora, Mucor, Aspergillus, Fusarium, Humicola, and
Trichoderma, and anamorphs and teleomorphs thereof.
[0211] In some embodiments, the host cell is a bacterium.
[0212] The present invention also provides an oligonucleotide
consisting essentially of at least 12 consecutive nucleotides of a
nucleic acid sequence selected from the group consisting of: SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, and SEQ
ID NO:9.
[0213] The present invention also provides a kit comprising at
least one oligonucleotide of the present invention.
[0214] The present invention also provides a method for producing a
protein of the present invention comprising culturing a cell that
has been transfected with a nucleic acid molecule comprising a
nucleic acid sequence encoding the protein, and expressing the
protein with the transfected cell.
[0215] In some embodiments, the method further comprises recovering
the protein from the cell or from a culture comprising the
cell.
[0216] The present invention also provides a genetically modified
organism wherein the organism has been genetically modified to
express at least one protein of the present invention.
[0217] In some embodiments, the genetically modified organism is a
microorganism.
[0218] In some embodiments, the microorganism is a filamentous
fungus.
[0219] In some embodiments, the filamentous fungus is from a genus
selected from the group consisting of: Chrysosporium, Thielavia,
Neurospora, Aureobasidium, Filibasidium, Piromyces, Corynascus,
Cryplococcus, Acremonium, Tolypocladium, Scytalidium,
Schizophyllum, Sporotrichum, Penicillium, Talaromyces, Gibberella,
Myceliophthora, Mucor, Aspergillus, Fusarium, Humicola, and
Trichoderma.
[0220] In some embodiments, the filamentous fungus is selected from
the group consisting of: Trichoderma reesei, Chrysosporium
lucknowense, Aspergillus japonicus, Penicillium canescens,
Penicillium solitum, Penicillium funiculosum, and Talaromyces
flavus.
[0221] In some embodiments, the genetically modified organism is a
plant.
[0222] The present invention also provides a recombinant enzyme
isolated from a genetically modified microorganism of the present
invention.
[0223] In some embodiments, the enzyme has been subjected to a
purification step.
[0224] The present invention also provides a crude fermentation
product produced by culturing a genetically modified microorganism
of the present invention wherein the crude fermentation product
contains at least one protein of the present invention.
[0225] The present invention also provides a method for degrading a
polypeptide or proteinaceous material, comprising contacting the
polypeptide or proteinaceous material with at least one isolated
protein of the present invention.
[0226] The present invention also provides a method for decreasing
the protease activity in a fungus comprising deleting or
inactivating a gene that hybridizes to a nucleic acid sequence of
the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0227] FIGS. 1A-C show: a schematic representation of circular
transformation vector pPInsT PyrE tel PyrG (A), the derivative
after in vivo linearization (B) and after integration at the
telomeric ends of C. lucknowense chromosomes (C).
[0228] FIG. 2 is a schematic representation of various telomeric
vectors described in the Examples (pyrE, orotate phosphoribosyl
transferase encoding gene of Aspergillus niger; pyrG,
orotidine-5'-phosphate-decarboxylase encoding gene of A. oryzae;
PgpdA, Promoter region of the glyceraldehyde-3-phosphate
dehydrogenase gene of A. nidulans; TtrpC, trpC terminator of A.
nidulans; Pcbh1, promoter of the cellobiohydrolase encoding gene of
C. lucknowense; Tcbh1, terminator of the cellobiohydrolase encoding
gene of C. lucknowense; glaA, the glucoamylase encoding gene of A.
niger; lac1, modified laccase encoding cDNA clone from Pycnoporus
cinnabarinus. Only the restriction sites essential for construction
and insert recovery (Bold) are indicated).
[0229] FIGS. 3A and B show the following digital images: (A) Colony
morphology of UV18#100.f.DELTA.pyr5.DELTA.pyr4[pAN52 pyrE tel pyrG]
on selective medium of after regeneration (left; ragged) and
propagule formation (right, smooth). (B) Southern analysis of
genomic DNA isolated from UV18#100.f.DELTA.pyr5.DELTA.pyr4 (9) and
four UV18#100.f.DELTA.pyr5.DELTA.pyr4[pAN52 pyrE tel pyrG]
transformants with a ragged (1-4) and smooth (5-8) phenotype.
Genomic DNA was separated on a 0.8% TEA-agarose gel and transferred
to a Nylon membrane. The filter was probed with .sup.32P-labeled
vector pAN52 pyrE tel pyrG.
[0230] FIGS. 4A-C show the following images: (A) Schematic
representation of the in vivo linearized derivative of the
telomeric vector pPgpdA-TtrpC pyrE tel pyrG. (B) The schematic
representation of situations after stable integration of the
telomeric vector within or near the telomeric regions of the C.
lucknowense chromosomes. (C) A digitized image of a Southern
analysis of NotI digested chromosomal DNA of five
UV18#100.f.DELTA.pyr5.DELTA.pyr4[pPgpdA-TtrpC pyrE tel pyrG]
transformants probed with specific probes for pyrE (left), pyrG
(middle) and PgpdA-TtrpC. The positive control is 10 ng of the
vector pPgpdA-TtrpC pyrE tel pyrG digested with I-CeuI and
NotI.
[0231] FIGS. 5A-D show ABTS oxidation assay plates of various
medium samples. (A) Medium samples of 12 randomly selected
UV18#100.f.DELTA.pyr5.DELTA.pyr4[pPcbh1-Tcbh1 pyrE tel pyrG] (top)
and 24 randomly selected
UV18#100.f.DELTA.pyr5.DELTA.pyr4[pPcbh1-lac1-TtrpC pyrE tel pyrG]
transformants. (B) Medium samples of a screening plate for laccase
activity. Propagules of UV18#100.f.DELTA.pyr5.DELTA.pyr4[pAN52 pyrE
tel pyrG] and UV18#100.f.DELTA.pyr5.DELTA.pyr4 [pPcbh1-lac1-TtrpC
pyrE tel pyrG] were mixed such that one well in six was expected to
have a positive hit. (C) Medium samples of 27 Pyr.sup.+
co-transformants of UV18#100.f.DELTA.pyr5 [pPcbh1-lac1-TtrpC
rescue/pBlue-pyrE].
[0232] FIG. 6 is a flow chart of the integration of the individual
steps for High Throughput Screening in Chrysosporium lucknowense C1
using the Allegro.TM. and Staccato.TM. robotic liquid handling
systems from Caliper Lifesciences (Hopkinton, USA).
[0233] FIG. 7 is a map of pPcbh1-glaA(II)-Fc.
[0234] FIG. 8 is a digitized image showing expression of a
glucoamylase-Fc (Gla-Fc) fusion in C. lucknowense. A Coomassie Blue
stained polyacrylamide gel of filtrate from a fermentation culture
(lane 5) and proteins purified by protein A chromatography (lanes
E1 through E5). As indicated by the gel analysis, the protein
eluting from the protein A column was highly enriched for the
Gla-Fc protein, with a purity exceeding 90%.
[0235] FIG. 9 is a digitized image showing in vitro stability of
antibodies against C. lucknowense fermentation culture filtrates as
analyzed by Western analysis. Human IgG1 was mixed with culture
filtrates of a .DELTA.alp1(single deletion) and a
.DELTA.alp1.DELTA.pep4.DELTA.alp2 (triple deletion) strain for the
times indicated.
[0236] FIG. 10 is a schematic drawing of expression plasmids used
to construct C. lucknowense strains expressing full-length human
antibodies.
[0237] FIG. 11 is a digitized image of a polyacrylamide gel
analysis of culture filtrate from a C. lucknowense strain
expressing full-length antibody. Lane 1 is unpurified culture
filtrate. Lane 2 indicates culture filtrate purified by protein A
chromatography.
[0238] FIG. 12 is a schematic drawing of two plasmids for use in
screening of libraries of libraries for antibody binding.
[0239] FIGS. 13-14 are schematic drawings showing a recombinant
vector strategy using recombination sites and showing fusion of
protein elements with processing sites.
[0240] FIGS. 15-18 are schematic drawings showing alternate
recombinant vector strategies using recombination sites.
[0241] FIG. 19 is a schematic drawing illustrating the use of a
double marker vector for screening of gene libraries and subsequent
recovery of DNA from hits.
[0242] FIG. 20 is a schematic drawing of plasmids expressing heavy
and light chains of human IgG fused to A. niger glucoamylase with a
kex2 site, pyrE, pyrG and telomeric sequence.
[0243] FIGS. 21A and B illustrate the detection of transformants
that produce the dimer of the IgG1 heavy chains by spotblot
analysis using an AP-conjugated anti-Fc antibody (A) and by ELISA
analysis using a protein A coated plate (B).
[0244] FIGS. 22A-22C show Protein A column purified antibodies
produced by transformants, as detected by Western blotting with
antibodies against heavy chain (A) and light chain (B) or by
protein staining (C). From left to right in panels A and B, M--kDa
marker, 1--antibody processing control strain
UV18#100f.DELTA.alp1[88/90]#58, 2--transformant
UV18#100f.DELTA.alp1[88+90 pyrE tel pyrG]#E8,
3--UV18#100f.DELTA.alp1[88+90 pyrE tel pyrG]#E2 and 4--negative
control strain UV18#100f.DELTA.alp1[pyrE tel pyrG]. From left to
right in panel C, M--kDa marker, then the first three fractions,
respectively, of 1--negative control strain
UV18#100f.DELTA.alp1[pyrE tel pyrG], 2--transformant
UV18#100f.DELTA.alp1[88+90 pyrE tel pyrG]#E2,
3--UV18#100f.DELTA.alp1[88+90 pyrE tel pyrG]#E2, 4--antibody
processing control strain UV18#100f.DELTA.alp1[88/90]#58 and 5--two
lanes of IgG control protein (14 and 28 ng).
[0245] FIG. 23 shows a partial nucleotide sequence of the
terminator region of cbh1 (Tcbh1). The C1 repetitive sequence (CRS)
is indicated in gray.
[0246] FIG. 24 shows a schematic representation of the eg2
expression vector Pcbh-eg2-Tcbh.
[0247] FIG. 25 shows a Southern blot on chromosomal DNA digested
with NcoI from UV18[EG3#20[pyr4] and cotransformants with
additional copies of an eg2 expression cassette with CRS (left
panel) and without CRS in Tcbh1 (right panel) using an internal eg2
(BstEII-EcoRV) fragment as a probe.
[0248] FIG. 26 shows a SDS-page gel on supernatant from
fermentations (10 .mu.l loaded on gel from 10.times. dilution).
[0249] FIG. 27 shows a schematic of the vector Pcbh1 glaA(II) heavy
(88) Tcbh1 Pcbh1 glaA(II) light (90) Tcbh1 pyrE tel pyrG. The
sequence of the complete vector is represented as SEQ ID NO:10. The
vector includes the following features at the indicated sequence
locations within SEQ ID NO:10:1-1324 pyrE flanking region;
1324-2171 pyrE; 3887-4217 pyrE flanking region; 4236-4811 hTEL;
4830-4834 I-CeuI; 4857-5432 hTEL; 5687-7030 pyrG flanking region;
7031-7932 pyrG; 7933-8498 pyrG flanking region; 8520-8703 Pgpd;
8704-8711 Tcbh1; 9500-10360 ampR; 11601-13397 Pcbh1; 13398-15286
Gla(GII) kex2; 15287-16730 heavy (88); 16731-17746 Tcbh1;
17757-19553 Pcbh1; 19554-21442 Gla(GII) kex2; 21443-22173 light
(90); and 22173-23188 Tcbh'1.
DETAILED DESCRIPTION OF THE INVENTION
[0250] In its broadest aspect, the invention is directed to
transformed filamentous fungi that generate transferable
reproductive elements in suspension, to libraries of such fungi,
and to methods of screening such libraries for biological
properties of interest, such as biochemical or biological activity
associated with expressed heterogeneous or heteromultimeric complex
proteins comprised of two or more domains, subunits or components
(multimeric proteins). Combination expression and screening methods
useful in the context of expressing and evaluating such complex
proteins are particularly described herein. The library of
low-viscosity filamentous fungi comprises fungi containing nucleic
acid sequences, each nucleic acid sequence encoding a heterologous
protein or proteins, which particularly includes heterogeneous or
heteromultimeric complex proteins comprised of two or more domains,
subunits or components, each of said nucleic acid sequences being
operably linked to an expression regulating region and optionally a
secretion signal encoding sequence and/or a carrier protein
encoding sequence. Preferably a transformed strain according to the
invention will secrete the heterologous protein(s).
[0251] The present invention combines expression technology with
screening technology in filamentous fungal hosts (including, but
not limited to, high throughput screening abilities) to produce,
express, and/or screen complex libraries (including libraries of
libraries) to obtain sufficient quantities of protein to allow for:
(i) identification of new or best candidate proteins among a pool,
including a very large pool of proteins; (ii) improvement of the
new or best candidates or a pool of candidates by directed
evolution techniques and/or synthetic DNA modifications to engineer
advantageous properties into the proteins; and (iii) manufacturing
the proteins produced and selected by these methods in: (a)
preclinical quantities, (b) clinical quantities, and/or (c)
production quantities for sale. These elements of the invention can
all be performed in the same, filamentous fungal system, or
alternatively, subsequent to (i) and/or (ii), or possibly (a)
and/or (b), the production of the proteins can be readily
transferred to a different system (e.g., a mammalian, yeast,
bacterial, plant, algae or other filamentous fungal system), for
example, a system that may already be approved by the FDA or
foreign equivalent and have a proven track record of safety, and
production capability. Details of the expression and screening
systems encompassed by this invention are described below.
[0252] By way of example of expression and screening of complex,
heterogenous/heteromultimeric proteins, the present inventors have
demonstrated herein that active antibodies can be expressed and
screened using the methods of the invention, and in particular, the
inventors have demonstrated the combination of: (i) expression
technology, (ii) screening technology and (iii) production of
active antibodies in a state or form that will allow for the rapid
production of sufficient quantities of antibodies (and other
proteins), all in a filamentous fungal host system of the
invention. The inventors have demonstrated and described the
ability to generate and express improved complex libraries (e.g.,
libraries of libraries) for selecting optimal or preferred
candidates, overcoming problems associated with library size versus
efficiency of screening and production, and overcoming problems
associated with the ability to achieve the end result of producing
usable quantities of selected proteins in a cost-effective and
commercially viable manner. The present invention allows for the
efficient and rapid production of sufficient quantities of selected
proteins to carry on research in a timely manner, which accordingly
enables rapid discovery of the best candidates for research,
commercial, and clinical applications. Prior to the present
invention, because such an efficient and flexible expression and
screening system was lacking, the best candidates from complex or
large libraries may have been overlooked and inferior candidates
brought forth instead as less effective drugs/agents. In addition,
drugs with intolerable side effects have been problematic, and new
indications for promising drug candidates were missed due to the
inefficiencies and economic considerations associated with prior
methods. The present invention provides a method that results in
more efficient and effective research and development strategies
for new drugs/agents, which can lower the cost of healthcare and
increase the supply of the drug/agent to more people, more
affordably, worldwide.
[0253] The characteristics of a fungal host cell suitable for
expression of a DNA library are different in many respects from the
characteristics of hosts suitable for industrial protein
manufacture. In general terms, a suitable fungal host for
high-throughput screening should meet numerous criteria; among them
are the following:
[0254] The host should be transformed with high efficiency.
[0255] The host should process intron-containing genes and carry
out any necessary splicing.
[0256] The host should post-translationally process the expressed
protein so that it is produced in an active form.
[0257] Where the library is to be assayed for a protein, the host
should produce the protein in high enough yield for detection by
the assay.
[0258] The host should accept a variety of expression regulatory
elements, for ease of use and versatility.
[0259] The host should permit the use of easily-selectable
markers.
[0260] The host cell cultures should be of low viscosity.
[0261] The host should be deficient in proteases and/or be amenable
to suppression of protease expression.
[0262] The host should permit screens for a wide variety of
exogenous protein activities or properties.
[0263] The host should also permit screening of protein activities
and/or characteristics from heterogeneous and heteromultimeric
protein complexes, such as antibodies and fragments/domains thereof
and other heterodimeric or heterogeneous or heteromultimeric
receptors.
[0264] The host should also allow large-scale fermentation of
selected proteins without further modifications or without the
requirement to transfer the nucleic acid sequences encoding the
proteins into a different host.
[0265] The hyphae in a culture of the host fungus should not be so
entangled as to prevent the isolation of single clones, and should
not be so entangled as to raise the viscosity to the point of
preventing efficient transfer and replication in a miniaturized
high throughput screening format (e.g., by micropipeting).
[0266] The host should not form surface mats, but should
preferentially grow as a submerged culture.
[0267] The host should allow the efficient production of submerged
spores or other propagules under the growth conditions provided in
the high throughput screen.
[0268] In cases where metabolites are being screened for, it would
be advantageous if the host cells secreted the metabolites into the
medium, where they could be readily detected and/or assayed.
Ideally, the host should secrete only the exogenous protein.
[0269] In cases where a protein is being assayed for, it would be
particularly advantageous if the host also expressed enough
heterologous protein to enable isolation and purification of the
protein. A host cell with this characteristic would make it
possible to further characterize all heterologous proteins of
interest merely by culturing the host cells, without the
time-consuming molecular biological manipulations needed to
transfer the gene to another organism. Preferably, the host should
be capable of secretion of the protein, as this would permit more
reliable and more varied assays.
[0270] In cases where the protein to be screened is comprised of
two or more heterogeneous components (e.g., immunoglobulins), it
would be advantageous if the host was versatile enough to
efficiently and effectively express large combinatorial
libraries.
[0271] It would also be advantageous if the host cell were amenable
to ready isolation of the heterologous DNA, so that further studies
and modifications of the genes themselves may be carried out.
[0272] In addition to these qualities of the host, the
transformation system should also exhibit certain characteristics.
The transformation frequency should be sufficiently high to
generate the numbers of transformants required for meaningful
screens, and should be highly adaptable to accommodate single,
double or multiple expression control regions. The present
invention provides novel fungal host cells and vectors for use with
such host cells, that meet the criteria above and substantially
improve the ability to express and screen for desired products
using such fungal host cells. In particular, as discussed above,
the present invention provides improved systems for the production
and screening of libraries comprising one, two or more variable
constituents and/or prepared from two or more sublibraries (e.g.,
for the expression and screening of immunoglobulin (including
fragments and derivatives of whole immunoglobulin proteins) and
other receptor or complex DNA libraries or libraries of
libraries).
[0273] The present invention employs filamentous fungi which
produce "transferable reproductive elements" when grown in
submerged culture. By "transferable reproductive element" is meant
a spore, propagule, hyphal fragment, protoplast, micropellet, or
other fungal element that is (1) readily separated from other such
elements in the culture medium, and (2) capable of reproducing
itself into a monoclonal culture. The fungi preferably also exhibit
a less pronounced filamentous phenotype and/or a compact growth
morphology, have low protease activity, and/or produce
low-viscosity cultures that are suitable for the physical
manipulations involved in high-throughput DNA library screening.
Particularly preferred are filamentous fungi that, even in the
absence of agitation, tend to grow as submerged cultures rather
than as surface mats. Other preferred filamentous fungi include
those that have improved homologous integration and/or reduced
non-homologous integration of exogenously introduced DNA, as
compared to a wild-type strain. Any combination of these features
is useful in filamentous fungi of the invention, but the invention
is not limited to the use of filamentous fungi with these
characteristics.
[0274] The term "yeast" as used in the context of yeast expression
systems generally refers to organisms of the order
Saccharomycetales, such as S. cerevisiae and Pichia pastoris. For
the purposes of this disclosure, the terms "fungi" and "fungal"
should be understood to refer to Basidiomycetes, Zygomycetes,
Oomycetes, and Chythridiomycetes, and Ascomycetes of the class
Euascomycetes, which are not of the order Saccharomycetales.
Filamentous fungi may be distinguished from yeast by their hyphal
elongation during vegetative growth, and obligately aerobic carbon
catabolism (vegetative growth in yeast is accomplished by budding
from a unicellular thallus, and yeast may employ fermentative
catabolism.)
[0275] The present invention takes advantage of the properties of
the transformation system disclosed in international patent
applications PCT/NL99/00618 and PCT/EP99/202516, and further
described in U.S. patent application Ser. No. 09/548,938, now U.S.
Pat. No. 6,573,086 and in U.S. patent application Ser. No.
09/834,434, now U.S. Pat. No. 7,122,330, and describes significant
improvements to these systems. These previous applications describe
a transformation system for filamentous fungal hosts including, but
not limited to, Chrysosporium lucknowense, Aspergillus sojae and
Trichoderma. These applications also disclose that mutant strains
are readily prepared which retain all the advantages of the
wild-type host cells, but which have partially lost their
filamentous phenotype and thus provide low-viscosity cultures.
[0276] While the present invention may, in some embodiments,
utilize fungal host strains such as those described above, one
aspect of the invention provides improved fungal host strains for
use in expression and screening systems. Particularly preferred
embodiments of the invention are related to improved fungal host
strains with low protease activity, with improved homologous
integration of exogenous DNA (improved homologous recombination),
and/or with reduced non-homologous integration of exogenous DNA
(reduced non-homologous recombination).
[0277] The fungi preferred for use in the invention express and
secrete large amounts of exogenous protein, producing a high
protein/biomass ratio relative to previously known filamentous
fungal hosts. The invention provides a transformation system that
exhibits high yields of transformants. Improved fungal host strains
of the invention are described in more detail below and in the
Examples.
[0278] The invention also provides libraries of transformant fungi
that efficiently express the protein products of heterologous cDNA
inserts, and especially genomic DNA inserts. In particular, the
invention provides libraries of transformant fungi that efficiently
express heterogeneous or heteromultimeric proteins (complex
proteins). As used herein, the terms "heterogeneous" and
"heteromultimeric" can be used interchangeably to describe proteins
that can be produced using the host cells and methods of the
invention. Heterogeneous or heteromultimeric proteins are defined
as any protein having two or more different or variable domains,
subunits, components or constituents (i.e., the domains, subunits,
components or constituents are variable, different, or
heterogeneous with respect to each other). Heteromultimeric may be
more commonly used to describe proteins with two or more different
subunits or constituents, while heterogeneous may be more commonly
used to describe single proteins with more than one domain or
variable region. As such, a heterogeneous protein can include a
single protein with more than one domain, and can include a protein
with at least one variable domain (the structure of the domain may
vary from protein to protein). Heteromultimeric proteins include,
but are not limited to, members of the immunoglobulin supergene
family (e.g., immunoglobulin, major histocompatibility complex, T
cell receptors, CD3, adhesion molecules), hormones and hormone
receptors, cytokines and cytokine receptors, other growth factors
and growth factor receptors, etc.
[0279] Other embodiments of the invention are related to a variety
of improved, self-replicating vectors that enable the construction
of complex libraries in fungal host strains as described herein.
The vectors enable very high transformation frequencies and have
allowed the successful integration of library construction and
screening in an automated set-up. Moreover, use of the improved
vectors of the invention allow for reduced variation in the
expression levels between independent or individual transformants,
and offer flexibility in transferring the libraries from one
organism to another. These vectors are described in detail below
and in the Examples.
[0280] In another aspect of the invention, the libraries of
transformed fungi, and particularly those utilizing the improved
fungal host strains and/or vectors described herein, may be used in
screening for activities or properties of any heterologous
proteins, or in screening for metabolites produced by the
transformed fungi as a consequence of exogenous protein activities,
or in screening for the heterologous DNA or for RNA transcripts
derived therefrom. It will be appreciated that the present
invention also enables high-throughput screening for metabolites of
non-transformed strains having the phenotypic characteristics
described above. It will be further appreciated that the present
invention enables the expression and high-throughput screening for
a variety of heterologous proteins, including many
heterogeneous/heteromultimeric complex proteins comprised of two or
more domains, subunits or components (multimeric proteins).
[0281] In yet another aspect of the invention, the libraries of
transformed fungi may be screened for useful properties of the
fungi themselves, such as for example high levels of production of
a particular expressed protein, protein complex, or metabolite.
This aspect of the invention is illustrated by a quantitative assay
for the expressed protein of interest, where the particular
transformant having the most favorable combination of protein
production, protein processing, and protein secretion would be
detected.
[0282] In another aspect of the invention, the libraries of
transformed fungi may be screened for the presence of DNA sequences
capable of hybridizing to a nucleic acid probe of interest.
[0283] In a particularly preferred aspect of the invention,
transformed fungi as described herein are used to express and
screen proteins produced from complex or combinatorial libraries
(e.g., libraries of libraries), which include a large variety of
heterogeneous or heteromultimeric proteins (e.g., immunoglobulins
and fragments thereof, and other receptors, as non-limiting
examples).
[0284] The expression and screening methods of the invention, and
the fungi employed therein, are useful for producing proteins
(including mammalian proteins and protein complexes), metabolites,
and DNA molecules having utility in a variety of applications,
including a variety of diagnostic and therapeutic applications for
use in humans. The methods of the invention are also useful for
producing nucleic acid and protein sequence information, and this
information itself is regarded as a valuable product of the claimed
methods.
[0285] Another embodiment of the invention relates to the
integration of the expression of complex or variable proteins with
high-throughput screening of the invention, as exemplified by, but
not limited to, immunoglobulin expression, to select improved,
targeted, or desired proteins and/or protein complexes having two
or more different or variable domains, subunits or constituents
(e.g., human monoclonal antibodies) by combining libraries encoding
one constituent (e.g., light chain variants) with libraries
encoding the other constituent (e.g., heavy chain variants), with
individual clones of the resulting combinatorial libraries
expressing both constituents (e.g., full-length or single chain
antibodies). Since the strain used for screening can also be used
to express these complex proteins, positive hits from the screen
can be cultivated to produce enough protein for further biochemical
and biological investigation. Isolation of the cloned DNA from
those strains and subsequent recloning for maximum expression will
lead to economical production of such proteins for potential
clinical applications, including diagnostic, prognostic, and
therapeutic applications. Moreover, the methods of the invention
can be used for molecular evolution of such proteins, which can be
used to produce highly effective reagents for such applications.
These methods of the invention are high throughput expression and
screening methods, which are invaluable for the rapid
identification of a variety of therapeutically beneficial
tools.
Preferred Host Strains for Expression and Screening
[0286] The present invention includes fungal strains that either
naturally exhibit decreased protease activity or that have been
mutated or selected to have low protease activity. In certain
embodiments, the invention includes a fungus that has less than
50%, less than 10%, or less than 1% of the protease activity as
compared to a non-mutated, non-selected or wild-type fungus. The
invention also includes fungal strains in which one or more
protease genes have been inactivated or disrupted, as described in
greater detail below.
[0287] Preferred filamentous fungi of the invention are
characterized by the low viscosity of the culture medium. Whereas a
typical industrial-grade filamentous fungus will typically produce
cultures with viscosities well over 200 centipoise (cP) and usually
over 1,000 cP, and can reach over 10,000 cP, the fungi of this
invention exhibit a culture viscosity of less than 200 cP,
preferably less than 100 cP, more preferably less than 60 cP, and
most preferably less than 10 cP after 48 or more hours of culturing
in the presence of adequate nutrients under optimal or near-optimal
growth conditions. The filamentous fungi of the invention usually
exhibit a morphology characterized by short, discrete,
non-entangled hyphae, or micropellets. Micropellets are slightly-
or non-entangled collections of hyphae arising from a single clone,
as distinct from pellets which are much larger and are derived from
multiple entangled clones. For example, the mutant UV18-25
Chrysosporium lucknowense strain (viscosity<10 cP) and the
morphologically similar mutant Trichoderma longibrachiatum X-252
strain (viscosity<60 cP) are characterized by the presence of
short, distinct, non-entangled hyphae between 100 and 200 microns
in length, and the low viscosity engineered mutant Aspergillus
sojae pclA is characterized by a compact form with considerable
branching and short hyphae (see, e.g., FIG. 14 of U.S. Pat. No.
7,122,330). Whereas the low-viscosity fungi described in WO
97/26330 are described as having "more extensive hyphal branching,"
some fungi of the present invention have equivalent or even
slightly reduced hyphal branching when compared to the non-mutant
strains. It appears that hyphal length plays the dominant role in
controlling the viscosity of the culture.
[0288] Particularly preferred fungal strains are characterized by
having a high exogenous secreted protein/biomass ratio. This ratio
is preferably greater than 1:1, more preferably greater than 2:1,
more preferably greater than 3:1, more preferably greater than 4:1,
more preferably greater than 5:1, and even more preferably 6:1 or
greater. Most preferably, the ratio is 8:1 or higher. Such high
ratios are advantageous in a high-throughput screening environment,
because they result in a higher concentration of exogenous protein,
allowing more sensitive and/or more rapid screening assays. This is
of particular benefit as the volume of the assay solution
decreases, for example upon going from 96-well plates to 384-well
plates, and thence to 1536-well plates. The methods of the present
invention are suitable for any of these microtiter plate formats,
and for most other HTS formats employing liquid samples.
[0289] It is contemplated that any filamentous fungus can be
converted, by the processes of mutation described herein, into
mutant strains suitable for use in the present invention. Among the
preferred genera of filamentous fungi are the Chrysosporium,
Thielavia, Neurospora, Aureobasidium, Filibasidium, Piromyces,
Cryplococcus, Acremonium, Tolypocladium, Scytalidium,
Schizophyllum, Sporotrichum, Penicillium, Gibberella,
Myceliophthora, Mucor, Aspergillus, Fusarium, Humicola, and
Trichoderma, and anamorphs and teleomorphs thereof. More preferred
are Chrysosporium, Myceliophthora, Trichoderma, Aspergillus, and
Fusarium. One preferred genus of fungus is Chrysosporium, although
the invention is in no way limited to this genus. The genus and
species of fungi can be defined by morphology consistent with that
disclosed in Barnett and Hunter, Illustrated Genera of Imperfect
Fungi, 3rd Edition, 1972, Burgess Publishing Company. A source
providing details concerning classification of fungi of the genus
Chrysosporium is Van Oorschot, C.A.N. (1980) "A revision of
Chrysosporium and allied genera" in Studies in Mycology No. 20,
Centraal Bureau voor Schimmelcultures (CBS), Baarn, The
Netherlands, pp. 1-36. According to these teachings the genus
Chrysosporium falls within the family Moniliaceae that belongs to
the order Hyphomycetales.
[0290] Another ready source providing information on fungal
nomenclature is the Budapest Treaty depositories, especially those
providing online databases (the following internet addresses employ
the http protocol). The ATCC (US) provides information at
www.atcc.org, the CBS (NE) at www.cbs.knaw.nl, and the VKM (RU) at
www.bdt.org.br.bdt.msdn.vkm/general. Another source is
NT.ars-grin.gov/fungaldatabases. All these institutions can provide
teaching on the distinguishing characteristics of fungal species.
An alternate taxonomy of the Ascomycota may be found at
www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Undef&id=4890.
According to this alternate taxonomy, the genus Chrysosporium
belongs to family Onygenaceae, order Onygenales, phylum
Ascomycota.
[0291] The definition of Chrysosporium includes but is not limited
to these strains: C. botryoides, C. carmichaelii, C.
crassitunicatum, C. europae, C. evolceannui, C. farinicola, C.
fastidium, C. filiforme, C. georgiae, C. globiferum, C. globiferum
var. articulatum, C. globiferum var. niveum, C. hirundo, C.
hispanicum, C. holmii, C. indicum, C. inops, C. keratinophilum, C.
kreiselii, C. kuzurovianum, C. lignorum, C. lobatum, C.
lucknowense, C. lucknowense Garg 27K, C. medium, C. medium var.
spissescens, C. mephiticum, C. merdarium, C. merdarium var. roseum,
C. minor, C. pannicola, C. parvum, C. parvum var. crescens, C.
pilosum, C. pseudomerdarium, C. pyriformis, C. queenslandicum, C.
sigleri, C. sulfureum, C. synchronum, C. tropicum, C. undulatum, C.
vallenarense, C. vespertilium, C. zonatum.
[0292] C. lucknowense is a species of Chrysosporium that is of
particular interest as it has provided a natural high producer of
cellulase proteins (international applications WO 98/15633,
PCT/NL99/00618, and U.S. Pat. Nos. 5,811,381 and 6,015,707).
Strains with international depository accession numbers ATCC 44006,
CBS 251.72, CBS 143.77, CBS 272.77, and VKM F-3500D are examples of
Chrysosporium lucknowense strains. Also included within the
definition of Chrysosporium are strains derived from Chrysosporium
predecessors including those that have mutated either naturally or
by induced mutagenesis. The methods of the invention, in one
embodiment, employ mutants of Chrysosporium, obtained by a
combination of irradiation and chemically-induced mutagenesis, that
tend to produce transferable reproductive elements in suspension,
and that exhibit a morphology characterized by short, discrete,
non-entangled hyphae ("compact growth"), and a phenotype
characterized by submerged growth and reduced viscosity of the
fermentation medium when cultured in suspension. In another
embodiment, the invention employs phenotypically similar mutants of
Trichoderma. In another embodiment, the invention employs
phenotypically similar mutants of Myceliophthora. In yet other
embodiments the invention employs phenotypically similar mutants of
Aspergillus sojae or Aspergillus niger. The invention also includes
any strains derived from predecessors of any fungi described herein
including those that have mutated either naturally or by induced
mutagenesis, as well as any derivatives or mutants of any of the
fungi described herein.
[0293] In one aspect, the present invention includes a fungus such
as C. lucknowense or a mutant or other derivative thereof, and more
particularly, from the fungal strain denoted herein as C1
(Accession No. VKM F-3500-D). The invention also includes proteins
expressed by the C1 fungus. As described in U.S. Pat. No. 6,015,707
or U.S. Pat. No. 6,573,086, each of which is incorporated herein by
reference for all purposes, a strain called C1 (Accession No. VKM
F-3500-D), was isolated from samples of forest alkaline soil from
Sola Lake, Far East of the Russian Federation. This strain was
deposited at the All-Russian Collection of Microorganisms of
Russian Academy of Sciences (VKM), Bakhurhina St. 8, Moscow,
Russia, 113184, under the terms of the Budapest Treaty on the
International Regulation of the Deposit of Microorganisms for the
Purposes of Patent Procedure on Aug. 29, 1996, as Chrysosporium
lucknowense Garg 27K, VKM-F 3500 D. Various mutant strains of C.
lucknowense C1 have been produced and these strains have also been
deposited at the All-Russian Collection of Microorganisms of
Russian Academy of Sciences (VKM), Bakhurhina St. 8, Moscow,
Russia, 113184, under the terms of the Budapest Treaty on the
International Regulation of the Deposit of Microorganisms for the
Purposes of Patent Procedure on Sep. 2, 1998. For example, Strain
C1 was mutagenized by subjecting it to ultraviolet light to
generate strain UV13-6 (Accession No. VKM F-3632 D). This strain
was subsequently further mutated with
N-methyl-N'-nitro-N-nitrosoguanidine to generate strain NG7C-19
(Accession No. VKM F-3633 D). This latter strain in turn was
subjected to mutation by ultraviolet light, resulting in strain
UV18-25 (VKM F-3631 D). Strain C1 was classified as a Chrysosporium
lucknowense based on morphological and growth characteristics of
the microorganism, as discussed in detail in U.S. Pat. No.
6,015,707 and U.S. Pat. No. 6,573,086. Additional information
concerning the generation of mutant C1 strains and their
characteristics can be found in U.S. Pat. No. 7,399,627.
[0294] In particular the anamorph form of Chrysosporium has been
found to be suited for the screening application according to the
invention. The metabolism of the anamorph renders it particularly
suitable for a high degree of expression. A teleomorph should also
be suitable as the genetic make-up of the anamorphs and teleomorphs
is identical. The difference between anamorph and teleomorph is
that one is the asexual state and the other is the sexual state;
the two states exhibit different morphology under certain
conditions.
[0295] Another example embodies genetically engineered mutant
strains of Aspergillus sojae. In one of these mutants a specific
endoprotease encoding gene was disrupted. This resulted in a
compact growth phenotype exhibiting enhanced branching and short
hyphae, and the formation of micropellets in submerged cultivation.
Moreover, the Aspergillus sojae referred to in this application may
be induced to exhibit efficient sporulation under specific
submerged cultivation conditions, which renders it especially
suitable for use in a high-throughput screening system. In this
case, the conditions conducive to formation of the transferable
reproductive elements simply consisted of a synthetic medium
containing 0.6 g/kg EDTA. The conducive conditions will vary from
one host to another, but it is evident that the conditions will
already be known if a host has been found to be suitable.
[0296] The term "mutant filamentous fungus" as used herein refers
simply to fungi not found in nature. The "mutations" that lead to
desirable phenotypic characteristics, such as a compact growth
form, low viscosity, reduced protease levels, submerged growth,
improved homologous integration of DNA, reduced non-homologous
integration of DNA, etc., may be introduced randomly by either
classical means, such as UV irradiation and chemical mutagenesis,
or by molecular biological methods such as cassette mutagenesis, or
may be deliberately introduced by genetic engineering methods.
Should a naturally occurring fungus be found to possess the
necessary properties, it will of course be usable in the methods of
the invention.
[0297] It is preferable to use non-toxigenic and non-pathogenic
fungal strains, of which a number are known in the art, as this
will reduce risks to the practitioner and will simplify the overall
screening process. In a preferred embodiment the fungi will also be
protease deficient, so as to minimize degradation of the exogenous
proteins, and/or amenable to suppression of protease production.
The use of protease deficient strains as expression hosts is well
known; see for example PCT application WO 96/29391. Protease
deficient strains may be produced by screening of mutants, or the
protease gene(s) may be "knocked out" or otherwise inactivated by
methods known in the art, as described for example by Christensen
and Hynes in U.S. Pat. No. 6,025,185 (Aspergillus oryzae with
non-functional areA gene).
[0298] It has been found that mutant fungal strains of the
invention, including Chrysosporium mutant strains, can be made that
have reduced expression of protease (e.g., extracellular protease),
thus making them even more suitable for the production of
proteinaceous products, especially if the proteinaceous product is
sensitive to protease activity. Thus the invention may also employ
a mutant fungal strain that produces less extracellular protease
than the corresponding non-mutant fungal strain, for example less
than C. lucknowense strain C1 (VKM F-3500 D). An example of such a
novel mutant strain is described in detail herein. In particular,
the protease activity (other than any selective protease intended
to cleave a secreted fusion protein) of such strains is less than
half the amount, more preferably less than 30% of the amount, and
more preferably less than about 10%, and more preferably less than
about 9%, and more preferably less than about 8%, and more
preferably less than about 7%, and more preferably less than about
6%, and more preferably less than about 5%, and more preferably
less than about 4%, and more preferably less than about 3%, and
more preferably less than about 2%, and more preferably less than
about 1% of the amount produced by the wild-type, or non-mutant
reference strain. The decreased protease activity can be measured
by known methods, such as by measuring the halo formed on skim milk
plates or by bovine serum albumin (BSA) degradation. Such strains
can be produced by selection of natural mutants, by random or
directed mutagenesis followed by screening, or by directed genetic
modification (genetic engineering), such as by gene deletion or
disruption.
[0299] By way of example, new mutant fungal strains useful in the
present invention are described herein, where the strains were
mutated and the selected for the characteristic of low protease
activity. In a further example, mutant Chrysosporium strains,
described below, were produced by the selection process described
above, and then further selective disruption of at least one, and
up to all three of the protease genes, alp1, pep4, and alp2.
Nucleotide and amino acid sequences from each of these genes are
provided herein. Random or classical mutagenesis and screening can
be used alone or in combination with genetic engineering to readily
produce additional low protease mutants. These protease mutants may
be obtained not only by disrupting actual protease encoding genes;
in addition, strains mutated in genes that regulate protease genes
may result in the desired type of strains. These regulatory genes
may include those encoding for protease-specific transcription
factors as well as broadly acting transcription factors. These low
protease strains are highly useful for the production of proteins,
and particularly large proteins and protein complexes, and are
described in more detail in the Examples section.
[0300] Exemplary low protease strains of the present invention,
include, but are not limited to, a mutant of strain UV18-25, which
has been mutated with ultraviolet light and selected for low
protease activity, denoted herein as UV18#100.f (deposited at the
Centraalbureau voor Schimmelcultures (CBS) in the Netherlands under
the terms of the Budapest Treaty on the International Regulation of
the Deposit of Microorganisms for the Purposes of Patent Procedure
on Dec. 5, 2007, as Chrysosporium lucknowense UV18#100.f, CBS
122188); a strain denoted UV18#100.f .DELTA.pyr5 .DELTA.alp1, a
mutant of UV18#100.f in which the protease gene alp1 has been
selectively disrupted (and/or .DELTA.alp2 or .DELTA.pep4); and a
strain denoted UV18#100.f .DELTA.pyr5 .DELTA.alp1 .DELTA.pep4
.DELTA.alp2, another mutant of UV18#1001 in which three protease
genes, alp1, pep4, and alp2 have been disrupted. Production of
these mutant strains is described in the Examples section. Each of
these strains represents a novel, improved fungal host strain for
use with the vectors and in the methods of the present
invention.
[0301] It may be desirable to inactivate other genes in the host
filamentous fungus, such as for example those encoding cellulases
and other abundant secreted proteins, in order to minimize
interference in the assay by host proteins. The genes encoding
secreted proteins may be deleted or mutated, or alternatively genes
controlling the induction system or other pathways involved in the
expression of unwanted proteins may be modified in such a way as to
reduce such expression. Where an endogenous promoter is employed in
the vectors of the invention (see below), it may be especially
desirable to inactivate genes for other proteins under control by
the same inducer. Fungi amenable to suppression of protease
secretion are those where protease expression is under the control
of a regulatory element that responds to environmental conditions,
such that these conditions (e.g., amino acid concentration) can be
manipulated to minimize protease production.
[0302] In another embodiment of the invention, it is desirable to
provide host filamentous fungi with an improved homologous
DNA-integration characteristic (e.g., exogenously introduced DNA)
and/or with reduced or decreased non-homologous DNA-integration
characteristics, as compared to typical or wild-type fungi. Such
strains are particularly useful for the expression and screening
methods described herein, since such strains can be used to ensure
integration of heterologous DNA (e.g., from DNA libraries) at a
directed location, and are likely to be more stable transformants
in which expression levels of the heterologous proteins can be
controlled (e.g., high expression can be achieved, whereby
expression levels from transformant to transformant is relatively
similar). Moreover, replicating vectors will have improved
stability characteristics or improved homologous integration in
these strains due to the absence of non-homologous integration.
Although these are valuable features in the expression of any
heterologous protein, such stable and controlled expression is
particularly useful when expressing and screening combinatorial
libraries, where two or more protein components must be expressed
and associated in order to evaluate a biological activity. Such
improvements to the strains may be introduced randomly by either
classical means, such as UV irradiation and chemical mutagenesis,
or by molecular biological methods such as cassette mutagenesis, or
may be deliberately introduced by genetic engineering methods.
Should a naturally-occurring fungus be found to possess the
necessary properties, it will of course be usable in the methods of
the invention.
Proteases
[0303] The present invention is also directed to C. lucknowense
strain C1 (VKM F-3500 D) enzymes with protease activity. For
example, the polypeptides denoted herein as Alp1 (SEQ ID NO:2),
Alp2 (SEQ ID NO:5), and Pep4 (SEQ ID NO:8) possess protease
activity. These enzymes participate in the hydrolysis of peptide
bonds that link amino acids together in a polypeptide chain.
Example J below demonstrates that cells deficient in each of these
enzymes exhibit reduced protease activity.
[0304] Proteases may be used in any method where it is desirable to
hydrolyze peptide bonds in polypeptides or proteinaceous materials
or any other method wherein enzymes of the same or similar function
are useful. For example, a protease of the present invention may be
incubated with a polypeptide or proteinaceous material to degrade
the material. Accordingly, the present invention includes the use
of at least one protease of the present invention, compositions
comprising at least one protease of the present invention in
methods for degrading polypeptides or proteinaceous materials. In
one embodiment, the method comprises contacting the polypeptides or
protein-containing materials with an effective amount of one or
more protease of the present invention.
[0305] In one embodiment, the knowledge of the nucleotide sequences
encoding the proteases of the invention is used to selectively
disrupt or deactivate these genes in the endogenous host. The
resulting protease-deficient strains are particularly useful in the
expression and screening methods described herein. Exemplary
protease deficient strains are discussed in detail above and in the
Examples.
Alp1
[0306] The enzyme denoted Alp1 is encoded by the genomic nucleic
acid sequence represented herein as SEQ ID NO:1 and the cDNA
sequence represented herein as SEQ ID NO:3. The Alp1 nucleic acid
sequence encodes a 392 amino acid sequence, represented herein as
SEQ ID NO:2. The signal peptide for Alp1 is located from positions
1 to about position 16 of SEQ ID NO:2, with the mature protein
spanning from about position 17 to position 392 of SEQ ID NO:2.
Within Alp1 is a catalytic domain (CD). The amino acid sequence
containing the CD of Alp1 spans from a starting point of about
position 39 of SEQ ID NO:2 to an ending point of about position 387
of SEQ ID NO:2. As demonstrated below, Alp1 exhibits protease
activity on substrates such as casein.
[0307] Alp1 has a molecular weight of 31 kDa and an isoelectric
point of 9.0. Alp1 exhibits optimal activity at a pH of about 10.5,
and exhibits at least 50% of maximum activity with a pH range of
6.5 to 11.7. Alp1 exhibits optimal activity at a temperature of
about 60.degree. C., and exhibits at least 50% of maximum activity
with a temperature range of 45.degree. C. to 67.degree. C.
Substrate specificities of Alp1 are illustrated in Table A below.
Enzyme activities and substrate specificities demonstrating enzyme
activity for the indicated substrates were determined using
standard assays known in the art.
TABLE-US-00001 TABLE A Activity Substrate (Units/mg) Casein, pH
7.0, 40.degree. C., 30 min, A275 1556 Casein, pH 9.0, 40.degree.
C., 30 min, A275 1620 Hemoglobin, pH 5.0, 40.degree. C., 30 min,
A275 140 n-Benzoyl-Phe-Val-Arg-p-Nitroanilide (trypsin-like
activity) 39.3 n-Succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide 8.1
(chymotrypsin-like activity)
n-Succinyl-Ala-Ala-Pro-Leu-p-Nitroanilide 1.3 (chymotrypsin-like
activity) p-Nph-Caproate, pH 7.3 (esterase activity) 1.5
Alp2
[0308] The enzyme denoted Alp2 is encoded by the genomic nucleic
acid sequence represented herein as SEQ ID NO:4 and the cDNA
sequence represented herein as SEQ ID NO:6. The Alp2 nucleic acid
sequence encodes a 534 amino acid sequence, represented herein as
SEQ ID NO:5. The signal peptide for Alp2 is located from positions
1 to about position 15 of SEQ ID NO:5, with the mature protein
spanning from about position 16 to position 534 of SEQ ID NO:5.
Alp2 possesses amino acid sequence homology (about 79%) with a
protease from Chaetomium thermophilum (GenBank Accession No.
ABH07518).
Pep4
[0309] The enzyme denoted Pep4 is encoded by the genomic nucleic
acid sequence represented herein as SEQ ID NO:7 and the cDNA
sequence represented herein as SEQ ID NO:9. The Pep4 nucleic acid
sequence encodes a 397 amino acid sequence, represented herein as
SEQ ID NO:8. The signal peptide for Pep4 is located from positions
1 to about position 18 of SEQ ID NO:8, with the mature protein
spanning from about position 19 to position 397 of SEQ ID NO:8.
Pep4 possesses amino acid sequence homology (about 79%) with a
protease from Neurospora crassa (Gen Bank Accession No.
AAA79878).
[0310] The present invention also provides enzyme combinations that
break down polypeptides or proteinaceous material. Such enzyme
combinations or mixtures can include a multi-enzyme composition
that contains at least one protein of the present invention in
combination with one or more additional proteins of the present
invention or one or more proteases, other enzymes or other proteins
from other microorganisms, plants, or similar organisms.
Synergistic enzyme combinations and related methods are
contemplated. The invention includes methods to identify the
optimum ratios and compositions of enzymes with which to degrade
each polypeptide or proteinaceous material. These methods entail
tests to identify the optimum enzyme composition and ratios.
[0311] Another embodiment of the present invention relates to a
composition comprising at least about 500 ng, and preferably at
least about 1 .mu.g, and more preferably at least about 5 .mu.g,
and more preferably at least about 10 .mu.g, and more preferably at
least about 25 .mu.g, and more preferably at least about 50 .mu.g,
and more preferably at least about 75 .mu.g, and more preferably at
least about 100 .mu.g, and more preferably at least about 250
.mu.g, and more preferably at least about 500 .mu.g, and more
preferably at least about 750 .mu.g, and more preferably at least
about 1 mg, and more preferably at least about 5 mg, of an isolated
protein comprising any of the proteins or homologues or fragments
thereof discussed herein. Such a composition of the present
invention may include any carrier with which the protein is
associated by virtue of the protein preparation method, a protein
purification method, or a preparation of the protein for use in any
method according to the present invention. For example, such a
carrier can include any suitable buffer, extract, or medium that is
suitable for combining with the protein of the present invention so
that the protein can be used in any method described herein
according to the present invention.
[0312] In one embodiment of the invention, one or more enzymes of
the invention is bound to a solid support, i.e., an immobilized
enzyme. As used herein, an immobilized enzyme includes immobilized
isolated enzymes, immobilized microbial cells which contain one or
more enzymes of the invention, other stabilized intact cells that
produce one or more enzymes of the invention, and stabilized
cell/membrane homogenates. Stabilized intact cells and stabilized
cell/membrane homogenates include cells and homogenates from
naturally occurring microorganisms expressing the enzymes of the
invention and preferably, from genetically modified microorganisms
as disclosed elsewhere herein. Thus, although methods for
immobilizing enzymes are discussed below, it will be appreciated
that such methods are equally applicable to immobilizing microbial
cells and in such an embodiment, the cells can be lysed, if
desired.
[0313] A variety of methods for immobilizing an enzyme are
disclosed in Industrial Enzymology 2nd Ed., Godfrey, T. and West,
S. Eds., Stockton Press, New York, N.Y., 1996, pp. 267-272;
Immobilized Enzymes, Chibata, I. Ed., Halsted Press, New York,
N.Y., 1978; Enzymes and Immobilized Cells in Biotechnology, Laskin,
A. Ed., Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif.,
1985; and Applied Biochemistry and Bioengineering, Vol. 4, Chibata,
I. and Wingard, Jr., L. Eds, Academic Press, New York, N.Y., 1983,
which are incorporated herein in their entirety.
[0314] Briefly, a solid support refers to any solid organic,
biopolymer or inorganic supports that can form a bond with an
enzyme without significantly effecting the activity of the enzyme.
Exemplary organic solid supports include polymers such as
polystyrene, nylon, phenol-formaldehyde resins, acrylic copolymers
(e.g., polyacrylamide), stabilized intact whole cells, and
stabilized crude whole cell/membrane homogenates. Exemplary
biopolymer supports include cellulose, polydextrans (e.g.,
Sephadex.RTM.), agarose, collagen and chitin. Exemplary inorganic
supports include glass beads (porous and nonporous), stainless
steel, metal oxides (e.g., porous ceramics such as ZrO.sub.2,
TiO.sub.2, Al.sub.2O.sub.3, and NiO) and sand. In one embodiment,
the solid support is selected from the group consisting of
stabilized intact cells and/or crude cell homogenates (e.g.,
produced from the microbial host cells expressing recombinant
enzymes, alone or in combination with natural enzymes). Preparation
of such supports requires a minimum of handling and cost.
Additionally, such supports provide excellent stability of the
enzyme.
[0315] Stabilized intact cells and/or cell/membrane homogenates can
be produced, for example, by using bifunctional crosslinkers (e.g.,
glutaraldehyde) to stabilize cells and cell homogenates. In both
the intact cells and the cell membranes, the cell wall and
membranes act as immobilizing supports. In such a system, integral
membrane proteins are in the "best" lipid membrane environment.
Whether starting with intact cells or homogenates, in this system
the cells are either no longer "alive" or "metabolizing", or
alternatively, are "resting" (i.e., the cells maintain metabolic
potential and active enzyme, but under the culture conditions are
not growing); in either case, the immobilized cells or membranes
serve as biocatalysts.
[0316] An enzyme of the invention can be bound to a solid support
by a variety of methods including adsorption, cross-linking
(including covalent bonding), and entrapment. Adsorption can be
through van del Waal's forces, hydrogen bonding, ionic bonding, or
hydrophobic binding. Exemplary solid supports for adsorption
immobilization include polymeric adsorbents and ion-exchange
resins. Solid supports in a bead form are particularly well-suited.
The particle size of an adsorption solid support can be selected
such that the immobilized enzyme is retained in the reactor by a
mesh filter while the substrate is allowed to flow through the
reactor at a desired rate. With porous particulate supports it is
possible to control the adsorption process to allow enzymes or
cells to be embedded within the cavity of the particle, thus
providing protection without an unacceptable loss of activity.
[0317] Cross-linking of an enzyme to a solid support involves
forming a chemical bond between a solid support and the enzyme. It
will be appreciated that although cross-linking generally involves
linking the enzyme to a solid support using an intermediary
compound, it is also possible to achieve a covalent bonding between
the enzyme and the solid support directly without the use of an
intermediary compound. Cross-linking commonly uses a bifunctional
or multifunctional reagent to activate and attach a carboxyl group,
amino group, sulfur group, hydroxy group or other functional group
of the enzyme to the solid support. The term "activate" refers to a
chemical transformation of a functional group which allows a
formation of a bond at the functional group. Exemplary amino group
activating reagents include water-soluble carbodiimides,
glutaraldehyde, cyanogen bromide, N-hydroxysuccinimide esters,
triazines, cyanuric chloride, and carbonyl diimidazole. Exemplary
carboxyl group activating reagents include water-soluble
carbodiimides, and N-ethyl-5-phenylisoxazolium-3-sulfonate.
Exemplary tyrosyl group activating reagents include diazonium
compounds. And exemplary sulfhydryl group activating reagents
include dithiobis-5,5'-(2-nitrobenzoic acid), and
glutathione-2-pyridyl disulfide. Systems for covalently linking an
enzyme directly to a solid support include Eupergit.RTM., a
polymethacrylate bead support available from Rohm Pharma
(Darmstadt, Germany), kieselguhl (Macrosorbs), available from
Sterling Organics, kaolinite available from English China Clay as
"Biofix" supports, silica gels which can be activated by
silanization, available from W.R. Grace, and high-density alumina,
available from UOP (Des Plaines, Ill.).
[0318] Entrapment can also be used to immobilize an enzyme.
Entrapment of an enzyme involves formation of, inter alia, gels
(using organic or biological polymers), vesicles (including
microencapsulation), semipermeable membranes or other matrices.
Exemplary materials used for entrapment of an enzyme include
collagen, gelatin, agar, cellulose triacetate, alginate,
polyacrylamide, polystyrene, polyurethane, epoxy resins,
carrageenan, and egg albumin. Some of the polymers, in particular
cellulose triacetate, can be used to entrap the enzyme as they are
spun into a fiber. Other materials such as polyacrylamide gels can
be polymerized in solution to entrap the enzyme. Still other
materials such as polyglycol oligomers that are functionalized with
polymerizable vinyl end groups can entrap enzymes by forming a
cross-linked polymer with UV light illumination in the presence of
a photosensitizer.
[0319] Further embodiments of the present invention include nucleic
acid molecules that encode a protein of the present invention, as
well as any homologues or fragments of such nucleic acid molecules.
A nucleic acid molecule of the present invention includes a nucleic
acid molecule comprising, consisting essentially of, or consisting
of, a nucleic acid sequence encoding any of the isolated proteins
disclosed herein, including a fragment or a homologue of such
proteins, described above. Nucleic acid molecules can include a
nucleic acid sequence that encodes a fragment of a protein that
does not have biological activity, and can also include portions of
a gene or polynucleotide encoding the protein that are not part of
the coding region for the protein (e.g., introns or regulatory
regions of a gene encoding the protein). Nucleic acid molecules can
include a nucleic acid sequence that is useful as a probe or primer
(oligonucleotide sequences).
[0320] Many of the enzymes and proteins of the present invention,
including those comprising, consisting essentially of, or
consisting of, any of SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8 may
be desirable targets for modification and/or used in the products,
cells, and processes described herein. These proteins have been
described in terms of function and amino acid sequence (and nucleic
acid sequence encoding the same) of representative wild-type
proteins. In one embodiment of the invention, homologues of a given
protein (which can include related proteins from other organisms or
modified forms of the given protein) are encompassed for use in the
invention. Homologues of a protein are described in detail in the
General Definitions below.
[0321] In one embodiment, a nucleic molecule of the present
invention includes a nucleic acid molecule comprising, consisting
essentially of, or consisting of, a nucleic acid sequence
represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:9, or fragments or homologues thereof.
Preferably, the nucleic acid sequence encodes a protein (including
fragments and homologues thereof) useful in the invention, or
encompasses useful oligonucleotides or complementary nucleic acid
sequences.
[0322] In one embodiment, a nucleic molecule of the present
invention includes a nucleic acid molecule comprising, consisting
essentially of, or consisting of, a nucleic acid sequence encoding
an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:5, SEQ
ID NO:8, or fragments or homologues thereof. Preferably, the
nucleic acid sequence encodes a protein (including fragments and
homologues thereof) useful in the invention, or encompasses useful
oligonucleotides or complementary nucleic acid sequences.
[0323] In one embodiment, such nucleic acid molecules include
isolated nucleic acid molecules that hybridize under moderate
stringency conditions, and more preferably under high stringency
conditions, and even more preferably under very high stringency
conditions, as described above, with the complement of a nucleic
acid sequence encoding a protein of the present invention (i.e.,
including naturally occurring allelic variants encoding a protein
of the present invention). Preferably, an isolated nucleic acid
molecule encoding a protein of the present invention comprises a
nucleic acid sequence that hybridizes under moderate, high, or very
high stringency conditions to the complement of a nucleic acid
sequence that encodes a protein comprising an amino acid sequence
represented by SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8.
[0324] One embodiment of the present invention relates to a
recombinant nucleic acid molecule which comprises the isolated
nucleic acid molecule described above which is operatively linked
to at least one expression control sequence. Recombinant nucleic
acid molecules are described in detail in the General Definitions
below. One or more recombinant molecules of the present invention
can be used to produce an encoded product (e.g., a protein) of the
present invention. Methods for producing an encoded product and
preferred host cells of the invention are described in detail
below.
[0325] Another aspect of the present invention relates to a
genetically modified microorganism that has been transfected with
one or more nucleic acid molecules of the present invention. The
present invention also encompasses a genetically modified
microorganism wherein one or more nucleic acid molecules of the
present invention has been disrupted in the organism. In one
embodiment, a nucleic acid molecule encoding an endogenous protease
may be disrupted. Additional embodiments include fungi in which the
alp1, alp2 or pep4 genes, or any combination of the genes, have
been disrupted. Genes corresponding to the alp1, alp2 or pep4 genes
disclosed herein, or any combination thereof, may also be disrupted
in other organisms. Genetically modified microorganisms and methods
of producing such organisms are described in detail below.
[0326] The invention also contemplates genetically modified plants
transformed with one or more nucleic acid molecules of the
invention. The plants may be used for production of the enzymes.
Methods to generate recombinant plants are known in the art. For
instance, numerous methods for plant transformation have been
developed, including biological and physical transformation
protocols. See, for example, Miki et al., "Procedures for
Introducing Foreign DNA into Plants" in Methods in Plant Molecular
Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds.
(CRC Press, Inc., Boca Raton, 1993) pp. 67-88. In addition, vectors
and in vitro culture methods for plant cell or tissue
transformation and regeneration of plants are available. See, for
example, Gruber et al., "Vectors for Plant Transformation" in
Methods in Plant Molecular Biology and Biotechnology, Glick, B. R.
and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp.
89-119.
[0327] Another embodiment of the present invention relates to an
isolated binding agent capable of selectively binding to a protein
of the present invention. Suitable binding agents may be selected
from an antibody, an antigen binding fragment, or a binding
partner. The binding agent selectively binds to an amino acid
sequence selected from SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8,
including to any fragment of any of the above sequences comprising
at least one antibody binding epitope. Antibodies, antigen binding
fragments, and binding partners are described in detail below.
Vectors Useful in the Invention
[0328] The present invention includes improved
expression/transforming vectors used to express and screen proteins
produced from complex or combinatorial libraries (e.g., libraries
of libraries), which include a large variety of heterogeneous or
heteromultimeric proteins (e.g., immunoglobulins and fragments
thereof, and other receptors, as non-limiting examples). The
vectors of the present invention allow for very high transformation
frequencies and, upon prolonged cultivation, the integration of the
linearized vector in the host cell's genome.
[0329] In a broad aspect, the vectors include a sequence that
promotes autonomous replication and enhances transformation in a
fungal host, and two selection markers that flank an expression
cassette in the vector, wherein the entire expression cassette
integrates into the genome of a fungal host transformed with the
vector. As discussed in more detail below, the vectors may include
additional elements including, but not limited to, telomeric
sequences, fusion partners, promoters, terminators, and other
expression regulatory elements.
[0330] Preferably a homologous expression-regulating region
enabling high expression in the selected host cell of the invention
is employed in the transforming vector. High expression-regulating
regions derived from a heterologous host, such as from Trichoderma
or Aspergillus, are well known in the art and can also be used. By
way of example, and not limitation, examples of proteins known to
be expressed in large quantities and thus providing suitable
expression regulating sequences for use in the present invention
are hydrophobin, protease, amylase, xylanase, pectinase, esterase,
beta-galactosidase, cellulase (e.g., endo-glucanase,
cellobiohydrolase) and polygalacturonase.
[0331] An expression-regulating region comprises a promoter
sequence operably linked to a nucleic acid sequence encoding the
protein to be expressed. The promoter is linked such that the
positioning vis-a-vis the initiation codon of the sequence to be
expressed allows expression. The promoter sequence can be
constitutive but preferably is inducible. Use of an inducible
promoter and appropriate induction media favors expression of genes
operably linked to the promoter. Any expression regulating sequence
from a homologous species, or from a heterologous strain capable of
permitting expression of a protein, is envisaged. The expression
regulating sequence is suitably a fungal expression-regulating
region, e.g., an ascomycete regulating region. Suitably the
ascomycete expression regulating region is a regulating region from
any of the following genera: Aspergillus, Trichoderma,
Chrysosporium, Myceliophthora, Humicola, Neurospora, Tolypocladium,
Fusarium, Penicillium, Talaromyces, or alternative sexual forms
thereof such as Emericella and Hypocrea. The cellobiohydrolase
promoter from Trichoderma; alcohol dehydrogenase A, alcohol
dehydrogenase R, glutamate dehydrogenase, TAKA amylase,
glucoamylase, and glyceraldehyde phosphate dehydrogenase promoters
from Aspergillus; phosphoglycerate and cross-pathway control
promoters of Neurospora; lipase and aspartic proteinase promoter of
Rhizomucor miehei; beta-galactosidase promoter of Penicillium
canescens; and cellobiohydrolase, endoglucanase, xylanase,
glyceraldehyde-3-phosphate dehydrogenase A, and protease promoters
from Chrysosporium are representative, but not limiting, examples.
An expression regulating sequence from the same genus as the host
strain is preferable, as it is more likely to be specifically
adapted to the host.
[0332] Natural expression-regulating sequences from strains of
Chrysosporium which express proteins in extremely large amounts,
are particularly preferred. Examples of such strains have been
deposited in accordance with the Budapest Treaty with the All
Russian Collection (VKM) depository institute in Moscow. Wild type
C1 strain has the number VKM F-3500 D, deposit date Aug. 29, 1996,
C1 UV13-6 mutant was deposited with number VKM F-3632 D, and
deposit date Sep. 2, 1998, C1 NG7C-19 mutant was deposited with
number VKM F-3633 D and deposit date Sep. 2, 1998 and C1 UV18-25
mutant was deposited with number VKM F-3631 D and deposit date Sep.
2, 1998. These strains are also preferred as sources for the
generation of low-viscosity mutants; indeed the VKM F-3631 D strain
already exhibits the necessary low viscosity phenotype. A
low-viscosity mutant Trichoderma strain, designated X-252, was
obtained after two rounds of irradiation of Trichoderma
longibrachiatum 18.2KK, which in turn was derived by mutation of
the QM 9414 strain of T. longibrachiatum (ATCC 26921). In other
embodiments the invention employs phenotypically similar mutants of
Aspergillus sojae and Aspergillus niger.
[0333] Preferably, where the host is a Chrysosporium, a
Chrysosporium promoter sequence is employed to ensure good
recognition thereof by the host. The analogous situation is
extended to other fungal genera. Certain heterologous
expression-regulating sequences also work as efficiently in
Chrysosporium as native Chrysosporium sequences. This allows
well-known constructs and vectors to be used in transformation of
Chrysosporium, and offers numerous other possibilities for
constructing vectors enabling good rates of transformation and
expression in this host. For example, standard Aspergillus
transformation techniques can be used as described for example by
Christiansen et al., in Bio/Technology 1988 6:1419-1422. Other
documents providing details of Aspergillus transformation vectors,
e.g., U.S. Pat. Nos. 4,816,405, 5,198,345, 5,503,991, 5,364,770,
5,705,358, 5,728,547, and 5,578,463, EP-B-215.594 (also for
Trichoderma) and their contents are incorporated by reference. As
extremely high expression rates for cellulase have been observed in
Chrysosporium strains, the expression regulating regions of
cellulase genes are particularly preferred.
[0334] In one embodiment, the vectors of the invention can comprise
a promoter sequence derived from a gene encoding an enzyme,
preferably a secreted enzyme. Examples of suitable enzymes from
which promoter sequences may be taken are the
carbohydrate-degrading enzymes (e.g., cellulases, xylanases,
mannanases, mannosidases, pectinases, amylases, e.g.,
glucoamylases, .alpha.-amylases, .alpha.- and
.beta.-galactosidases, .alpha.- and .beta.-glucosidases,
.beta.-glucanases, chitinases, chitosanases), proteases
(endoproteases, amino-proteases, amino- and carboxy-peptidases),
other hydrolases (lipases, esterases, phytases), oxidoreductases
(catalases, glucose-oxidases) and transferases (transglycosylases,
transglutaminases, isomerases and invertases). Several examples
from Chrysosporium lucknowense are presented in Table B.
[0335] A nucleic acid construct will preferably comprise a nucleic
acid expression regulatory region from the host genus, such as
Chrysosporium, more preferably from the host species, such as
Chrysosporium lucknowense or a derivative thereof, operably linked
to a nucleic acid sequence encoding a protein to be expressed.
Particularly preferred nucleic acid constructs will comprise an
expression regulatory region from a host, such as Chrysosporium,
associated with cellulase or xylanase expression, preferably
cellobiohydrolase expression, most preferably expression of the 55
kDa cellobiohydrolase (CBH1) described in Table A. As additional
examples, the Chrysosporium promoter sequences of hydrophobin,
protease, amylase, xylanase, esterase, pectinase,
beta-galactosidase, cellulase (e.g., endoglucanase,
cellobiohydrolase), chitinase, and polygalacturonase are also
considered to fall within the scope of the invention.
TABLE-US-00002 TABLE B Characteristics of selected enzymes from
Chrysosporium lucknowense Stability Highest pH at which >50%
Highest pH at which >70% 20 h, 50.degree. C. activity is
retained activity is retained pH 7.5/8 No. of amino RBB Other RBB
Other % of max activity Sample acids CMCase CMCase substrates
CMCase CMCase substrates remaining 30 kD alkaline 12.5 12.0
protease 30 kD Xyl (alkaline) 333 10.0 8.5 80 51 kDXyl 8.0 7.5 60
kDXyl 9.5 9.0 85 30 kD endo (EG3) 247 45 kD endo 7.0 8.0 6.5 7.0 75
55 kD endo 247 8.0 8.0 7.0 7.0 55 25 kD(21.8 kD)endo 225 7.5 10.0
6.5 9.0 80 (EG5) 43 kD(39.6 kD*)endo 395 8.0 8.0 7.2 7.2 (EG6) 45
kD a,i)-Gal/1)-Gluc 6.8 5.7 48 kDCBH 5.2 7.5 8.0 5.0 6.8 55 kDCBHI
526 8.0 9.0 7.4 8.5 70 65 kDPGU 8.0 7.3 90 kD protease 9.0 9.0 100
kD esterase 9.0 9.0 Notes: *molecular weights by MALDI; all others
by SDS PAGE xyl = xylanase endo = endoglucanase gal = galactosidase
glue = glucosidase CBN = cellbiohydrolase PGU =
polygalacturonase
[0336] Any of the promoters or regulatory regions of expression of
enzymes disclosed in Table B, for example, can be suitably
employed, although to be clear, the invention is not limited in any
way to the use of these promoters or regulatory regions. The
nucleic acid sequences of these promoters and regulatory regions
can readily be obtained from a Chrysosporium strain. Methods by
which promoter sequences can be determined are numerous and well
known in the art. Promoter sequences are generally found
immediately preceding the ATG start codon at the beginning of the
relevant gene. For example, promoter sequences can be identified by
deleting sequences upstream of the relevant gene, using recombinant
DNA techniques, and examining the effects of these deletions on
expression of the gene. Also, for example, promoter sequences can
often be inferred by comparing the sequence of regions upstream of
the relevant gene with consensus promoter sequences.
[0337] For example, the promoter sequences of C1 endoglucanases
were identified in this manner (see PCT/NL99/00618) by cloning the
corresponding genes. Preferred promoters according to the invention
are the 55 kDa cellobiohydrolase (CBH1), glyceraldehyde-3-phosphate
dehydrogenase A, and the 30 kDa xylanase (XylF) promoters from
Chrysosporium, as these enzymes are expressed at high level by
their own promoters. The promoters of the carbohydrate-degrading
enzymes of Chrysosporium lucknowense in particular, especially C.
lucknowense GARG 27K, can advantageously be used for expressing
libraries of proteins in other fungal host organisms.
[0338] Particular embodiments of nucleic acid sequences according
to the invention are known for Chrysosporium, Aspergillus and
Trichoderma. Promoters for Chrysosporium are described in
PCT/NL99/00618. The prior art provides a number of expression
regulating regions for use in Aspergillus, e.g., U.S. Pat. Nos.
4,935,349; 5,198,345; 5,252,726; 5,705,358; and 5,965,384; and PCT
application WO 93/07277. Expression in Trichoderma is disclosed in
U.S. Pat. No. 6,022,725. The contents of these patents are hereby
incorporated by reference in their entirety.
[0339] The hydrophobin gene is a fungal gene that is highly
expressed. It is thus suggested that the promoter sequence of a
hydrophobin gene, preferably from Chrysosporium, may be suitably
applied as expression regulating sequence in a suitable embodiment
of the invention. Trichoderma reesei and Trichoderma harzianum gene
sequences for hydrophobin have been disclosed for example in the
prior art as well as a gene sequence for Aspergillus fumigatus and
Aspergillus nidulans and the relevant sequence information is
hereby incorporated by reference (Nakari-Setala et al., Eur. J.
Biochem. 1996, 235:248-255; Parta et al., Infect. Immun. 1994
62:4389-4395; Munoz et al., Curr. Genet. 1997, 32:225-230; and
Stringer et al., Mol. Microbiol. 1995 16:33-44). Using this
sequence information a person skilled in the art can obtain the
expression regulating sequences of Chrysosporium hydrophobin genes
without undue experimentation following standard techniques such as
those suggested above. A recombinant Chrysosporium strain according
to the invention can comprise a hydrophobin-regulating region
operably linked to the sequence encoding the heterologous
protein.
[0340] An expression regulating sequence can also additionally
comprise an enhancer or silencer. These are also well known in the
prior art and are usually located some distance away from the
promoter. The expression regulating sequences can also comprise
promoters with activator binding sites and repressor binding sites.
In some cases such sites may also be modified to eliminate this
type of regulation. For example, filamentous fungal promoters in
which creA sites are present have been described. The creA sites
can be mutated to ensure that the glucose repression normally
resulting from the presence of creA is eliminated. Use of such a
promoter enables production of the library of proteins encoded by
the nucleic acid sequences regulated by the promoter in the
presence of glucose. The method is exemplified in WO 94/13820 and
WO 97/09438. These promoters can be used either with or without
their creA sites. Mutants in which the creA sites have been mutated
can be used as expression regulating sequences in a recombinant
strain according to the invention and the library of nucleic acid
sequences it regulates can then be expressed in the presence of
glucose. Such Chrysosporium promoters ensure depression in an
analogous manner to that illustrated in WO 97/09438. The identity
of creA sites is known from the prior art. Alternatively, it is
possible to apply a promoter with CreA binding sites that have not
been mutated in a host strain with a mutation elsewhere in the
repression system, e.g., in the creA gene itself, so that the
strain can, notwithstanding the presence of creA binding sites,
produce the library of proteins in the presence of glucose.
[0341] Terminator sequences are also expression-regulating
sequences and these are operably linked to the 3' termini of the
sequences to be expressed. A variety of known fungal terminators
are likely to be functional in the host strains of the invention.
Examples are the A. nidulans trpC terminator, A. niger
alpha-glucosidase terminator, A. niger glucoamylase terminator,
Mucor miehei carboxyl protease terminator (see U.S. Pat. No.
5,578,463), and the Trichoderma reesei cellobiohydrolase
terminator. Chrysosporium terminator sequences, e.g., the EG6
terminator, will of course function well in Chrysosporium.
[0342] One suitable terminator sequence is the terminator region
from the cbh1 gene of C. lucknowense (Tcbh1), the sequence of which
is represented as SEQ ID NO:11. The Tcbh1 sequence was found to
contain an approximately 340 bp repetitive sequence designated the
C1 Repetitive Sequence (CRS; represented as SEQ ID NO:12). FIG. 23
illustrates the position of the CRS within the Tcbh1 sequence. The
presence of one of more copies of the CRS in an expression vector,
either within a larger terminator region or in place of a
terminator region, may increase the amount of protein encoded by
the expression vector produced by an organism that expresses the
vector. The presence of one of more copies of the CRS in an
expression vector may also allow an increase in the number of
expression cassettes that become integrated in a host cell's
genomic DNA when the cell is transfected with the vector. As a
result, the CRS may increase the copy number of a gene contained
within the expression vector. Example I below provides one
non-limiting example of how the presence of the CRS in a vector
leads to a 2-fold increase in enzyme activity produced by fungal
strains transfected with a vector encoding the enzyme, as compared
to those strains transfected with a vector without the CRS.
[0343] In certain embodiments of the present invention, an
expression vector may contain one or more copies of the CRS. In
some embodiments, the expression vector contains 1, 2, 3, 4, 5, 6,
7, 8, 9, 10 or more copies of the CRS. The CRS may be present in
the expression vector as part of a terminator sequence or as an
independent sequence element within the vector.
[0344] A suitable transformation vector for use according to the
invention may optionally have the exogenous nucleic acid sequences
to be expressed operably linked to a sequence encoding a signal
sequence. A signal sequence is an amino acid sequence which, when
operably linked to the amino acid sequence of an expressed protein,
enables secretion of the protein from the host organism. Such a
signal sequence may be one associated with a heterologous protein
or it may be one native to the host. The nucleic acid sequence
encoding the signal sequence must be positioned in frame to permit
translation of the signal sequence and the heterologous proteins.
Signal sequences may be particularly preferred where the invention
is being used in conjunction with directed molecular evolution, and
a single, secreted exogenous protein is being evolved, or for the
expression and screening of combinatorial libraries.
[0345] It will be understood that it is less advantageous to
incorporate a signal sequence in a vector that is to be used to
express certain libraries, as this will decrease the probability of
expressing the protein of interest. In a genomic library prepared
by randomly shearing the DNA and cloning into a vector, the
probability that one would obtain an in frame fusion of a gene in
the library to the signal sequence is low. Also, even where an
in-frame fusion has been obtained, the chosen signal sequence may
not work with all genes. For these reasons it may be preferable not
to employ a signal sequence when screening a genomic DNA library,
but rather to screen for the activity or presence of intracellular
exogenous protein. Analysis of the activity or presence of
intracellular proteins may be accomplished by pretreating the
transformant library with enzymes that convert the fungal cells to
protoplasts, followed by lysis. The procedure has been described by
van Zeyl et al., J. Biotechnol. 59:221-224 (1997). This procedure
has been applied to Chrysosporium to allow colony PCR from
Chrysosporium transformants grown in microtiter plates.
[0346] Any signal sequence capable of permitting secretion of a
protein from a Chrysosporium strain is envisaged. Such a signal
sequence is preferably a fungal signal sequence, more preferably an
Ascomycete signal sequence. Suitable signal sequences can be
derived from eukaryotes generally, preferably from yeasts or from
any of the following genera of fungi: Aspergillus, Trichoderma,
Chrysosporium, Myceliophthora, Pichia, Neurospora, Rhizomucor,
Hansenula, Humicola, Mucor, Tolypocladium, Fusarium, Penicillium,
Saccharomyces, Talaromyces or alternative sexual forms thereof such
as Emericella and Hypocrea. Signal sequences that are particularly
useful are those natively associated with cellobiohydrolase,
endoglucanase, beta-galactosidase, xylanase, pectinase, esterase,
hydrophobin, protease or amylase. Examples include amylase or
glucoamylase of Aspergillus or Humicola, TAKA amylase of
Aspergillus oryzae, a-amylase of Aspergillus niger, carboxyl
peptidase of Mucor (U.S. Pat. No. 5,578,463), a lipase or
proteinase from Rhizomucor miehei, cellobiohydrolase of
Trichoderma, beta-galactosidase of Penicillium canescens CBH1 from
Chrysosporium, and the alpha mating factor of Saccharomyces.
[0347] Alternatively the signal sequence can be from an amylase or
subtilisin gene of a strain of Bacillus. A signal sequence from the
same genus as the host strain is extremely suitable as it is most
likely to be specifically adapted to the specific host; thus when
Chrysosporium lucknowense is the host, the signal sequence is
preferably a signal sequence of Chrysosporium. Chrysosporium
strains C1, UV13-6, NG7C-19 and UV18-25 secrete proteins in
extremely large amounts, and signal sequences from these strains
are of particular interest. Signal sequences from filamentous fungi
and yeast may be useful, as well as signal sequences of non-fungal
origin.
[0348] As yet another alternative, the exogenous gene in a library
to be expressed can be fused to a fusion partner (fusion segment)
to produce a fusion protein. Suitable fusion partners for use with
the present invention include, but are not limited to, fusion
partners that can: enhance a protein's stability; enhance or permit
secretion of a protein from the host cell; provide other enzymatic
activity; and/or assist purification of a protein from a host cell
(e.g., by affinity chromatography). A suitable fusion partner can
be a protein or domain or fragment thereof of any size that has the
desired function (e.g., imparts increased stability, solubility,
action or activity; provides other enzymatic activity; and/or
simplifies purification of a protein). Fusion partners can be
joined to amino and/or carboxyl termini of the exogenous protein
that is expressed by a DNA library, and can be susceptible to
cleavage in order to enable straight-forward recovery of the
expressed exogenous protein. The fusion partners are expressed
in-frame with the heterologous protein in the expression cassette
of the transformation vector. Preferred fusion partners for use in
the present invention include any protein that is readily expressed
by a fungal host cell of the present invention and which imparts
the desired property to the fusion protein, and particularly, which
enables or facilitates the secretion, screening and/or recovery of
the protein. A variety of suitable proteins will be apparent for
use in the present invention and include various fungal proteins,
including, but not limited to, cellulases, xylanases, mannanases,
mannosidases, pectinases, amylases, (e.g., glucoamylases,
.alpha.-amylases), .alpha.- and .beta.-galactosidases, .alpha.- and
.beta.-glucosidases, .beta.-glucanases, chitinases, and
chitosanases. In one embodiment, preferred fusion partners are
small proteins or peptides and/or have high rates of secretion from
the fungal host, which make them particularly useful for expression
and screening of proteins expressed by complex DNA libraries. The
fusion partner need not be a fungal protein, although such proteins
may be preferred for stable expression in the host cells.
[0349] In the case of expression of multimeric proteins (e.g.,
proteins having two or more domains, subunits or components), it is
not necessary to use the same fusion partner for expression of each
domain, subunit or component of the protein. For example, fusion of
a carrier with two separable domains can be used to express a
multimeric protein such as an immunoglobulin. Alternatively each
protein can be expressed fused to a different carrier partner. In
yet another embodiment, two components of a multimeric protein
expressed according to the invention can be fused to each other
both with and without a fusion partner as described above.
[0350] The fusion construct as described above is preferably
designed to include a protein-processing site (cleavage site), such
as (but not limited to) a kex2 cleavage site. The presence of this
site results in cleavage of the fusion partner from the protein
expressed by the library insert, so that free proteins are
produced. Such processing sites are not limited to kex2 cleavage
sites, as other sequences can be used that will render the fusion
protein susceptible to cleavage. In one embodiment of the
invention, it is desirable to construct a DNA library so that the
encoded proteins have limited or modified sequences that resemble
the processing sites used in the fusion protein, in order to
enhance stability and yields of the expressed protein. For example,
DNA libraries for immunoglobulins can be modified in regions other
than the variable region to be screened, to remove processing sites
that might be targets for the fungal host proteases.
[0351] A transformed recombinant host fungus according to any of
the embodiments of the invention, or particularly the vector used
to transform the host fungus, can further comprise a selectable
marker. Such a selectable marker permits selection of transformed
or transfected cells. A selectable marker often encodes a gene
product providing a specific type of resistance foreign to the
non-transformed strain. This can be resistance to heavy metals,
antibiotics or biocides in general. Prototrophy is also a useful
selectable marker of the non-antibiotic variety. Auxotrophic
markers generate nutritional deficiencies in the host cells, and
genes correcting those deficiencies can be used for selection.
Examples of commonly used resistance and auxotrophic selection
markers are amdS (acetamidase), hph (hygromycin
phosphotransferase), pyrG (orotidine-5'-phosphate decarboxylase),
pyr4 (orotidine-5'-phosphate decarboxylase), pyrE (orotate
phosphoribosyl transferase), pyr5 (orotate phosphoribosyl
transferase), trpC (anthranilate synthase), argB (ornithine
carbamoyltransferase), sC (sulphate adenyltransferase), bar
(phosphinothricin acetyltransferase), niaD (nitrate reductase),
Sh-ble (bleomycin-phleomycin resistance), mutant acetolactate
synthase (sulfonylurea resistance), and neomycin phosphotransferase
(aminoglycoside resistance). Preferred selection markers in
Chrysosporium are pyr5 (orotate phosphoribosyl transferase) and
pyr4 (orotidine-5'-phosphate decarboxylase). Selection can be
carried out by cotransformation where the selection marker is on a
separate vector or where the selection marker is on the same
nucleic acid fragment as the protein-encoding sequence for the
heterologous protein.
[0352] In a preferred embodiment, the expression/transformation
vector includes at least two selectable markers. In a particularly
preferred embodiment, described in more detail below, the
transformation vector comprises two selectable markers, located
flanking the expression cassette in the vector. The present
inventors have found that this vector design is useful to select
for transformants in which the entire expression cassette is
integrated. During prolonged cultivation, integration of the
expression vector into the genome occurs. When a single marker is
used, portions of the expression cassette distal to the marker fail
to integrate in a significant fraction of transformants. The use of
two markers flanking the cassette ensures that the entire cassette
integrates in most transformants.
[0353] A further improvement of the transformation frequency may be
obtained by the use of the AMA1 replicator sequence (Autonomous
Maintenance in Aspergillus), which can be useful, for example, in
Aspergillus niger (Verdoes et al., Gene 146:159-165 (1994)). This
sequence results in a 10- to 100-fold increase in the
transformation frequency in a number of different filamentous
fungi. Furthermore, in some embodiments, the introduced DNA can be
retained autonomously in the fungal cells, in a multiple-copy
fashion, without integration into the fungal genome. In some
aspects of the invention, this may be beneficial for a high
throughput screening method of the present invention, as the
non-integrative state may reduce variations in the level of gene
expression between different transformants (however, as described
below, the inventors have designed novel vectors for use in the
fungal hosts of the invention that allow for integration of vectors
while reducing variation in the level of gene expression between
different transformants). Moreover, as the introduced DNA is not
recombined into the host DNA, no unwanted mutations in the host
genome will occur. Uniform levels of exogenous gene expression may
be obtained by use of autonomously replicating vectors such as
AMA1. The Examples section below and U.S. Pat. No. 7,122,330
describe the production and use of a vector comprising an AMA1
replicator sequence. In the experiments described, this vector was
not maintained in C. lucknowense. Accordingly, in applications
where a non-integrating vector is preferred and C. lucknowense is
the host cell, alternate strategies for improving transformation
efficiency and maintenance of an autologous plasmid may be
employed. However, it is anticipated that use of autonomously
replicating vectors such as AMA1 will be useful in other fungal
hosts.
[0354] In a preferred alternative, autonomous replication in fungi
can be promoted by telomeric sequences (see e.g., A. Aleksenko and
L. Ivanova, Mol. Gen. Genet. 1998 260:159-164.), although the
invention is not limited to the use of telomeric sequences (other
sequences that achieve the goal of autonomous replication may be
employed). In particular, human and fungal telomeric sequences have
been shown to promote autonomous replication and enhance
transformation in various filamentous fungi like A. nidulans
(Aleksenko and Ivanova, ibid.), Nectria haematococca (Kistler H C
and Benny U. Gene 117, 81-89 (1992)), Podospora anserin (Javerzat
J-P, Bhattacherjee V, Barreau C. Nucleic Acids Research 21, 497-504
(1993)) and Fusarium oxysporum (Powell W A, Kistler H C. J.
Bacteriol. 172, 3163-3171 (1990).). The present inventors have used
human telomeric sequences to develop dedicated exemplary vectors to
obtain transformation frequencies that allow complex library
construction in fungi. This has enabled the further development and
optimization of an efficient DNA transfer protocol and its
integration with a robotic handling system for high throughput
screening in C. lucknowense. The use of telomeric sequences in the
vector increased the number of transformants by 50-100-fold. The
majority of transformants maintained the vector as a
non-integrating linear DNA molecule, although after prolonged
cultivation, the vector eventually integrated.
[0355] In a further improvement of the telomeric vector described
above, the present inventors have designed a vector in which the
promoter and terminator sequences are flanked by two selection
markers to exclude integration events in which part of the linear
vector is lost during integration. Using this vector and the
appropriate selective medium, transformants having stable
integration of a single copy of the vector within or near the end
of the telomeric region of the host chromosomes was achieved with
high frequency. These transformants are stable over several
generations, even in the absence of selective conditions. Such
transformants will result in a more uniform expression pattern of
the expressed protein, which is highly desirable in high throughput
screening assays. Accordingly, it is a further embodiment of the
invention to construct expression vectors wherein the expression
cassette is flanked by selectable markers.
[0356] Vectors that are particularly suitable for use in the
expression and screening methods described herein also contain
sequences that allow for replication and efficient transfer and/or
selection of the vector to or from another host. In one embodiment,
vectors useful in the invention are designed to be efficiently
transferred to a bacterial phage or from a bacterial phage (or to
and from another organism of interest). For example, in the
immunoglobulin screening methods described herein, phage display
libraries for immunoglobulins can be constructed using vectors that
are readily used to generate derivative vectors used to transform
fungal hosts as described herein, wherein the hosts are immediately
ready for expression and screening using the methods described
herein. Ideally, such vectors can be easily retrieved from the
fungal host and used to identify the gene encoding the preferred
protein or protein complex, allowing the creation and/or expression
of the corresponding gene in the fungal host or in mammalian cells
such as CHO cells to offer maximum flexibility for the creation and
handling of DNA libraries and particularly, in evolution
methods.
[0357] Yet another embodiment of the invention relates to vectors
that allow specific integration into the fungal DNA of independent
vectors expressing the different constituents of the protein
complexes desired, using different mutually compatible selection
markers for each of the independent vectors. A preferred embodiment
in this approach is the use of mutant fungal host strains where the
fraction of site-specifically integrated vector copies is improved
by either reducing non-homologous recombination, such as by
targeted or selected modification of genes involved in
recombination processes (e.g., genes homologous to various genes
found in Neurosporra and other fungi or other organisms e.g., Ku70,
KU80 and Lig4; see Ishibashi et al., Proc Natl Acad Sci USA. 2006
Oct. 3; 103(40):14871-6 or Ninomiya et al., Proc Natl Acad Sci USA.
2004 Aug. 17; 101(33):12248-53), or by increasing homologous
integration in strains. These integrative vector/strain
combinations represent another approach to obtain strains with a
more limited variation in expression level within the collection of
transformants obtained in the library of libraries designed for
multidomain protein expression. In a further embodiment, one can
use such mutant strains with modified recombination abilities and
produce further improved mutants that also have improved ability to
maintain self-replicating vectors that are used to transform such
strains (e.g., by stabilizing integrative vectors and replicative
vectors so that the strains are more stable and can be predictably
and reproducibly transformed).
[0358] Improved vectors and strains according to the present
invention utilizing many of the approaches described above are
described in detail in the Examples and the Figures. One example is
the vector pPcbh1 glaA(II) heavy(88) Tcbh1 Pcbh1 glaA(II) light(90)
Tcbh1 PyrE tel PyrG, which represents a vector comprising many of
the elements described above as well as encoding the heavy and
light chains of an immunoglobulin molecule as gla fusion proteins
(FIG. 27). This vector was deposited at the Centraalbureau voor
Schimmelcultures (CBS) in the Netherlands under the terms of the
Budapest Treaty on the International Regulation of the Deposit of
Microorganisms for the Purposes of Patent Procedure on Dec. 5,
2007, as E. coli JM109 containing the plasmid pPcbh1 glaA(II)
heavy(88) Tcbh1 Pcbh1 glaA(II) light(90) Tcbh1 PyrE tel PyrG,
Accession # CBS 122187.
[0359] This vector includes the heavy and light chain fusion
proteins each flanked on one end by the cbh1 promoter and on the
end by the cbh1 terminator sequence. Also included in the vector
are the pyrE and pyrG selection markers flanking human telomeric
sequences (tel). The complete nucleic acid sequence of pPcbh1
glaA(II) heavy(88) Tcbh1 Pcbh1 glaA(II) light(90) Tcbh1 PyrE tel
PyrG is represented as SEQ ID NO:10. The coding sequence for the
selection marker pyrE is represented as SEQ ID NO:13, and the
corresponding pyrE amino acid sequence is represented as SEQ ID
NO:14. The coding sequence for the selection marker pyrG is
represented as SEQ ID NO:15, and the corresponding pyrG amino acid
sequence is represented as SEQ ID NO:16. The vector also contains
two copies of a human telomeric sequence (hTel), represented as SEQ
ID NO:17. In some embodiments, vectors of the present invention
comprise a fragment of hTel, wherein the fragment comprises nucleic
acids 1-422 of SEQ ID NO:17. In some embodiments, the sequences
encoding the immunoglobulin heavy and light chains (positions 15287
to 16730 and positions 21443 to 22173 of SEQ ID NO:10,
respectively) may be removed from the vector and/or replaced with
an additional protein-encoding sequence as described elsewhere in
this application. The vector also includes two Gla fusion protein
encoding sequences with kex2 cleavage sites (positions 13398 to
15286 and positions 19554 to 21442 of SEQ ID NO:10, respectively).
Table C below lists the elements present in the vector and the
positions of each within SEQ ID NO:10, as depicted in FIG. 27.
TABLE-US-00003 TABLE C Elements of the vector Pcbh1 glaA(II) heavy
(88) Tcbh1 Pcbh1 glaA(II) light (90) Tcbh1 pyrE tel pyrG. Element
or Feature Position within SEQ ID NO: 10 pyrE flanking region
1-1324 pyrE 1324-2171 pyrE flanking region 3887-4217 hTel 4236-4811
I-Ceul 4830-4834 hTel 4857-5432 pyrG flanking region 5687-7030 pyrG
complement 7031-7932 pyrG flanking region 7933-8498 Pgpd 8520-8703
Tcbh1 8704-8711 Ampicillin resistance gene 9500-10360 Pcbh1
11601-13397 Gla(GII) kex2 13398-15286 Immunoglobulin heavy chain
15287-16730 Tcbh1 16731-17746 Pcbh1 17757-19553 Gla(GII) kex2
19554-21442 Immunoglobulin light chain 21443-22173 Tcbh1
22173-23188
[0360] Exemplary vectors that are particularly useful for the
expression and screening of complex proteins, such as
immunoglobulins, are described herein. Such vectors are generally
characterized as being self-replicating vectors (but also include
integrating and non-integrating vectors, as well as vectors that
are initially maintained as autonomous vectors but will integrate
after extended periods of cultivation), comprising promoter and
terminator sequences as described above, selectable marker
sequences as described above, and other sequences useful for
allowing replication in E. coli, yeast, and/or efficient transfer
to and/or from a bacterial phage. Particularly preferred vectors
for use in the expression and screening of complex libraries as
described herein further include telomeric sequences as described
above, although any sequence that improves transformation
efficiency and stability of a vector can be used. Other
particularly preferred vectors include the use of double dominant
selection markers flanking the expression cassette, resulting in
stable transformants with uniform expression as described above.
The vectors preferably contain one or more cloning sites for
cloning of the DNA from an expression DNA library which is linked
to a sequence encoding a fusion partner for production and
appropriate expression (e.g., secretion) of fusion proteins by the
vector (described above).
[0361] An example of a preferred self-replicating vector for use in
the expression and screening of large and/or complex libraries
(e.g., combinatorial libraries) is illustrated in FIG. 1 and also
in FIG. 19. In one embodiment, such an exemplary vector comprises
an expression cassette for expression of the DNA library insert
including promoter and terminator sequences (which can optionally
include DNA encoding a fusion partner as described above so that
the insert is expressed as a fusion protein), selectable markers
flanking the expression cassette, restriction enzyme sites to
facilitate excision and recovery of the expression cassette,
telomeric sequences to enhance transformation efficiency and
stability, and optionally, sequences allowing for expression and
selection in another host organism, such as E. coli.
[0362] Additional preferred vectors used in the expression and
screening for protein complexes containing two or more
heterogeneous components, such as immunoglobulins, are illustrated
in FIG. 12. While these figures illustrate the use of the vector to
express the heavy and light chains of immunoglobulins, it will be
apparent that sequences encoding proteins for other complex
proteins (e.g., other heteromultimeric receptors) could be used in
such vectors and with similar vector designs. FIG. 12A shows a
vector in which libraries of immunoglobulin heavy and light chain
variable regions (V.sub.H and V.sub.L, respectively) are together
cloned into a C. lucknowense-specific replicating vector so that
glucoamylase fusions to a single-chain Fv are produced. FIG. 12B
shows a vector in which full length antibody chains are expressed
as glucoamylase fusions, each from their own promoter. Numerous
possibilities exist for the latter vector. The light and heavy
chains could be placed in either order in either orientation.
Additionally, although both chains in this figure are shown
utilizing cbh1 promoter and terminator sequences, distinct
promoters and terminators could be used to minimize repetitive
sequences in the vector. Also, in addition to using individual
expression cassettes for the light and heavy chains, the light and
heavy chains could be fused at the gene level, introducing
processing sites between the carrier and the different chains
(e.g., see FIG. 14). In addition, as discussed above, the fusion
partner is not limited to glucoamylase, as many other suitable
fusion partners could be utilized. In addition, fusion protein
processing sites can be introduced to allow for cleavage of the
expressed protein from the fusion partner. The vectors use human
telomeric sequences (hTel) for replication in C1 and contain two
selective markers pyrE and pyrG flanking the expression constructs,
and sequences allowing replication in E. coli. Again, the
selectable markers and telomeric sequences can be modified based
upon vector design and host fungus. In addition, the vector can be
designed to include additional elements to facilitate the transfer
of the vector from a fungal host to a bacterial phage and/or from a
bacterial phage to the fungal host (e.g., using Gateway.RTM.
technology; see Examples and FIGS. 14-18).
[0363] Additional examples of preferred vectors used in the
expression and screening for protein complexes containing two or
more heterogeneous components, such as immunoglobulins, are
illustrated in FIG. 20.
[0364] In these examples, upon introduction into C. lucknowense,
the vectors spontaneously linearize between the human telomeric
sequences (hTel). The use of doubly marked pyr4-pyr5 mutants of C1
ensures that integrants contain the entire expression construct.
Individual transformants are then separated and screened for
binding in a high-throughput fashion. The expression constructs
from transformants of interest can be isolated by purifying genomic
DNA from those constructs, digesting with NotI, ligating to
recircularize, then transforming E. coli and selecting for
ampicillin resistance. The resulting E. coli transformants will
contain plasmids carrying the antibody expression construct. In the
case where full-length antibodies are screened, the expression
constructs can be subcloned directly into expression vectors to
allow high-level expression of the antibodies as described herein.
When a single-chain Fv is used in the expression screening, the
plasmids can be deconstructed and relevant sequences spliced into
full-length heavy and light chains for expression as described
elsewhere herein.
Libraries and Proteins for Expression and Screening
[0365] As used herein the term "heterologous protein" is a protein
or polypeptide not normally expressed or secreted by the host
strain used for expression and screening according to the
invention. A heterologous protein can also include proteins that
are native to the host strain, species, or genus, but that are
under the control of a promoter other than its own and/or in a
different genomic locus. A heterologous protein may be of
prokaryotic origin, or it may be derived from a virus, fungus,
plant, insect, or higher animal such as a mammal. For
pharmaceutical screening purposes, quite often a preference will
exist for human proteins, thus a preferred embodiment will be a
host wherein the DNA library is of human origin. Such embodiments
are therefore also considered suitable examples of the
invention.
[0366] The present inventors have found that the fungal systems for
expression and screening of DNA libraries described herein is
useful not only to express and screen simple, monomeric proteins,
but also to express and screen more complex proteins and/or protein
complexes (heterogeneous or heteromultimeric proteins). For
example, a particularly useful embodiment of the present invention
is the screening of complex, combinatorial DNA libraries, where the
protein expressed by the DNA is composed of two or more domains,
constituents or subunits. Exemplary proteins that can be expressed
and screened by such combinatorial libraries (libraries of
libraries) include, but are not limited to, a variety of
heterodimeric or heteromultimeric receptors and proteins, such as
members of the immunoglobulin supergene family (e.g.,
immunoglobulin, major histocompatibility complex, T cell receptors,
CD3, adhesion molecules), hormones and hormone receptors, cytokines
and cytokine receptors, other growth factors and growth factor
receptors, etc. Many of these proteins are involved in the
regulation of significant physiological processes, and the ability
to study these processes and design or identify diagnostic and
therapeutic reagents based on the properties of such proteins has
far reaching value, particularly in the area of clinical
applications.
[0367] In one preferred embodiment of the present invention, the
heterologous protein to be expressed and screened for using the
fungal system described herein is an immunoglobulin (antibody).
According to the present invention, immunoglobulins or antibodies
are characterized in that they comprise immunoglobulin domains and
as such, they are members of the immunoglobulin superfamily of
proteins. Generally speaking, an antibody molecule comprises two
types of chains, although heavy chain antibodies as produced in
llamas are a different type of antibody molecule consisting of one
type of chain. For most antibodies, one type of chain is referred
to as the heavy or H chain and the other is referred to as the
light or L chain. The two chains are present in an equimolar ratio,
with each antibody molecule typically having two H chains and two L
chains. The two H chains are linked together by disulfide bonds and
each H chain is linked to a L chain by a disulfide bond. There are
only two types of L chains referred to as lambda (.lamda.) and
kappa (.kappa.) chains. In contrast, there are five major H chain
classes referred to as isotypes. The five classes include
immunoglobulin M (IgM or .mu.), immunoglobulin D (IgD or .delta.),
immunoglobulin G (IgG or .gamma.), immunoglobulin A (IgA or
.alpha.), and immunoglobulin E (IgE or .epsilon.). The distinctive
characteristics between such isotypes are defined by the constant
domain of the immunoglobulin and are discussed in detail below.
Human immunoglobulin molecules comprise nine isotypes, IgM, IgD,
IgE, four subclasses of IgG including IgG1 (.gamma.1), IgG2
(.gamma.2), IgG3 (.gamma.3) and IgG4 (.gamma.4), and two subclasses
of IgA including IgA1 (.alpha.1) and IgA2 (.alpha.2).
[0368] Each H or L chain of an immunoglobulin molecule comprises
two regions referred to as L chain variable domains (V.sub.L
domains) and L chain constant domains (C.sub.L domains), and H
chain variable domains (V.sub.H domains) and H chain constant
domains (C.sub.H domains). A complete C.sub.H domain comprises
three sub-domains (C.sub.H1, C.sub.H2, C.sub.H3) and a hinge
region. Together, one H chain and one L chain can form an arm of an
immunoglobulin molecule having an immunoglobulin variable region. A
complete immunoglobulin molecule comprises two associated (e.g.,
di-sulfide linked) arms. Thus, each arm of a whole immunoglobulin
comprises a V.sub.H+L region, and a C.sub.H+L region. As used
herein, the term "variable region" or "V region" refers to a
V.sub.H+L region (also known as an Fv fragment), a V.sub.L region
or a V.sub.H region. Also as used herein, the term "constant
region" or "C region" refers to a C.sub.H+L region, a C.sub.L
region or a C.sub.H region.
[0369] Limited digestion of an immunoglobulin with a protease may
produce two fragments. An antigen binding fragment is referred to
as an Fab, an Fab', or an F(ab')2 fragment. A fragment lacking the
ability to bind to antigen is referred to as an Fc fragment. An Fab
fragment comprises one arm of an immunoglobulin molecule containing
a L chain (V.sub.L+C.sub.L domains) paired with the V.sub.H region
and a portion of the C.sub.H region (C.sub.H1 domain). An Fab'
fragment corresponds to an Fab fragment with part of the hinge
region attached to the C.sub.H1 domain. An F(ab')2 fragment
corresponds to two Fab' fragments that are normally covalently
linked to each other through a di-sulfide bond, typically in the
hinge regions.
[0370] The C.sub.H domain defines the isotype of an immunoglobulin
and confers different functional characteristics depending upon the
isotype. For example, .mu. constant regions enable the formation of
pentameric aggregates of IgM molecules and a constant regions
enable the formation of dimers.
[0371] The antigen specificity of an immunoglobulin molecule is
conferred by the amino acid sequence of a variable, or V, region.
As such, V regions of different immunoglobulin molecules can vary
significantly depending upon their antigen specificity. Certain
portions of a V region are more conserved than others and are
referred to as framework regions (FW regions). In contrast, certain
portions of a V region are highly variable and are designated
hypervariable regions. When the V.sub.L and V.sub.H domains pair in
an immunoglobulin molecule, the hypervariable regions from each
domain associate and create hypervariable loops that form the
antigen binding sites. Thus, the hypervariable loops determine the
specificity of an immunoglobulin and are termed
complementarity-determining regions (CDRs) because their surfaces
are complementary to antigens.
[0372] Further variability of V regions is conferred by
combinatorial variability of gene segments that encode an
immunoglobulin V region. Immunoglobulin genes comprise multiple
germline gene segments that somatically rearrange to form a
rearranged immunoglobulin gene that encodes an immunoglobulin
molecule. V.sub.L regions are encoded by a L chain V gene segment
and J gene segment (joining segment). V.sub.H regions are encoded
by a H chain V gene segment, D gene segment (diversity segment) and
J gene segment (joining segment).
[0373] Both a L chain and H chain V gene segment contain three
regions of substantial amino acid sequence variability. Such
regions are referred to as L chain CDR1, CDR2 and CDR3, and H chain
CDR1, CDR2 and CDR3, respectively. The length of an L chain CDR1
can vary substantially between different V.sub.L regions. For
example, the length of CDR1 can vary from about 7 amino acids to
about 17 amino acids. In contrast, the lengths of L chain CDR2 and
CDR3 typically do not vary between different V.sub.L regions. The
length of a H chain CDR3 can vary substantially between different
V.sub.H regions. For example, the length of CDR3 can vary from
about 1 amino acid to about 20 amino acids. Each H and L chain CDR
region is flanked by FW regions.
[0374] In one embodiment, immunoglobulin DNA libraries useful in
the present include libraries encoding humanized antibodies.
Humanized antibodies are molecules having an antigen binding site
derived from an immunoglobulin from a non-human species, the
remaining immunoglobulin-derived parts of the molecule being
derived from a human immunoglobulin. The antigen binding site may
comprise either complete variable regions fused onto human constant
domains or only the complementarity determining regions (CDRs)
grafted onto appropriate human framework regions in the variable
domains. Humanized antibodies can be produced, for example, by
modeling the antibody variable domains, and producing the
antibodies using genetic engineering techniques, such as CDR
grafting (described below). A description various techniques for
the production of humanized antibodies is found, for example, in
Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-55;
Whittle et al., (1987) Prot. Eng. 1:499-505; Co et al., (1990) J.
Immunol. 148:1149-1154; Co et al., (1992) Proc. Natl. Acad. Sci.
USA 88:2869-2873; Carter et al., (1992) Proc. Natl. Acad. Sci.
89:4285-4289; Routledge et al., (1991) Eur. J. Immunol.
21:2717-2725 and PCT Patent Publication Nos. WO 91/09967; WO
91/09968 and WO 92/113831. Such techniques may be used in
connection with the expression and screening methods described
herein to produce and select optimized or evolved humanized
antibodies.
[0375] The invention is especially useful for the expression and
screening of genetically-engineered antibodies or antigen binding
fragments thereof, including single chain antibodies, humanized
antibodies (discussed above), antibodies that can bind to more than
one epitope (e.g., bi-specific antibodies), or antibodies that can
bind to one or more different antigens (e.g., bi- or multi-specific
antibodies). Genetically engineered antibodies include those
produced by standard recombinant DNA techniques involving the
manipulation and re-expression of DNA encoding antibody variable
and/or constant regions. Particular examples include, chimeric
antibodies, where the V.sub.H and/or V.sub.L domains of the
antibody come from a different source as compared to the remainder
of the antibody, and CDR grafted antibodies (and antigen binding
fragments thereof), in which at least one CDR sequence and
optionally at least one variable region framework amino acid is
(are) derived from one source and the remaining portions of the
variable and the constant regions (as appropriate) are derived from
a different source. Construction of chimeric and CDR-grafted
antibodies are described, for example, in European Patent
Applications: EP-A 0194276, EP-A 0239400, EP-A 0451216 and EP-A
0460617.
[0376] For example it is possible to clone a particular antibody
and then identify the variable region genes encoding the desired
antibody, including the sequences encoding the CDRs. From here,
modified antibodies and antigen binding fragments can be engineered
by preparing one or more replicable expression vectors containing
at least the DNA sequence encoding the variable domain of the
antibody heavy or light chain and optionally other DNA sequences
encoding remaining portions of the heavy and/or light chains as
desired, and transforming/transfecting an appropriate host cell as
described herein, in which expression of the antibody will occur
and in which the expressed antibodies can be screened for a
desirable characteristic, which is typically antigen binding. There
are numerous publications, including patent specifications,
detailing techniques suitable for the preparation of antibodies by
manipulation of DNA, creation of expression vectors and
transformation of appropriate cells, for example as reviewed by
Mountain A and Adair, J R in Biotechnology and Genetic Engineering
Reviews (ed. Tombs, M P, 10, Chapter 1, 1992, Intercept, Andover,
UK) and in the aforementioned European Patent Applications.
Methods, employing, for example, phage display technology (see for
example U.S. Pat. No. 5,969,108, U.S. Pat. No. 5,565,332, U.S. Pat.
No. 5,871,907, U.S. Pat. No. 5,858,657) or the selected lymphocyte
antibody method of U.S. Pat. No. 5,627,052 may also be used for the
production of antibodies and/or antigen fragments of the invention,
as will be readily apparent to the skilled individual. These
methods can be used to engineer DNA libraries suitable for
expression and screening in conjunction with the fungal systems
described herein.
[0377] Antibodies to be expressed and screened using the expression
and screening systems and methods of the present invention can
include whole antibodies (e.g., monoclonal antibodies), as well as
functional equivalents of whole antibodies, such as antigen binding
fragments in which one or more antibody domains are truncated or
absent (e.g., Fv, Fab, Fab', or F(ab')2 fragments, CH fragments, Fc
fragments), as well as genetically-engineered antibodies or antigen
binding fragments thereof, including single chain antibodies, heavy
chain antibodies, humanized antibodies (discussed above),
antibodies that can bind to more than one epitope (e.g.,
bi-specific antibodies), or antibodies that can bind to one or more
different antigens (e.g., bi- or multi-specific antibodies).
Antibodies useful in the invention include catalytic antibodies
(e.g., antibodies which exhibit catalytic activity; see Catalytic
Antibodies, Ehud Keinan (Editor), ISBN: 3-527-30688-9, January
2005), neutralizing antibodies, and any antibody type or derivative
or fragment thereof that is useful in any research, diagnostic,
therapeutic, or other clinical application.
[0378] Antibodies and fragments or functional equivalents thereof,
as discussed above, may be from any species. Examples include
human, mouse, rat, goat, hamster, sheep, horse, monkey, llama and
the like. The structure of antibodies and the production thereof in
these species are well known in the art.
[0379] As another example of the use of the invention to screen for
complex proteins, a heterologous protein can include a major
histocompatibility protein (MHC), expressed alone or more
preferably, in complex with a library of peptides. The
identification of peptide epitopes associated with particular T
cell receptors is often still a bottle neck in studying T cells and
their antigenic targets in, for example, autoimmunity,
hypersensitivity, and cancer. In many clinical situations, when
pathological T cells are identified, only the major
histocompatibility complex (MHC), but not the specific peptide
portion of the antigen that is recognized by the T cell, is known.
Having a rapid method to identify these peptides would aid in the
identification of the protein source of the antigens driving the T
cell responses. These peptides would help also in creating tools to
monitor the frequency and functional state of the T cells as well
as the development of therapeutic reagents to control them.
[0380] MHC proteins are generally classified into two categories:
class I and class II MHC proteins. An MHC class I protein is an
integral membrane protein comprising a glycoprotein heavy chain,
also referred to herein as the .alpha. chain, which has three
extracellular domains (i.e., .alpha..sub.1, .alpha..sub.2, and
.alpha..sub.3) and two intracellular domains (i.e., a transmembrane
domain (TM) and a cytoplasmic domain (CYT)). The heavy chain is
non-covalently associated with a soluble subunit called
.beta.2-microglobulin (.beta.2m). An MHC class II protein is a
heterodimeric integral membrane protein comprising one .alpha.
chain and one .beta. chain in noncovalent association. The .alpha.
chain has two extracellular domains (.alpha..sub.1 and
.alpha..sub.2), and two intracellular domains (a TM domain and a
CYT domain). The .beta. chain contains two extracellular domains
(.beta..sub.1 and .beta..sub.2), and two intracellular domains (a
TM domain and CYT domain). Many human and other mammalian MHC
molecules are well known in the art and any MHC Class I or Class II
molecules can be used in the present invention. MHC molecules are
cell surface receptors that complex with peptides, the complex of
which can be recognized by a T cell receptor. A peptide binding
groove of a class I protein can comprise portions of the
.alpha..sub.1 and .alpha..sub.2 domains of the heavy chain capable
of forming two .beta.-pleated sheets and two .alpha. helices.
Inclusion of a portion of the .beta.2-microglobulin chain
stabilizes the complex. While for most versions of MHC Class II
molecules, interaction of the .alpha. and .beta. chains can occur
in the absence of a peptide, the two chain complex of MHC Class I
is unstable until the binding groove is filled with a peptide. A
peptide binding groove of a class II protein can comprise portions
of the .alpha..sub.1 and .beta..sub.1 domains capable of forming
two .beta.-pleated sheets and two .alpha. helices. A first portion
of the .alpha..sub.1 domain forms a first .beta.-pleated sheet and
a second portion of the .alpha..sub.1 domain forms a first .alpha.
helix. A first portion of the .beta..sub.1 domain forms a second
.beta.-pleated sheet and a second portion of the .beta..sub.1
domain forms a second .alpha. helix. The X-ray crystallographic
structure of class II protein with a peptide engaged in the binding
groove of the protein shows that one or both ends of the engaged
peptide can project beyond the MHC protein (Brown et al., pp.
33-39, 1993, Nature, Vol. 364). Thus, the ends of the .alpha..sub.1
and .beta..sub.1 .alpha. helices of class II form an open cavity
such that the ends of the peptide bound to the binding groove are
not buried in the cavity. Moreover, the X-ray crystallographic
structure of class II proteins shows that the N-terminal end of the
MHC .beta. chain apparently projects from the side of the MHC
protein in an unstructured manner since the first 4 amino acid
residues of the .beta. chain could not be assigned by X-ray
crystallography. Methods of linking peptides to MHC complexes,
which could be developed to create libraries for use in the present
invention, is described, for example, in U.S. Pat. No.
5,820,866.
[0381] The present invention is useful for effectively and
efficiently creating, developing, and selecting reagents useful in
vaccines. The vaccines comprise one or more antigens or antibodies
or fragments thereof that can be identified, designed and/or
developed/evolved using the expression and screening systems
described herein. Indeed, the expression and screening system of
the invention will allow the identification, creation and
production of large quantities of vaccines in a more cost-effective
and time-effective (short timeframe) manner than currently
available methods for vaccine production. Vaccines to be produced
using the methods of the invention include vaccines for any
conventional or adapted use, and include both prophylactic and
therapeutic vaccines.
[0382] When it is desirable to stimulate an immune response, the
term "antigen" can be used interchangeably with the term
"immunogen", and is used herein to describe a protein, peptide,
cellular composition, organism or other molecule which elicits a
humoral and/or cellular immune response (i.e., is antigenic), such
that administration of the immunogen to an animal (e.g., via a
vaccine of the present invention) mounts an antigen-specific immune
response against the same or similar antigens that are encountered
within the tissues of the animal. Therefore, to vaccinate an animal
against a particular antigen means, in one embodiment, that an
immune response is elicited against the antigen or immunogenic or
toleragenic portion thereof, as a result of administration of the
antigen. Vaccination preferably results in a protective or
therapeutic effect, wherein subsequent exposure to the antigen (or
a source of the antigen) elicits an immune response against the
antigen (or source) that reduces or prevents a disease or condition
in the animal. The concept of vaccination is well known in the art.
The immune response that is elicited by administration of a
therapeutic composition of the present invention can be any
detectable change in any facet of the immune response (e.g.,
cellular response, humoral response, cytokine production), as
compared to in the absence of the administration of the
vaccine.
[0383] An immunogenic domain (portion, fragment, epitope) of a
given antigen can be any portion of the antigen (i.e., a peptide
fragment or subunit or an antibody epitope or other conformational
epitope) that contains at least one epitope that acts as an
immunogen when administered to an animal. For example, a single
protein can contain multiple different immunogenic domains.
Immunogenic domains need not be linear sequences within a protein,
in the case of a humoral response.
[0384] An epitope is defined herein as a single immunogenic site
within a given antigen that is sufficient to elicit an immune
response, or a single toleragenic site within a given antigen that
is sufficient to suppress, delete or render inactive an immune
response. Those of skill in the art will recognize that T cell
epitopes are different in size and composition from B cell
epitopes, and that epitopes presented through the Class I MHC
pathway differ from epitopes presented through the Class II MHC
pathway. Epitopes can be linear sequence or conformational epitopes
(conserved binding regions) depending on the type of immune
response. An antigen can be as small as a single epitope, or
larger, and can include multiple epitopes. As such, the size of an
antigen can be as small as about 5-12 amino acids (e.g., a peptide)
and as large as: a full length protein, including a multimer and
fusion proteins, chimeric proteins, whole cells, whole
microorganisms, or portions thereof (e.g., lysates of whole cells
or extracts of microorganisms).
[0385] These are only a few exemplary uses for the expression and
screening of DNA libraries for complex proteins described herein.
One can readily use the tools and system described herein to screen
other libraries for the development and identification of
therapeutically and clinically important proteins.
[0386] Expression of a library of human genes, derived from a
genomic human DNA library, in the filamentous fungi of the
invention is expected to be efficient for several reasons. It is
now known that the average size of human genes is 3,000-5,000 bp,
and that human introns average about 75 to about 150 bp (total
range 40->50,000). Filamentous fungi have introns of 40-75 bp,
but they can deal with introns up to 500 bp in length. On average,
human genes carry 3-5 introns per gene (M. Deutsch, M. Long, Nucl.
Acids Res. 1999 27:3219-3228; Table D). Human signal sequences are
also known to function in filamentous fungi. For these reasons, it
is likely that a large number of human genes can be expressed and
secreted at high levels by the methods of this invention.
TABLE-US-00004 TABLE D Introns Average intron Organism per gene
size (nt) (range) Intron structure Animal/Plant 3-5 75-150 GTnnGt .
. . CtxAC . . . yAG (40->50000) 80% under 150 nt Fungi 3 40-75
GTAnGy . . . CtxAC . . . yAG (40-500) Yeast 0.01 50-60 GTATGT . . .
TACTAAC . . . yAG (?-?)
[0387] The methods of the invention are thus expected to be useful
for expression of DNA libraries derived from both prokaryotic and
eukaryotic genomes. Indeed, as described herein, the inventors have
demonstrated the use of the antibody to express and screen for
human immunoglobulin proteins as well as other proteins such as
enzymes. As described above, the methods are capable of expression
and discovery of both secreted and intracellular proteins, giving
ready access to an extremely large number of genes and
proteins.
[0388] A further aspect of the invention includes the construction
and screening of fungal mutant libraries, and fungal mutant
libraries prepared by the methods disclosed herein. The libraries
may be obtained by transformation of the fungal hosts according to
this invention with any means of integrative or non-integrative
transformation, using methods known to those skilled in the art.
This library of fungi based on the preferred host strains may be
handled and screened for desired properties or activities of
exogenous proteins, including any heterologous proteins described
herein, in miniaturized and/or high-throughput format screening
methods. By property or activity of interest is meant any physical,
physicochemical, chemical, biological, or catalytic property, or
any improvement, increase, or decrease in such a property,
associated with an exogenous protein of a library member. The
library may also be screened for metabolites or for a property or
activity associated with a metabolite, produced as a result of the
presence of exogenous and/or endogenous proteins. The library may
also be screened for fungi producing increased or decreased
quantities of such protein or metabolites.
[0389] In another aspect of this invention, the library of
transformed fungi may be screened for the presence of fungal
metabolites having desirable properties. Examples of such
metabolites include polyketides, alkaloids, and terpenoid natural
products. It is anticipated that multiple genes or gene clusters
(operons) may be transferred to the host cells of the invention,
and that non-protein products generated by the action of the
encoded enzymes will then be generated in the host cells. For
example, it has been shown that DNA encoding the proteins necessary
for production of lovastatin can be transferred to Aspergillus
oryzae (U.S. Pat. No. 5,362,638; see also U.S. Pat. No.
5,849,541).
[0390] In another embodiment of the invention, the library of
transformed fungi may be screened for the presence of DNA that
hybridizes to a nucleic acid probe of interest. In this embodiment,
expression and/or secretion of exogenous proteins is not essential,
although it will often still be desirable. Where protein expression
is not needed, it will be appreciated that regulatory sequences are
not needed in the vector.
[0391] In yet another embodiment of the invention, the library of
transformed fungi may be screened for some desirable property of
the fungi themselves, such as for example tolerance to a physically
or chemically extreme environment, or the ability to produce,
modify, degrade or metabolize a substance of interest. Such
desirable properties may or may not be ascribable to the presence
of a single exogenous protein. This embodiment will be of
particular utility when employed as part of a process of directed
evolution.
[0392] The heterologous DNA may be genomic DNA or cDNA, prepared
from biological specimens by methods well known in the art. The
biological specimen may be an environmental sample (for example,
soil, compost, forest litter, seawater, or fresh water), or an
extracted, filtered, or centrifuged or otherwise concentrated
sample therefrom. Mixed cultures of microorganisms derived from
environmental samples may be employed as well. The biological
sample may also be derived from any single species of organism,
such as a cultured microorganism, or plant, insect, or other animal
such as a mammal. In addition, the heterologous DNA may be
synthetic or semi-synthetic, for example random DNA sequences or
DNA comprising naturally-occurring segments which have been
shuffled, mutated, or otherwise altered or engineered. An example
of a semi-synthetic nucleic library is found in Wagner et al., WO
00/0632. DNA from environmental samples (or mixed cultures derived
therefrom) will be advantageous for the discovery of novel
proteins, while the use of DNA from a single species will be
advantageous in that (1) an appropriate vector may be more
judiciously chosen, and (2) the practitioner will be directed to
related or similar species for further screening if a protein of
interest is identified. Genetically engineered libraries can
include libraries from a variety of sources, and can include
combinatorial libraries as described herein.
[0393] The heterologous DNA used in the libraries can be further
modified to increase the efficiency of expression and screening of
the proteins in the fungal system. For example, the DNA libraries
can be optimized for the codon usage of the fungal host. In
addition, as discussed above, heterologous DNA can be engineered to
minimize the presence of processing sites, or when fusion proteins
are created that include processing sites, the sites can be
engineered to avoid sites that may be similar or duplicated in the
heterologous DNA, to avoid inadvertent processing of the
heterologous protein. The heterologous DNA can be engineered to
reduce glycosylation sites or to result in perfectly glycosylated
proteins when produced by the fungal host.
[0394] Compared to traditional fungal hosts, transformation,
expression and secretion rates are exceedingly high when using a
Chrysosporium strain exhibiting the compact mycelial morphology of
strain UV18-25, and mutants thereof. Thus a recombinant strain
according to the invention will preferably exhibit such morphology.
The invention however also covers non-recombinant strains or
otherwise engineered strains of fungi exhibiting this
characteristic.
[0395] An attractive embodiment of the invention would employ a
recombinant Chrysosporium strain exhibiting a viscosity below that
of strain NG7C-19, preferably below that of UV18-25 under
corresponding or identical culture conditions. We have determined
that the viscosity of a culture of UV18-25 is below 10 cP as
opposed to that of previously known Trichoderma reesei being of the
order 200-600 cP, and with that of traditional Aspergillus niger
being of the order 1500-2000 cP under optimal culture conditions
during the middle to late stages of fermentation. Accordingly the
invention may employ any engineered or mutant filamentous fungus
exhibiting this low-viscosity characteristic, such as the
Chrysosporium UV18-25 (VKM F-3631D) strain, the Trichoderma X 252
strain, or A. sojae pclA (derived from ATCC 11906) or A. niger
pclA.
[0396] The fluidity of filamentous fungal cultures can vary over a
wide range, from nearly solid to a free-flowing liquid. Viscosity
can readily be quantitated by Brookfield rotational viscometry, use
of kinematic viscosity tubes, falling ball viscometer or cup type
viscometer. Fermentation broths are non-Newtonian fluids, and the
apparent viscosity will be dependent to some extent upon the shear
rate (Goudar et al., Appl. Microbiol. Biotechnol. 1999 51:310-315).
This effect is however much less pronounced for the low-viscosity
cultures employed in the present invention.
Methods of the Invention
[0397] The improved vectors and protease-deficient fungal strains
discussed above greatly increase the efficiency of the screening
and expression of proteins, particularly proteins expressed by
complex DNA libraries. Accordingly, the present invention includes
the screening and expression methods described below wherein either
an improved vector or a fungus described above (or a combination
thereof) is employed to express or screen for a protein or
plurality of proteins. The improved vectors and fungal strains
described above also allow for the expression and screening of DNA
libraries not only to express and screen simple, monomeric
proteins, but also to express and screen more complex proteins
and/or protein complexes (heterogeneous or heteromultimeric
proteins).
[0398] The use of such low viscosity cultures in the screening of
an expression library according to the method of the invention is
highly advantageous. The screening of DNA libraries expressed in
filamentous fungi has heretofore been limited to relatively slow
and laborious methods. In general, once fungi have been transformed
(and the transformants optionally selected for), it has been
necessary to prepare spores or conidia, or to mechanically disrupt
the mycelia, in order to disperse the library of transformed fungi
into individual organisms or reproductive elements. This dispersal
is necessary so that the separated organisms can be cultured into
clonal colonies or cultures. The spores, conidia, or mycelial
fragments are then diluted and "plated out" in standard culture
dishes, and the individual colonies are inspected for color,
alterations to the substrate, or other detectable indication of the
presence of the protein activity or property being sought. In
another approach, secreted proteins are blotted from the colonies
onto a membrane, and the membrane is probed or examined for an
indication of the presence of the protein activity or property of
interest. Use of membranes has proved useful where proteolytic
degradation of exogenous protein is a problem (Asgeirsdottir et
al., Appl. Environ. Microbiol. 1999, 65:2250-2252). Such procedures
are labor-intensive and have not proven amenable to automation, and
as a result high-throughput screening of fungally-expressed
proteins has not heretofore been accomplished with conventional
filamentous fungi. For purposes of this disclosure, high-throughput
screening refers to any partially- or fully-automated screening
method or process to search through large or fairly large numbers
of substances for desired activity, resulting in less costly and
faster processes. In one aspect, high-throughput screening refers
to any partially- or fully-automated screening method that is
capable of evaluating the proteins expressed by at least about 2,
3, 4, 5, 6, 7, 8, 9 or 10, or more transformants per day, about 100
or more transformants per day, about 250 or more transformants per
day, about 500 or more transformants per day, about 750 or more
transformants per day, about 1,000 or more transformants per day,
and particularly to those methods capable of evaluating 5,000 or
more transformants per day, and most particularly to methods
capable of evaluating 10,000 or more transformants per day. The
invention, however, is not limited to screening large or very large
numbers of transformants. For example, the present invention is
highly useful for expression and screening of smaller libraries of
proteins that have been prescreened by another method (e.g., phage
display), followed by production of libraries of pre-screened
libraries. The present invention enables the identification of the
best candidate proteins from a library of any size, including very
small to very large.
[0399] The automated high-throughput screening of a library of
transformed fungi according to the present invention, accordingly,
may be carried out in a number of known ways. Methods that are
known to be applicable to bacteria or yeast may in general be
applied to the low-viscosity fungi of the present invention. This
is made possible by the presence of transferable reproductive
elements in combination with the low-viscosity phenotype, a
consequence of the relatively non-entangled morphology of the
hyphae of the mutant fungi employed. In essence, the mutant fungi,
and/or their transferable reproductive elements, behave very much
like individual bacteria or yeast during the mechanical
manipulations involved in automated high-throughput screening. This
is in contrast to wild-type fungi, and most industrially-adapted
fungi as well, which produce highly entangled mycelia which do not
permit the ready separation of the individual organisms from one
another.
[0400] For example, a dilute suspension of transformed fungi
according to the present invention may be aliquotted out through a
mechanical micropipette into the wells of a 96-well microplate. It
is anticipated that liquid-handling apparatus capable of pipetting
into 384- or 1536-well microplates can also be adapted to the task
of automated dispersal of the organisms into microplates. The
concentration of the suspended organisms can be adjusted as desired
to control the average number of organisms (or other transferable
reproductive elements) per well. It will be appreciated that where
multiple individual organisms are aliquotted into wells, the
identification of the desired protein activity or property in that
well will be followed by dilution of the contents of the well and
culturing the organisms present into individual clonal colonies or
cultures. In this manner the throughput of the system may be
increased, at the cost of the need for subsequent resolution of the
contents of each well that presents a "hit".
[0401] In addition, transformed fungi according to the present
invention may also be grown and/or screened in larger culture
plates, test tubes, shake flasks, small fermentors, etc. to obtain
higher protein quantities than from micro titer wells.
[0402] In an alternative embodiment, a cell sorter may be
interposed in the fluid path, which is capable of directing the
flow of the culture to the wells of the microplate upon the
detection of an organism or other transferable reproductive element
in the detector cell. This embodiment permits the reasonably
accurate dispensation of one organism per well. The use of an
optically-detectable marker, such as green fluorescent protein, to
identify transformants is particularly useful in this embodiment,
as it permits the automated selection of transformants by a
fluorescence-activated cell sorter.
[0403] In yet another embodiment, colonies growing on solid media
can be picked by a robotic colony picker, and the organisms
transferred by the robot to the wells of a microtiter plate.
Well-separated colonies will give rise to single clones in each
well.
[0404] The dispersed organisms are then permitted to grow into
clonal cultures in the microplate wells. Inducers, nutrients, etc.
may be added as desired by the automated fluid dispensing system.
The system may also be used to add any reagents required to enable
the detection of the protein activity or property of interest. For
example, chromogenic or fluorogenic substrates can be added so as
to permit the spectroscopic or fluorometric detection of an enzyme
activity. The low viscosity and submerged growth properties of the
cultures in the wells of a microtiter plate permit the rapid
diffusion of such reagents into the culture, greatly enhancing the
sensitivity and reliability of the assay. Diffusion of oxygen and
nutrients is also greatly enhanced, facilitating rapid growth and
maximal expression and secretion of exogenous peptides. Certain
assays, such as the scintillation proximity assay, rely on the
diffusion of soluble components so as to arrive at an equilibrium
state; again the low viscosity of the fungal cultures of the
present invention makes this high throughput assay possible.
Finally, in a highly automated system it will be desirable to
automatically pick, aspirate, or pipette clonal cultures of
interest from their wells in the microtiter plate, and the low
viscosity and submerged growth habit of the cultures will make this
possible. All of the above operations would be difficult or
impossible given the viscosity of traditional filamentous fungal
cultures, especially cultures growing as surface mats in the
minimally stirred, shear-free conditions of a microtiter plate
well.
[0405] In another embodiment, single cells are passed through a
microfluidic apparatus, and the property or activity of interest is
detected optically (Wada et al., WO 99/67639). Low viscosity is
essential to the operation of a microfluidics device, and cultures
of the low-viscosity mutant fungi of the present invention are
expected to be amenable to microfluidic manipulation. Short et al.,
in U.S. Pat. No. 6,174,673, have described how fluorogenic
substrates may be employed to detect an enzyme activity of
interest, and how host cells expressing such an activity may be
isolated with a fluorescence-activated cell sorter. The methods of
the present invention are compatible with this method of
identification of expressed proteins.
[0406] In one embodiment, where transformants carry a fluorescent
protein as a marker, the fluorescence may be quantitated and
employed as a measure of the amount of gene expression and/or
expressed protein present in a given culture. In this embodiment,
it is possible not only to detect an exogenous protein of interest,
but to estimate the specific activity of the protein, as described
by Blyna et al., in WO 00/78997. This embodiment will be
particularly preferred where the screening method of the invention
is employed as part of a process of directed evolution.
[0407] In those cases where a greater viscosity is acceptable, a
gel-forming matrix may provide certain advantages when culturing
fungi, and conducting biochemical assays, in a microplate format,
as described by Bochner in U.S. Pat. No. 6,046,021.
[0408] Another class of high-throughput screens is by photometric
analysis, by digital imaging spectroscopy, of large numbers of
individual colonies growing on a solid substrate. See for example
Youvan et al., 1994, Meth. Enzymol. 246:732-748. In this method,
changes in the overall absorption or emission spectra of
specialized reagents are indicative of the presence of a
heterologous protein activity or property of interest. The ready
dispersal of individual organisms attendant upon the use of
low-viscosity mutants also enables the use of filamentous fungi in
this method. The tendency for colonies of the mutant fungi of the
invention to exhibit less lateral growth, and to produce smooth,
compact, and well-defined colonies on solid media, is also
advantageous in such a screening system. Furthermore, the superior
expression and secretion characteristics of fungi as compared to
bacteria provide greater quantities of protein for spectral
analysis.
[0409] An automated microorganism-handling tool is described in
Japanese patent application publication number 11-304666. This
device is capable of the transfer of microdroplets containing
individual cells, and it is anticipated that the fungal strains of
the present invention, by virtue of their morphology, will be
amenable to micromanipulation of individual clones with this
device.
[0410] An automated microbiological high-throughput screening
system is described in Beydon et al., J. Biomol. Screening 5:13-21
(2000). The robotic system is capable of transferring droplets with
a volume of 400 nl to agar plates, and processing 10,000 screening
points per hour, and has been used to conduct yeast two-hybrid
screens. It is anticipated that the fungal hosts of the present
invention will be as amenable as yeast to high-throughput screening
with systems of this type.
[0411] As an alternative to microtiter plates, transformants can be
grown on plates and, in the form of microcolonies, assayed
optically as described in WO 00/78997.
[0412] In one embodiment, fungal cultures used in the expression
and screening methods described herein are conducted under
conditions that inhibit proteases. For example, in addition to or
as an alternative to using the various fungal strains described
herein with low protease activity, culture conditions can include
protease inhibition, such as by adding protease inhibitors to
culture or screening media and reagents.
[0413] Various other techniques can be used in high throughput
methods to detect properties of heterologous proteins expressed by
the fungal system of the invention, including, but not limited to,
Western blot, immunoblot, enzyme-linked immunosorbant assay
(ELISA), radioimmunoassay (RIA), immunoprecipitation, surface
plasmon resonance, chemiluminescence, fluorescent polarization,
phosphorescence, immunohistochemical analysis, matrix-assisted
laser desorption/ionization time-of-flight (MALDI-TOF) mass
spectrometry, microcytometry, microarray, microscopy, fluorescence
activated cell sorting (FACS), flow cytometry, or protein
microchip, microarray, or any cell-based bioassays.
[0414] The development of high throughput screens in general is
discussed by Jayawickreme and Kost, Curr. Opin. Biotechnol.
8:629-634 (1997). A high throughput screen for rarely transcribed
differentially expressed genes is described in von Stein et al.,
Nucleic Acids Res. 35: 2598-2602 (1997).
[0415] The low protease Chrysosporium strain UV18#100.f and
variants of the strain wherein protease genes (e.g., alp1, pep4
and/or alp2) have been disrupted illustrate various aspects of the
invention exceedingly well. The invention, however, may employ
other mutant or otherwise engineered strains of filamentous fungi
that produce transferable reproductive elements in suspension and
exhibit low viscosity in culture. For example, various mutants of
Chrysosporium strain UV18-25 having low protease activity (e.g.,
UV18#100f .DELTA.alp1, UV18#100f .DELTA.pyr5 .DELTA.alp1,
UV18#100.f .DELTA.alp1.DELTA.pep4 .DELTA.alp2, etc.) are described
herein. The specific morphology of the fungi may not be critical;
the present inventors have observed short, non-entangled mycelia in
these two strains but other morphologies, such as close and
extensive hyphal branching, may also lead to reduced viscosity.
Fungal strains according to the invention are preferred if they
exhibit optimal growth conditions at neutral pH and temperatures of
25-43.degree. C. Such screening conditions are advantageous for
maintaining the activity of exogenous proteins, in particular those
susceptible to degradation or inactivation at acidic pH. Most
mammalian proteins, and human proteins in particular, have evolved
to function at physiological pH and temperature, and screening for
the normal activity of a human enzyme is best carried out under
those conditions. Proteins intended for therapeutic use will have
to function under such conditions, which also makes these the
preferred screening conditions. Chrysosporium strains exhibit
precisely this characteristic, growing well at neutral pH and
35-40.degree. C., while other commonly employed fungal host species
(e.g., Aspergillus and Trichoderma) grow best at acidic pH, and in
some embodiments, at lower temperatures, and may be less suitable
for this reason.
[0416] Another application of the method of the present invention
is in the process of "directed evolution," wherein novel
protein-encoding DNA sequences are generated, the encoded proteins
are expressed in a host cell, and those sequences encoding proteins
exhibiting a desired characteristic are selected, mutated, and
expressed again. The process is repeated for a number of cycles
until a protein with the desired characteristics is obtained. Gene
shuffling, protein engineering, error-prone PCR, site-directed
mutagenesis, and combinatorial and random mutagenesis are examples
of processes through which novel DNA sequences encoding exogenous
proteins can be generated. U.S. Pat. Nos. 5,223,409, 5,780,279 and
5,770,356 provide teaching of directed evolution. See also Kuchner
and Arnold, Trends in Biotechnology, 15:523-530 (1997);
Schmidt-Dannert and Arnold, Trends in Biotech., 17:135-136 (1999);
Arnold and Volkov, Curr. Opin. Chem. Biol., 3:54-59 (1999); Zhao et
al., Manual of Industrial Microbiology and Biotechnology, 2.sup.nd
Ed., (Demain and Davies, eds.) pp. 597-604, ASM Press, Washington
D.C., 1999; Arnold and Wintrode, Encyclopedia of Bioprocess
Technology: Fermentation, Biocatalysis, and Bioseparation,
(Flickinger and Drew, eds.) pp. 971-987, John Wiley & Sons, New
York, 1999; Minshull and Stemmer, Curr. Opin. Chem. Biol.
3:284-290; and Rajpal et al., PNAS 2005 102: 8467-8471. Directed
evolution is particularly useful for screening combinatorial
libraries as described herein. For example, using directed
evolution to screen for antibodies is highly desirable, since such
methods can mimic the affinity maturation processes through which
naturally occurring antibodies progress, and more importantly, can
be used to develop and screen for high affinity or high avidity
antibodies that bind to particular antigens, including various
therapeutic antibodies that are engineered to have desirable
characteristics (e.g., neutralizing antibodies, catalytic
antibodies, etc.).
[0417] An application of combinatorial mutagenesis is disclosed in
Hu et al., Biochemistry. 1998 37:10006-10015. U.S. Pat. No.
5,763,192 describes a process for obtaining novel protein-encoding
DNA sequences by stochastically generating synthetic sequences,
introducing them into a host, and selecting host cells with the
desired characteristic. Methods for effecting artificial gene
recombination (DNA shuffling) include random priming recombination
(Z. Shao, et al., Nucleic Acids Res., 26:681-683 (1998)), the
staggered extension process (H. Zhao et al., Nature Biotech.,
16:258-262 (1998)), and heteroduplex recombination (A. Volkov et
al., Nucleic Acids Res., 27:e18 (1999)). Error-prone PCR is yet
another approach (Song and Rhee, Appl. Environ. Microbiol.
66:890-894 (2000)).
[0418] The present invention makes use of any of these and other
genetic engineering approaches to modify DNA for directed evolution
and screening methods described herein, including, but not limited
to, engineering DNA for optimal host expression (e.g., codon
optimization); modification of genes to avoid or inhibit protease
degradation or protein processing, particularly by the host cells;
modification of DNA to provide higher expression levels of
proteins, better functionality, and/or improved stability. Genetic
engineering methods are well-known in the art and are encompassed
by the present invention.
[0419] There are two widely-practiced methods of carrying out the
selection step in a directed evolution process, although the
invention is not limited to these approaches. In one method, the
protein activity of interest is somehow made essential to the
survival of the host cells. For example, if the activity desired is
a cellulase active at pH 8, a cellulase gene could be mutated and
introduced into the host cells. The transformants are grown with
cellulose as the sole carbon source, and the pH raised gradually
until only a few survivors remain. The mutated cellulase gene from
the survivors, which presumably encodes a cellulase active at
relatively high pH, is subjected to another round of mutation, and
the process is repeated until transformants that can grow well on
cellulose at pH 8 are obtained. Thermostable variants of enzymes
can likewise be evolved, by cycles of gene mutation and
high-temperature culturing of host cells (Liao et al., Proc. Natl.
Acad. Sci. USA 1986 83:576-580; Giver et al., Proc. Natl. Acad.
Sci. USA. 1998 95:12809-12813). For purposes of this application,
mutation of DNA sequences encoding exogenous proteins may be
accomplished by any of several methods employed for directed
evolution, for example by gene shuffling, in vivo recombination, or
cassette mutagenesis.
[0420] The chief advantage of this method is the massively parallel
nature of the "survival of the fittest" selection step. Millions,
or billions, of unsuccessful mutations are simultaneously
eliminated from consideration without the need to evaluate them
individually. However, it is not always possible to link an enzyme
activity of interest to the survival of the host. For example where
the desired protein property is selective binding to a target of
interest, making the binding property essential to survival is
likely to be difficult. Also, survival under forced conditions such
as high temperature or extreme pH is likely to be dependent upon
multiple factors, and a desirable mutation will not be selected for
and will be lost if the host cell is unable to survive for reasons
unrelated to the properties of the mutant protein.
[0421] An alternative to the massively parallel "survival of the
fittest" approach is serial screening. In this approach, individual
transformants are screened by traditional methods, such as
observation of cleared or colored zones around colonies growing on
indicator media, colorimetric or fluorometric enzyme assays,
immunoassays, binding assays, etc. See for example Joo et al.,
Nature 399:670-673 (1999), where a cytochrome P450 monooxygenase
not requiring NADH as a cofactor was evolved by cycles of mutation
and screening; May et al., Nature Biotech. 18:317-320 (2000), where
a hydantoinase of reversed stereoselectivity was evolved in a
similar fashion; and Miyazaki et al., J. Mol. Biol. 297:1015-1026
(2000), where a thermostable subtilisin was evolved.
[0422] The screening approach has clear advantages over a simple
"survival screen," especially if it can be carried out in a
high-throughput manner that approaches the throughput of the
massively parallel "survival screen" technique. For example, a
degree of parallelism has been introduced by employing such
measures as digital imaging of the transformed organisms (Joo et
al., Chemistry & Biology, 6:699-706 (1999)) or digital
spectroscopic evaluation of colonies (Youvan et al., 1994, Meth.
Enzymol. 246:732-748). Serial assays can be automated by the use of
cell sorting (Fu et al., Nature Biotech., 17:1109-1111 (1999)). A
well-established approach to high-throughput screening involves the
automated evaluation of expressed proteins in microtiter plates,
using commercially available plate readers, and the method of the
present invention is well-suited to the application of this mode of
high-throughput screening to directed evolution.
[0423] In this embodiment of the invention, a gene encoding a
protein of interest is mutated by any known method of generating a
plurality of mutants, the mutant protein-encoding DNA is introduced
by means of a suitable expression vector into a low-viscosity
filamentous fungal host according to the present invention, and the
transformants are optionally selected for and cultured. The host
cells are then dispersed as described previously into the wells of
a microtiter plate, or otherwise spatially separated into
resolvable locations, so as to provide individual monoclonal
cultures (or polyclonal cultures having fewer than about 100
different clones). The cells are preferably dispersed into the
wells of a micro-titer plate. The protein encoded by the mutant DNA
is preferably secreted into the medium in the wells of the
microtiter plates. Each of the dispersed cultures is screened for
the protein activity of interest, and those most strongly
exhibiting the desired property are selected. The gene encoding the
protein of interest in the selected cultures is mutated again, the
mutant DNA is again introduced into the low-viscosity fungal host,
and the transformants are re-screened. The mutating and
re-screening process is repeated until the value of the property of
interest reaches a desired level.
[0424] In an alternative embodiment, directed evolution is carried
out by mutation and reproduction of the gene of interest in another
organism, such as E. coli, followed by transfer of the mutant genes
to a filamentous fungus according to the present invention for
screening. In one embodiment, phage display technology can be
combined with screening in a filamentous fungal host. The use of
phage display libraries for immunoglobulin screening may be a
particularly advantageous combination with the methods of the
present invention.
[0425] It will be readily appreciated by those skilled in the art
that a protein that appears to be of interest based upon the
screening assay will not necessarily have all the other properties
required for commercial utility. For example, the possession of
enzymatic activity, however high the specific activity, will not
indicate that the mutant enzyme has the requisite thermal or pH
stability, or detergent or protease resistance, or
non-immunogenicity, or other property that might be desirable or
necessary in a commercially viable product. There is a need for
methods of readily determining whether an identified protein has
commercially useful properties.
[0426] The prior art approaches to screening have not provided a
solution to this need, because the host organisms (bacteria and
yeast) were not adapted to the production of isolable quantities of
protein. It has heretofore been necessary to transfer potentially
useful genes from one organism to another, as one proceeded through
DNA library preparation, gene expression, screening, expression of
research quantities of gene products, and over-expression in
industrially suitable production strains. The mutant filamentous
fungi of the present invention, on the other hand, are excellent
overproducers and secretors of exogenous proteins, especially when
employed with the vectors disclosed herein. Sufficient protein may
be isolated not only for purposes of characterization, but for
evaluation in application trials. Indeed, the strains used in the
screening method of the invention are suitable for industrial
production as well, since they possess desirable production
properties such as low viscosity, high expression rates, and very
high protein/biomass ratios.
[0427] Accordingly, in a preferred embodiment of the present
invention, the method further comprises culturing a clonal colony
or culture identified according to the method of the invention,
under conditions permitting expression and secretion of the
exogenous library protein (or a precursor thereof), and recovering
the subsequently produced protein to obtain the protein of
interest. As discussed above, expression and secretion of a library
protein may be facilitated by creating an in-frame fusion of the
cloned gene with the gene for a heterologous protein (or a fragment
thereof) with its corresponding signal sequence, or with the signal
sequence from a third protein, all operably linked to an expression
regulating sequence. By this approach a fusion protein is created
that contains heterologous amino acid sequences upstream of the
library protein. Subsequently, this fusion precursor protein may be
isolated and recovered using purification techniques known in the
art. The method may optionally comprise subjecting the secreted
fusion protein precursor to a cleavage step to generate the library
protein of interest. The cleavage step can be carried out with
Kex-2, a Kex-2 like protease, or another selective protease, when
the vector is engineered so that a protease cleavage site links a
well-secreted protein carrier and the protein of interest.
[0428] The ready availability of mutant protein, directly from the
screening host organism, has not previously been possible with
prior art screening hosts. The present invention thus provides an
advantage, in that the mutant proteins deemed of interest based
upon the high-throughput screen can be isolated in sufficient
quantities (milligrams) for further characterization and even
larger quantities (grams to kilograms) for application trials. This
particular embodiment of the invention thus permits the
practitioner to select mutant proteins for the next round of
directed evolution based upon any number of desirable properties,
and not merely upon the one property detected in the
high-throughput screen. The more stringent selection criteria made
possible by the present invention should lead to a more efficient
and cost-effective directed evolution process.
[0429] The method of production of a recombinant mutant filamentous
fungal strain according to the invention comprises introducing a
library of DNA sequences comprising nucleic acid sequences encoding
heterologous proteins into a low-viscosity mutant filamentous
fungus according to the invention, the nucleic acid sequences being
operably linked to an expression regulating region. The
introduction of the DNA sequences may be carried out in any manner
known per se for transforming filamentous fungi. Those skilled in
the art will appreciate that there are several well-established
methods, such as CaCl.sub.2-polyethylene glycol stimulated DNA
uptake by fungal protoplasts (Johnstone et al., EMBO J., 1985,
4:1307-1311). A protoplast transformation method is described in
U.S. Pat. No. 7,122,330 and the Examples section below. Alternative
protoplast or spheroplast transformation methods are known and can
be used as have been described in the prior art for other
filamentous fungi. Vectors suitable for multicopy integration of
heterologous DNA into the fungal genome are well-known; see for
example Giuseppin et al., WO 91/00920. The use of autonomously
replicating plasmids has long been known as an efficient
transformation tool for fungi (Gems et al., Gene 1991 98:61-67;
Verdoes et al., Gene 1994 146:159-165; Aleksenko and Clutterbuck,
Fungal Genetics Biol. 1997 21:373-387; Aleksenko et al., Mol. Gen.
Genet. 1996 253:242-246). Details of such methods can be found in
many of the cited references, and they are thus incorporated by
reference.
GENERAL DEFINITIONS
[0430] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry, nucleic acid chemistry, and immunology, which are
well known to those skilled in the art. Such techniques are
explained fully in the literature, such as, Methods of Enzymology,
Vol. 194, Guthrie et al., eds., Cold Spring Harbor Laboratory Press
(1990); Molecular Cloning: A Laboratory Manual, second edition
(Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual,
third edition (Sambrook and Russell, 2001), (jointly referred to
herein as "Sambrook"); Current Protocols in Molecular Biology (F.
M. Ausubel et al., eds., 1987, including supplements through 2001);
PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);
Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York; Harlow and Lane (1999) Using
Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (jointly referred to herein as
"Harlow and Lane"), Beaucage et al., eds., Current Protocols in
Nucleic Acid Chemistry John Wiley & Sons, Inc., New York,
2000); and Vaccines, S. Plotkin and W. Orenstein, eds., 3.sup.rd
edition (1999).
[0431] As used herein, reference to an isolated protein or
polypeptide in the present invention, including any of the proteins
disclosed herein, such as any of the proteases described above,
includes full-length proteins, fusion proteins, or any fragment or
homologue of such a protein. More specifically, an isolated
protein, such as an enzyme according to the present invention, is a
protein (including a polypeptide or peptide) that has been removed
from its natural milieu (i.e., that has been subject to human
manipulation) and can include purified proteins, partially purified
proteins, recombinantly produced proteins, synthetically produced
proteins, proteins complexed with lipids, soluble proteins, and
isolated proteins associated with other proteins, for example. As
such, "isolated" does not reflect the extent to which the protein
has been purified. Preferably, an isolated protein of the present
invention is produced recombinantly. In addition, and by way of
example, a "C. lucknowense protein" or "C. lucknowense enzyme"
refers to a protein (generally including a homologue of a naturally
occurring protein) from Chrysosporium lucknowense or to a protein
that has been otherwise produced from the knowledge of the
structure (e.g., sequence) and perhaps the function of a naturally
occurring protein from Chrysosporium lucknowense. In other words, a
C. lucknowense protein includes any protein that has substantially
similar structure and function of a naturally occurring C.
lucknowense protein or that is a biologically active (i.e., has
biological activity) homologue of a naturally occurring protein
from C. lucknowense as described in detail herein. As such, a C.
lucknowense protein can include purified, partially purified,
recombinant, mutated/modified and synthetic proteins. According to
the present invention, the terms "modification" and "mutation" can
be used interchangeably, particularly with regard to the
modifications/mutations to the amino acid sequence of a C.
lucknowense protein (or nucleic acid sequences) described herein.
An isolated protein according to the present invention can be
isolated from its natural source, produced recombinantly or
produced synthetically.
[0432] According to the present invention, the terms "modification"
and "mutation" can be used interchangeably, particularly with
regard to the modifications/mutations to the primary amino acid
sequences of a protein or peptide (or nucleic acid sequences)
described herein. The term "modification" can also be used to
describe post-translational modifications to a protein or peptide
including, but not limited to, methylation, farnesylation,
carboxymethylation, geranyl geranylation, glycosylation,
phosphorylation, acetylation, myristoylation, prenylation,
palmitation, and/or amidation. Modifications can also include, for
example, complexing a protein or peptide with another compound.
Such modifications can be considered to be mutations, for example,
if the modification is different than the post-translational
modification that occurs in the natural, wild-type protein or
peptide.
[0433] As used herein, the term "homologue" is used to refer to a
protein or peptide which differs from a naturally occurring protein
or peptide (i.e., the "prototype" or "wild-type" protein) by minor
modifications to the naturally occurring protein or peptide, but
which maintains the basic protein and side chain structure of the
naturally occurring form. Such changes include, but are not limited
to: changes in one or a few amino acid side chains; changes one or
a few amino acids, including deletions (e.g., a truncated version
of the protein or peptide), insertions and/or substitutions;
changes in stereochemistry of one or a few atoms; and/or minor
derivatizations, including but not limited to: methylation,
glycosylation, phosphorylation, acetylation, myristoylation,
prenylation, palmitation, amidation and/or addition of
glycosylphosphatidyl inositol. A homologue can have either
enhanced, decreased, or substantially similar properties as
compared to the naturally occurring protein or peptide. A homologue
can include an agonist of a protein or an antagonist of a
protein.
[0434] Homologues can be the result of natural allelic variation or
natural mutation. A naturally occurring allelic variant of a
nucleic acid encoding a protein is a gene that occurs at
essentially the same locus (or loci) in the genome as the gene
which encodes such protein, but which, due to natural variations
caused by, for example, mutation or recombination, has a similar
but not identical sequence. Allelic variants typically encode
proteins having similar activity to that of the protein encoded by
the gene to which they are being compared. One class of allelic
variants can encode the same protein but have different nucleic
acid sequences due to the degeneracy of the genetic code. Allelic
variants can also comprise alterations in the 5' or 3' untranslated
regions of the gene (e.g., in regulatory control regions). Allelic
variants are well known to those skilled in the art.
[0435] Homologues can be produced using techniques known in the art
for the production of proteins including, but not limited to,
direct modifications to the isolated, naturally occurring protein,
direct protein synthesis, or modifications to the nucleic acid
sequence encoding the protein using, for example, classic or
recombinant DNA techniques to effect random or targeted
mutagenesis.
[0436] Modifications in protein homologues, as compared to the
wild-type protein, either agonize, antagonize, or do not
substantially change, the basic biological activity of the
homologue as compared to the naturally occurring protein.
Modifications of a protein, such as in a homologue, may result in
proteins having the same biological activity as the naturally
occurring protein, or in proteins having decreased or increased
biological activity as compared to the naturally occurring protein.
Modifications which result in a decrease in protein expression or a
decrease in the activity of the protein, can be referred to as
inactivation (complete or partial), down-regulation, or decreased
action of a protein. Similarly, modifications which result in an
increase in protein expression or an increase in the activity of
the protein, can be referred to as amplification, overproduction,
activation, enhancement, up-regulation or increased action of a
protein.
[0437] According to the present invention, an isolated protein,
including a biologically active homologue or fragment thereof, has
at least one characteristic of biological activity of a wild-type,
or naturally occurring, protein. As discussed above, in general,
the biological activity or biological action of a protein refers to
any function(s) exhibited or performed by the protein that is
ascribed to the naturally occurring form of the protein as measured
or observed in vivo (i.e., in the natural physiological environment
of the protein) or in vitro (i.e., under laboratory conditions).
The biological activity of a protein of the present invention can
include an enzyme activity (catalytic activity and/or substrate
binding activity), such as protease activity or any other activity
disclosed herein. Methods of detecting and measuring the biological
activity of a protein of the invention are known in the art. Such
assays include, but are not limited to, measurement of enzyme
activity (e.g., catalytic activity), measurement of substrate
binding, and the like. It is noted that an isolated protein of the
present invention (including homologues) is not required to have a
biological activity such as catalytic activity. A protein can be a
truncated, mutated or inactive protein, or lack at least one
activity of the wild-type enzyme, for example. Inactive proteins
may be useful in some screening assays, for example, or for other
purposes such as antibody production.
[0438] Methods to measure protein expression levels of a protein
according to the invention include, but are not limited to: western
blotting, immunocytochemistry, flow cytometry or other
immunologic-based assays; assays based on a property of the protein
including but not limited to, ligand binding or interaction with
other protein partners. Binding assays are also well known in the
art. For example, a BIAcore machine can be used to determine the
binding constant of a complex between two proteins. The
dissociation constant for the complex can be determined by
monitoring changes in the refractive index with respect to time as
buffer is passed over the chip (O'Shannessy et al., Anal. Biochem.
212:457-468 (1993); Schuster et al., Nature 365:343-347 (1993)).
Other suitable assays for measuring the binding of one protein to
another include, for example, immunoassays such as enzyme linked
immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), or
determination of binding by monitoring the change in the
spectroscopic or optical properties of the proteins through
fluorescence, UV absorption, circular dichroism, or nuclear
magnetic resonance (NMR).
[0439] Homologues of a protein encompassed by the present invention
can comprise, consist essentially of, or consist of, in one
embodiment, an amino acid sequence that is at least about 35%
identical, and more preferably at least about 40% identical, and
more preferably at least about 45% identical, and more preferably
at least about 50% identical, and more preferably at least about
55% identical, and more preferably at least about 60% identical,
and more preferably at least about 65% identical, and more
preferably at least about 70% identical, and more preferably at
least about 75% identical, and more preferably at least about 80%
identical, and more preferably at least about 85% identical, and
more preferably at least about 90% identical, and more preferably
at least about 95% identical, and more preferably at least about
96% identical, and more preferably at least about 97% identical,
and more preferably at least about 98% identical, and more
preferably at least about 99% identical, or any percent identity
between 35% and 99%, in whole integers (i.e., 36%, 37%, etc.), to
an amino acid sequence disclosed herein that represents the amino
acid sequence of an enzyme or protein according to the invention
(including a biologically active domain of a full-length protein).
Preferably, the amino acid sequence of the homologue has a
biological activity of the wild-type or reference protein or of a
biologically active domain thereof (e.g., a catalytic domain).
[0440] In one embodiment, a protein of the present invention
comprises, consists essentially of, or consists of an amino acid
sequence that is less than 100% identical to an amino acid sequence
described herein (e.g., an amino acid sequence selected from: SEQ
ID NO:2, SEQ ID NO:5, SEQ ID NO:8) (i.e., a homologue). In another
aspect of the invention, a homologue according to the present
invention has an amino acid sequence that is less than about 99%
identical to any of such amino acid sequences, and in another
embodiment, is less than about 98% identical to any of such amino
acid sequences, and in another embodiment, is less than about 97%
identical to any of such amino acid sequences, and in another
embodiment, is less than about 96% identical to any of such amino
acid sequences, and in another embodiment, is less than about 95%
identical to any of such amino acid sequences, and in another
embodiment, is less than about 94% identical to any of such amino
acid sequences, and in another embodiment, is less than about 93%
identical to any of such amino acid sequences, and in another
embodiment, is less than about 92% identical to any of such amino
acid sequences, and in another embodiment, is less than about 91%
identical to any of such amino acid sequences, and in another
embodiment, is less than about 90% identical to any of such amino
acid sequences, and so on, in increments of whole integers.
[0441] As used herein, unless otherwise specified, reference to a
percent (%) identity refers to an evaluation of homology which is
performed using: (1) a BLAST 2.0 Basic BLAST homology search using
blastp for amino acid searches and blastn for nucleic acid searches
with standard default parameters, wherein the query sequence is
filtered for low complexity regions by default (described in
Altschul, S. F., Madden, T. L., Schaaffer, A. A., Zhang, J., Zhang,
Z., Miller, W. & Lipman, D. J. (1997) "Gapped BLAST and
PSI-BLAST: a new generation of protein database search programs."
Nucleic Acids Res. 25:3389-3402, incorporated herein by reference
in its entirety); (2) a BLAST 2 alignment (using the parameters
described below); (3) PSI-BLAST with the standard default
parameters (Position-Specific Iterated BLAST; and/or (4) CAZy
homology determined using standard default parameters from the
Carbohydrate Active EnZymes database (Coutinho, P. M. &
Henrissat, B. (1999) Carbohydrate-active enzymes: an integrated
database approach. In "Recent Advances in Carbohydrate
Bioengineering", H. J. Gilbert, G. Davies, B. Henrissat and B.
Svensson eds., The Royal Society of Chemistry, Cambridge, pp.
3-12).
[0442] It is noted that due to some differences in the standard
parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific
sequences might be recognized as having significant homology using
the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic
BLAST using one of the sequences as the query sequence may not
identify the second sequence in the top matches. In addition,
PSI-BLAST provides an automated, easy-to-use version of a "profile"
search, which is a sensitive way to look for sequence homologues.
The program first performs a gapped BLAST database search. The
PSI-BLAST program uses the information from any significant
alignments returned to construct a position-specific score matrix,
which replaces the query sequence for the next round of database
searching. Therefore, it is to be understood that percent identity
can be determined by using any one of these programs.
[0443] Two specific sequences can be aligned to one another using
BLAST 2 sequence as described in Tatusova and Madden, (1999),
"Blast 2 sequences--a new tool for comparing protein and nucleotide
sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein
by reference in its entirety. BLAST 2 sequence alignment is
performed in blastp or blastn using the BLAST 2.0 algorithm to
perform a Gapped BLAST search (BLAST 2.0) between the two sequences
allowing for the introduction of gaps (deletions and insertions) in
the resulting alignment. For purposes of clarity herein, a BLAST 2
sequence alignment is performed using the standard default
parameters as follows.
For blastn, using 0 BLOSUM62 matrix: [0444] Reward for match=1
[0445] Penalty for mismatch=-2 [0446] Open gap (5) and extension
gap (2) penalties [0447] gap x_dropoff (50) expect (10) word size
(11) filter (on) For blastp, using 0 BLOSUM62 matrix: [0448] Open
gap (11) and extension gap (1) penalties [0449] gap x_dropoff (50)
expect (10) word size (3) filter (on).
[0450] A protein of the present invention can also include proteins
having an amino acid sequence comprising at least 10 contiguous
amino acid residues of any of the sequences described herein (i.e.,
10 contiguous amino acid residues having 100% identity with 10
contiguous amino acids of SEQ ID NO:2). In other embodiments, a
homologue of a protein amino acid sequence includes amino acid
sequences comprising at least 20, or at least 30, or at least 40,
or at least 50, or at least 75, or at least 100, or at least 125,
or at least 150, or at least 175, or at least 150, or at least 200,
or at least 250, or at least 300, or at least 350 contiguous amino
acid residues of any of the amino acid sequence represented
disclosed herein. Even small fragments of proteins without
biological activity are useful in the present invention, for
example, in the preparation of antibodies against the full-length
protein or in a screening assay (e.g., a binding assay). Fragments
can also be used to construct fusion proteins, for example, where
the fusion protein comprises functional domains from two or more
different proteins (e.g., a domain from one protein linked to a
catalytic domain from another protein). In one embodiment, a
homologue has a measurable or detectable biological activity
associated with the wild-type protein (e.g., enzymatic
activity).
[0451] According to the present invention, the term "contiguous" or
"consecutive", with regard to nucleic acid or amino acid sequences
described herein, means to be connected in an unbroken sequence.
For example, for a first sequence to comprise 30 contiguous (or
consecutive) amino acids of a second sequence, means that the first
sequence includes an unbroken sequence of 30 amino acid residues
that is 100% identical to an unbroken sequence of 30 amino acid
residues in the second sequence. Similarly, for a first sequence to
have "100% identity" with a second sequence means that the first
sequence exactly matches the second sequence with no gaps between
nucleotides or amino acids.
[0452] In another embodiment, a protein of the present invention,
including a homologue, includes a protein having an amino acid
sequence that is sufficiently similar to a natural amino acid
sequence that a nucleic acid sequence encoding the homologue is
capable of hybridizing under moderate, high or very high stringency
conditions (described below) to (i.e., with) a nucleic acid
molecule encoding the natural protein (i.e., to the complement of
the nucleic acid strand encoding the natural amino acid sequence).
Preferably, a homologue of a protein of the present invention is
encoded by a nucleic acid molecule comprising a nucleic acid
sequence that hybridizes under low, moderate, or high stringency
conditions to the complement of a nucleic acid sequence that
encodes a protein comprising, consisting essentially of, or
consisting of, any amino acid sequence described herein, including,
but not limited to, an amino acid sequence represented by any of:
SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8. Such hybridization
conditions are described in detail below.
[0453] A nucleic acid sequence complement of nucleic acid sequence
encoding a protein of the present invention refers to the nucleic
acid sequence of the nucleic acid strand that is complementary to
the strand that encodes the protein. It will be appreciated that a
double stranded DNA which encodes a given amino acid sequence
comprises a single strand DNA and its complementary strand having a
sequence that is a complement to the single strand DNA. As such,
nucleic acid molecules of the present invention can be either
double-stranded or single-stranded, and include those nucleic acid
molecules that form stable hybrids under stringent hybridization
conditions with a nucleic acid sequence that encodes an amino acid
sequence such as SEQ ID NO:2, and/or with the complement of the
nucleic acid sequence that encodes an amino acid sequence such as
SEQ ID NO:2. Methods to deduce a complementary sequence are known
to those skilled in the art. It should be noted that since nucleic
acid sequencing technologies are not entirely error-free, the
sequences presented herein, at best, represent apparent sequences
of the proteins of the present invention.
[0454] As used herein, reference to hybridization conditions refers
to standard hybridization conditions under which nucleic acid
molecules are used to identify similar nucleic acid molecules. Such
standard conditions are disclosed, for example, in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs
Press, 1989. Sambrook et al., ibid., is incorporated by reference
herein in its entirety (see specifically, pages 9.31-9.62). In
addition, formulae to calculate the appropriate hybridization and
wash conditions to achieve hybridization permitting varying degrees
of mismatch of nucleotides are disclosed, for example, in Meinkoth
et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid.,
is incorporated by reference herein in its entirety.
[0455] More particularly, moderate stringency hybridization and
washing conditions, as referred to herein, refer to conditions
which permit isolation of nucleic acid molecules having at least
about 70% nucleic acid sequence identity with the nucleic acid
molecule being used to probe in the hybridization reaction (i.e.,
conditions permitting about 30% or less mismatch of nucleotides).
High stringency hybridization and washing conditions, as referred
to herein, refer to conditions which permit isolation of nucleic
acid molecules having at least about 80% nucleic acid sequence
identity with the nucleic acid molecule being used to probe in the
hybridization reaction (i.e., conditions permitting about 20% or
less mismatch of nucleotides). Very high stringency hybridization
and washing conditions, as referred to herein, refer to conditions
which permit isolation of nucleic acid molecules having at least
about 90% nucleic acid sequence identity with the nucleic acid
molecule being used to probe in the hybridization reaction (i.e.,
conditions permitting about 10% or less mismatch of nucleotides).
As discussed above, one of skill in the art can use the formulae in
Meinkoth et al., ibid. to calculate the appropriate hybridization
and wash conditions to achieve these particular levels of
nucleotide mismatch. Such conditions will vary, depending on
whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated
melting temperatures for DNA:DNA hybrids are 10.degree. C. less
than for DNA:RNA hybrids. In particular embodiments, stringent
hybridization conditions for DNA:DNA hybrids include hybridization
at an ionic strength of 6.times.SSC (0.9 M Na.sup.+) at a
temperature of between about 20.degree. C. and about 35.degree. C.
(lower stringency), more preferably, between about 28.degree. C.
and about 40.degree. C. (more stringent), and even more preferably,
between about 35.degree. C. and about 45.degree. C. (even more
stringent), with appropriate wash conditions. In particular
embodiments, stringent hybridization conditions for DNA:RNA hybrids
include hybridization at an ionic strength of 6.times.SSC (0.9 M
Na.sup.+) at a temperature of between about 30.degree. C. and about
45.degree. C., more preferably, between about 38.degree. C. and
about 50.degree. C., and even more preferably, between about
45.degree. C. and about 55.degree. C., with similarly stringent
wash conditions. These values are based on calculations of a
melting temperature for molecules larger than about 100
nucleotides, 0% formamide and a G+C content of about 40%.
Alternatively, T.sub.m can be calculated empirically as set forth
in Sambrook et al., supra, pages 9.31 to 9.62. In general, the wash
conditions should be as stringent as possible, and should be
appropriate for the chosen hybridization conditions. For example,
hybridization conditions can include a combination of salt and
temperature conditions that are approximately 20-25.degree. C.
below the calculated T.sub.m of a particular hybrid, and wash
conditions typically include a combination of salt and temperature
conditions that are approximately 12-20.degree. C. below the
calculated T.sub.m of the particular hybrid. One example of
hybridization conditions suitable for use with DNA:DNA hybrids
includes a 2-24 hour hybridization in 6.times.SSC (50% formamide)
at about 42.degree. C., followed by washing steps that include one
or more washes at room temperature in about 2.times.SSC, followed
by additional washes at higher temperatures and lower ionic
strength (e.g., at least one wash as about 37.degree. C. in about
0.1.times.-0.5.times.SSC, followed by at least one wash at about
68.degree. C. in about 0.1.times.-0.5.times.SSC).
[0456] The minimum size of a protein and/or homologue of the
present invention is a size sufficient to have biological activity
or, when the protein is not required to have such activity,
sufficient to be useful for another purpose associated with a
protein of the present invention, such as for the production of
antibodies that bind to a naturally occurring protein. In one
embodiment, the protein of the present invention is at least 20
amino acids in length, or at least about 25 amino acids in length,
or at least about 30 amino acids in length, or at least about 40
amino acids in length, or at least about 50 amino acids in length,
or at least about 60 amino acids in length, or at least about 70
amino acids in length, or at least about 80 amino acids in length,
or at least about 90 amino acids in length, or at least about 100
amino acids in length, or at least about 125 amino acids in length,
or at least about 150 amino acids in length, or at least about 175
amino acids in length, or at least about 200 amino acids in length,
or at least about 250 amino acids in length, and so on up to a full
length of each protein, and including any size in between in
increments of one whole integer (one amino acid). There is no
limit, other than a practical limit, on the maximum size of such a
protein in that the protein can include a portion of a protein or a
full-length protein, plus additional sequence (e.g., a fusion
protein sequence), if desired.
[0457] The present invention also includes a fusion protein that
includes a domain of a protein of the present invention (including
a homologue) attached to one or more fusion segments, which are
typically heterologous in sequence to the protein sequence (i.e.,
different than protein sequence). Suitable fusion segments for use
with the present invention include, but are not limited to,
segments that can: enhance a protein's stability; provide other
desirable biological activity; and/or assist with the purification
of the protein (e.g., by affinity chromatography). A suitable
fusion segment can be a domain of any size that has the desired
function (e.g., imparts increased stability, solubility, action or
biological activity; and/or simplifies purification of a protein).
Fusion segments can be joined to amino and/or carboxyl termini of
the domain of a protein of the present invention and can be
susceptible to cleavage in order to enable straight-forward
recovery of the protein. Fusion proteins are preferably produced by
culturing a recombinant cell transfected with a fusion nucleic acid
molecule that encodes a protein including the fusion segment
attached to either the carboxyl and/or amino terminal end of a
domain of a protein of the present invention. Accordingly, proteins
of the present invention also include expression products of gene
fusions (for example, used to overexpress soluble, active forms of
the recombinant protein), of mutagenized genes (such as genes
having codon modifications to enhance gene transcription and
translation), and of truncated genes (such as genes having membrane
binding domains removed to generate soluble forms of a membrane
protein, or genes having signal sequences removed which are poorly
tolerated in a particular recombinant host).
[0458] In one embodiment of the present invention, any of the amino
acid sequences described herein can be produced with from at least
one, and up to about 20, additional heterologous amino acids
flanking each of the C- and/or N-terminal ends of the specified
amino acid sequence. The resulting protein or polypeptide can be
referred to as "consisting essentially of" the specified amino acid
sequence. According to the present invention, the heterologous
amino acids are a sequence of amino acids that are not naturally
found (i.e., not found in nature, in vivo) flanking the specified
amino acid sequence, or that are not related to the function of the
specified amino acid sequence, or that would not be encoded by the
nucleotides that flank the naturally occurring nucleic acid
sequence encoding the specified amino acid sequence as it occurs in
the gene, if such nucleotides in the naturally occurring sequence
were translated using standard codon usage for the organism from
which the given amino acid sequence is derived.
[0459] In accordance with the present invention, an isolated
nucleic acid molecule is a nucleic acid molecule (polynucleotide)
that has been removed from its natural milieu (i.e., that has been
subject to human manipulation) and can include DNA, RNA, or
derivatives of either DNA or RNA, including cDNA. As such,
"isolated" does not reflect the extent to which the nucleic acid
molecule has been purified. Although the phrase "nucleic acid
molecule" primarily refers to the physical nucleic acid molecule,
and the phrase "nucleic acid sequence" primarily refers to the
sequence of nucleotides on the nucleic acid molecule, the two
phrases can be used interchangeably, especially with respect to a
nucleic acid molecule, or a nucleic acid sequence, being capable of
encoding a protein. An isolated nucleic acid molecule of the
present invention can be isolated from its natural source or
produced using recombinant DNA technology (e.g., polymerase chain
reaction (PCR) amplification, cloning) or chemical synthesis.
Isolated nucleic acid molecules can include, for example, genes,
natural allelic variants of genes, coding regions or portions
thereof, and coding and/or regulatory regions modified by
nucleotide insertions, deletions, substitutions, and/or inversions
in a manner such that the modifications do not substantially
interfere with the nucleic acid molecule's ability to encode a
protein of the present invention or to form stable hybrids under
stringent conditions with natural gene isolates. An isolated
nucleic acid molecule can include degeneracies. As used herein,
nucleotide degeneracy refers to the phenomenon that one amino acid
can be encoded by different nucleotide codons. Thus, the nucleic
acid sequence of a nucleic acid molecule that encodes a protein of
the present invention can vary due to degeneracies. It is noted
that a nucleic acid molecule of the present invention is not
required to encode a protein having protein activity. A nucleic
acid molecule can encode a truncated, mutated or inactive protein,
for example. In addition, nucleic acid molecules of the invention
are useful as probes and primers for the identification, isolation
and/or purification of other nucleic acid molecules. If the nucleic
acid molecule is an oligonucleotide, such as a probe or primer, the
oligonucleotide preferably ranges from about 5 to about 50 or about
500 nucleotides, more preferably from about 10 to about 40
nucleotides, and most preferably from about 15 to about 40
nucleotides in length.
[0460] According to the present invention, reference to a gene
includes all nucleic acid sequences related to a natural (i.e.
wild-type) gene, such as regulatory regions that control production
of the protein encoded by that gene (such as, but not limited to,
transcription, translation or post-translation control regions) as
well as the coding region itself. In another embodiment, a gene can
be a naturally occurring allelic variant that includes a similar
but not identical sequence to the nucleic acid sequence encoding a
given protein. Allelic variants have been previously described
above. The phrases "nucleic acid molecule" and "gene" can be used
interchangeably when the nucleic acid molecule comprises a gene as
described above.
[0461] Preferably, an isolated nucleic acid molecule of the present
invention is produced using recombinant DNA technology (e.g.,
polymerase chain reaction (PCR) amplification, cloning, etc.) or
chemical synthesis. Isolated nucleic acid molecules include any
nucleic acid molecules and homologues thereof that are part of a
gene described herein and/or that encode a protein described
herein, including, but not limited to, natural allelic variants and
modified nucleic acid molecules (homologues) in which nucleotides
have been inserted, deleted, substituted, and/or inverted in such a
manner that such modifications provide the desired effect on
protein biological activity or on the activity of the nucleic acid
molecule. Allelic variants and protein homologues (e.g., proteins
encoded by nucleic acid homologues) have been discussed in detail
above.
[0462] A nucleic acid molecule homologue (i.e., encoding a
homologue of a protein of the present invention) can be produced
using a number of methods known to those skilled in the art (see,
for example, Sambrook et al.). For example, nucleic acid molecules
can be modified using a variety of techniques including, but not
limited to, by classic mutagenesis and recombinant DNA techniques
(e.g., site-directed mutagenesis, chemical treatment, restriction
enzyme cleavage, ligation of nucleic acid fragments and/or PCR
amplification), or synthesis of oligonucleotide mixtures and
ligation of mixture groups to "build" a mixture of nucleic acid
molecules and combinations thereof. Another method for modifying a
recombinant nucleic acid molecule encoding a protein is gene
shuffling (i.e., molecular breeding) (See, for example, U.S. Pat.
No. 5,605,793 to Stemmer; Minshull and Stemmer; 1999, Curr. Opin.
Chem. Biol. 3:284-290; Stemmer, 1994, P.N.A.S. USA 91:10747-10751,
all of which are incorporated herein by reference in their
entirety). This technique can be used to efficiently introduce
multiple simultaneous changes in the protein. Nucleic acid molecule
homologues can be selected by hybridization with a gene or
polynucleotide, or by screening for the function of a protein
encoded by a nucleic acid molecule (i.e., biological activity).
[0463] The minimum size of a nucleic acid molecule of the present
invention is a size sufficient to encode a protein (including a
fragment or homologue of a full-length protein) having biological
activity, sufficient to encode a protein comprising at least one
epitope which binds to an antibody, or sufficient to form a probe
or oligonucleotide primer that is capable of forming a stable
hybrid with the complementary sequence of a nucleic acid molecule
encoding a natural protein (e.g., under moderate, high, or high
stringency conditions). As such, the size of the nucleic acid
molecule encoding such a protein can be dependent on nucleic acid
composition and percent homology or identity between the nucleic
acid molecule and complementary sequence as well as upon
hybridization conditions per se (e.g., temperature, salt
concentration, and formamide concentration). The minimal size of a
nucleic acid molecule that is used as an oligonucleotide primer or
as a probe is typically at least about 12 to about 15 nucleotides
in length if the nucleic acid molecules are GC-rich and at least
about 15 to about 18 bases in length if they are AT-rich. There is
no limit, other than a practical limit, on the maximal size of a
nucleic acid molecule of the present invention, in that the nucleic
acid molecule can include a portion of a protein encoding sequence,
a nucleic acid sequence encoding a full-length protein (including a
gene), including any length fragment between about 20 nucleotides
and the number of nucleotides that make up the full length cDNA
encoding a protein, in whole integers (e.g., 20, 21, 22, 23, 24, 25
. . . nucleotides), or multiple genes, or portions thereof.
[0464] The phrase "consisting essentially of", when used with
reference to a nucleic acid sequence herein, refers to a nucleic
acid sequence encoding a specified amino acid sequence that can be
flanked by from at least one, and up to as many as about 60,
additional heterologous nucleotides at each of the 5' and/or the 3'
end of the nucleic acid sequence encoding the specified amino acid
sequence. The heterologous nucleotides are not naturally found
(i.e., not found in nature, in vivo) flanking the nucleic acid
sequence encoding the specified amino acid sequence as it occurs in
the natural gene or do not encode a protein that imparts any
additional function to the protein or changes the function of the
protein having the specified amino acid sequence.
[0465] In one embodiment, the polynucleotide probes or primers of
the invention are conjugated to detectable markers. Detectable
labels suitable for use in the present invention include any
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels in the present invention include biotin for staining
with labeled streptavidin conjugate, magnetic beads (e.g.,
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein, Texas red,
rhodamine, green fluorescent protein, and the like), radiolabels
(e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C or .sup.32P), enzymes
(e.g., horse radish peroxidase, alkaline phosphatase and others
commonly used in an ELISA), and colorimetric labels such as
colloidal gold or colored glass or plastic (e.g., polystyrene,
polypropylene, latex, etc.) beads. Preferably, the polynucleotide
probes are immobilized on a substrate such as: artificial
membranes, organic supports, biopolymer supports and inorganic
supports.
[0466] According to the present invention, a recombinant nucleic
acid molecule typically comprises a recombinant vector and any one
or more of the isolated nucleic acid molecules as described herein.
According to the present invention, a recombinant vector is an
engineered (i.e., artificially produced) nucleic acid molecule that
is used as a tool for manipulating a nucleic acid sequence of
choice and/or for introducing such a nucleic acid sequence into a
host cell. The recombinant vector is therefore suitable for use in
cloning, sequencing, and/or otherwise manipulating the nucleic acid
sequence of choice, such as by expressing and/or delivering the
nucleic acid sequence of choice into a host cell to form a
recombinant cell. Such a vector typically contains heterologous
nucleic acid sequences, that is, nucleic acid sequences that are
not naturally found adjacent to nucleic acid sequence to be cloned
or delivered, although the vector can also contain regulatory
nucleic acid sequences (e.g., promoters, untranslated regions)
which are naturally found adjacent to nucleic acid sequences of the
present invention or which are useful for expression of the nucleic
acid molecules of the present invention (discussed in detail
below). The vector can be either RNA or DNA, either prokaryotic or
eukaryotic, and typically is a plasmid. The vector can be
maintained as an extrachromosomal element (e.g., a plasmid) or it
can be integrated into the chromosome of a recombinant host cell,
although it is preferred if the vector remain separate from the
genome for most applications of the invention. The entire vector
can remain in place within a host cell, or under certain
conditions, the plasmid DNA can be deleted, leaving behind the
nucleic acid molecule of the present invention. An integrated
nucleic acid molecule can be under chromosomal promoter control,
under native or plasmid promoter control, or under a combination of
several promoter controls. Single or multiple copies of the nucleic
acid molecule can be integrated into the chromosome. A recombinant
vector of the present invention can contain at least one selectable
marker.
[0467] In one embodiment, a recombinant vector used in a
recombinant nucleic acid molecule of the present invention is an
expression vector. As used herein, the phrase "expression vector"
is used to refer to a vector that is suitable for production of an
encoded product (e.g., a protein of interest, such as an enzyme of
the present invention). In this embodiment, a nucleic acid sequence
encoding the product to be produced (e.g., the protein or homologue
thereof) is inserted into the recombinant vector to produce a
recombinant nucleic acid molecule. The nucleic acid sequence
encoding the protein to be produced is inserted into the vector in
a manner that operatively links the nucleic acid sequence to
regulatory sequences in the vector that enable the transcription
and translation of the nucleic acid sequence within the recombinant
host cell.
[0468] Typically, a recombinant nucleic acid molecule includes at
least one nucleic acid molecule of the present invention
operatively linked to one or more expression control sequences
(e.g., transcription control sequences or translation control
sequences). As used herein, the phrase "recombinant molecule" or
"recombinant nucleic acid molecule" primarily refers to a nucleic
acid molecule or nucleic acid sequence operatively linked to a
transcription control sequence, but can be used interchangeably
with the phrase "nucleic acid molecule", when such nucleic acid
molecule is a recombinant molecule as discussed herein. According
to the present invention, the phrase "operatively linked" refers to
linking a nucleic acid molecule to an expression control sequence
in a manner such that the molecule is able to be expressed when
transfected (i.e., transformed, transduced, transfected, conjugated
or conduced) into a host cell. Transcription control sequences are
sequences that control the initiation, elongation, or termination
of transcription. Particularly important transcription control
sequences are those that control transcription initiation, such as
promoter, enhancer, operator and repressor sequences. Suitable
transcription control sequences include any transcription control
sequence that can function in a host cell or organism into which
the recombinant nucleic acid molecule is to be introduced.
[0469] Recombinant nucleic acid molecules of the present invention
can also contain additional regulatory sequences, such as
translation regulatory sequences, origins of replication, and other
regulatory sequences that are compatible with the recombinant cell.
In one embodiment, a recombinant molecule of the present invention,
including those that are integrated into the host cell chromosome,
also contains secretory signals (i.e., signal segment nucleic acid
sequences) to enable an expressed protein to be secreted from the
cell that produces the protein. Suitable signal segments include a
signal segment that is naturally associated with the protein to be
expressed or any heterologous signal segment capable of directing
the secretion of the protein according to the present invention. In
another embodiment, a recombinant molecule of the present invention
comprises a leader sequence to enable an expressed protein to be
delivered to and inserted into the membrane of a host cell.
Suitable leader sequences include a leader sequence that is
naturally associated with the protein, or any heterologous leader
sequence capable of directing the delivery and insertion of the
protein to the membrane of a cell.
[0470] According to the present invention, the term "transfection"
is generally used to refer to any method by which an exogenous
nucleic acid molecule (i.e., a recombinant nucleic acid molecule)
can be inserted into a cell. The term "transformation" can be used
interchangeably with the term "transfection" when such term is used
to refer to the introduction of nucleic acid molecules into
microbial cells or plants and describes an inherited change due to
the acquisition of exogenous nucleic acids by the microorganism
that is essentially synonymous with the term "transfection."
Transfection techniques include, but are not limited to,
transformation, particle bombardment, electroporation,
microinjection, lipofection, adsorption, infection and protoplast
fusion.
[0471] An encoded product is produced by expressing a nucleic acid
molecule as described herein under conditions effective to produce
the protein. A preferred method to produce an encoded protein is by
transfecting a host cell with one or more recombinant molecules to
form a recombinant cell. Suitable host cells to transfect include,
but are not limited to, any bacterial, fungal (e.g., filamentous
fungi or yeast), plant, insect, or animal cell that can be
transfected. Host cells can be either untransfected cells or cells
that are already transfected with at least one other recombinant
nucleic acid molecule.
[0472] Suitable cells (e.g., a host cell or production organism)
include any microorganism (e.g., a bacterium, a protist, an alga, a
fungus, or other microbe), and is preferably a bacterium, a yeast
or a filamentous fungus. Suitable bacterial genera include, but are
not limited to, Escherichia, Bacillus, Lactobacillus, Pseudomonas
and Streptomyces. Suitable bacterial species include, but are not
limited to, Escherichia coli, Bacillus subtilis, Bacillus
licheniformis, Lactobacillus brevis, Pseudomonas aeruginosa and
Streptomyces lividans. Suitable genera of yeast include, but are
not limited to, Saccharomyces, Schizosaccharomyces, Candida,
Hansenula, Pichia, Kluyveromyces, and Phaffia. Suitable yeast
species include, but are not limited to, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Candida albicans, Hansenula polymorphs,
Pichia pastoris, P. canadensis, Kluyveromyces marxianus and Phaffia
rhodozyma.
[0473] Suitable fungal genera include, but are not limited to,
Chrysosporium, Thielavia, Neurospora, Aureobasidium, Filibasidium,
Piromyces, Corynascus, Cryplococcus, Acremonium, Tolypocladium,
Scytalidium, Schizophyllum, Sporotrichum, Penicillium, Gibberella,
Myceliophthora, Mucor, Aspergillus, Fusarium, Humicola, and
Trichoderma, and anamorphs and teleomorphs thereof. Suitable fungal
species include, but are not limited to, Aspergillus niger,
Aspergillus nidulans, Aspergillus japonicus, Absidia coerulea,
Rhizopus oryzae, Chrysosporium lucknowense, Neurospora crassa,
Neurospora intermedia, Trichoderma reesei, Penicillium canescens,
Penicillium solitum, Penicillium funiculosum, and Talaromyces
flavus. In one embodiment, the host cell is a fungal cell of the
species Chrysosporium lucknowense. In one embodiment, the host cell
is a fungal cell of Strain C1 (VKM F-3500-D) or a mutant strain
derived therefrom (e.g., UV13-6 (Accession No. VKM F-3632 D);
NG7C-19 (Accession No. VKM F-3633 D); or UV18-25 (VKM F-3631D)).
Host cells can be either untransfected cells or cells that are
already transfected with at least one other recombinant nucleic
acid molecule. Additional embodiments of the present invention
include any of the genetically modified cells described herein.
[0474] In one embodiment, one or more protein(s) expressed by an
isolated nucleic acid molecule of the present invention are
produced by culturing a cell that expresses the protein (i.e., a
recombinant cell or recombinant host cell) under conditions
effective to produce the protein. In some instances, the protein
may be recovered, and in others, the cell may be harvested in
whole, either of which can be used in a composition.
[0475] Microorganisms used in the present invention (including
recombinant host cells or genetically modified microorganisms) are
cultured in an appropriate fermentation medium. An appropriate, or
effective, fermentation medium refers to any medium in which a cell
of the present invention, including a genetically modified
microorganism (described below), when cultured, is capable of
expressing enzymes useful in the present invention. Such a medium
is typically an aqueous medium comprising assimilable carbon,
nitrogen and phosphate sources. Such a medium can also include
appropriate salts, minerals, metals and other nutrients.
Microorganisms and other cells of the present invention can be
cultured in conventional fermentation bioreactors. The
microorganisms can be cultured by any fermentation process which
includes, but is not limited to, batch, fed-batch, cell recycle,
and continuous fermentation. The fermentation of microorganisms
such as fungi may be carried out in any appropriate reactor, using
methods known to those skilled in the art. For example, the
fermentation may be carried out for a period of 1 to 14 days, or
more preferably between about 3 and 10 days. The temperature of the
medium is typically maintained between about 25 and 50.degree. C.,
and more preferably between 28 and 40.degree. C. The pH of the
fermentation medium is regulated to a pH suitable for growth and
protein production of the particular organism. The fermentor can be
aerated in order to supply the oxygen necessary for fermentation
and to avoid the excessive accumulation of carbon dioxide produced
by fermentation. In addition, the aeration helps to control the
temperature and the moisture of the culture medium. In general the
fungal strains are grown in fermentors, optionally centrifuged or
filtered to remove biomass, and optionally concentrated,
formulated, and dried to produce an enzyme(s) or a multi-enzyme
composition that is a crude fermentation product. Particularly
suitable conditions for culturing filamentous fungi are described,
for example, in U.S. Pat. No. 6,015,707 and U.S. Pat. No.
6,573,086, supra.
[0476] Depending on the vector and host system used for production,
resultant proteins of the present invention may either remain
within the recombinant cell; be secreted into the culture medium;
be secreted into a space between two cellular membranes; or be
retained on the outer surface of a cell membrane. The phrase
"recovering the protein" refers to collecting the whole culture
medium containing the protein and need not imply additional steps
of separation or purification. Proteins produced according to the
present invention can be purified using a variety of standard
protein purification techniques, such as, but not limited to,
affinity chromatography, ion exchange chromatography, filtration,
electrophoresis, hydrophobic interaction chromatography, gel
filtration chromatography, reverse phase chromatography,
concanavalin A chromatography, chromatofocusing and differential
solubilization.
[0477] Proteins of the present invention are preferably retrieved,
obtained, and/or used in "substantially pure" form. As used herein,
"substantially pure" refers to a purity that allows for the
effective use of the protein in any method according to the present
invention. For a protein to be useful in any of the methods
described herein or in any method utilizing enzymes of the types
described herein according to the present invention, it is
substantially free of contaminants, other proteins and/or chemicals
that might interfere or that would interfere with its use in a
method disclosed by the present invention (e.g., that might
interfere with enzyme activity), or that at least would be
undesirable for inclusion with a protein of the present invention
(including homologues) when it is used in a method disclosed by the
present invention (described in detail below). Preferably, a
"substantially pure" protein, as referenced herein, is a protein
that can be produced by any method (i.e., by direct purification
from a natural source, recombinantly, or synthetically), and that
has been purified from other protein components such that the
protein comprises at least about 80% weight/weight of the total
protein in a given composition (e.g., the protein of interest is
about 80% of the protein in a solution/composition/buffer), and
more preferably, at least about 85%, and more preferably at least
about 90%, and more preferably at least about 91%, and more
preferably at least about 92%, and more preferably at least about
93%, and more preferably at least about 94%, and more preferably at
least about 95%, and more preferably at least about 96%, and more
preferably at least about 97%, and more preferably at least about
98%, and more preferably at least about 99%, weight/weight of the
total protein in a given composition.
[0478] It will be appreciated by one skilled in the art that use of
recombinant DNA technologies can improve control of expression of
transfected nucleic acid molecules by manipulating, for example,
the number of copies of the nucleic acid molecules within the host
cell, the efficiency with which those nucleic acid molecules are
transcribed, the efficiency with which the resultant transcripts
are translated, and the efficiency of post-translational
modifications. Additionally, the promoter sequence might be
genetically engineered to improve the level of expression as
compared to the native promoter. Recombinant techniques useful for
controlling the expression of nucleic acid molecules include, but
are not limited to, integration of the nucleic acid molecules into
one or more host cell chromosomes, addition of vector stability
sequences to plasmids, substitutions or modifications of
transcription control signals (e.g., promoters, operators,
enhancers), substitutions or modifications of translational control
signals (e.g., ribosome binding sites), modification of nucleic
acid molecules to correspond to the codon usage of the host cell,
and deletion of sequences that destabilize transcripts.
[0479] As used herein, a genetically modified microorganism can
include a genetically modified bacterium, yeast, filamentous
fungus, or other microbe. Such a genetically modified microorganism
has a genome which is modified (i.e., mutated or changed) from its
normal (i.e., wild-type or naturally occurring) form such that the
desired result is achieved (i.e., increased or modified activity
and/or production of a protein or deletion or inactivation of a
protein. Genetic modification of a microorganism can be
accomplished using classical strain development and/or molecular
genetic techniques. Such techniques known in the art and are
generally disclosed for microorganisms, for example, in Sambrook et
al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Labs Press or Molecular Cloning: A Laboratory Manual, third
edition (Sambrook and Russell, 2001), (jointly referred to herein
as "Sambrook"). The references of Sambrook, ibid., are incorporated
by reference herein in its entirety. A genetically modified
microorganism can include a microorganism in which nucleic acid
molecules have been inserted, deleted or modified (i.e., mutated;
e.g., by insertion, deletion, substitution, and/or inversion of
nucleotides), in such a manner that such modifications provide the
desired effect within the microorganism.
[0480] According to the present invention, the phrase "selectively
binds to" refers to the ability of an antibody, antigen binding
fragment or binding partner of the present invention to
preferentially bind to specified proteins. More specifically, the
phrase "selectively binds" refers to the specific binding of one
protein to another (e.g., an antibody, fragment thereof, or binding
partner to an antigen), wherein the level of binding, as measured
by any standard assay (e.g., an immunoassay), is statistically
significantly higher than the background control for the assay. For
example, when performing an immunoassay, controls typically include
a reaction well/tube that contain antibody or antigen binding
fragment alone (i.e., in the absence of antigen), wherein an amount
of reactivity (e.g., non-specific binding to the well) by the
antibody or antigen binding fragment thereof in the absence of the
antigen is considered to be background. Binding can be measured
using a variety of methods standard in the art including enzyme
immunoassays (e.g., ELISA), immunoblot assays, etc.).
[0481] Antibodies are characterized in that they comprise
immunoglobulin domains and as such, they are members of the
immunoglobulin superfamily of proteins. An antibody of the
invention includes polyclonal and monoclonal antibodies, divalent
and monovalent antibodies, bi- or multi-specific antibodies, serum
containing such antibodies, antibodies that have been purified to
varying degrees, and any functional equivalents of whole
antibodies. Isolated antibodies of the present invention can
include serum containing such antibodies, or antibodies that have
been purified to varying degrees. Whole antibodies of the present
invention can be polyclonal or monoclonal. Alternatively,
functional equivalents of whole antibodies, such as antigen binding
fragments in which one or more antibody domains are truncated or
absent (e.g., Fv, Fab, Fab', or F(ab).sub.2 fragments), as well as
genetically-engineered antibodies or antigen binding fragments
thereof, including single chain antibodies or antibodies that can
bind to more than one epitope (e.g., bi-specific antibodies), or
antibodies that can bind to one or more different antigens (e.g.,
bi- or multi-specific antibodies), may also be employed in the
invention. Methods for the generation and production of antibodies
are well known in the art.
[0482] Monoclonal antibodies may be produced according to the
methodology of Kohler and Milstein (Nature 256:495-497, 1975).
Non-antibody polypeptides, sometimes referred to as binding
partners, are designed to bind specifically to a protein of the
invention. Examples of the design of such polypeptides, which
possess a prescribed ligand specificity are given in Beste et al.,
(Proc. Natl. Acad. Sci. 96:1898-1903, 1999), incorporated herein by
reference in its entirety. In one embodiment, a binding agent of
the invention is immobilized on a substrate such as: artificial
membranes, organic supports, biopolymer supports and inorganic
supports such as for use in a screening assay.
[0483] Each publication or reference cited herein is incorporated
herein by reference in its entirety.
[0484] The following examples are provided for the purpose of
illustration and are not intended to limit the scope of the present
invention.
Examples
[0485] Additional examples of fungal strains, library development
and expression systems can be found in U.S. Pat. No. 7,122,330,
which is incorporated herein by reference in its entirety.
A. Construction of Improved C. lucknowense Host Strains
[0486] As described in U.S. Pat. No. 7,122,330, morphological
variants of the original C. lucknowense strain were isolated during
the course of a strain development program for neutral cellulase
production. These mutants, of which the prototypical strain is
designated UV18-25, are characterized by hypersecretion of protein,
low viscosity in fermentation, and hyphal fragmentation. The
morphology not only enables the high-level production of proteins,
but also the separation of monoclonal elements, called
"propagules", from mixed populations. The formation of propagules
also results in low viscosity that allows manipulation of cultures
using robotic liquid handling systems.
[0487] Strain UV18-25, in addition to producing high levels of
neutral cellulase, is also characterized by the production of high
levels of extracellular proteases. The presence of these proteases
often confounds the production of heterologous proteins, presumably
by degrading those proteins before they can accumulate to high
levels. In order to isolate improved strains for the production of
heterologous proteins, low protease variants of UV18-25 were
isolated.
[0488] The mutants were produced by mutagenesis, followed by
selection for low protease producers. Colonies that were
characterized by reduced clearing of skim milk in agar plates were
isolated and characterized. One such colony, which had
.sup..about.10% of the protease activity of UV18-25 against casein,
was designated UV18#100.f.
[0489] The residual extracellular proteases and the conditions
under which they were expressed were extensively studied. It was
determined that three proteases, whose genes are designated alp1,
pep4, and alp2, were responsible for the bulk of the residual
activity. Using "reverse genetics" and genomics methods, the
corresponding genes were isolated and disrupted with the pyr5 gene,
encoding the orotate phosphoribosyl transferase of C. lucknowense.
The proteases were sequential removed by introducing the disrupted
genes into a pyr5 mutant of UV18#100.f requiring uracil and uridine
for growth, selecting pyrimidine prototrophs, screening for the
lack of the targeted protease, and removing the pyr5 gene from the
disruption construct. This ultimately led to pyr5 mutant strains of
UV18#100.f containing deletions of one or more of these proteases.
The same gene disruption strategy was used to isolate protease
deletion variants of other C. lucknowense host strains. Strain
UV18#100.f .DELTA.pyr5 .DELTA.alp1 and UV18#100.f .DELTA.pyr5
.DELTA.alp1.DELTA.pep4 .DELTA.alp2 were used to test in vitro
stability and expression of antibodies in C. lucknowense. These
strains produced less than 1% of the extracellular protease
activity of strain UV18-25.
Host Strains with Reduced Non-Homologous Recombination
[0490] For the efficient development of the screening methods
described herein, host strains with mutations in one of the genes
essential for non-homologous recombination were generated using
disruption of the C. lucknowense homologue of the human Ku70 gene.
This strain showed a highly increased fraction of transformants
resulting from homologous recombination. This strain has useful
attributes for methods using both the replicative or integrative
transformation vectors as described herein. Other strains can be
produced using similar methods or by classical mutagenesis and are
encompassed by this invention.
[0491] The mutated phenotype from this type of host strain may also
be beneficial for utilization in classical mutagenesis
programs.
B. Production of a Dedicated Vector for Efficient Library
Construction and High Throughput Screening in the Hyphal Fungus
Chrysosporium Lucknowense
Materials and Methods
Transformation
[0492] Protoplasts of strains UV18#100.f .DELTA.pyr5 and UV18#100.f
.DELTA.pyr5.DELTA.pyr4 were isolated after incubation of mycelium
with Caylase C4 (Cayla, Toulouse, France), and, transformants were
selected for pyrimidine prototrophy on selective solid or liquid
minimal medium. The transformed protoplasts were regenerated in
osmotically stabilized (0.67 M sucrose) medium, and after
regeneration, were plated on selective solid medium to determine
the transformation frequency. The remaining protoplasts were seeded
into 100 volumes selective medium and incubated in shake flasks
until propagules were formed. The propagules were filtered through
Miracloth (Merck Biosciences, Darmstadt, Germany), concentrated by
centrifugation, dissolved in physiological salt solution and
glycerol was added to a final concentration of 20%. Finally the
propagule mix was fractionated and stored at -80.degree. C.
[0493] Transformation of E. coli was done by electroporation
according to standard procedures described by the supplier (BioRad,
Veenendaal, The Netherlands).
Medium, Strains and Cultivation Conditions
[0494] C. lucknowense strains were cultivated on minimal medium
(MM). To cultivate the auxotrophic mutants of C. lucknowense
(.DELTA.pyr5 and .DELTA.pyr5.DELTA.pyr4), uridine and uracil were
added to the medium. To stimulate fast propagule formation in
microtiter plates, a medium optimized for propagule formation
(PFO), in which the glucose concentration was reduced 100-fold, was
used. This resulted in a uniform distribution of the number of
propagules/ml in deep-well microtiter plates after a short
cultivation period even when the initial seeding density was as low
as approximately 20 spores/ml. To maintain a constant medium pH
during the production of secreted proteins in microtiter plate
cultivations, production medium (PM) was used. In PM, the nitrogen
source present in MM was replaced by (NH.sub.4).sub.2SO.sub.4 and
the original KH.sub.2PO.sub.4 concentration was increased
5-fold.
[0495] Prior to a seeding in microtiter plates, an actual colony
count was conducted. The frozen propagule mixture was thawed on ice
and a small sample was plated on selective solid medium. Based on
these numbers, an appropriate aliquot of the propagule mixture was
then added to the propagule formation optimized medium to give a
final concentration of approximately five propagules per well when
seeded to 96 deep-well microtiter plates. Plates were placed in an
incubator shaker (Multitron, Infors, Bottmingen, Switzerland) for 3
days at 35.degree. C. and a rotation speed of 850 rpm. Three .mu.L
of the newly formed propagules mixture were transferred to a new
plate containing 0.22 mL medium in each well. After 4 days of
incubation the mycelium fragments were pelleted by centrifugation
and medium samples (10-20 .mu.L) were taken for activity
measurements.
Chemicals
[0496] Restriction enzymes and T4 DNA ligase were purchased from
MBI Fermentas (St. Leon, Germany). SuperTaq polymerase was
purchased from SphaeroQ (Leiden, The Netherlands).
[.alpha.-.sup.32P]dCTP was obtained from GE Healthcare (Little
Chalfont, UK). All medium chemicals were obtained from
Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
DNA Manipulation
[0497] For the recovery of the expression cassette and the flanking
sequence responsible for plasmid replication and selection in E.
coli from a fungal transformant the following rescue procedure was
set up. One microgram of genomic DNA of the identified transformant
was digested to completion with Nod then self-ligated using T4 DNA
ligase. The ligated DNA was used for E. coli transformation by
electroporation using standard conditions and plated to LB-agar
plates containing 50 mg/L ampicillin.
[0498] For Southern analysis chromosomal DNA was separated on a
0.8% TEA-agarose gel and transferred to a Nylon membrane. The
filters were hybridized under stringent conditions with a
.sup.32P-labeled probe using the Rediprime.TM. II random Prime
labeling system from GE Healthcare.
Vector Constructions
[0499] The following vectors were constructed to test whether DNA
elements like AMA and telomeres could promote autonomous
replication in C. lucknowense and result in higher transformation
frequencies. Heterologous fungal genes that could complement the
auxotrophic mutations in the acceptor strains of C. lucknowense
were used to limit sequence homology between the vectors to be
constructed and the genomic DNA of the fungal host strain. The pyrE
gene of A. niger was cloned as a 5.2 kb SstII fragment in
pBluescript (Stratagene Europe, Amsterdam, The Netherlands),
yielding pBlue-pyrE. The expression cassette conferring kanamycin
resistance as present between the two inverted 0.6 kb stretches of
the human telomeric repeats.sup.11 was replaced by a synthetic
linker containing the recognition site of the meganuclease I-CeuI.
The modified cassette was cloned as a 1.2 kb HindIII fragment in
pBlue-pyrE yielding pBlue-pyrE tel. The AMA sequence was isolated
as a partial HindIII fragment from pHELP.sup.7 and cloned into the
corresponding site of pBlue-pyrE, yielding pBlue-pyrE AMA.
[0500] The following vectors were constructed to test the
characteristics of the developed telomeric vector for robotic high
throughput screening in C. lucknowense. The vector pPgpdA-TtrpC
pyrE tel pyrG (FIG. 2) was constructed using a derivative of
pAN52-4.sup.16 called pAN52-NotI.DELTA.ss (unpublished results). A
3.8 kb NotI-BamHI pyrE fragment derived from pBlue-pyrE was
inserted in pAN52-NotI.DELTA.ss. In this vector, pAN52 pyrE, a
blunt ended 1.2 kb telomere fragment was cloned in the StuI site
yielding pAN52 pyrE tel. Finally, the pyrG gene from Aspergillus
oryzae, present on a subcloned 2.8 kb SalI-XhoI fragment, was
inserted in the XhoI sites of pAN52 pyrE tel.
[0501] The vector pPcbh1-glaA-Tcbh1 pyrE tel pyrG (FIG. 2) contains
the full genome sequence of glaA, the glucoamylase (GLA) encoding
gene of A. niger (accession number #232690). The vector
pPcbh1-lac1-TtrpC pyrE tel pyrG (FIG. 2) contains a modified
laccase encoding cDNA clone from Pycnoporus cinnabarinus.sup.16.
This expression cassette was obtained from pLac1-B in which the 21
amino acids of the laccase signal peptide were replaced by the
preprosequence of GLA from A. niger.sup.16.
Construction of C. lucknowense Host Strains
[0502] As discussed above, the main prerequisites for a successful
library construction and screening procedure in fungi are: i) a
very high transformation frequency, ii) stable and high level
expression of the genes cloned in the expression vector and iii) an
efficient procedure to re-isolate the DNA fragment that encodes the
(enzymatic) activity screened.
[0503] The strain UV18#100.f .DELTA.pyr5 was generated by
transforming strain UV18#100.f with the pyr5 gene disrupted with
the A. nidulans amdS gene, selecting for strains able to grow with
acetamide as sole nitrogen source, and screening for pyrimidine
auxotrophy. Subsequent counterselection for the spontaneous loss of
the amdS marker on fluoro-acetamide containing medium resulted in
the strain UV18#100.f.DELTA.pyr5. The uridine requirement could be
resolved by the introduction of an integrative vector containing
either the pyr5 gene of C1 (pBlue-pyr5) or the A. nidulans ortholog
pyrE (pBlue-pyrE). The transformation frequency was similar and in
both cases transformants have the same phenotype as the parental
strain UV18#100.f.
[0504] In order to test library construction and screening in C.
lucknowense, a second specific auxotrophic mutation in the uridine
biosynthetic pathway of C1 was introduced by the inactivation of
the pyr4 gene. The pyr4 gene encodes the enzyme
orotidine-5'-phosphate-decarboxylase. This enzyme converts the
reaction product of the pyr5 encoded orotate
phosphoribosyltransferase catalyzed reaction into
uridine-5-phosphate. By a similar approach as described for
UV18#100.f .DELTA.pyr5, the strain UV18#100.f
.DELTA.pyr5.DELTA.pyr4 was generated.
Increased Transformation Frequency
[0505] To test the ability of either the AMA sequence or the human
telomeric sequence to increase the transformation frequency and to
facilitate autonomous replication in C. lucknowense these genetic
elements were introduced in the vector pBlue-pyrE.
[0506] The strain UV18#100.f .DELTA.pyr5 was transformed with equal
amounts of the vectors pBlue-pyrE, pBlue-pyrE AMA1 and pBlue-pyrE
tel. A 50 to 100-fold increase in the number of pyr.sup.+
transformants was observed for the telomere containing vector.
Southern analysis of chromosomal DNA isolated from a set of
transformants obtained with the pBlue-pyrE AMA1 vector indicated
that the vector did integrate. The morphology of all these
transformants was large and smooth.
[0507] In contrast, more than 90% of the pBlue-pyrE tel
transformants displayed a ragged-type colony morphology (FIG. 3A).
Southern analysis indicated that the telomeric vector in these C.
lucknowense transformants was maintained as a non-integrated linear
DNA molecule (FIG. 3B). Such ragged-type phenotype was described
previously for A. niger transformants having an autonomously
replicating vector copy of an AMA containing vector.sup.8. The
primary C. lucknowense transformants were cultivated in selective
liquid medium, the propagules were isolated and plated on selective
medium agar-plates (FIG. 3A). A gradual decrease in the number of
slow growing, ragged-type colonies indicative for the presence of
the linear autonomously replicating vector and the concomitant
increase in the number of smooth-type, fast growing colonies
indicative for integration, was observed. The suggestion that after
prolonged cultivation the linear plasmid finally tends to integrate
in the C1 genome was verified by Southern analysis (FIG. 3B).
Similar observations were also reported for a telomere vector in A.
nidulans.sup.11. The transformation procedure was further optimized
by introducing an additional regeneration step in osmotically
stabilized medium. Transformation frequencies of up to 13,000
primary transformants per microgram of plasmid DNA were
obtained.
Telomeric Vectors for Complex Library Construction in C1
[0508] AMA containing vectors have been used for gene cloning by
complementation in A. nidulans.sup.19 and A. niger.sup.8. The
disadvantage of this "instant gene bank" approach was the
instability of the vector and the uncontrolled recombination
between the AMA vector and the complementing DNA fragments.
Therefore DNA rescue from AMA derived plasmid molecules via
transformation of E. coli with total genomic DNA of the A. niger
transformants was not always possible.sup.8. The initial high
transformation frequency and the controlled stabilization of the
telomeric vector after propagule formation as found in C.
lucknowense fit the demands for the construction of large, complex
libraries. The next step was to test the use of the telomeric
vector for library construction and screening in C. lucknowense. In
the first instance the inventors analyzed the complementation of
the pyr4 deletion. Chromosomal DNA of C1 was partially digested
with Sau3A and fragments from 2-6 kb were isolated and inserted in
the unique BamHI site of pPgpdA-TtrpC pyrE tel (FIG. 2). This
vector contains in addition to the genetic elements present in
pBlue-pyrE tel an expression cassette of the A. nidulans
glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) promoter and
A. nidulans trpC terminator. The primary library in E. coli
DH5.alpha. consisted of 190,000 transformants. The transformants
were pooled and an aliquot was plated on LB agar plates
supplemented with carbenicillin. Cells were harvested after an
overnight incubation at 37.degree. C. and plasmid DNA was isolated.
Seventy .mu.g of plasmid DNA was used to transform strain
UV18#100.f .DELTA.pyr5.DELTA.pyr4. Simultaneously, the same amount
of plasmid DNA was also transformed into strain UV18#100.f
.DELTA.pyr5 to determine the transformation frequency. About
330,000 Pyr.sup.+ transformants were obtained in UV18#100.f
.DELTA.pyr5 as host and 14 Pyr.sup.+ transformants were obtained on
selective agar plates using UV18#100.f pyr5.DELTA.pyr4 as host. By
PCR and sequence analysis we could show that in the latter case all
Pyr.sup.+ transformants contained an insert with the complete C1
pyr4 gene.
[0509] Analysis of a set of Pyr.sup.+ transformants of UV18#100.f
.DELTA.pyr5 obtained with the vector pPgpdA-Ttrpc pyrE tel showed
that during the integration process part of the linear vector could
be lost and that sequences closer to the dominant selection marker
(pyrE) were retained more frequently. Based on these observations
the vector was redesigned by introducing a second dominant
selection marker, the pyrG gene of A. nidulans. In this vector,
assigned as pPgpdA-TtrpC pyrE tel pyrG (FIG. 2) the promoter and
terminator sequences were then flanked by two selection markers.
This vector was introduced into strain UV18#100.f
.DELTA.pyr5.DELTA.pyr4. The transformation frequency and ratio of
colonies displaying a ragged or smooth colony morphology was
comparable to that obtained with the previous telomeric vector
pBlue-pyrE tel. Twenty colonies from the pool of propagules of
UV18#100.f .DELTA.pyr5.DELTA.pyr4[pPgpdA-TtrpC pyrE tel pyrG] that
grew on selective medium were selected for further analysis. PCR
and Southern analysis demonstrated that in all these transformants
the expression cassette (PgpdA-TtrpC) was present and intact. Based
on the hybridization patterns of chromosomal DNA probed with the
fragments for pyrE, pyrG and the expression cassette we could
deduce the position of integration (FIG. 4). The vector had
integrated into the genome at the end of a chromosome in all
transformants. Recombination occurred via one of telomeric regions
of pPgpdA-TtrpC pyrE tel pyrG resulting in a situation that either
the pyrG (type I) or pyrE (type II) gene was located at the most
distal part of a chromosome (FIG. 4B). The difference in size of
the restriction fragments of both type I and II transformants shows
that integration occurs at different chromosomes. The integrated
vectors were genetically stable over several generations even under
non-selective cultivation conditions (medium supplied with uridine)
in liquid medium. The advantage of this stabilization step during
propagule formation in liquid medium is that with our newly
designed vector single copy integration occurs within or near the
telomeric regions of the C1 chromosomes at a very high frequency.
This aspect is important for HTS as it could result in a more
uniform expression pattern compared to other integrative
vectors.
Validation of Reporter Gene Expression in the Telomeric Vectors
[0510] Two reporter constructs were cloned in the backbone of the
pPgpdA-TtrpC pyrE tel pyrG vector (FIG. 2) to evaluate the use of
the telomeric vector containing two auxotrophic selection markers
for expression and screening. In this procedure the gpdA promoter
was replaced with the promoter for the cellobiohydrolase (CBH1)
encoding gene cbh1 of C. lucknowense to maximize the expression
levels for the reporter genes. A high level of expression is
essential as the inventors have shown that the integrated telomeric
vector is present as a single copy in C. lucknowense. The reporter
genes used were the glaA gene of A. niger and the laccase encoding
gene (lac1) of P. cinnabarinus. For both enzymatic activities a
robot compatible assay was available. The two reporter vectors
pPcbh1-lac1-TtrpC pyrE tel pyrG and pPcbh1-glaA-Tcbh1 pyrE tel pyrG
and a control vector (pPcbh1-Tcbh1 pyrE tel pyrG) were introduced
separately into strain UV18#100.f .DELTA.pyr5.DELTA.pyr4. The
transformation frequencies of the reporter containing vectors were
comparable to those obtained with the control vector. Eighteen
colonies derived from the propagule pool of UV18#100.f
.DELTA.pyr5.DELTA.pyr4[pPcbh1-glaA-Tcbh1 pyrE tel pyrG]
transformants were randomly selected for further analysis. All
contained the Pcbh1-glaA-Tcbh1 expression cassette and produced
glucoamylase. In a glucoamylase activity assay based on
p-nitrophenyl-.beta.-maltoside 17 transformants had an increased
absorbency value at 405 nm value (.SIGMA..sub.E405 nm:
1.46.+-.1.10) that was at least 5-fold higher than the value of the
control strain UV18#100.f .DELTA.pyr5.DELTA.pyr4[pPcbh1-Tcbh1 pyrE
tel pyrG] (E405 nm: 0.13). Upon introduction of the vector
pPcbh1-lac1-TtrpC pyrE tel pyrG into strain UV18#100.f
.DELTA.pyr5.DELTA.pyr4 laccase positive transformants were
identified by monitoring the oxidation of ABTS
(2,2-azino-bis-[3-ethylthiazoline-6-sulfonat]) (FIG. 5A). Although
there is still variation in the production levels both in
glucoamylase and laccase case, this variation in expression profile
is significantly less than the variation obtained with traditional,
ectopic integrative vectors at a single copy level. The variation
in reporter gene expression with the telomeric vector might still
be the results of the site of integration. This could be a combined
effect of the integration at different ends of the chromosomes and
the orientation of the expression cassette. In conclusion, using
the telomeric vector containing two auxotrophic selection markers
more than 95% of the propagules express the reporter genes at
clearly detectable level compared to the host strain.
Fungal High Throughput Screening and Insert Recovery
[0511] To demonstrate the screening procedure in a fungal
high-throughput screening setting using the C. lucknowense system
and to demonstrate the recovery procedure of the inserts of
interest, propagules from a ABTS positive
UV18#100.f.DELTA.pyr5.DELTA.pyr4 [pPcbh1-lac1-TtrpC pyrE tel pyrG]
transformant were mixed with propagules from a UV18#1001
.DELTA.pyr5.DELTA.pyr4[pPcbh1-Tcbh1 pyrE tel pyrG] transformant. In
the described proof of concept experiment propagules of a laccase
transformant (oxidation of ABTS) were mixed with propagules of the
control transformant in a ratio of 1:40. An aliquot, containing
approximately the number of ABTS positive propagules expected to be
present in two 96 wells plates, was plated directly on selective
agar plates and 37 ABTS positive colonies were found. The propagule
mixture was sampled into the wells of microtiter plates using the
Allegro system (Caliper Lifesciences, Hopkinton, USA). When
suitable cultivation conditions are applied the propagules will
first form mycelium pellets which are then converted in a high
density propagules mixture. From this propagule mixtures small
aliquots were transferred robotically to a new plate containing
production medium. The culture supernatants from the resulting
daughter cultures were assayed using a fully automated ABTS
oxidation assay in the Staccato robotic system (Caliper
Lifesciences, Hopkinton, USA). Thirty-five ABTS positive wells were
identified in the two plates seeded with the mixture of propagules
(FIG. 5B).
[0512] To verify and identify the ABTS positive clone in the
propagule mixture a second screening was performed for six positive
hits. Firstly, the number of propagules in the well that
corresponds to a hit was determined by making serial dilutions and
performing colony counting on selective agar plates. The samples
were then diluted to an average seeding density of one propagule
per well and seeded in the wells of two microtiter plates. The
number of pellets formed in each well was scored by visual
inspection after two days of cultivation and this number was
interpreted as the number of propagules seeded per well. Medium
samples were taken after 96 h of cultivation and ABTS oxidation was
measured. Using this set up many ABTS positive wells that were
seeded with a single propagule were identified (results not
shown).
[0513] The inventors have tested the recovery of the reporter genes
from the positive C. lucknowense transformants by PCR using a
combination of a specific primer in the promoter and in the
terminator region and total genomic DNA as template. However, based
on the integration pattern of the vector at the telomeric ends of
the C1 chromosomes as described above, the complete expression
cassette and the backbone for propagation and selection in E. coli
should be present on a defined NotI fragment (FIG. 4). Therefore
beside the PCR approach, the recovery of the essential parts of the
integrated telomere vector should be possible via transformation of
E. coli with genomic DNA isolated from the positive transformants.
The advantages of this approach are that it eliminates the chances
of introducing unwanted mutations by PCR and that the recovered
vector contains an expression cassette that can be used directly
for final hit verification. Several mycelium pellets of ABTS
positive wells that were seeded with a single propagule were used
to inoculate a shake flask medium culture and from the formed
mycelium genomic DNA was isolated. Using a set of Pcbh1 and TtrpC
derived primers a specific lac1 containing fragment could be
amplified by PCR in all samples. Genomic DNA isolated from a
verified positive hit was treated with NotI, self-ligated, and the
ligation mixture was introduced into E. coli. Plasmid DNA from
ampicillin-resistant colonies was analyzed for the presence of the
expression cassette. According to PCR and restriction analysis
eleven out of 12 colonies tested contained the expected plasmid,
pPcbh1-lac1-TtrpC rescue (FIG. 5D).
[0514] To verify the advantages of the approach described above,
the strain UV18#100.f .DELTA.pyr5 was co-transformed with a mixture
of the vectors pPcbh1-lac1-TtrpC rescue and pBlue-pyrE.
Transformants were selected by growth on minimal agar plates.
Individual transformants were tested for their ability to oxidize
ABTS. Twenty percent of the Pyr.sup.+ transformants were ABTS
positive (FIG. 5C). Within this set of transformants the variation
in expression level was much more pronounced compared to a set of
transformants obtained with the expression cassette present on the
telomeric vector (FIG. 20A). As for the production levels obtained
in this case in some of the UV18#100.f .DELTA.pyr5 [pPlac1T
rescue/pBlue-pyrE] transformants a more than 20-fold higher laccase
activity was measured. These high levels might be the result of
multiple copy integration of the vector, the integration in the
genome at a position that facilitates high expression levels or a
combination of both. Accordingly, the re-isolated insert DNA
encodes the enzymatic activity. In addition, a first generation
production strain is produced.
[0515] The experiments presented show the successful use of the
telomeric vector for library construction and high frequency
transformation and the subsequent screening of genomic and of
artificial cDNA libraries in a high throughput fashion (FIG. 6).
The results support the inventors' concept that this vector, in
combination with the screening strategy, will be a powerful tool
for the screening of evolved libraries. The need to reduce the
variation between individual transformants is provided by the
design of the telomeric vector and the DNA stabilization procedure.
The actual use of a production strain as host for screening and the
use of the rescued plasmid without further modification shortens
the time required between screening and product development.
REFERENCES FOR EXAMPLE B
[0516] 1. Short J M, Keller M. High throughput screening for novel
enzymes. U.S. Pat. No. 6,174,673 (2001). [0517] 2. Dalboge H,
Heldt-Hansen H P. A novel method for efficient expression cloning
of fungal enzyme genes. Mol. Gen. Genet. 243, 253-260 (1994).
[0518] 3. Punt P J, van Zeyl C, van den Hondel CAMJJ. High
throughput screening of expressed libraries in filamentous fungi.
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Transformation of filamentous fungi based on hygromycin B and
phleomycin resistance markers. Methods in Enzymology 216, 447-457
(1993). [0520] 5. Stinchcomb D T, Struhl K, Davies R W. Isolation
and characterization of a yeast chromosomal replicator. Nature 282,
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Ter-Avanesyan M D, Rhee J S and Rhee S K. A novel autonomously
replicating sequence (ARS) for multiple integration in the yeast
Hansenula polymorphs DL-1. J. Bacteriol. 178, 4420-4428 (1996).
[0522] 7. Gems D, Johnstone I L, Clutterbuck A J. An autonomously
replicating plasmid transforms Aspergillus nidulans at high
frequency. Gene 98, 61-67 (1991). [0523] 8. Verdoes J C, Punt P J,
van der Berg P, Debets F, Stouthamer A H, van den Hondel CAMJJ.
Characterization of an efficient gene cloning strategy for
Aspergillus niger based on an autonomously replicating plasmid:
cloning of the nicB gene of A. niger. Gene 146, 159-165 (1994).
[0524] 9. Fierro F, Kosalkova K, Gutierrez S, Martin J F.
Autonomously replicating plasmids carrying the AMA1 region in
Penicillium chrysogenum. Curr. Genet. 29, 482-489 (1996). [0525]
10. Bruckner B, Unkles S E, Weltring K, Kinghorn J R.
Transformation of Gibberella fujikuroi: effect of the Aspergillus
nidulans AMA1 sequence on frequency and integration. Curr. Genet.
22, 313-316 (1992). [0526] 11. Aleksenko A, Ivanova L. In vivo
linearization and autonomous replication of plasmids containing
human telomeric DNA in Aspergillus nidulans. Mol. Gen. Genet. 260,
159-164 (1998). [0527] 12. Kistler H C, Benny U. Autonomously
replicating plasmids and chromosomes rearrangements during
transformation of Nectria haematococca. Gene 117, 81-89 (1992).
[0528] 13. Javerzat J-P, Bhattacherjee V, Barreau C. Isolation of
telomeric DNA from the filamentous fungus Podospora anserina and
construction of a self replicating linear plasmid showing high
transformation frequency. Nucleic Acids Research 21, 497-504
(1993). [0529] 14. Powell W A, Kistler H C. In vivo rearrangement
of foreign DNA by Fusarium oxysporum produces linear
self-replicating plasmids. J. Bacteriol. 172, 3163-3171 (1990).
[0530] 15. Bennett J W, Lasure L L. Growth Media. In: More Gene
Manipulation in Fungi. Bennett J W, Lasure L L (eds.), pp 444-445.
San Diego: Academic Press, Inc. (1991). [0531] 16. Record E, Punt P
J, Chamkha M, Labat M, van den Hondel CAMJJ, Asther M. Expression
of Pycnoporus cinnabarinus laccase gene in Aspergillus niger and
characterization of the recombinant enzyme. Eur. J. Biochem. 269,
602-609 (2002). [0532] 17. Hynes M J, Corrick C M, King J A.
Isolation of genomic clones containing the amdS gene of Aspergillus
nidulans and their use in the analysis of structural and regulatory
mutations. Mol. Cell. Biol. 3, 1430-1439 (1983). [0533] 18. Hynes M
J, Pateman J A. The genetic analysis of regulation of amidase
synthesis in Aspergillus nidulans. II. Mutants resistant to
fluoroacetamide. Mol. Gen. Genet. 108, 107-116 (1970). [0534] 19.
Gems D, Aleksenko A, Beleky 1, Robertson S, Ramsden M, Vinetski Y,
Clutterbuck A J. An "instant gene bank" method for the gene cloning
by mutant complementation. Mol. Gen. Genet. 242, 467-471 (1994). C.
Expression of a Glucoamylase-Fc Fusion Protein in C.
lucknowense
[0535] The plasmid pPcbh1-glaA(II)-Fc was constructed in which the
catalytic domain of the glucoamylase gene from Aspergillus niger
was fused to an Fc gene. Expression of the fusion gene was driven
by the cbh1 promoter of C. lucknowense. This plasmid is shown in
FIG. 7. The large NotI fragment was excised from the plasmid and
purified, and this DNA, was cotransformed with another DNA fragment
containing the pyr5 gene of C. lucknowense into strain UV18#1001
pyr5 .DELTA.alp1. Pyrimidine prototrophic strains were selected and
screened for the presence of glucoamylase DNA. Among the
transformants was a strain that produced about 1 gram per liter of
the predicted glucoamylase-Fc fusion protein. The protein was
readily purified from culture filtrates of the transformant strain
to a purity greater than 90% as shown in FIG. 8. These data
indicate that high levels of human Fc can be stably produced as a
glucoamylase fusion protein and that the Fc portion of the molecule
properly dimerizes and folds as indicated by the binding to protein
A.
D. In Vitro Stability of Human IgG1 Against C. lucknowense Culture
Filtrates
[0536] Human IgG1 was incubated with culture filtrate from C.
lucknowense strains deleted for alp1 (single protease-deleted
mutant) or alp1, pep4, and alp2 (triple protease-deleted mutant).
Samples were removed after 0, 4, 8, and 24 hours of incubation and
subsequently subjected to Western blot analysis using an
Fc-specific anti-human IgG1 antibody. The results of this type of
analysis are shown in FIG. 9. As seen the antibody is reasonably
stable against culture filtrate from the single mutant and more
stable against culture filtrate from the triple mutant. In the
latter case, no sign of degradation of the heavy chain is seen
after 24 hours of incubation. Western analyses against light-chain
specific antibodies showed similar results.
E. Expression of Full-Length Antibodies in C. lucknowense
[0537] Plasmids expressing the light and heavy chains of a human
IgG1 were separately constructed, as shown in FIG. 10. Each plasmid
contained genes for the heavy or light chain of the antibody fused
to the catalytic subunit of A. niger glucoamylase. Between the
glucoamylase gene and the antibody gene is a kex2 cleavage site.
The presence of this site results in cleavage of the glucoamylase
from the antibody chain so that free glucoamylase and antibody are
produced. Each glucoamylase-antibody fusion construct was driven by
the cbh1 promoter. The light chain-expressing plasmid contained the
pyr5 gene as a selection marker. The heavy chain-expressing plasmid
contained the A. nidulans amdS gene as a selection marker. C.
lucknowense strains are naturally amdS-negative. The amdS gene
encodes an amidase, and introduction of this gene into C.
lucknowense allows the strain to grow on acetamide as sole nitrogen
source. The large NotI fragments were separately isolated and
co-transformed into UV18#100.f pyr5 .DELTA.alp1, selecting for
Pyr5-positive AmdS-positive transformants. The transformants were
further screened for production of glucoamylase and subsequently
for the production of both heavy and light antibody chains.
[0538] A number of transformants expressed both the heavy and light
chains of the human IgG1. One such transformant was analyzed in
detail. Filtrates from fermentation cultures contained
approximately 200-400 mg of full-length IgG1 measured by
quantitative Western analysis. The antibody was readily purified by
chromatography on protein A (FIG. 11), and cell-based bioactivity
assays, performed on protein A-purified antibody, indicated that
about 200 mg of active IgG was present per liter of culture
filtrate. These results indicate that full-length antibodies can be
expressed at high titer in a dimerized, properly folded, and active
form in C. lucknowense strains.
F. Screening for Improved Antibody Affinity Using C.
lucknowense
[0539] Based on data indicating the antibodies can be stably
expressed and proof-of-concept experiments showing that
high-throughput screening of expression libraries can be
accomplished utilizing C. lucknowense host strains, it is possible
to produce libraries of libraries containing variants of both heavy
and light chains co-expressed in the same host strains. The
screening technology will allow arraying of monoclonal cultures,
each expressing a distinct antibody into wells of microtiter
dishes. Using ELISA or other assays, the antibodies expressed by
the separate clones can be assayed for binding and the best binding
antibodies screened for. The cultures selected from the screening
procedure can be further cultivated for more detailed analysis. DNA
from the most promising clones can be retrieved, isolated and
sequenced. Using the known sequences, C. lucknowense strains
optimized for expression of the antibody can be generated to
produce large quantities of those antibodies.
[0540] FIG. 12 shows plasmids that can be used for screening of
libraries of libraries for antibody binding. Two alternatives are
shown. FIG. 12A (left) shows a vector in which libraries of heavy
and light chain variable regions (V.sub.H and V.sub.L,
respectively) are together cloned into a C1-specific replicating
vector so that glucoamylase fusions to a single-chain Fv are
produced. FIG. 12B (right) shows a vector in which full-length
antibody chains are expressed as glucoamylase fusions, each from
their own promoter. Numerous possibilities exist for the latter
vector. In another aspect, the light and heavy chains could be
placed in either order in either orientation. Additionally,
although both chains in FIG. 12 are shown utilizing cbh1 promoter
and terminator sequences, distinct promoters and terminators could
be used to minimize repetitive sequences in the vector. In one
aspect, a fusion partner-heavy-light chain fusion protein can be
produced in which the three components (fusion partner, heavy and
light-chain) are separated by proteolytic processing sites (see
FIGS. 13-14). These vectors use human telomeric sequences (hTel)
for replication in C. lucknowense and contain two selective markers
pyrE and pyrG flanking the expression constructs, and sequences
allowing replication in E. coli. Upon introduction into C.
lucknowense, the vectors spontaneously linearize between the human
telomeric sequences (hTel). The use of doubly marked pyr4-pyr5
mutants of C. lucknowense ensures that integrants contain the
entire expression construct. Individual transformants are then
separated and screened for binding in a high-throughput fashion.
The expression constructs from transformants of interested can be
isolated by purifying genomic DNA from those constructs, digesting
with NotI, ligating to recircularize, then transforming E. coli and
selecting for ampicillin resistance. The resulting E. coli
transformants will contain plasmids carrying the antibody
expression construct. In the case where full-length antibodies are
screened, the expression constructs can be subcloned directly into
expression vectors to allow high-level expression of the antibodies
as described in Example E. When a single-chain Fv is used in the
expression screening, the plasmids can be deconstructed and
relevant sequences spliced into full-length heavy and light chains
for expression as described in Example E. As an alternative to the
replicative vectors, individual vectors carrying the heavy and
light chain genes were generated in each using the pyrE or pyrG
selection marker.
G. Additional Vectors for Antibody Expression and Screening
[0541] One non-limiting example of site-specific recombination
technology is Gateway.RTM. cloning. "Gateway.RTM. cloning" is a
cloning method based on the site-specific recombination of lambda
bacteriophage. Briefly, the Gateway.RTM. technology (Invitrogen,
Carlsbad, Calif.), uses a lambda recombinase to catalyze in vitro
recombination events between sequences flanked by 25-bp att sites.
Often, those sequences are present on two separate plasmids, as
when moving an insert from a starting "entry" construct to a
"destination" vector. There are also systems for cloning PCR
fragments directly into Gateway.RTM. plasmids using recombinases.
This technology is described, for example, in Mabashi et al.,
Biosci Biotechnol Biochem. 2006 August; 70(8):1882-9. Various
vectors can be produced using any suitable in vitro recombination
strategy, of which the Gateway.RTM. technology is only one,
non-limiting example. The design of vectors that may be produced
using in vitro recombination strategies is described herein.
[0542] Specifically, the present invention also encompasses the
creation of vectors for transformation of fungal host cells as
described herein that are designed to allow the efficient transfer
of the expression cassette to and/or from a lambda bacteriophage.
Exemplary designs for such vectors are illustrated in FIGS. 15-18.
While these figures illustrate the use of the vectors to introduce
immunoglobulin heavy and light chains, the vectors can similarly be
used to express other heterodimeric and heteromultimeric proteins.
As discussed above, the technology used to create or implement such
vectors can make use of any suitable recombination strategy.
[0543] FIGS. 15-16 illustrate a vector of the present invention in
which two separate lambda bacteriophage vectors, one comprising an
immunoglobulin heavy chain construct and one comprising an
immunoglobulin light chain construct, DNA fragments are excised
from the lambda bacteriophage vectors and ligated into a fungal
expression vector according to the present invention. In this
non-limiting example, the 3' end of one construct contains the same
att site as the 5' end of the second construct (see attR2 and
attL2), such that by a RL (right-left) recombination event, the two
constructs are ligated into the fungal vector next to one another,
flanked by the remaining att sites from each of the two phage
vectors (see attL1 and attR3). The expression cassette can then be
excised again for transfer into a phage vector, or excised using
distal restriction enzyme sites for, for example, ligation into an
E. coli vector. Although both protein chains in this figure are
shown utilizing cbh1 promoter and terminator sequences (see FIG.
16), distinct promoters and terminators could be used to minimize
repetitive sequences in the vector.
[0544] In a second non-limiting example, different right and left
att sites are used in each bacteriophage vector, so that the two
constructs are excised and then ligated into the fungal vector as
separate expression cassettes. This is illustrated in FIGS. 17-18.
In this example, each cassette is flanked by different restriction
sites (A and B in FIGS. 17 and 18) which will facilitate, if
desired, the separate excision of each DNA expression component
from the vector. Alternatively, single restriction sites flanking
the entire expression cassette are available to facilitate
recovery, cloning, and/or transfer of the DNA.
[0545] In a third non-limiting example, a strategy similar to that
shown in FIGS. 15-16 is used, except that the fusion components
(e.g., the fusion partner, heavy chain and light chain) are linked
to one another via processing sites. This construct provides
additional flexibility in manipulating the components of the
vectors as a unit or as individual components and can enhance
isolation, cloning, and DNA manipulation strategies. This design is
illustrated in FIGS. 13-14.
H. Expression of Full-Length Antibodies in C. lucknowense
[0546] The NotI fragment of the telomere vector flanked with pyrE
and pyrG selection markers (example B; FIG. 2) was introduced in
the unique NotI site of a vector containing both the expression
cassette of the heavy and light chain each fused to the catalytic
subunit of A. niger glucoamylase (GLA). Between the GLA encoding
gene (glaA) and the antibody genes a kex2 cleavage site is present.
The plasmids (FIGS. 20A and B) with the expression cassettes for
expressing both the heavy and the light chains of the human IgG1
were used to demonstrate a fungal high throughput robotic screening
procedure for improved antibody variants in C. lucknowense. The
plasmids were introduced into strain
UV18#100.f.DELTA.pyr5.DELTA.pyr4.DELTA.alp1.
[0547] The primary set of transformants was regenerated at 45 rpm
and 33.degree. C. in minimal medium supplemented with 0.67 M
sucrose. After 40 hours the mixture was inoculated in shake flasks
in minimal medium and incubated at 35.degree. C. for 144 hours. The
propagules were harvested by filtration and centrifugation. Diluted
aliquots of the resuspended propagule mixtures were plated on
selective agar plates. Wells of two microtiter plates containing
medium optimized for propagule formation were seeded with small
mycelium samples of two times 96 individual colonies. After three
days, small aliquots of the formed propagule mixtures were
transferred to new plates (daughters) containing production medium.
Plates were placed in an incubator shaker (35.degree. C. and a
rotation speed of 850 rpm) and after 96 hours culture supernatant
was collected. The large majority of transformants produced the
dimer of the IgG1 heavy chains as detected by spotblot analysis
using an AP-conjugated anti-Fc antibody (FIG. 21A) and by ELISA
analysis using a protein A coated plate (FIG. 21B).
[0548] Two antibody producing transformants obtained after the
introduction of the telomeric vector were analyzed in more detail
in fed-batch fermentation simultaneously with a positive-control
antibody producing strain and a negative control non-antibody
producing strain. Both the heavy and light chains of IgG were
detected by Western blot analysis. Filtrates from these controlled
fermentation cultures withdrawn after 72 h of glucose feed
contained approximately 600 mg of GLA. Antibody was purified from
the filtrates using protein A chromatography and the presence of
both the light and the heavy chains was verified by Western blot
analysis. Protein A column purified antibodies were denatured by
heating at 95.degree. C. prior to Western blotting. FIGS. 22A and B
shows, from left to right, M--kDa marker, 1--antibody-producing
control strain UV18#100f.DELTA.alp1[88/90]#58, 2--transformant
UV18#100f.DELTA.alp1 [88+90 pyrE tel pyrG]#E8,
3--UV18#100f.DELTA.alp1[88+90 pyrE tel pyrG]#E2 and 4--negative
control strain UV18#100f.DELTA.alp1[pyrE tel pyrG]. Western blot
detection was performed with antibodies specific for the Ig heavy
chain (FIG. 22A) or Ig light chain (FIG. 22B).
[0549] Absorption measurements at 280 nm indicated that
approximately 150 mg purified antibodies were obtained per liter of
fermentation broth. A quantification based on glucoamylase activity
indicates that about 200 mg of full-length antibody is produced per
liter of fermentation supernatant.
[0550] 80 ml of each fermentation end sample (96 hours for samples
1-3, 120 hours for sample 4; glucose feed was started after 24
hours of fermentation) was purified on protein A column, this was
eluted in eight different fractions of 1 ml each. Glucoamylase
activity was detected in two fractions, which were pooled and total
protein concentration was determined by absorption at 280 nm.
Subsequently, 100 ml portions of the end-of-fermentation (EOF)
sample were purified using the same protocol. In four eluted
fractions glucoamylase activity was detected, and protein
concentration was measured using by absorption at 280 nm. The
antibody yields are presented in Table E below. All calculations
are based on a 100% recovery from the protein A column.
TABLE-US-00005 TABLE E Calculated Ab yield Calculated Ab yield
Calculated Ab yield (mg/l) in EOF (mg/l) in EOF (mg/l) in EOF
medium based on medium based on medium based on 2 ml protein A 80
ml protein A 100 ml protein A purified sample purified sample
purified sample 1: control strain 0 2 0.5 2: ab pyrEtelpyrG E2 110
55 46 3: ab pyrEtelpyrG E8 150 38 52 4: ab #58 200 103 330
[0551] Protein A column purified antibodies from the first three
fractions above, along with positive and negative controls, were
denatured by heating at 95.degree. C., ran on a gel, then
visualized by standard protein staining techniques; FIG. 22C shows,
from left to right, M--kDa marker, then the first three fractions,
respectively, of 1--negative control strain
UV18#100f.DELTA.alp1[pyrE tel pyrG], 2--transformant
UV18#100f.DELTA.alp1[88+90 pyrE tel pyrG]#E2,
3--UV18#100f.DELTA.alp1[88+90 pyrE tel pyrG]#E2, 4--antibody
processing control strain UV18#100f.DELTA.alp1[88/90]#58 and 5--two
lanes of IgG control protein (14 and 28 ng).
I. The Effect of C1 Repetitive Sequence (CRS)
[0552] The effect of a C1 repetitive sequence (CRS), as present in
the terminator region of the cbh1 terminator, was analyzed. DNA
fragments containing the egg expression cassette (Pcbh1-egg-Tcbh1)
were introduced with and without the CRS in a pyr4 deletion mutant
of a Eg3 production strain. Based on the model system the presence
of the CRS in Tcbh1 leads to 1) an increase in the average
beta-glucanase and CMCase activity in microtiter plate and shake
flasks cultures; 2) a set of transformants with the highest
enzymatic activities; 3) 15-20% increase in total extracellular
protein content under controlled fermentation conditions; and 4) an
increase in the number of integrated expression cassettes. In the
new Eg3/Eg2 production strains both the beta-glucanase and the
CMCase have been increased approximately 2-fold compared to the
current Eg3 production strain.
[0553] A C1 Repetitive Sequence (CRS) of approximately 340 bp was
identified in the terminator region the cbh1 gene (Tcbh1) of C.
lucknowense. FIG. 23 shows a partial nucleotide sequence of the
terminator region of cbh1, with the CRS (SEQ ID NO:12) indicated in
gray. In all C1 expression vectors containing the Tcbh1, this CRS
was present. Compared to other filamentous fungi like Aspergillus,
the number of integrated copies of the expression cassette in C1 is
rather low. Therefore the effect of the CRS on integration was
analyzed using the endoglucanase 2 (Eg2) encoding gene (egg) as
model.
Transformation and Selection
[0554] From the expression vector pPcbh-eg2-Tcbh (FIG. 24), the
expression cassette with the CRS was isolated as 4.4 kb NotI
fragment. The expression cassette without a large portion of the
CRS was isolated as a 4.1 kb NotI-Bcl1 fragment. The strain
UV18[EG3#20].DELTA.pyr4 was selected as the C1 host strain for
transformation, although any C1 strain should be adequate for
purposes of this example. The expression cassette with and without
CRS (10 .mu.g) were each independently introduced by
co-transformation with the pyr4 gene (2 .mu.g) as selection marker.
Approximately 90 transformants with and without CRS were obtained
and purified on selective minimal medium.
Cultivation and Enzyme Activities
[0555] Microtiter plates containing propagule formation optimized
(PFO) medium were inoculated with these transformants and incubated
for 72 hours. From these cultures, small propagule samples were
transferred to new microplates containing minimal production
medium. Supernatant was analyzed after 96 hours of incubation at
35.degree. C. for .beta.-glucanase (Azo-barley) and CMC-ase
(AzoCMC) activity.
[0556] The results indicated that, compared to the control strains
UV18[EG3#20][pyr4], the number of transformants with a more than
2-fold increase in activity was higher in the collection of
transformants with the CRS than within the set without the CRS
(Table F).
TABLE-US-00006 TABLE F Percentages of Samples with Elevated
Activity Azobarley AzoCMC-ase (>2 x background) (>2 x
background) +CRS 76% 56%* .DELTA.CRS 20% 43% *only the samples with
>2-fold increase in beta-glucanase activity were tested
[0557] Based on these assays, the best producers were selected for
further analysis in shake flask cultures. Shake flasks containing
50 ml synthetic minimal medium (NH.sub.4, 55 mM PO.sub.4, cas, p/s,
biotin, 0.5% glucose) were inoculated with 5.times.10.sup.7 spores.
After 120 hours, medium samples were analyzed for total
extracellular protein content, .beta.-glucanase and CMC-ase
activity. Only minor differences in specific .beta.-glucanase
activity were detected (results not shown) whereas clear
differences in CMC-ase activity levels were found (Table G). In all
strains with introduced copies of the eg2 expression cassette, the
CMC-ase activity is 2 to 4 fold higher than in the control strains
(data are expressed in values relative to the control strains, the
averages of which were assigned values of one). Strains
co-transformed with the eg2 expression cassette with the CRS (Pcbh
eg2 Tcbh/pyr4) displayed a higher activity than strains transformed
with the expression cassette without the CRS (Pcbh eg2
TcbhACRS/pyr4).
Southern Blot Analysis of eg2 Transformants
[0558] Chromosomal DNA was isolated from transformants in order to
determine the copy number of the introduced eg2 expression cassette
and the integration patterns. Chromosomal DNA was digested with the
restriction enzyme NcoI, size fractionated by electrophoresis in an
agarose gel, and finally transferred to a nylon membrane. The blot
was hybridized with a radioactive labeled internal DNA fragment
(BstEII-EcoRV) of the eg2 encoding gene (FIG. 25).
[0559] An estimation of the copy number of the eg2 gene was made
based on the intensity of the hybridizing fragments (Table G). The
copy number is less in the transformants in which the eg2
expression cassette without the CRS was introduced. There is no
clear correlation between egg copy number and CMCase activity. Most
likely, the site of integration plays an important role.
TABLE-US-00007 TABLE G Data overview eg2 multicopy transformants of
UV18[Eg3#20].DELTA.pyr4 Relative CMC-ase Relative Total Relative
Specific copy 120 hr Protein Activity # (u/ml) 120 hr units/mg C1
(wt) UV18[EG3#20] UV18[pyr4]#2 UV18[pyr4]#6 UV18[EG3#20]
[Pcbh-eg2-Tcbh/pyr4]#33 4 3.54 0.96 3.76 [Pcbh-eg2-Tcbh/pyr4]#35
3.38 0.86 4.00 [Pcbh-eg2-Tcbh/pyr4]#36 7 4.77 1.07 4.53
[Pcbh-eg2-Tcbh/pyr4]#44 4 3.08 0.96 3.29 [Pcbh-eg2-Tcbh/pyr4]#84 4
3.38 0.98 3.53 [Pcbh-eg2-Tcbh/pyr4]#91 4 2.62 0.89 3.00
UV18[EG3#20] [Pcbh-eg2-TcbMCRS/pyr4]#23 2 4.92* 1.37 3.65
[Pcbh-eg2-TcbMCRS/pyr4]#39 2 4.92* 1.23 4.06
[Pcbh-eg2-TcbMCRS/pyr4]#53 3.69 1.57 2.41
[Pcbh-eg2-TcbMCRS/pyr4]#57 2.46 1.21 2.06
[Pcbh-eg2-TcbMCRS/pyr4]#63 4 2.92 1.26 2.35
[Pcbh-eg2-TcbMCRS/pyr4]#74 3.54 1.25 2.88
[Pcbh-eg2-TcbMCRS/pyr4]#79 2 2.46 1.25 2.00 *measured in different
assay without internal controls. Activity assays and total protein
measurements were performed in supernatant from shake flask
cultures after 120 hours of incubation.
Controlled Fed Batch Fermentation in Pharma Medium.
[0560] Two strains from each set of transformants were selected for
additional controlled fermentation runs in production medium. As
shown in Table H, a 2-fold increase in specific .beta.-glucanase
and CMCase activity was determined in both types of egg multicopy
strains compared to the pyr4 transformed host strain. However, in
fermentor culture supernatant of strains with the CRS, the
.beta.-glucanase activity, CMCase activity and total extracellular
protein content is 15-20% higher than in the supernatant from
strains without the CRS. A PageBlue colored SDS-PAGE gel loaded
with end of fermentation (EOF) samples is shown in FIG. 26. In
samples of fermentor 1 and 2, an increase in a 40 kDa protein band
(Eg2) was observed.
TABLE-US-00008 TABLE H Relative Beta- Relative Relative Relative
Beta Relative glucanase CM Case Total Relative Glucanase CMCase
activity STRAIN: (AzoBarley) (AzoCMC) biomass Extracellular
(AzoBarley) (AzoCMC) UV18[EG3#20].DELTA.pyr4 U/ml U/ml (g) Protein
g/L u/mg/protein U/mg protein [pyr4]#7 1 1 1 1 1 1 [Pcbh-eg2- 1.75
1.50 1.44 0.82 2.14 1.83 Tcbh/pyr4]#33 [Pcbh-eg2- 1.92 1.51 1.66
0.77 2.49 1.96 Tcbh/pyr4]#36 [Pcbh-eg2- 1.40 1.27 1.81 0.62 2.22
2.04 Tcbh.DELTA.CRS/pyr4]#39 Pcbh-eg2- 1.52 1.06 1.74 0.59 2.57
1.79 Tcbh.DELTA.CRS/pyr4]#53
[0561] The results indicate that presence of one CRS in the
expression cassette has a positive effect on the number of copies
of the expression cassette that integrate and thus on the
productivity. The effect of multiple copies of CRS on integration,
stability and productivity can be tested via a similar approach.
New expression cassettes may be constructed with two CRS, the
second CRS introduced in the upstream region of the promoter. In
addition, multiple expression cassettes in one construct can be
flanked by two CRS. The above assays may then be repeated with
strains transformed with vectors containing these multiple CRS
expression cassettes.
J. Assay for Protease Activity
[0562] The following assay may be used to determine the protease
activity of enzymes and protease-deficient strains of the present
invention.
[0563] The protease activity is measured using N,N-dimethylcaseine
(Sigma, C 9801) as a substrate. The procedure was fully automated
using a Cobas Mira Plus autoanalyser (Roche).
[0564] Reagents include N,N-dimethylcaseine (5 g/l in 0.1 M
NaAc/HAc (pH=5.5), 5 g/l in 0.1 M K.sub.2HPO.sub.4 (pH=7.0), or 5
g/l in 0.1 M Na.sub.2B.sub.4O.sub.2.10H.sub.2O (pH=8.5)); Starter 1
(0.1 M N.alpha..sub.2B.sub.4O.sub.2.10H.sub.2O (pH=9.3)+0.5 g/l
Na.sub.2SO.sub.3); and Starter 2 (5% TNBS (2,4,6, Trinitrobenzene
Sulfonic Acid, Pierce #28997) 2.times. diluted with water).
[0565] 2 .mu.l of sample (+13 .mu.l water) is mixed with 75 .mu.l
N,N-dimethylcaseine and incubated at 37.degree. C. for 1050 (or
900) seconds. The reaction is stopped by addition of 185 .mu.l
starter 1 and 5 .mu.l starter 2. The absorption at 405 nm is
measured after 200 seconds. As a standard, a known concentration of
glycine is used. Samples are also incubated with water (instead of
N,N-dimethylcaseine) to measure the background activity. The sample
data are corrected for this background activity. One unit of
protease is the amount (in .mu.mol) of peptide bonds cleaved per
minute per ml sample at 37.degree. C. at a given pH.
[0566] Using the assay above, the protease activity of
protease-deficient strains of C. lucknowense was determined, and
the results provided in Table I. The results demonstrate the
reduced protease activity of strains UV18#100.f,
UV18#100.f.DELTA.alp1, UV18#100.f.DELTA.alp1.DELTA.pep4, and
UV18#100.f.DELTA.alp1.DELTA.pep4.DELTA.alp2 as compared to the
parent strain UV18-25.
TABLE-US-00009 TABLE I Protease Activities Protease Activity Strain
pH 5.5 pH 7.0 pH 8.5 UV18-25 306 415 793 UV18#100.f 104 80 81
UV18#100.f.DELTA.alp1 38 48 18 UV18#100.f.DELTA.alp1.DELTA.pep4 30
51 21 UV18#100.f.DELTA.alp1.DELTA.pep4.DELTA.alp2 8 11 1
[0567] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims.
Sequence CWU 1
1
1714200DNAChrysosporium
lucknowenseCDS(1588)..(1869)CDS(1976)..(2161)CDS(2230)..(2402)CDS(2480)..-
(2825)CDS(2943)..(3131) 1gcatggaaat tcccctcccc ccacggccag
acttggacca aggaaaagag ataccacctg 60ccgaacgtgg ctctcgctcc agcatttcga
gagcgtacct cagccaacca ctcggctccc 120cgtgtcgagc gatcggcact
tgcggccttt gcaatgcccc atccttgaac tccaccaaat 180aggctaccac
cacaccaccc ctccatttct tgttcctcgg cttcctcgct cgaggtaccg
240atccagggtg ggccgattgc gatggtgcca ttgctgccct tgctttggct
tcacctaggc 300gatgtcacgt tcagatatag tccgcaggct ttaccccaga
tcctctgatt gccgatctcg 360gccatgacct ctggttgttt cacaagcaca
cagggtcagt cgcccccgtt gcgcctctgt 420acagtctgta cagaccttct
cagctgaatg tttccgagac tagagactaa aatctgaatc 480actttggccc
agagagaggg ttcgcgaagt cccacacacc cttctagaag gagagaccag
540agccacgaaa catgaagcct gatcgcttat tttttttttt ttttttggcc
ccggagtgcc 600cgcggtcacg gtactttggg gttatgacag gctgtttgac
ttccatggat aatccccttt 660aattatttag gctgaccact caccggacct
gtttcgcctg tgcaacttca ccagtcggag 720gtcatgctca aattgtcagt
cagatacttt atacatactc tgtgtacaac ataccacaac 780acacacgcac
acacatagaa agtacataca tgctggatcg gaacccacca cgccttgtac
840atacacccac acacccctcc ccacacccct cttcccggca cttttcgcgc
cagagatcgt 900cgcctttgcc ccttaggcaa gttcacccgt tatgttaggt
aaccctctcg acggggccgc 960ctgcggatgt tggcgcatgc ttgacacgcc
cggtcgtgcg gcgttgctag tcctcgaaag 1020tcaggtattg cacccggaac
ccctgatcac aagcacttga tcacggcggg agcacccgcg 1080cgcctgaacg
ggaccccagc caatgccgga ccagaggccg aagcgggaag gtgtcttgct
1140ttctggcctg cccttttctt tcaacaatgg gcaatacggg tcagcgaaac
ccttcctagt 1200cctcgcagca aactcgagct gctatcagat tcccgggaag
cggcctgcca cagccgctca 1260acccggcctt ggcatggcca ggcggccctt
tcatgtgtcg aaagcggcag gtcatcagca 1320cagatctcga gggtgggaaa
gagagggggg ggaggggcga tgctggggcg atgctgctag 1380gagccgcatt
cggggagggg gccctgctgt tcatccatat cccaggatga tgcgagattg
1440aagcaagata aataacacgg cttccccctc ccctttcgat ccggaccaga
ccatcgtctc 1500caacacccca aagtcgatcc gacaagtccc aatccacccc
gcccgcccct ccctccgtcg 1560ccgtcccggt cttccgattt cgtcaag atg cac ttc
tcc acc gct ctc ctg gcc 1614 Met His Phe Ser Thr Ala Leu Leu Ala 1
5 ttc ctg ccc gcc gcc ctc gcg gcc cct act gcc gag acc ctc gac aag
1662Phe Leu Pro Ala Ala Leu Ala Ala Pro Thr Ala Glu Thr Leu Asp Lys
10 15 20 25 cgc gcc ccg atc ctg act gct cgc gct ggc cag gtc gtc ccg
ggc aag 1710Arg Ala Pro Ile Leu Thr Ala Arg Ala Gly Gln Val Val Pro
Gly Lys 30 35 40 tac atc atc aag ctc cgc gac gga gcc agc gac gat
gtc ctt gag gcc 1758Tyr Ile Ile Lys Leu Arg Asp Gly Ala Ser Asp Asp
Val Leu Glu Ala 45 50 55 gcc atc ggc aag ctc cgc tcc aag gcc gac
cac gtc tac cgc ggc aag 1806Ala Ile Gly Lys Leu Arg Ser Lys Ala Asp
His Val Tyr Arg Gly Lys 60 65 70 ttc agg ggc ttt gcc ggc aag ctc
gag gat gac gtc ctt gac gcc atc 1854Phe Arg Gly Phe Ala Gly Lys Leu
Glu Asp Asp Val Leu Asp Ala Ile 75 80 85 cgt ctt ctc ccc gaa
gtgagtccgc gtcccggaaa gaaatagagc gagcggggga 1909Arg Leu Leu Pro Glu
90 gagagtgaag ggcgaaaaga gccgtgtttt gttaaccgct tgtcttttct
ttctctcttg 1969caatag gtc gag tac gtc gag gag gag gcc atc ttc acc
atc aac gcg 2017 Val Glu Tyr Val Glu Glu Glu Ala Ile Phe Thr Ile
Asn Ala 95 100 105 tac acc tcg cag tcc aac gcc ccc tgg ggc ctt gcg
cgc ctc tcg tcc 2065Tyr Thr Ser Gln Ser Asn Ala Pro Trp Gly Leu Ala
Arg Leu Ser Ser 110 115 120 aag acc gcg ggc tcc acc acc tac acc tac
gac acc agc gcc ggc gag 2113Lys Thr Ala Gly Ser Thr Thr Tyr Thr Tyr
Asp Thr Ser Ala Gly Glu 125 130 135 140 ggc acc tgt gcc tat gtg atc
gac acg ggc atc tac act agc cac tcc 2161Gly Thr Cys Ala Tyr Val Ile
Asp Thr Gly Ile Tyr Thr Ser His Ser 145 150 155 gtatgtctcg
cggttacctc ccctttcgga agaaggggca tccatatgct gacccctcct 2221gatcacag
gac ttc ggc ggc cgt gcc act ttc gcc gcc aac ttc gtc gac 2271 Asp
Phe Gly Gly Arg Ala Thr Phe Ala Ala Asn Phe Val Asp 160 165 170 agc
tct aac acc gat ggc aac ggc cac ggc acc cac gtc gcc ggc acc 2319Ser
Ser Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr 175 180
185 atc ggc ggc acc acg tac ggt gtt gcc aag aag acc aag ctc tac gcc
2367Ile Gly Gly Thr Thr Tyr Gly Val Ala Lys Lys Thr Lys Leu Tyr Ala
190 195 200 gtc aag gtt ctc ggc tcc gac ggc tct ggc acc ac
gtatgcctcg 2412Val Lys Val Leu Gly Ser Asp Gly Ser Gly Thr Thr 205
210 cacccgcgca cccgcacacc cgcccggccg ttatcttctg actgacattc
ctctttctcc 2472tctctag t tct ggt gtc att gct ggc atc aac ttc gtc
gct gac gac gcg 2522 Ser Gly Val Ile Ala Gly Ile Asn Phe Val Ala
Asp Asp Ala 215 220 225 ccc aag cgc agc tgc ccc aag ggc gtc gtc gcc
aac atg tcg ctc ggc 2570Pro Lys Arg Ser Cys Pro Lys Gly Val Val Ala
Asn Met Ser Leu Gly 230 235 240 ggt agc tac tcg gcc tcc atc aac aac
gcc gcc gcc gcc ctc gtc agg 2618Gly Ser Tyr Ser Ala Ser Ile Asn Asn
Ala Ala Ala Ala Leu Val Arg 245 250 255 260 tcg ggc gtc ttc ctg gcc
gtc gcc gcc ggc aac gag aac cag aac gcc 2666Ser Gly Val Phe Leu Ala
Val Ala Ala Gly Asn Glu Asn Gln Asn Ala 265 270 275 gcc aac tcg tcg
ccc gcc tcc gag gcg tcc gcc tgc acc gtc ggc gcc 2714Ala Asn Ser Ser
Pro Ala Ser Glu Ala Ser Ala Cys Thr Val Gly Ala 280 285 290 acc gac
agg aac gac gcc aag gcc agc tac tcc aac tac ggc agc gtc 2762Thr Asp
Arg Asn Asp Ala Lys Ala Ser Tyr Ser Asn Tyr Gly Ser Val 295 300 305
gtc gat atc cag gcc ccc ggc tcc aac atc ctg agc acc tgg atc ggc
2810Val Asp Ile Gln Ala Pro Gly Ser Asn Ile Leu Ser Thr Trp Ile Gly
310 315 320 agc acc tct gct acc gtaagccccc cctcccccca cccaccccca
gcctttggcg 2865Ser Thr Ser Ala Thr 325 acattcccgc cccgtattta
tttctccggg gtgggggaga aacaaaacaa aatagctaac 2925atgagatgca ctctcag
aac acc atc tcg ggt acc tcg atg gcc tcc ccc 2975 Asn Thr Ile Ser
Gly Thr Ser Met Ala Ser Pro 330 335 340 cac att gcc ggc ctc ggt gcc
tac ctc ctg gcc ctc gag ggc tcc aag 3023His Ile Ala Gly Leu Gly Ala
Tyr Leu Leu Ala Leu Glu Gly Ser Lys 345 350 355 acc cct gcc gag ctc
tgc aac tac atc aag tcg acc ggc aac gcc gcc 3071Thr Pro Ala Glu Leu
Cys Asn Tyr Ile Lys Ser Thr Gly Asn Ala Ala 360 365 370 atc act ggc
gtt ccc agc ggc acc acc aac cgc atc gcc ttc aac ggc 3119Ile Thr Gly
Val Pro Ser Gly Thr Thr Asn Arg Ile Ala Phe Asn Gly 375 380 385 aac
ccc tct gcc tgaattgttt cccgcgatcc gggacaaaat ggggcatgag 3171Asn Pro
Ser Ala 390 cacttcctgc acctcttctt attctagagg attcgggagt ggggagccgg
caaaaaaagg 3231aggtggtgga ggaggaggag gaggagataa cggccggggt
cttctccgag cgaatgaggg 3291ctgcatattc tcttgttcat ttttttggtt
catgtctatt atggttttac gcattttatt 3351ctagttggga cagagtcacg
atgcgggtcc gaggggcgcc gatcggggtt cctgcccacc 3411tccccagcgt
ctaaataact ttcatagacg aggaaatgat gagatctcat gagcggaccg
3471cgaaggcctg gactgacttc tatcgtgact aattatgtga atcatgaggg
cggaatgaga 3531gagatgatat gtcagaatac gcatacttaa ggtgcaattg
ctggcgggca attgcggcgt 3591cacttttgct tttcgacatg atatcatgtc
tccttaatcc aagtagttaa taattagtct 3651ataaataatt tgtctataat
tttgtctatt gcctgaagaa ataagcgatt ttgcaaattc 3711tggtatgtag
agtacaggtc aagtattgga gaggaaggaa ggaagcggta tgtttctcat
3771attgacaagt gacaggagca agcttcttcc tagaatctta gcaaggaaat
gttgaaaatt 3831aagaaagcag aatagaaaca aggactaata gagcaattga
ttgactcaat caatcgttaa 3891ttatgagtcg aagataggtt ctcaaaactt
tttcaaatta gttttgggag gacatgcccg 3951agccatgtaa aacgggcgag
gtacctcggt atgttaatgg ggttgcgtaa tgcttggctg 4011tcgaggatac
tagtaattgt atcgtgtttg tcagaatacc tacttaggtg caattgctgg
4071aagcagcaat tgcggcgtcg cttttgcagt ttcacagtgt tgaagaggtg
agggcaaaca 4131tgtatcgcat atcttggggg tcggtttacc aagagagata
tcatatctaa cccctaagag 4191atgtgtatt 42002392PRTChrysosporium
lucknowense 2Met His Phe Ser Thr Ala Leu Leu Ala Phe Leu Pro Ala
Ala Leu Ala 1 5 10 15 Ala Pro Thr Ala Glu Thr Leu Asp Lys Arg Ala
Pro Ile Leu Thr Ala 20 25 30 Arg Ala Gly Gln Val Val Pro Gly Lys
Tyr Ile Ile Lys Leu Arg Asp 35 40 45 Gly Ala Ser Asp Asp Val Leu
Glu Ala Ala Ile Gly Lys Leu Arg Ser 50 55 60 Lys Ala Asp His Val
Tyr Arg Gly Lys Phe Arg Gly Phe Ala Gly Lys 65 70 75 80 Leu Glu Asp
Asp Val Leu Asp Ala Ile Arg Leu Leu Pro Glu Val Glu 85 90 95 Tyr
Val Glu Glu Glu Ala Ile Phe Thr Ile Asn Ala Tyr Thr Ser Gln 100 105
110 Ser Asn Ala Pro Trp Gly Leu Ala Arg Leu Ser Ser Lys Thr Ala Gly
115 120 125 Ser Thr Thr Tyr Thr Tyr Asp Thr Ser Ala Gly Glu Gly Thr
Cys Ala 130 135 140 Tyr Val Ile Asp Thr Gly Ile Tyr Thr Ser His Ser
Asp Phe Gly Gly 145 150 155 160 Arg Ala Thr Phe Ala Ala Asn Phe Val
Asp Ser Ser Asn Thr Asp Gly 165 170 175 Asn Gly His Gly Thr His Val
Ala Gly Thr Ile Gly Gly Thr Thr Tyr 180 185 190 Gly Val Ala Lys Lys
Thr Lys Leu Tyr Ala Val Lys Val Leu Gly Ser 195 200 205 Asp Gly Ser
Gly Thr Thr Ser Gly Val Ile Ala Gly Ile Asn Phe Val 210 215 220 Ala
Asp Asp Ala Pro Lys Arg Ser Cys Pro Lys Gly Val Val Ala Asn 225 230
235 240 Met Ser Leu Gly Gly Ser Tyr Ser Ala Ser Ile Asn Asn Ala Ala
Ala 245 250 255 Ala Leu Val Arg Ser Gly Val Phe Leu Ala Val Ala Ala
Gly Asn Glu 260 265 270 Asn Gln Asn Ala Ala Asn Ser Ser Pro Ala Ser
Glu Ala Ser Ala Cys 275 280 285 Thr Val Gly Ala Thr Asp Arg Asn Asp
Ala Lys Ala Ser Tyr Ser Asn 290 295 300 Tyr Gly Ser Val Val Asp Ile
Gln Ala Pro Gly Ser Asn Ile Leu Ser 305 310 315 320 Thr Trp Ile Gly
Ser Thr Ser Ala Thr Asn Thr Ile Ser Gly Thr Ser 325 330 335 Met Ala
Ser Pro His Ile Ala Gly Leu Gly Ala Tyr Leu Leu Ala Leu 340 345 350
Glu Gly Ser Lys Thr Pro Ala Glu Leu Cys Asn Tyr Ile Lys Ser Thr 355
360 365 Gly Asn Ala Ala Ile Thr Gly Val Pro Ser Gly Thr Thr Asn Arg
Ile 370 375 380 Ala Phe Asn Gly Asn Pro Ser Ala 385 390
31159DNAChrysosporium lucknowense 3atgcacttct ccaccgctct cctggccttc
ctgcccgccg ccctcgcggc ccctactgcc 60gagaccctcg acaagcgcgc cccgatcctg
actgctcgcg ctggccaggt cgtcccgggc 120aagtacatca tcaagctccg
cgacggagcc agcgacgatg tccttgaggc cgccatcggc 180aagctccgct
ccaaggccga ccacgtctac cgcggcaagt tcaggggctt tgccggcaag
240ctcgaggatg acgtccttga cgccatccgt cttgtcgagt acgtcgagga
ggaggccatc 300ttcaccatca acgcgtacac ctcgcagtcc aacgccccct
ggggccttgc gcgcctctcg 360tccaagaccg cgggctccac cacctacacc
tacgacacca gcgccggcga gggcacctgt 420gcctatgtga tcgacacggg
catctacact agccactcga cttcggcggc cgtgccactt 480tcgccgccaa
cttcgtcgac agctctaaca ccgatggcaa cggccacggc acccacgtcg
540ccggcaccat cggcggcacc acgtacggtg ttgccaagaa gaccaagctc
tacgccgtca 600aggttctcgg ctccgacggc tctggcacct tctggtgtca
ttgctggcat caacttcgtc 660gctgacgacg cgcccaagcg cagctgcccc
aagggcgtcg tcgccaacat gtcgctcggc 720ggtagctact cggcctccat
caacaacgcc gccgccgccc tcgtcaggtc gggcgtcttc 780ctggccgtcg
ccgccggcaa cgagaaccag aacgccgcca actcgtcgcc cgcctccgag
840gcgtccgcct gcaccgtcgg cgccaccgac aggaacgacg ccaaggccag
ctactccaac 900tacggcagcg tcgtcgatat ccaggccccc ggctccaaca
tcctgagcac ctggatcggc 960agcacctctg aacaccatct cgggtacctc
gatggcctcc ccccacattg ccggcctcgg 1020tgcctacctc ctggccctcg
agggctccaa gacccctgcc gagctctgca actacatcaa 1080gtcgaccggc
aacgccgcca tcactggcgt tcccagcggc accaccaacc gcatcgcctt
1140caacggcaac ccctctgcc 115944325DNAChrysosporium
lucknowenseCDS(1759)..(2130)CDS(2189)..(3418) 4gcccttacgc
gtaagtatta cagatcgcgt tcgggcaact ccctagaatc atggagcaca 60gcatcggctc
gatcgcccaa caacttccta gcattgactt caaaaaggag acatgttggt
120tacaacgatc gatgtttaca aaactcgacc attcctattt cctcgtatgc
tcccaacgct 180cttcgaacct gacacactac cacgcattgt ccccagcata
tctaccaaca tcattgaaaa 240tgctgcggca ttgttattgt ctaggctttt
ttcttttaag caaacgtcgt ggatacccta 300gatagagaat acgacggata
atgttttcac aagatctgtg gcatcaagga tatgctccac 360gaacctgacc
tggcaacttg gttacagtcc gcgggttatt aaagtagtct cgacttttgc
420gcccgagact aaggtgggca accaaagggg gctggtttcg catagccgaa
tgaatcatct 480ctcagtcgat cgatttggag cgagtgttgc tccggaggtc
cagtatgacc tataatattg 540caacagctga cagctgatat tccgcctggt
gaatatgaca ccgataggct gttcaacgaa 600tcaccttttg cccgatggag
gaactactac gctgtgcaat aattatttat gcacaaagtg 660cagtgcgcaa
tgtttttgac ggatactcgc actggtactc cgtccactcg cggaatccag
720ggtctgctgg gatcacggtc gagtcagacg gacaatgcga tgcgacccct
tgcacagctt 780cgtcgctcgt gacccatcat ccgtccctga cagggagcgg
cgggttggca ggggtggaga 840tgacgccccg cccttgcact acttggcttg
gcggtgacgt gcccggaatc agatctgacc 900tcaacgtgca agcagtcaac
agcggacagc gggtcaagat gtttgacagc ttatcaagtt 960ccggctgcat
tgagccaaca gaggaaatcg ctgcctcgct cccccggaac tgcacctcca
1020accgagcttt tacgacctgg tctgccagtg caagccagtg gagaatgcag
ggcagccgag 1080cccagggcaa gccaaggcag ccaaccgtct ggtacgcggt
gtggacggga ccaagtcatg 1140tacccagtaa ctcccagtag gtaacagccg
ggggtccgaa agcggtggga gctcaactgg 1200ccgcagatcc cggacagtca
gcctcgccgc ggcatctgcg tgcgacgcct ttgtttcacc 1260gtacaataga
cgctttggtg cgcaacttct gccggaaata agaatcgttt acgtatgctg
1320cacgctgcct gcctacctat cttatggagc ttcgcaactg aggagtcttg
cacttcgccc 1380actgcacttg gtgatggcgg tatcaggcta tcagcggcct
ggtgactgtc cgataagcgc 1440tggtccaccc gttatctccc ttccccagag
acaacacgcc cggagcaggc tcatacgtga 1500ttggatgccc cgccttgact
agtccgcatt aggtgaaagc gggccaaacc agcattggtc 1560tgaggcaact
gccagctgcg ccttccgtta aagcagctcc ggtcctctcc cacgctcgct
1620gctcctctct tcttccttca tcaaaccatc ttcctcttcc tgttcgccga
atcaagggaa 1680gaggtgacag acttgctttt tatcccccga gtttctcgaa
gtcgcaattt ttacggcttc 1740gcctctcttt cgttcacc atg aga ggc ctc gtc
gca ttc tcg ctc gca gcc 1791 Met Arg Gly Leu Val Ala Phe Ser Leu
Ala Ala 1 5 10 tgc gtt tcg gca gcg ccg agc ttc aag acc gag acc atc
aac ggc gag 1839Cys Val Ser Ala Ala Pro Ser Phe Lys Thr Glu Thr Ile
Asn Gly Glu 15 20 25 cat gcc ccc att ctc tcc tcg tcg aat gcc gag
gtc gtc ccc aac tcg 1887His Ala Pro Ile Leu Ser Ser Ser Asn Ala Glu
Val Val Pro Asn Ser 30 35 40 tat atc atc aag ttc aag aag cat gtg
gat gag agc tcg gcc tcc gcc 1935Tyr Ile Ile Lys Phe Lys Lys His Val
Asp Glu Ser Ser Ala Ser Ala 45 50 55 cac cat gcc tgg atc caa gac
atc cat acc tcg cgc gag aaa gtt cgt 1983His His Ala Trp Ile Gln Asp
Ile His Thr Ser Arg Glu Lys Val Arg 60 65 70 75 caa gac ttg aag aag
cgc ggc cag gtg ccg ctg ctt gac gac gtc ttc 2031Gln Asp Leu Lys Lys
Arg Gly Gln Val Pro Leu Leu Asp Asp Val Phe 80 85 90 cat ggc ctc
aag cac acc tac aag att ggc caa gag ttc ctt ggc tac 2079His Gly Leu
Lys His Thr Tyr Lys Ile Gly Gln Glu Phe Leu Gly Tyr 95 100 105 tct
ggc cat ttt gat gac gaa acc atc gag caa gtc cgg agg cac ccc 2127Ser
Gly His Phe Asp Asp Glu Thr Ile Glu Gln Val Arg Arg His Pro 110
115
120 gat gtaagttgac tccagggcga ttgggctctt tccacatgct gatccacacg
2180Asp ggcgacag gtg gag tac att gag cgc gac agc att gtc cac acg
atg cgt 2230 Val Glu Tyr Ile Glu Arg Asp Ser Ile Val His Thr Met
Arg 125 130 135 gtc acc gag gaa aca tgc gat ggc gag ctt gag aag gcc
gct cct tgg 2278Val Thr Glu Glu Thr Cys Asp Gly Glu Leu Glu Lys Ala
Ala Pro Trp 140 145 150 ggc ttg gcc cgt atc tcg cac cga gat acg ctc
ggc ttc tcg acg ttc 2326Gly Leu Ala Arg Ile Ser His Arg Asp Thr Leu
Gly Phe Ser Thr Phe 155 160 165 170 aac aag tat ctg tat gcc gcc gag
ggc agt gag ggc gtt gac gcg tac 2374Asn Lys Tyr Leu Tyr Ala Ala Glu
Gly Ser Glu Gly Val Asp Ala Tyr 175 180 185 gtc atc gac acc ggt acc
aac att gag cac gtc gac ttc gag ggt cgc 2422Val Ile Asp Thr Gly Thr
Asn Ile Glu His Val Asp Phe Glu Gly Arg 190 195 200 gcc aaa tgg ggc
aag acc atc ccc gca ggc gat gct gat gtt gac ggc 2470Ala Lys Trp Gly
Lys Thr Ile Pro Ala Gly Asp Ala Asp Val Asp Gly 205 210 215 aac ggc
cac gga acc aac tgc tcc ggc acc atc gct ggc aag aag tac 2518Asn Gly
His Gly Thr Asn Cys Ser Gly Thr Ile Ala Gly Lys Lys Tyr 220 225 230
ggc gtc gcc aag aag gcg aat gtc tat gcc gtc aag gtt ctg cgc tcc
2566Gly Val Ala Lys Lys Ala Asn Val Tyr Ala Val Lys Val Leu Arg Ser
235 240 245 250 aac ggc tcc ggc acc atg gct gat gtc gtt gcc ggt gtc
gaa tgg gct 2614Asn Gly Ser Gly Thr Met Ala Asp Val Val Ala Gly Val
Glu Trp Ala 255 260 265 gcc aag tcc cat ctg gag cag gtg cag gct gcc
aag gat ggc aag cgc 2662Ala Lys Ser His Leu Glu Gln Val Gln Ala Ala
Lys Asp Gly Lys Arg 270 275 280 aag ggc ttc aag ggt tcc gtc gcc aac
atg tcg ctt gga ggc ggc aag 2710Lys Gly Phe Lys Gly Ser Val Ala Asn
Met Ser Leu Gly Gly Gly Lys 285 290 295 acc agg gcc ctc gat gac act
gtg aac gct gct gtc tct gtc ggt atc 2758Thr Arg Ala Leu Asp Asp Thr
Val Asn Ala Ala Val Ser Val Gly Ile 300 305 310 cac ttc gcc gtc gcc
gcc ggc aac gac aac gcc gat gcc tgc aac tac 2806His Phe Ala Val Ala
Ala Gly Asn Asp Asn Ala Asp Ala Cys Asn Tyr 315 320 325 330 tcg cct
gct gcg gcc gag aag gct gtc acg gtt ggt gcc tcg gct att 2854Ser Pro
Ala Ala Ala Glu Lys Ala Val Thr Val Gly Ala Ser Ala Ile 335 340 345
gac gac agc cgt gcc tac ttc tcc aac tac ggc aag tgc acc gac atc
2902Asp Asp Ser Arg Ala Tyr Phe Ser Asn Tyr Gly Lys Cys Thr Asp Ile
350 355 360 ttc gcc ccc ggc ctg agc atc ctc tcc acc tgg atc ggc agc
aag tat 2950Phe Ala Pro Gly Leu Ser Ile Leu Ser Thr Trp Ile Gly Ser
Lys Tyr 365 370 375 gcc acc aac acc atc tct ggc acc tcg atg gcc tcc
ccc cat att gct 2998Ala Thr Asn Thr Ile Ser Gly Thr Ser Met Ala Ser
Pro His Ile Ala 380 385 390 ggt ctg ctg gcc tac tat ctc tcg ctc cag
ccc gcc acc gat tcg gag 3046Gly Leu Leu Ala Tyr Tyr Leu Ser Leu Gln
Pro Ala Thr Asp Ser Glu 395 400 405 410 tac tcg gtc gct ccc atc acc
cct gag aag atg aag tcg aac ttg ctc 3094Tyr Ser Val Ala Pro Ile Thr
Pro Glu Lys Met Lys Ser Asn Leu Leu 415 420 425 aag atc gcc acc cag
gat gcc ctt act gac atc ccc gac gag acg ccc 3142Lys Ile Ala Thr Gln
Asp Ala Leu Thr Asp Ile Pro Asp Glu Thr Pro 430 435 440 aat ctg ctc
gcc tgg aac ggc ggt ggc tgc aac aac tat acc gcc atc 3190Asn Leu Leu
Ala Trp Asn Gly Gly Gly Cys Asn Asn Tyr Thr Ala Ile 445 450 455 gtc
gag gct ggc ggc tac aag gcc aag aag aag acc acg act gac aag 3238Val
Glu Ala Gly Gly Tyr Lys Ala Lys Lys Lys Thr Thr Thr Asp Lys 460 465
470 gtt gac att ggc gcc tcg gtc tct gag ctt gag aag ctt atc gag cac
3286Val Asp Ile Gly Ala Ser Val Ser Glu Leu Glu Lys Leu Ile Glu His
475 480 485 490 gat ttt gag gtc atc tct ggc aag gtt gtc aag ggc gtc
tcg tcg ttt 3334Asp Phe Glu Val Ile Ser Gly Lys Val Val Lys Gly Val
Ser Ser Phe 495 500 505 gcg gac aag gcc gag aag ttc tct gag aag att
cac gag ctg gtc gat 3382Ala Asp Lys Ala Glu Lys Phe Ser Glu Lys Ile
His Glu Leu Val Asp 510 515 520 gag gaa ctc aag gag ttt ctt gag gac
atc gct gcc taagccttag 3428Glu Glu Leu Lys Glu Phe Leu Glu Asp Ile
Ala Ala 525 530 acatgccatc ggcgttgtga gcgtgcagcc tgagatgcgt
attttggtct gggattaggg 3488gatagggatt gttttttttt tcgtctctgc
ttttcttggt ctattggacg ggcatacatc 3548gacacggtgg gtgttccttg
tactggatgg gtaacatggg attcggacgg acgcaagatt 3608gtctggcttg
gtttgcattt gagggtagtc gcgttcgttc agctccatca ttgatcagtt
3668ctcattcgtc cactgacggc ctcgcatcaa tcaggccgtc cactctgccc
gtctctcgaa 3728atgcggctgg ccgtggtcca ttctgctcct tccgctcgtg
ttctccgcgg cagctctgat 3788ccagttgtgc tttccctagg aacccgcggg
ccccctcccc ctccccttcc cttcccttcc 3848cttcccttcc ctctctcgcc
cccggtcccc ctgtgtactc ggcccttgtt tctccatgat 3908ggcttccatc
tatctcgctc tcctctcccc cgctcccgtt tggcctaggg tctttcgtcc
3968tctgtcgtat ccgcggaacc cacattgtgc cacgaacctg tgcatctcat
ctctcttggc 4028ataggtttag ctacacagtt ttactgttga ttagtctcct
ctccctgaca aactgttacc 4088gcgtcgctgt ttcggtgcga ctgcaccgtt
acgcgggcac cgccttgttc caccgtttca 4148atgctgcgtc ggcccagcct
caagtcgagg acaaacctca aacgccgcaa gtccaccttg 4208tcaacgcagg
gggttgtcct tgagcatcac gacccggcgc tgggcacaac gggatgctca
4268tattgcggct taggaggcct acgtcagagc ccagagccgt atcatggcag aacccgc
43255534PRTChrysosporium lucknowense 5Met Arg Gly Leu Val Ala Phe
Ser Leu Ala Ala Cys Val Ser Ala Ala 1 5 10 15 Pro Ser Phe Lys Thr
Glu Thr Ile Asn Gly Glu His Ala Pro Ile Leu 20 25 30 Ser Ser Ser
Asn Ala Glu Val Val Pro Asn Ser Tyr Ile Ile Lys Phe 35 40 45 Lys
Lys His Val Asp Glu Ser Ser Ala Ser Ala His His Ala Trp Ile 50 55
60 Gln Asp Ile His Thr Ser Arg Glu Lys Val Arg Gln Asp Leu Lys Lys
65 70 75 80 Arg Gly Gln Val Pro Leu Leu Asp Asp Val Phe His Gly Leu
Lys His 85 90 95 Thr Tyr Lys Ile Gly Gln Glu Phe Leu Gly Tyr Ser
Gly His Phe Asp 100 105 110 Asp Glu Thr Ile Glu Gln Val Arg Arg His
Pro Asp Val Glu Tyr Ile 115 120 125 Glu Arg Asp Ser Ile Val His Thr
Met Arg Val Thr Glu Glu Thr Cys 130 135 140 Asp Gly Glu Leu Glu Lys
Ala Ala Pro Trp Gly Leu Ala Arg Ile Ser 145 150 155 160 His Arg Asp
Thr Leu Gly Phe Ser Thr Phe Asn Lys Tyr Leu Tyr Ala 165 170 175 Ala
Glu Gly Ser Glu Gly Val Asp Ala Tyr Val Ile Asp Thr Gly Thr 180 185
190 Asn Ile Glu His Val Asp Phe Glu Gly Arg Ala Lys Trp Gly Lys Thr
195 200 205 Ile Pro Ala Gly Asp Ala Asp Val Asp Gly Asn Gly His Gly
Thr Asn 210 215 220 Cys Ser Gly Thr Ile Ala Gly Lys Lys Tyr Gly Val
Ala Lys Lys Ala 225 230 235 240 Asn Val Tyr Ala Val Lys Val Leu Arg
Ser Asn Gly Ser Gly Thr Met 245 250 255 Ala Asp Val Val Ala Gly Val
Glu Trp Ala Ala Lys Ser His Leu Glu 260 265 270 Gln Val Gln Ala Ala
Lys Asp Gly Lys Arg Lys Gly Phe Lys Gly Ser 275 280 285 Val Ala Asn
Met Ser Leu Gly Gly Gly Lys Thr Arg Ala Leu Asp Asp 290 295 300 Thr
Val Asn Ala Ala Val Ser Val Gly Ile His Phe Ala Val Ala Ala 305 310
315 320 Gly Asn Asp Asn Ala Asp Ala Cys Asn Tyr Ser Pro Ala Ala Ala
Glu 325 330 335 Lys Ala Val Thr Val Gly Ala Ser Ala Ile Asp Asp Ser
Arg Ala Tyr 340 345 350 Phe Ser Asn Tyr Gly Lys Cys Thr Asp Ile Phe
Ala Pro Gly Leu Ser 355 360 365 Ile Leu Ser Thr Trp Ile Gly Ser Lys
Tyr Ala Thr Asn Thr Ile Ser 370 375 380 Gly Thr Ser Met Ala Ser Pro
His Ile Ala Gly Leu Leu Ala Tyr Tyr 385 390 395 400 Leu Ser Leu Gln
Pro Ala Thr Asp Ser Glu Tyr Ser Val Ala Pro Ile 405 410 415 Thr Pro
Glu Lys Met Lys Ser Asn Leu Leu Lys Ile Ala Thr Gln Asp 420 425 430
Ala Leu Thr Asp Ile Pro Asp Glu Thr Pro Asn Leu Leu Ala Trp Asn 435
440 445 Gly Gly Gly Cys Asn Asn Tyr Thr Ala Ile Val Glu Ala Gly Gly
Tyr 450 455 460 Lys Ala Lys Lys Lys Thr Thr Thr Asp Lys Val Asp Ile
Gly Ala Ser 465 470 475 480 Val Ser Glu Leu Glu Lys Leu Ile Glu His
Asp Phe Glu Val Ile Ser 485 490 495 Gly Lys Val Val Lys Gly Val Ser
Ser Phe Ala Asp Lys Ala Glu Lys 500 505 510 Phe Ser Glu Lys Ile His
Glu Leu Val Asp Glu Glu Leu Lys Glu Phe 515 520 525 Leu Glu Asp Ile
Ala Ala 530 61602DNAChrysosporium lucknowense 6atgagaggcc
tcgtcgcatt ctcgctcgca gcctgcgttt cggcagcgcc gagcttcaag 60accgagacca
tcaacggcga gcatgccccc attctctcct cgtcgaatgc cgaggtcgtc
120cccaactcgt atatcatcaa gttcaagaag catgtggatg agagctcggc
ctccgcccac 180catgcctgga tccaagacat ccatacctcg cgcgagaaag
ttcgtcaaga cttgaagaag 240cgcggccagg tgccgctgct tgacgacgtc
ttccatggcc tcaagcacac ctacaagatt 300ggccaagagt tccttggcta
ctctggccat tttgatgacg aaaccatcga gcaagtccgg 360aggcaccccg
atgtggagta cattgagcgc gacagcattg tccacacgat gcgtgtcacc
420gaggaaacat gcgatggcga gcttgagaag gccgctcctt ggggcttggc
ccgtatctcg 480caccgagata cgctcggctt ctcgacgttc aacaagtatc
tgtatgccgc cgagggcagt 540gagggcgttg acgcgtacgt catcgacacc
ggtaccaaca ttgagcacgt cgacttcgag 600ggtcgcgcca aatggggcaa
gaccatcccc gcaggcgatg ctgatgttga cggcaacggc 660cacggaacca
actgctccgg caccatcgct ggcaagaagt acggcgtcgc caagaaggcg
720aatgtctatg ccgtcaaggt tctgcgctcc aacggctccg gcaccatggc
tgatgtcgtt 780gccggtgtcg aatgggctgc caagtcccat ctggagcagg
tgcaggctgc caaggatggc 840aagcgcaagg gcttcaaggg ttccgtcgcc
aacatgtcgc ttggaggcgg caagaccagg 900gccctcgatg acactgtgaa
cgctgctgtc tctgtcggta tccacttcgc cgtcgccgcc 960ggcaacgaca
acgccgatgc ctgcaactac tcgcctgctg cggccgagaa ggctgtcacg
1020gttggtgcct cggctattga cgacagccgt gcctacttct ccaactacgg
caagtgcacc 1080gacatcttcg cccccggcct gagcatcctc tccacctgga
tcggcagcaa gtatgccacc 1140aacaccatct ctggcacctc gatggcctcc
ccccatattg ctggtctgct ggcctactat 1200ctctcgctcc agcccgccac
cgattcggag tactcggtcg ctcccatcac ccctgagaag 1260atgaagtcga
acttgctcaa gatcgccacc caggatgccc ttactgacat ccccgacgag
1320acgcccaatc tgctcgcctg gaacggcggt ggctgcaaca actataccgc
catcgtcgag 1380gctggcggct acaaggccaa gaagaagacc acgactgaca
aggttgacat tggcgcctcg 1440gtctctgagc ttgagaagct tatcgagcac
gattttgagg tcatctctgg caaggttgtc 1500aagggcgtct cgtcgtttgc
ggacaaggcc gagaagttct ctgagaagat tcacgagctg 1560gtcgatgagg
aactcaagga gtttcttgag gacatcgctg cc 160275871DNAChrysosporium
lucknowenseCDS(2211)..(2312)CDS(2369)..(2525)CDS(2596)..(3527)
7gctggctcac cgttatttgc tcccgcagga agtccaggtc ctcctcgcag ttggacaaac
60tctgcttcgc agcctgcaac tttgactcaa ggagcgcctc ggcctcgtcg attgggtaag
120acagcatgac gttggcctgc caaatgtcag cctctagaag cacactccca
ctctcgttgg 180aaaggttcct accccaagcc acaagtaaac ctcgtccgtc
ggcggtatct cggccttcgc 240atagagagtg tcgttcaatt cgaatgttgt
ctctatcgga tcagattcgc cctgccacaa 300tcaaccgccg atcagcacca
tggccgctca tcgagagtgg caacgcctcg ccctaccgtc 360ctcagcttca
aaaagcggac agcctccagc gttttccgaa tgtcgggcat tttgtccttt
420agtcccgcta ccctccgctg caggttctgc tccatgaact ggtatttcct
gcacgccgac 480cacgtatcag ccgaacgccg tccgtcaagg ctggatttca
atcttaaccg ggagagctca 540cgcaatcatc tcttggaacc gacgcagcgt
cggctcaaca tctgctcgtg acgtgacata 600gtcctcgacc ttgtcgacga
atggcgcata cggaatgcca cgaggattgg agggtgtggc 660gtccctgtct
cgtgcaggtg gtcagtcagc aataacagcc agagtgcata tgctagaatg
720gcgcccgcgg gggagggaaa gtttggttac cttgctgctg cttccttgtc
tgtgctcgcc 780atcttggaca aattctcaca tgttgcagtg gaaggatact
gcaagcgact gttaacccga 840gccaacggag tgacgtcggg tttggtacct
agtttaggtc aagccgttct caagctgctg 900gccaaaaatt catggcgggg
tcgagtgggc agcgaggtac tcctcgtagg gagcaaggtg 960aagatgtggg
gtagcagggg tcgacgctac aaagtacttt gtatccggat tgctgtgtgg
1020tacgaagcgc ccgtgtgttg gatgctctct gtatgtacgg agtactgtac
ctttctccat 1080gcgctgcccc attctctatt tggttgcacc tgcttcgttc
gtagtgtatg tacagcagta 1140caactatcta cgacacctgc actgactagt
gcgtagaatt ctttagtttc tcgagtacgg 1200cgctaacgct tcgcgcagca
agcaccttct tctgattgtg ttactgtgct caaacctcgc 1260cagccagctg
cggtgctcca caagcccggc cgtgcccaac cgccatttgc atcccggtcc
1320catgaatctg tggacgaccc atccctctct gtaccgcgtc gcggtatcag
cccagaatga 1380tagcgggaag acaaacgcag tgattcggat tacgctcgca
ggaaatgggg ggagtagctt 1440gatagctctc cacggcgagg gtgtctcagg
ctgaggtgtc aactagttgt atgtacactc 1500aggacgaggc attctgcgtt
ttgaaacacc aatcttccaa taccggaggt gttgtatgca 1560ggatcacttg
aatatgtttg cacccattat tactgtacct ggatgattcg gacagggcga
1620gcatgattgg tcgccccgtt ttgtcaccgc attcgcagcg tcggcgggaa
gcagccacgt 1680agagcactgc caaacgtttc aagagacacc ccatatggag
taaattggag taatctgtat 1740ccttcagagc cgtcaatcaa actattgttt
ctcagcagga tggcccgttg ctcatggggg 1800atgtaccctg gtaggtagtt
cgttgttgat gacttccttg gatgagcctg ctgcgcatga 1860aggtgccggg
gccccaggtt gggtgcctaa aactaactgt aaacagacgc acggtggcga
1920cgacgtagcc gaaccggtgt agcgagcttt ccccggccac tacgtaatcg
gggcgatgca 1980ctgcaggaac acctcacacc tgacctaccc ccttcgcctc
cgcatccgtc ccaacccgct 2040tccccaacct ttccatcaac tacttccgag
actcgacatc accttttcgc gtcgtgtctc 2100atcgtcgtta tcatcaccat
cggcgataga tttgttcgct tcgatcgtcg catcgccttg 2160acttccattc
gtccttcacg ccgaccgacc ggaccagaca gtcgcccaaa atg aag 2216 Met Lys 1
gat gct ttt ttg ctg acc gca gct gtg ctg ctc ggc tcc gcc cag gga
2264Asp Ala Phe Leu Leu Thr Ala Ala Val Leu Leu Gly Ser Ala Gln Gly
5 10 15 gca gtt cac aaa atg aag ctg cag aag atc cct ctc tct gag cag
ctt 2312Ala Val His Lys Met Lys Leu Gln Lys Ile Pro Leu Ser Glu Gln
Leu 20 25 30 gtacgtctga ccccgttcaa gcacgcgtca gcggctactg accttatcgc
gtccag gag 2371 Glu 35 gcg gtt ccc atc aac acc cag ctc gag cat ctc
ggc caa aaa tac atg 2419Ala Val Pro Ile Asn Thr Gln Leu Glu His Leu
Gly Gln Lys Tyr Met 40 45 50 ggg ttg cgc cca cgt gaa tct caa gcc
gat gcc atc ttt aag ggc atg 2467Gly Leu Arg Pro Arg Glu Ser Gln Ala
Asp Ala Ile Phe Lys Gly Met 55 60 65 gtt gcc gac gtc aag ggc aac
cat cct att ccc atc tcc aac ttc atg 2515Val Ala Asp Val Lys Gly Asn
His Pro Ile Pro Ile Ser Asn Phe Met 70 75 80 aac gca cag t
gtatgtgacg ccactgtggt ggcatggatg gctcgtcctc 2565Asn Ala Gln 85
aattcggaga ctgacactgg agcaccctag ac ttc tcc gag atc acg att gga
2618 Tyr Phe Ser Glu Ile Thr Ile Gly 90 aca ccc cct cag tca ttc aag
gtg gtc ctc gat acc ggt agc tcc aac 2666Thr Pro Pro Gln Ser Phe Lys
Val Val Leu Asp Thr Gly Ser Ser Asn 95 100 105 110 ctg tgg gtt cca
tca gtc gag tgc ggc tcg att gct tgt tac ctg cac 2714Leu Trp Val Pro
Ser Val Glu Cys Gly Ser Ile Ala Cys Tyr Leu His 115 120 125 tcg aag
tat gac tca tct
gcc tcg tcc acc tac aag aag aac gga acc 2762Ser Lys Tyr Asp Ser Ser
Ala Ser Ser Thr Tyr Lys Lys Asn Gly Thr 130 135 140 tcg ttc gag atc
cgc tac ggg tca ggc agc ctg agc ggg ttt gtc tct 2810Ser Phe Glu Ile
Arg Tyr Gly Ser Gly Ser Leu Ser Gly Phe Val Ser 145 150 155 cag gac
aca gtg tcc atc ggc gat atc act atc cag ggc cag gac ttt 2858Gln Asp
Thr Val Ser Ile Gly Asp Ile Thr Ile Gln Gly Gln Asp Phe 160 165 170
gcc gag gcg acc agc gag ccc ggt ctt gcc ttt gcc ttt ggc cgt ttc
2906Ala Glu Ala Thr Ser Glu Pro Gly Leu Ala Phe Ala Phe Gly Arg Phe
175 180 185 190 gac ggt atc ctt ggc ctt ggc tac gac cgg atc tca gtc
aac ggc atc 2954Asp Gly Ile Leu Gly Leu Gly Tyr Asp Arg Ile Ser Val
Asn Gly Ile 195 200 205 gtc ccg cct ttt tac aag atg gtc gag cag aag
ctc atc gat gag ccc 3002Val Pro Pro Phe Tyr Lys Met Val Glu Gln Lys
Leu Ile Asp Glu Pro 210 215 220 gtc ttc gcc ttc tac ctg gcc gat acc
aat ggc cag tct gag gtt gtc 3050Val Phe Ala Phe Tyr Leu Ala Asp Thr
Asn Gly Gln Ser Glu Val Val 225 230 235 ttt ggc ggt gtt gac cac gac
aag tac aag ggc aag atc acc acc att 3098Phe Gly Gly Val Asp His Asp
Lys Tyr Lys Gly Lys Ile Thr Thr Ile 240 245 250 ccg ttg agg cgc aag
gcc tac tgg gag gtt gac ttc gat gcc att tct 3146Pro Leu Arg Arg Lys
Ala Tyr Trp Glu Val Asp Phe Asp Ala Ile Ser 255 260 265 270 tac ggc
gac gac act gcc gag ctt gag aac act ggc atc atc ctg gac 3194Tyr Gly
Asp Asp Thr Ala Glu Leu Glu Asn Thr Gly Ile Ile Leu Asp 275 280 285
acc ggt act tct ctg atc gct ctg ccc agc cag ctc gcc gag atg ctc
3242Thr Gly Thr Ser Leu Ile Ala Leu Pro Ser Gln Leu Ala Glu Met Leu
290 295 300 aac gct cag atc ggc gct aag aag agc tac act ggc cag tac
acc atc 3290Asn Ala Gln Ile Gly Ala Lys Lys Ser Tyr Thr Gly Gln Tyr
Thr Ile 305 310 315 gac tgc aac aag cgc gac tcc ctc aag gat gtc acg
ttc aac ctg gct 3338Asp Cys Asn Lys Arg Asp Ser Leu Lys Asp Val Thr
Phe Asn Leu Ala 320 325 330 ggc tac aat ttc acg ctc ggc ccc tac gac
tac gtt ctc gag gtc cag 3386Gly Tyr Asn Phe Thr Leu Gly Pro Tyr Asp
Tyr Val Leu Glu Val Gln 335 340 345 350 ggc agc tgc att tct acc ttt
atg ggc atg gat ttc ccg gct cct act 3434Gly Ser Cys Ile Ser Thr Phe
Met Gly Met Asp Phe Pro Ala Pro Thr 355 360 365 ggg cca ctt gcg atc
ctg ggc gat gcc ttc ctc cgg agg tat tac tcc 3482Gly Pro Leu Ala Ile
Leu Gly Asp Ala Phe Leu Arg Arg Tyr Tyr Ser 370 375 380 att tat gac
ctt ggc gcc gac acc gtc ggt ctg gct gag gcc aag 3527Ile Tyr Asp Leu
Gly Ala Asp Thr Val Gly Leu Ala Glu Ala Lys 385 390 395 tgattgaagg
atgggcggca gggaaagacg atgggtaata cggggagtct gggaatcggg
3587ctttggactg tggtctgtat ctagttgctc aagagagttg tcgtttgatt
ttgttatagg 3647atctgtctag gaaccttagc aggagtgaaa ttttttcgtg
tacgagcatc ggcgggctga 3707agtggtttga taacaagtct ggacttgagt
acgcaggcag ttgcacaatc tgcttcgccg 3767aggagagcaa aggcgtcctc
tttgaaaaag cctacctacg cgtcacaggg gtataatttt 3827ttgagtttga
cctacgccct gtcccatacc aaccgcgtcc caatccccgt caacccttgc
3887aatgtcatta cccgtggatg tatcacgtag cagaagccga catcccacac
gcttcaacct 3947tcctatccag acaatgacat ggtaagctca ttttttaaag
gtcgccgtcc tccctccctt 4007cacgtgattc attttccttg cgccttgtgg
cgcatcccct gacttcatgc cgtacggatc 4067aaagggtgca aacttgcccc
gcacctcttt tctgccgcca tcatcatcac catcatcgcc 4127gtttgtcgcc
tgcgcagcat gtagcacgga cgacgccttg ctgtagtcaa acggctcctg
4187ctcggcatcg tcatcatggc cttcctcctg ttcgcccgag gtctgttcgt
cggctgccga 4247ggtcgcggcg gaggcagatg tctgctgctg ctgctgctgc
tgctgcttct gggctttctt 4307ggcggctcga agtgccttcc tggcttgagc
cttgagttcc tttgctccct ttatgtctcc 4367gttttgagcc agttgctctg
ccaagagctg agcacgcttg aactcttctc gagcagcctt 4427cttggcttgt
ttctttgcct gcttggcggc cttgtcatca ccaccctcaa cttcctgctc
4487gacactagga gacttcgggt ggtctttgcc tgcggaacta tctccaccca
tctcgatgtc 4547ggaaactgct tcggcttcgg atgctgactc aacatcaaca
tccctagact tccgctttcg 4607accagccttc agagtgaaac cttcttcttc
gagaacaggg agacccttgg tgtcttgttc 4667agcgacacgc ctgatgaagg
atgctttttt gctgaccgca gctgtgctgc tcggctccgc 4727ccagggagca
gttcacaaaa tgaagctgca gaagatccct ctctctgagc agcttgaggc
4787ggttcccatc aacacccagc tcgagcatct cggccaaaaa tacatggggt
tgcgcccacg 4847tgaatctcaa gccgatgcca tctttaaggg catggttgcc
gacgtcaagg gcaaccatcc 4907tattcccatc tccaacttca tgaacgcaca
gtacttctcc gagatcacga ttggaacacc 4967ccctcagtca ttcaaggtgg
tcctcgatac cggtagctcc aacctgtggg ttccatcagt 5027cgagtgcggc
tcgattgctt gttacctgca ctcgaagtat gactcatctg cctcgtccac
5087ctacaagaag aacggaacct cgttcgagat ccgctacggg tcaggcagcc
tgagcgggtt 5147tgtctctcag gacacagtgt ccatcggcga tatcactatc
cagggccagg actttgccga 5207ggcgaccagc gagcccggtc ttgcctttgc
ctttggccgt ttcgacggta tccttggcct 5267tggctacgac cggatctcag
tcaacggcat cgtcccgcct ttttacaaga tggtcgagca 5327gaagctcatc
gatgagcccg tcttcgcctt ctacctggcc gataccaatg gccagtctga
5387ggttgtcttt ggcggtgttg accacgacaa gtacaagggc aagatcacca
ccattccgtt 5447gaggcgcaag gcctactggg aggttgactt cgatgccatt
tcttacggcg acgacactgc 5507cgagcttgag aacactggca tcatcctgga
caccggtact tctctgatcg ctctgcccag 5567ccagctcgcc gagatgctca
acgctcagat cggcgctaag aagagctaca ctggccagta 5627caccatcgac
tgcaacaagc gcgactccct caaggatgtc acgttcaacc tggctggcta
5687caatttcacg ctcggcccct acgactacgt tctcgaggtc cagggcagct
gcatttctac 5747ctttatgggc atggatttcc cggctcctac tgggccactt
gcgatcctgg gcgatgcctt 5807cctccggagg tattactcca tttatgacct
tggcgccgac accgtcggtc tggctgaggc 5867caag 58718397PRTChrysosporium
lucknowense 8Met Lys Asp Ala Phe Leu Leu Thr Ala Ala Val Leu Leu
Gly Ser Ala 1 5 10 15 Gln Gly Ala Val His Lys Met Lys Leu Gln Lys
Ile Pro Leu Ser Glu 20 25 30 Gln Leu Glu Ala Val Pro Ile Asn Thr
Gln Leu Glu His Leu Gly Gln 35 40 45 Lys Tyr Met Gly Leu Arg Pro
Arg Glu Ser Gln Ala Asp Ala Ile Phe 50 55 60 Lys Gly Met Val Ala
Asp Val Lys Gly Asn His Pro Ile Pro Ile Ser 65 70 75 80 Asn Phe Met
Asn Ala Gln Tyr Phe Ser Glu Ile Thr Ile Gly Thr Pro 85 90 95 Pro
Gln Ser Phe Lys Val Val Leu Asp Thr Gly Ser Ser Asn Leu Trp 100 105
110 Val Pro Ser Val Glu Cys Gly Ser Ile Ala Cys Tyr Leu His Ser Lys
115 120 125 Tyr Asp Ser Ser Ala Ser Ser Thr Tyr Lys Lys Asn Gly Thr
Ser Phe 130 135 140 Glu Ile Arg Tyr Gly Ser Gly Ser Leu Ser Gly Phe
Val Ser Gln Asp 145 150 155 160 Thr Val Ser Ile Gly Asp Ile Thr Ile
Gln Gly Gln Asp Phe Ala Glu 165 170 175 Ala Thr Ser Glu Pro Gly Leu
Ala Phe Ala Phe Gly Arg Phe Asp Gly 180 185 190 Ile Leu Gly Leu Gly
Tyr Asp Arg Ile Ser Val Asn Gly Ile Val Pro 195 200 205 Pro Phe Tyr
Lys Met Val Glu Gln Lys Leu Ile Asp Glu Pro Val Phe 210 215 220 Ala
Phe Tyr Leu Ala Asp Thr Asn Gly Gln Ser Glu Val Val Phe Gly 225 230
235 240 Gly Val Asp His Asp Lys Tyr Lys Gly Lys Ile Thr Thr Ile Pro
Leu 245 250 255 Arg Arg Lys Ala Tyr Trp Glu Val Asp Phe Asp Ala Ile
Ser Tyr Gly 260 265 270 Asp Asp Thr Ala Glu Leu Glu Asn Thr Gly Ile
Ile Leu Asp Thr Gly 275 280 285 Thr Ser Leu Ile Ala Leu Pro Ser Gln
Leu Ala Glu Met Leu Asn Ala 290 295 300 Gln Ile Gly Ala Lys Lys Ser
Tyr Thr Gly Gln Tyr Thr Ile Asp Cys 305 310 315 320 Asn Lys Arg Asp
Ser Leu Lys Asp Val Thr Phe Asn Leu Ala Gly Tyr 325 330 335 Asn Phe
Thr Leu Gly Pro Tyr Asp Tyr Val Leu Glu Val Gln Gly Ser 340 345 350
Cys Ile Ser Thr Phe Met Gly Met Asp Phe Pro Ala Pro Thr Gly Pro 355
360 365 Leu Ala Ile Leu Gly Asp Ala Phe Leu Arg Arg Tyr Tyr Ser Ile
Tyr 370 375 380 Asp Leu Gly Ala Asp Thr Val Gly Leu Ala Glu Ala Lys
385 390 395 91191DNAChrysosporium lucknowense 9atgaaggatg
cttttttgct gaccgcagct gtgctgctcg gctccgccca gggagcagtt 60cacaaaatga
agctgcagaa gatccctctc tctgagcagc ttgaggcggt tcccatcaac
120acccagctcg agcatctcgg ccaaaaatac atggggttgc gcccacgtga
atctcaagcc 180gatgccatct ttaagggcat ggttgccgac gtcaagggca
accatcctat tcccatctcc 240aacttcatga acgcacagta cttctccgag
atcacgattg gaacaccccc tcagtcattc 300aaggtggtcc tcgataccgg
tagctccaac ctgtgggttc catcagtcga gtgcggctcg 360attgcttgtt
acctgcactc gaagtatgac tcatctgcct cgtccaccta caagaagaac
420ggaacctcgt tcgagatccg ctacgggtca ggcagcctga gcgggtttgt
ctctcaggac 480acagtgtcca tcggcgatat cactatccag ggccaggact
ttgccgaggc gaccagcgag 540cccggtcttg cctttgcctt tggccgtttc
gacggtatcc ttggccttgg ctacgaccgg 600atctcagtca acggcatcgt
cccgcctttt tacaagatgg tcgagcagaa gctcatcgat 660gagcccgtct
tcgccttcta cctggccgat accaatggcc agtctgaggt tgtctttggc
720ggtgttgacc acgacaagta caagggcaag atcaccacca ttccgttgag
gcgcaaggcc 780tactgggagg ttgacttcga tgccatttct tacggcgacg
acactgccga gcttgagaac 840actggcatca tcctggacac cggtacttct
ctgatcgctc tgcccagcca gctcgccgag 900atgctcaacg ctcagatcgg
cgctaagaag agctacactg gccagtacac catcgactgc 960aacaagcgcg
actccctcaa ggatgtcacg ttcaacctgg ctggctacaa tttcacgctc
1020ggcccctacg actacgttct cgaggtccag ggcagctgca tttctacctt
tatgggcatg 1080gatttcccgg ctcctactgg gccacttgcg atcctgggcg
atgccttcct ccggaggtat 1140tactccattt atgaccttgg cgccgacacc
gtcggtctgg ctgaggccaa g 11911023188DNAArtifical
Sequencemisc_feature(969)..(969)n is a, c, g, or t 10gcggccgcca
ccgcgggaac aatcttctct catgctccgg atatgggcaa ggccaagaac 60gcggctgtgg
agttcaagaa actcttccgc aacaacaacc ccactacatc tgcaatcaat
120agctatcggt acggaccccc cgtccacgtt gcctccatgc aaggggaggt
tgaatttcgt 180gacgtgtcgt tccggtatcc cacacgtctg gaacagcctg
ttctacgcca cttgaacctc 240accgtcaagc caggactaat atgtcgctct
ggtggggtcg agtggtagtg gcaagagtac 300gattgttgcc ctgttggaac
ggttttatga agcgcaggtg ggcgagattt atattgacgg 360acgcaacatc
aaagcgttgg acaagaagtc gtatcgcagt catttggcgc tggtcagtca
420ggaaccatcg ctgttccatg gcactatccg ggagaacatt ctcctgggtt
gtacggataa 480ggaacatgtg tcggaggata tggtggtcag agcgtgcaga
gatgcgaata tttatgattt 540catcatgtcg ttgccgtgag tcctccctgt
ccctttcctc tttgtggtat atatgcggtt 600actgatagag aaacagacaa
ggctttgaca ccctcgttgg taacaagggt ggcatgttgt 660caggtggaca
gaaacagcgt atcgcgattg cgcgtgcgtt gattcggaac ccgcgcattt
720tgttgttgga cgaggcgact tccgcgctgg attctgagtc ggagaaggtg
gtgcaggctg 780cgctggacgc ggctgccaag gggaggacca ctattgcggt
ggcgcatcgg ctaagtacga 840ttcaacgggc ggatatgatc tatttcttag
agcagggaga ggtgattgag tgtgggacac 900ataaggagct gttgaggagg
aggggacggt attatgagat ggtgaatttg cagactttga 960ggtgatgana
ccattgactt ggtgggtggt ncatgggtta atgtgaaggc gttagtggta
1020atgtatatta atggtgagat gggctttgat tgggtttaat tggaatctgt
atattttcag 1080atggagtcaa cttttgaatg gccaatatat cctcggcgat
accgtcggag ataagataag 1140aataatcgca cactattccc aaagcatact
ggtacatact gcattcggct agtgcggggt 1200gcttacctca tccacccgaa
tgagcccaac ttttttgtct caatcaataa ttgcatccaa 1260attcccccgc
aacttccccc tccaaccccg tgtctatacc actccctcca cacccacaca
1320atcacaatgg ctctccctgc ctacaagacc gccttcctgg agtctctcgt
cggccaacgt 1380gctgactttc ggcaccttca ccctgaagtc gggtcgccgt
gcgtcacccc tccaacaccg 1440gcattatcgc aatcggaaga cttaccactg
tatacagact ccccctactt cttcaacgcc 1500ggcatcttca acaccgcctc
tctcctctcc gccctctcca ccatggccca caccatcatc 1560accttcctcg
ctgagaaccc ttccatcccc aagcccgacg tcatgcttcg ggtaaaaaac
1620cccctctttc cccaataccc cacttccact caacaaccca taaataacta
acaaaaaccc 1680cctaaacagc cccgcataca aaggcatccc cctcgcgtgc
gccaccctcc ttgaactcaa 1740ccgcatcgac cccgccacct ggggcagcgt
gtcctacagc tacaaccgca aagaagccaa 1800ggatcacggc gaaggcggca
acattgtcgg cgccgctctg aagggcaaga ccgtgcttgt 1860gatcgacgat
gtcatcacgg ccggtaccgc catgcgtgag accctcaacc tggtcgccaa
1920ggagggcggc aaggtcgtcg gattcactgt tgctctggac cgcttggaga
agatgcccgg 1980acccaaggac gagaacggtg tcgaggacga taagcccaga
atgagtgcta tgggtcagat 2040ccgtaaggag tatggtgtgc ccacgacgag
tattgttact ctggatgatt tgatcaagtt 2100gatgcaggcg aagggcaatg
aggccgatat gaagcggttg gaggagtata gggctaagta 2160tcaggctagt
gattagtcgg tttcattgac cgattgtttg ggtgggtgtg agaggttagg
2220ttaggttgtg ggcgtaggaa tgaaaagctg tatacatagg ggcctgaaga
ggtgcgtaga 2280gacggtcgtg agatgtttta tgtcaaaatc ttgaacaaat
gacaccttaa aaaagacccc 2340ttggtttcag ctgaattagc ccggaaagat
gctcggcacg ccatgagtct agcccactca 2400gtgggcaccc gtttcccaca
tttgaagtgg ccgacgctta tttggctgag gctgtggcct 2460ggaaaggcac
tatggcgtgc tgcggtacaa ggccggggct ggcgtacgaa ccacgacgcc
2520cgaagggaac tcttcggtct tactactact atgtccccag ttgacccccc
gaggagagtg 2580cgtgattgat tggttgtaga tgccgagctc tgggcatctt
atgaatccat tggttggcgg 2640ctcattaccg ctgtatatcg tctggatcgc
gcggttactn tgaaaatgga tcgttataag 2700cagtagtaac taacaatctc
gcacggatca tgtccttgcc gctaaacccc ccaagtacat 2760cggaaatcgg
ccccagctca ggcacatgct atgatgcttt tttattttga aagtcagcta
2820gtctggatgg catttctttt gctccccaag cgtggatttt cctggacaaa
tagtccgcta 2880atcaaaattc actactcagt ctcacacaag acatcttaga
ccattatatt tgggatgcgg 2940cacagttgca tgcagctaga ctggataatt
tataagcaac cattcgttat gatacatgga 3000gtgacttcaa ataataaatt
cgccctgctg ctatgttctc gttcctagcc actactccat 3060tctactgaat
tgtactattt tctttttaac gcatactcca tgttcgtttc taaaactaga
3120acagaatcta aaacactcaa taaatgtcca ccgctggcac actcccaccc
cctcccgacg 3180gtggatttca cgcctgggcc caagtagtct ccggccactt
agtggttgcg gtaacatggg 3240gctatgcagc cagcttcggc gtcttccaag
accattatga agtcactcta ccgcaaccct 3300cgtccgatat ttcgtggatc
ggtggcttcc aggtcttctg tctcctattc atgtcccctc 3360tctccgggcg
cgcgacggat gccggacaag ctcggcttgt cgtcggcatt ggcgcatttt
3420tgttgttact tggaaccttc atgaccagcc tagcaagggt ctattggcaa
ctatttctct 3480cccaggggct ctgcattggt atcggccagg gacttatgtg
gttgcctagt gtgactctcg 3540tttccacgta ctttgtccga cgccgtgtgt
tcgccgtcac ggctgctgcc accggaacca 3600gcacaggggg gattatcttt
ccagctatga tctcaataca ctcgactcgg ctttcacctt 3660cgtgaaagaa
tcgccgtctg cagagggtaa gaggcggagt agctttatcg cttctgttct
3720ttatgaaaga cataggtgga cgattcatga ccttctgctg atgcagcaga
tactttcctc 3780ccttcacgaa gctcttctac gggagttgta tgatgtgatg
atggattgct acgaaggaaa 3840acctcggccc atcggtccgt gatgtacgta
ctggagaatc atatcgaaat ggatctttcg 3900acactgaaat acgtcgagcc
tgctccgctt ggaagcggcg aggagcctcg tcctgtcaca 3960actaccaaca
tggagtacga taagggccag ttccgccagc tcattaagag ccagttcatg
4020ggcgttggca tgatggccgt catgcatctg tacttcaagt acaccaacgc
tcttctgatc 4080cagtcgatca tccgctgaag gcgctttcga atctggttaa
gatccacgtc ttcgggaagc 4140cagcgactgg tgacctccag cgtcccttta
aggcttgcca acagctttct cagccagggc 4200cagcccaaga ccgacaagga
gcttatcgat ttcgaacccc cgcgccgcct ttgcgagggt 4260ggagttgcct
tagggttagg gttagggtta gggttagggt tagggttagg gttagggtta
4320gggttagggt tagggttagg gttagggtta gggttagggt tagggttagg
gttagggtca 4380gggtcagggt agggtcaggg gtagggtcag gggtaggggt
agggtcaggg ttagggttag 4440ggttagggtt agggttaggg ttagggttag
ggtcagggtt agggttaggg ttaggggtag 4500gggtaggggt agggttaggg
ttagggttag ggttagggtt agggttaggg ttagggtcag 4560ggtcagggtc
aggggtaggg tagggtaggg ttaggggtta gggttagggt tagggttagg
4620gttagggtta gggttaaggg ttaagggtta agggnnnnnn nnnnnnnnnn
nnnnnnnnnn 4680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4800nnnnnnntgg gaattcgcgg
cctaactata acggtcctaa ggtagcgagg ccgcgaattc 4860ccannnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
4920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 4980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnccct
taacccttaa cccttaaccc 5040taaccctaac cctaacccta accctaaccc
taacccctaa ccctacccta ccctacccct 5100gaccctgacc ctgaccctaa
ccctaaccct aaccctaacc ctaaccctaa ccctaaccct 5160acccctaccc
ctacccctaa ccctaaccct aaccctgacc ctaaccctaa ccctaaccct
5220aaccctaacc ctaaccctaa ccctgaccct acccctaccc ctgaccctac
ccctgaccct 5280accctgaccc tgaccctaac cctaacccta accctaaccc
taaccctaac cctaacccta 5340accctaaccc taaccctaac cctaacccta
accctaaccc taaccctaac cctaacccta 5400aggcaactcc accctcgcaa
aggcggcgcg ggggttcgaa atcgataagc tcctccctcc 5460agaacgccga
gaagaactgg aggggtggtg tcaaggagga gtaagctcct tattgaagtc
5520ggaggacgga gcggtgtcaa gaggatattc ttcgactctg tattatagat
aagatgatga 5580ggaattggag gtagcatagc ttcatttgga tttgctttcc
aggctgagac tctagcttgg 5640agcatagagg gtcctttggc tttcaatatt
ctcaagtatc tcgagatctt attcacatat 5700cccttagata
cgatcaaaac ccgcattcag tctcgcgaat attcgcagtt cctgagaacc
5760aacacaggaa ccagcatatg gagacaccca gggatattcc gcggccttta
ccaaggtatc 5820gcgagcgtaa ccgctgcctc atttcccaca ggttggtgat
acccaacatt atccttaccc 5880aactcatatt cattgagcaa aaaataacgg
gtcaaagcgg gcgcattctt catcacatac 5940gagtatgcac agtcaggact
ccaagtcata catcaaaaac ttggaacgca tgagtctagc 6000tcagctcggc
tcttttccga tttttgcgca gcctccgttg cggatcttgc tgcttgcgga
6060gtctttgctc cggctgatgc attaaaacac aacgcgcaga tgatccaatc
acaccatcca 6120gacgcatcag cacctgtagc tgggggaagg gtaggtggtg
tagcccagaa agcgacacgg 6180ctagctttca agaagtttat caaccctaga
cagctttgga gcggataccc ggctcttgtg 6240gcgcatagct tgccagtgtc
ggcgattcag atgcctctgt acgagtcttt tcggtatcga 6300atttttgaat
atagattcgg agatcgagag aaggtgctag aaagaccaag agagtatgga
6360aagaaagagg cccattcgac aatcggagag gctgcagcga cagccgcaat
aagtgctgcg 6420gtctcgggcg gtatagctag tgtcttgaca gcacccatgg
atatggtccg gacacgaatc 6480atgctcgatg ctgcagatac aactgcacct
cagaagaaaa ggatgatcaa taccgtacgg 6540gagattgtac gaacagatgg
cccgagagga ctattccgag ggtgtgctat caacacgttt 6600atggccgctg
tcggatcagg attatacttt ggtctctacg agagcaccaa atggtggcta
6660ggctctgact cgatggataa ttgtgccgtg ttagagtagg gggtggtggg
aaatcttgta 6720tataattgtg attgtttgta cgatagtgac cgactgtaca
ttagtgatac cccactctaa 6780gaaaatagac caatctccag ctgcaccttc
agacactccg gtacaaattc tcgtctatgt 6840tggagattgt tgtgactttg
aaacatgacc cttgaccctg attttgaatt tgtccatata 6900tcgaggcagg
tgtcttattc gtacggagag ggtatctgtc gtagacacat agtagtagtc
6960atttcgagtg ctgaatttat aaatcgcatc atacttgcga catactgcca
taaaaggagt 7020acgtatccac cactacttat tgcgcaccaa cacgcttcag
gtatgcatcc catccctcct 7080tctggtactg cttcgccgcc tccacgggat
caggagcagc ataaattcca cggccagcaa 7140taataaagtc ggcaccgcgt
ccaacagccg actcaggagt ttggtactgc tgtcccagct 7200tgtcaccctt
cgaggagagg ttgacacctg tcgtgaagac gacaaaatct tcctcctccg
7260aaggcgagct aacttcagac tgaacctcgc caaggtgacg tgtcgagacg
aatcccatca 7320caaacttctt atacttccga gcatagtcaa cagaagaagt
agtatattga ccggtagcca 7380aagatccctt ggaggtcatc tccgcaagga
tcaaaaggcc cctctcggag ccgtagggga 7440agtcctcggc cgaagcagtc
tgggccagag cctcgacgat accctcaccg ggcagaatac 7500tgcagttgat
gatgtgggcc cactcagaga tacgcagagt gccgccatgg tactgctttt
7560ggactgtgtt tccgatatcg atgaacttgc gatcttcgaa gatgaggaaa
ttgtgcttct 7620ctgcaagggc cttcagaccg gtgatggttt cttcgctgaa
atcggagagg atatcgatgt 7680gagttttgat cacggcaatg tacggaccga
gtcctgttat ataatccacc attaaccatt 7740actagatcac atgtaagtgg
catccccggt gcgcatacgg tcagccaaat ccagcagctc 7800tttggtggtt
gtcacgtcgg cggaaacggt gacattggtt ttcttggcct cggcaacctc
7860gaagagcttc tttacgagcg cattggggtg cttgctagcg cgtgcgctgt
aggtcaattg 7920cgacttggaa gacatgttgg cgatggaggg gtagcgcggg
gttctgcaaa tattgtataa 7980atgagcactt agtggttgaa actggcttat
tagtaggtta gtacttcgag ttttcagtaa 8040ttagacaaaa taatcaggat
gtccaactac taactcttga tatatggaat gaaatgtaga 8100tacaaactgc
acgacaattg ccgcgaaaaa ttaaattgaa tctatggagg ggactgtcat
8160gcactagcca cacgtttctc cgcctgtggg gtgagccaca tgcctcattt
tgaccaaaca 8220catcgatgca gtcacatgta gataagatta gggcctatcc
ttagggtacc gtccacgcgg 8280ggattatgcc tggctttttg cctgcttttg
atatcctttc agggacatag caataagccc 8340aaccctatcg gccataataa
tgtcaattcc agcagcggct tgggtctaga atatttaatc 8400agctaccgag
caacttctac atcacagttt gaaagcacta aggatcaata tagaagcact
8460taccttcgca ttttctggta tattgttctg agatccatag gatctccatg
gacgcgtgac 8520gtcgagtacc atttaattct atttgtgttt gatcgagacc
taatacagcc cctacaacga 8580ccatcaaagt cgtatagcta ccagtgagga
agtggactca aatcgacttc agcaacatct 8640cctggataaa ctttaagcct
aaactataca gaataagata ggtggagagc ttataccgag 8700ctcgcggccg
caggtaccag gcaattcgcc ctatagtgag tcgtattacg cgcgctcact
8760ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
ttaatcgcct 8820tgcagcacat ccccctttcg ccagctggcg taatagcgaa
gaggcccgca ccgatcgccc 8880ttcccaacag ttgcgcagcc tgaatggcga
atgggacgcg ccctgtagcg gcgcattaag 8940cgcggcgggt gtggtggtta
cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 9000cgctcctttc
gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
9060tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
tcgaccccaa 9120aaaacttgat tagggtgatg gttcacgtag tgggccatcg
ccctgataga cggtttttcg 9180ccctttgacg ttggagtcca cgttctttaa
tagtggactc ttgttccaaa ctggaacaac 9240actcaaccct atctcggtct
attcttttga tttataaggg attttgccga tttcggccta 9300ttggttaaaa
aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
9360gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
ttgtttattt 9420ttctaaatac attcaaatat gtatccgctc atgagacaat
aaccctgata aatgcttcaa 9480taatattgaa aaaggaagag tatgagtatt
caacatttcc gtgtcgccct tattcccttt 9540tttgcggcat tttgccttcc
tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 9600gctgaagatc
agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
9660atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
taaagttctg 9720ctatgtggcg cggtattatc ccgtattgac gccgggcaag
agcaactcgg tcgccgcata 9780cactattctc agaatgactt ggttgagtac
tcaccagtca cagaaaagca tcttacggat 9840ggcatgacag taagagaatt
atgcagtgct gccataacca tgagtgataa cactgcggcc 9900aacttacttc
tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
9960ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
cataccaaac 10020gacgagcgtg acaccacgat gcctgtagca atggcaacaa
cgttgcgcaa actattaact 10080ggcgaactac ttactctagc ttcccggcaa
caattaatag actggatgga ggcggataaa 10140gttgcaggac cacttctgcg
ctcggccctt ccggctggct ggtttattgc tgataaatct 10200ggagccggtg
agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
10260tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
acgaaataga 10320cagatcgctg agataggtgc ctcactgatt aagcattggt
aactgtcaga ccaagtttac 10380tcatatatac tttagattga tttaaaactt
catttttaat ttaaaaggat ctaggtgaag 10440atcctttttg ataatctcat
gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 10500tcagaccccg
tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
10560tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
ggatcaagag 10620ctaccaactc tttttccgaa ggtaactggc ttcagcagag
cgcagatacc aaatactgtc 10680cttctagtgt agccgtagtt aggccaccac
ttcaagaact ctgtagcacc gcctacatac 10740ctcgctctgc taatcctgtt
accagtggct gctgccagtg gcgataagtc gtgtcttacc 10800gggttggact
caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
10860tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
cctacagcgt 10920gagctatgag aaagcgccac gcttcccgaa gggagaaagg
cggacaggta tccggtaagc 10980ggcagggtcg gaacaggaga gcgcacgagg
gagcttccag ggggaaacgc ctggtatctt 11040tatagtcctg tcgggtttcg
ccacctctga cttgagcgtc gatttttgtg atgctcgtca 11100ggggggcgga
gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
11160tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
ggataaccgt 11220attaccgcct ttgagtgagc tgataccgct cgccgcagcc
gaacgaccga gcgcagcgag 11280tcagtgagcg aggaagcgga agagcgccca
atacgcaaac cgcctctccc cgcgcgttgg 11340ccgattcatt aatgcagctg
gcacgacagg tttcccgact ggaaagcggg cagtgagcgc 11400aacgcaatta
atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
11460ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
aaacagctat 11520gaccatgatt acgccaagcg cgcaattaac cctcactaaa
gggaacaaaa gctggagctc 11580caccgcggtg gcggccggcc gcggatcnnn
nnnnnnnnnn nnnnnaaggt atccgatttg 11640gggaacgtcg atgaaagtat
tgcaaaagtg acgagagttg cgcaactaac tcgctgccga 11700agaagctgcg
gaagaaagag aacaccgaaa gtggaataac gttacggatg tcctgacctc
11760aaagttgaaa ccagcccttc ctgctctatt tgggaaagcg gcttgccctt
gaatgcgctg 11820cactgtggca cgactaccag tgatcgggag gagcaaacta
ccctggtccg ttccttggtg 11880gggcggcact aggcccaact tagggtgatc
ggaggtcgat gccgcggtcc tcgttggtct 11940gggctcttct catttcccgg
tttgcacccc ccgttgcacc tgctgatcgc ccgccaacgc 12000cgatgaggtt
gcgcccagac cgacaatcac cgcggctgca ttcccaagta tattgaagat
12060ggcaccaggt acccggtttt gcgtcccagt cgtttggtgc caaatttggg
agtttttgag 12120cctcaagatc tggggaaatc gacctcaact tccatacaag
ttaaagtcgc acacacggcg 12180agttccacga agagacacat ttttttctga
aggcctctct ccccgcacat cagaaaccac 12240caaataccaa gactgcagaa
gccggggtaa gtgggccacc gggactacac taaaatgcgg 12300ggagaagcga
gatccgttgc gaagggaagg gatggggtgt gctgcggctt tctccgctct
12360cgtgcgcctt ttgcttgaat ctagtgtaca ccagggtagg ctccgaagga
gtatctacgg 12420cagcgctgtt cgtgctgcgt tgagagtcag ggcggagacg
agcaggcgac aggagcctcg 12480caccggcact tcggatcgca tttgcgcgga
gcgtcaaata cgctcttctg cggtcatcag 12540agagcatcgt gaaccaaggt
tcttccgcag ggcggcctgg gcttcgcaga gtcgcactcg 12600gcggacgcct
tccgtgtcac ccctgataac ctggctgccg cgcccagact cctccaatga
12660ggtgtgtggt tgccctcgcc gacccttcag caaccttaat cgcttccatc
gcacggctcc 12720acgtcctcga acgatgccct cagtccgtgc ccggccgtgg
caaccataac gtgacatcgc 12780cgcccagcct actagccgct atcgaccggt
taggcttgtc accgcagcgc ccattctcca 12840tcgggcctct actctgatcc
acctcaccca ccgcaagcac tagcgagcct caccagagtg 12900caagcgacac
gacccgcttg gcccttcgtc cttgactatc tcccagacct cttgccatct
12960tgccgacgcc gccccctttt ttttctcctc cccctgccgg caggtcggtg
gccccagtcc 13020cgagatggca ttgctccgtt gtccatgacg acccatcatt
cgatggctga ctggcacact 13080cgtcttgttt gagcatcgac ggcccgcggc
ccgtctccca cggtacggaa cctcgttgta 13140cagtacctct cgtaatgata
cccaacaccg gggccgagcg ctgggagggc ggcgttcccg 13200agaagccggg
aaggcggctg gccggctgac ctttgtgact tggcgatgga tgcggccatg
13260gagaatgtcc gtccgaagcg acgcgacaat tagcctggct accatcgata
taaattgggt 13320gattcccagc tcttgatggg cgtgtcttct gcctggcagc
cctcgtcttc agatcaagca 13380actgtgtgct gatcctcttc cgtcatgggc
ttccgatctc tactcgccct gagcggcctc 13440gtctgcacag ggttggcaaa
tgtgatttcc aagcgcgcga ccttggattc atggttgagc 13500aacgaagcga
ccgtggctcg tactgccatc ctgaataaca tcggggcgga cggtgcttgg
13560gtgtcgggcg cggactctgg cattgtcgtt gctagtccca gcacggataa
cccggactgt 13620atgtttcgag ctcagattta gtatgagtgt gtcattgatt
gattgatgct gactggcgtg 13680tcgtttgttg tagacttcta cacctggact
cgcgactctg gtctcgtcct caagaccctc 13740gtcgatctct tccgaaatgg
agataccagt ctcctctcca ccattgagaa ctacatctcc 13800gcccaggcaa
ttgtccaggg tatcagtaac ccctctggtg atctgtccag cggcgctggt
13860ctcggtgaac ccaagttcaa tgtcgatgag actgcctaca ctggttcttg
gggacggccg 13920cagcgagatg gtccggctct gagagcaact gctatgatcg
gcttcgggca gtggctgctt 13980gtatgttctc cacccccttg cgtctgatct
gtgacatatg tagctgactg gtcaggacaa 14040tggctacacc agcaccgcaa
cggacattgt ttggcccctc gttaggaacg acctgtcgta 14100tgtggctcaa
tactggaacc agacaggata tggtgtgttt gttttatttt aaatttccaa
14160agatgcgcca gcagagctaa cccgcgatcg cagatctctg ggaagaagtc
aatggctcgt 14220ctttctttac gattgctgtg caacaccgcg cccttgtcga
aggtagtgcc ttcgcgacgg 14280ccgtcggctc gtcctgctcc tggtgtgatt
ctcaggcacc cgaaattctc tgctacctgc 14340agtccttctg gaccggcagc
ttcattctgg ccaacttcga tagcagccgt tccggcaagg 14400acgcaaacac
cctcctggga agcatccaca cctttgatcc tgaggccgca tgcgacgact
14460ccaccttcca gccctgctcc ccgcgcgcgc tcgccaacca caaggaggtt
gtagactctt 14520tccgctcaat ctataccctc aacgatggtc tcagtgacag
cgaggctgtt gcggtgggtc 14580ggtaccctga ggacacgtac tacaacggca
acccgtggtt cctgtgcacc ttggctgccg 14640cagagcagtt gtacgatgct
ctataccagt gggacaagca ggggtcgttg gaggtcacag 14700atgtgtcgct
ggacttcttc aaggcactgt acagcgatgc tgctactggc acctactctt
14760cgtccagttc gacttatagt agcattgtag atgccgtgaa gactttcgcc
gatggcttcg 14820tctctattgt ggtaagtcta cgctagacaa gcgctcatgt
tgacagaggg tgcgtactaa 14880cagaagtagg aaactcacgc cgcaagcaac
ggctccatgt ccgagcaata cgacaagtct 14940gatggcgagc agctttccgc
tcgcgacctg acctggtctt atgctgctct gctgaccgcc 15000aacaaccgtc
gtaactccgt cgtgcctgct tcttggggcg agacctctgc cagcagcgtg
15060cccggcacct gtgcggccac atctgccatt ggtacctaca gcagtgtgac
tgtcacctcg 15120tggccgagta tcgtggctac tggcggcacc actacgacgg
ctacccccac tggatctggc 15180agcgtgacct cgaccagcaa gaccaccgcg
actgctagca agaccagcac cagtacgtca 15240tcaacctcct gtaccactcc
cacggcgaat gtgatatcca agcgcgcgcc agtaccccca 15300ggagaagatt
ccaaagatgt agccgcccca cacagacagc cactcacctc ttcagaacga
15360tccaagcgcg aggtccagct cgtcgagagc ggcggtggcc tcgtccagcc
cggtcgcagc 15420ctccgcctca gctgcgccgc cagcggcttc accttcgacg
actacgccat gcactgggtc 15480cgtcaagccc ctggcaaggg cctggagtgg
gtctccgcca tcacctggaa cagcggccac 15540atcgactacg ccgacagcgt
cgagggccgc ttcaccatca gccgcgacaa cgccaagaac 15600agcctctacc
tccagatgaa cagcctccgc gccgaggaca ccgccgtcta ctactgcgcc
15660aaggtctcct acctcagcac cgccagcagc ctcgactact ggggccaggg
caccctcgtc 15720accgtgtcca gcgccagcac caagggtccc agcgtcttcc
ccctcgcccc cagcagcaag 15780agcaccagcg gcggcactgc cgccctcggc
tgcctcgtca aggactactt ccccgagccc 15840gtcacggtct cctggaattc
cggtgccctc acctccggcg tccacacctt ccccgccgtc 15900ctccagagca
gcggcctcta cagcctcagc agcgtcgtca ccgtccccag cagctccctc
15960ggcacccaga cctacatctg caacgtcaac cacaagccca gcaacaccaa
ggtcgacaaa 16020accgtcgagc ccaagagctg cgacaagacc cacacctgcc
ccccctgccc tgcccccgag 16080ctgctcggcg gtccctccgt cttcctcttc
ccccccaagc ccaaggacac cctcatgatc 16140agccgcaccc ccgaggtcac
ctgcgtcgtc gtcgacgtgt cccacgagga cccggaggtc 16200aagttcaact
ggtacgtcga cggtgtcgag gtccacaacg ctaagaccaa gccccgcgag
16260gagcagtaca acagcaccta ccgcgtcgtg tccgtcctca ccgtcctcca
ccaggactgg 16320ctcaacggca aggaatacaa gtgcaaggtc tccaacaagg
ccctccccgc tcccatcgag 16380aagaccatca gcaaggccaa gggccagccc
cgggagcccc aggtctacac cctccccccc 16440tcgcgggagg agatgaccaa
gaaccaggtc tccctcacct gcctggtcaa gggcttctac 16500cccagcgaca
tcgccgtcga gtgggagagc aacggccagc ccgagaacaa ctacaagacc
16560actccccccg tcctcgacag cgacggcagc ttcttcctct acagcaagct
caccgtcgac 16620aagagccgct ggcagcaggg caacgtcttc agctgcagcg
tcatgcacga ggccctccac 16680aaccactaca cccagaagag cctcagcctc
tcccccggca agtaagggcc cagatcctaa 16740gtaagtaaac gaacctctct
gaaggaggtt ctgagacacg cgcgattctt ctgtatatag 16800ttttattttt
cactctggag tgcttcgctc caccagtaca taaacctttt ttttcacgta
16860acaaaatggc ttcttttcag accatgtgaa ccatcttgat gccttgacct
cttcagttct 16920cactttaacg tanttcgcgt tagtctgtat gtcccagttg
catgtagttg agataaatac 16980ccctggaagt gggtctgggc ctttgtggga
cggagccctc tttctgtggt ctggagagcc 17040cgctctctac cgcctacctt
cttaccacag tacactactc acacattgct gaactgaccc 17100atcataccgt
actttatcct gttaattcgt ggtgctgtcg actattctat ttgctcaaat
17160ggagagcaca ttcatcggcg cagggataca cggtttatgg accccaagag
tgtaaggact 17220attattagta atattatatg cctctaggcg ccttaacttc
aacaggcgag cactactaat 17280caacttttgg tagacccaat tacaaacgac
catacgtgcc ggaaattttg ggattccgtc 17340cgctctcccc aaccaagcta
gaagaggcaa cgaacagcca atcccggtgc taattaaatt 17400atatggttca
ttttttttaa aaaaattttt tcttcccatt ttcctctcgc ttttcttttt
17460cgcatcgtag ttgatcaaag tccaagtcaa gcgagctatt tgtgctatag
ctcggtggct 17520ataatcagta cagcttagag aggctgtaaa ggtatgatac
cacagcagta ttcgcgctat 17580aagcggcact cctagactaa ttgttacggt
ctacagaagt aggtaataaa agcgttaatt 17640gttctaaata ctagaggcac
ttagagaagc tatctaaata tatattgacc ctagcttatt 17700atccctatta
gtaagttagt tagctctaac ctatagatag ccaaatgcgg ccggccgcgg
17760atcnnnnnnn nnnnnnnnnn naaggtatcc gatttgggga acgtcgatga
aagtattgca 17820aaagtgacga gagttgcgca actaactcgc tgccgaagaa
gctgcggaag aaagagaaca 17880ccgaaagtgg aataacgtta cggatgtcct
gacctcaaag ttgaaaccag cccttcctgc 17940tctatttggg aaagcggctt
gcccttgaat gcgctgcact gtggcacgac taccagtgat 18000cgggaggagc
aaactaccct ggtccgttcc ttggtggggc ggcactaggc ccaacttagg
18060gtgatcggag gtcgatgccg cggtcctcgt tggtctgggc tcttctcatt
tcccggtttg 18120caccccccgt tgcacctgct gatcgcccgc caacgccgat
gaggttgcgc ccagaccgac 18180aatcaccgcg gctgcattcc caagtatatt
gaagatggca ccaggtaccc ggttttgcgt 18240cccagtcgtt tggtgccaaa
tttgggagtt tttgagcctc aagatctggg gaaatcgacc 18300tcaacttcca
tacaagttaa agtcgcacac acggcgagtt ccacgaagag acacattttt
18360ttctgaaggc ctctctcccc gcacatcaga aaccaccaaa taccaagact
gcagaagccg 18420gggtaagtgg gccaccggga ctacactaaa atgcggggag
aagcgagatc cgttgcgaag 18480ggaagggatg gggtgtgctg cggctttctc
cgctctcgtg cgccttttgc ttgaatctag 18540tgtacaccag ggtaggctcc
gaaggagtat ctacggcagc gctgttcgtg ctgcgttgag 18600agtcagggcg
gagacgagca ggcgacagga gcctcgcacc ggcacttcgg atcgcatttg
18660cgcggagcgt caaatacgct cttctgcggt catcagagag catcgtgaac
caaggttctt 18720ccgcagggcg gcctgggctt cgcagagtcg cactcggcgg
acgccttccg tgtcacccct 18780gataacctgg ctgccgcgcc cagactcctc
caatgaggtg tgtggttgcc ctcgccgacc 18840cttcagcaac cttaatcgct
tccatcgcac ggctccacgt cctcgaacga tgccctcagt 18900ccgtgcccgg
ccgtggcaac cataacgtga catcgccgcc cagcctacta gccgctatcg
18960accggttagg cttgtcaccg cagcgcccat tctccatcgg gcctctactc
tgatccacct 19020cacccaccgc aagcactagc gagcctcacc agagtgcaag
cgacacgacc cgcttggccc 19080ttcgtccttg actatctccc agacctcttg
ccatcttgcc gacgccgccc cctttttttt 19140ctcctccccc tgccggcagg
tcggtggccc cagtcccgag atggcattgc tccgttgtcc 19200atgacgaccc
atcattcgat ggctgactgg cacactcgtc ttgtttgagc atcgacggcc
19260cgcggcccgt ctcccacggt acggaacctc gttgtacagt acctctcgta
atgataccca 19320acaccggggc cgagcgctgg gagggcggcg ttcccgagaa
gccgggaagg cggctggccg 19380gctgaccttt gtgacttggc gatggatgcg
gccatggaga atgtccgtcc gaagcgacgc 19440gacaattagc ctggctacca
tcgatataaa ttgggtgatt cccagctctt gatgggcgtg 19500tcttctgcct
ggcagccctc gtcttcagat caagcaactg tgtgctgatc ctcttccgtc
19560atgggcttcc gatctctact cgccctgagc ggcctcgtct gcacagggtt
ggcaaatgtg 19620atttccaagc gcgcgacctt ggattcatgg ttgagcaacg
aagcgaccgt ggctcgtact 19680gccatcctga ataacatcgg ggcggacggt
gcttgggtgt cgggcgcgga ctctggcatt 19740gtcgttgcta gtcccagcac
ggataacccg gactgtatgt ttcgagctca gatttagtat 19800gagtgtgtca
ttgattgatt gatgctgact ggcgtgtcgt ttgttgtaga cttctacacc
19860tggactcgcg actctggtct cgtcctcaag accctcgtcg atctcttccg
aaatggagat 19920accagtctcc tctccaccat tgagaactac atctccgccc
aggcaattgt ccagggtatc 19980agtaacccct ctggtgatct gtccagcggc
gctggtctcg gtgaacccaa gttcaatgtc 20040gatgagactg cctacactgg
ttcttgggga cggccgcagc gagatggtcc ggctctgaga 20100gcaactgcta
tgatcggctt cgggcagtgg ctgcttgtat gttctccacc cccttgcgtc
20160tgatctgtga catatgtagc tgactggtca ggacaatggc tacaccagca
ccgcaacgga 20220cattgtttgg cccctcgtta ggaacgacct gtcgtatgtg
gctcaatact ggaaccagac 20280aggatatggt gtgtttgttt tattttaaat
ttccaaagat gcgccagcag agctaacccg 20340cgatcgcaga tctctgggaa
gaagtcaatg gctcgtcttt ctttacgatt gctgtgcaac 20400accgcgccct
tgtcgaaggt agtgccttcg cgacggccgt cggctcgtcc tgctcctggt
20460gtgattctca ggcacccgaa attctctgct acctgcagtc cttctggacc
ggcagcttca 20520ttctggccaa cttcgatagc agccgttccg gcaaggacgc
aaacaccctc ctgggaagca 20580tccacacctt tgatcctgag gccgcatgcg
acgactccac cttccagccc tgctccccgc 20640gcgcgctcgc caaccacaag
gaggttgtag actctttccg ctcaatctat accctcaacg 20700atggtctcag
tgacagcgag gctgttgcgg tgggtcggta ccctgaggac acgtactaca
20760acggcaaccc
gtggttcctg tgcaccttgg ctgccgcaga gcagttgtac gatgctctat
20820accagtggga caagcagggg tcgttggagg tcacagatgt gtcgctggac
ttcttcaagg 20880cactgtacag cgatgctgct actggcacct actcttcgtc
cagttcgact tatagtagca 20940ttgtagatgc cgtgaagact ttcgccgatg
gcttcgtctc tattgtggta agtctacgct 21000agacaagcgc tcatgttgac
agagggtgcg tactaacaga agtaggaaac tcacgccgca 21060agcaacggct
ccatgtccga gcaatacgac aagtctgatg gcgagcagct ttccgctcgc
21120gacctgacct ggtcttatgc tgctctgctg accgccaaca accgtcgtaa
ctccgtcgtg 21180cctgcttctt ggggcgagac ctctgccagc agcgtgcccg
gcacctgtgc ggccacatct 21240gccattggta cctacagcag tgtgactgtc
acctcgtggc cgagtatcgt ggctactggc 21300ggcaccacta cgacggctac
ccccactgga tctggcagcg tgacctcgac cagcaagacc 21360accgcgactg
ctagcaagac cagcaccagt acgtcatcaa cctcctgtac cactcccacg
21420gcgaatgtga tatccaagcg cgcgccagta cccccaggag aagattccaa
agatgtagcc 21480gccccacaca gacagccact cacctcttca gaacgatcca
agcgcgacat ccagatgacc 21540cagagcccca gcagcctcag cgccagcgtc
ggtgaccgcg tcaccatcac ctgccgcgcc 21600agccagggca tccgcaacta
cctcgcctgg tatcagcaga agcccggcaa ggcccccaag 21660ctcctcatct
acgccgccag caccctccag agcggcgtcc ccagccgctt cagcggctcc
21720ggcagcggca ccgacttcac cctcaccatc agcagcctcc agcccgagga
cgtcgccacc 21780tactactgcc agcgctacaa ccgtgccccc tacaccttcg
gccagggcac caaggtcgag 21840atcaagggcc agcccaaggc cgctcccagc
gtcaccctct tccccccctc gagcgaggag 21900ctgcaggcca acaaggccac
cctcgtctgc ctcatcagcg acttctaccc cggtgccgtc 21960accgtcgcct
ggaaggccga cagcagcccc gtcaaggctg gcgtcgagac caccaccccc
22020agcaagcaga gcaacaacaa gtacgccgcc tccagctacc tcagcctcac
ccccgagcag 22080tggaagagcc acaagagcta cagctgccag gtcacccacg
agggcagcac cgtcgagaag 22140accgtcgccc ccaccgagtg cagctaaggg
cccagatcct aagtaagtaa acgaacctct 22200ctgaaggagg ttctgagaca
cgcgcgattc ttctgtatat agttttattt ttcactctgg 22260agtgcttcgc
tccaccagta cataaacctt ttttttcacg taacaaaatg gcttcttttc
22320agaccatgtg aaccatcttg atgccttgac ctcttcagtt ctcactttaa
cgtanttcgc 22380gttagtctgt atgtcccagt tgcatgtagt tgagataaat
acccctggaa gtgggtctgg 22440gcctttgtgg gacggagccc tctttctgtg
gtctggagag cccgctctct accgcctacc 22500ttcttaccac agtacactac
tcacacattg ctgaactgac ccatcatacc gtactttatc 22560ctgttaattc
gtggtgctgt cgactattct atttgctcaa atggagagca cattcatcgg
22620cgcagggata cacggtttat ggaccccaag agtgtaagga ctattattag
taatattata 22680tgcctctagg cgccttaact tcaacaggcg agcactacta
atcaactttt ggtagaccca 22740attacaaacg accatacgtg ccggaaattt
tgggattccg tccgctctcc ccaaccaagc 22800tagaagaggc aacgaacagc
caatcccggt gctaattaaa ttatatggtt catttttttt 22860aaaaaaattt
tttcttccca ttttcctctc gcttttcttt ttcgcatcgt agttgatcaa
22920agtccaagtc aagcgagcta tttgtgctat agctcggtgg ctataatcag
tacagcttag 22980agaggctgta aaggtatgat accacagcag tattcgcgct
ataagcggca ctcctagact 23040aattgttacg gtctacagaa gtaggtaata
aaagcgttaa ttgttctaaa tactagaggc 23100acttagagaa gctatctaaa
tatatattga ccctagctta ttatccctat tagtaagtta 23160gttagctcta
acctatagat agccaaat 23188111012DNAChrysosporium
lucknowensemisc_feature(191)..(191)n is a, c, g, or t 11aagtaaacga
acctctctga aggaggttct gagacacgcg cgattcttct gtatatagtt 60ttatttttca
ctctggagtg cttcgctcca ccagtacata aacctttttt ttcacgtaac
120aaaatggctt cttttcagac catgtgaacc atcttgatgc cttgacctct
tcagttctca 180ctttaacgta nttcgcgtta gtctgtatgt cccagttgca
tgtagttgag ataaataccc 240ctggaagtgg gtctgggcct ttgtgggacg
gagccctctt tctgtggtct ggagagcccg 300ctctctaccg cctaccttct
taccacagta cactactcac acattgctga actgacccat 360cataccgtac
tttatcctgt taattcgtgg tgctgtcgac tattctattt gctcaaatgg
420agagcacatt catcggcgca gggatacacg gtttatggac cccaagagtg
taaggactat 480tattagtaat attatatgcc tctaggcgcc ttaacttcaa
caggcgagca ctactaatca 540acttttggta gacccaatta caaacgacca
tacgtgccgg aaattttggg attccgtccg 600ctctccccaa ccaagctaga
agaggcaacg aacagccaat cccggtgcta attaaattat 660atggttcatt
ttttttaaaa aaattttttc ttcccatttt cctctcgctt ttctttttcg
720catcgtagtt gatcaaagtc caagtcaagc gagctatttg tgctatagct
cggtggctat 780aatcagtaca gcttagagag gctgtaaagg tatgatacca
cagcagtatt cgcgctataa 840gcggcactcc tagactaatt gttacggtct
acagaagtag gtaataaaag cgttaattgt 900tctaaatact agaggcactt
agagaagcta tctaaatata tattgaccct agcttattat 960ccctattagt
aagttagtta gctctaacct atagatagcc aaatgcggcc gg
101212352DNAChrysosporium lucknowense 12atggttcatt ttttttaaaa
aaattttttc ttcccatttt cctctcgctt ttctttttcg 60catcgtagtt gatcaaagtc
caagtcaagc gagctatttg tgctatagct cggtggctat 120aatcagtaca
gcttagagag gctgtaaagg tatgatacca cagcagtatt cgcgctataa
180gcggcactcc tagactaatt gttacggtct acagaagtag gtaataaaag
cgttaattgt 240tctaaatact agaggcactt agagaagcta tctaaatata
tattgaccct agcttattat 300ccctattagt aagttagtta gctctaacct
atagatagat gcatgcggcc gc 35213714DNAAspergillus nigerCDS(1)..(714)
13atg gct ctc cct gcc tac aag acc gcc ttc ctg gag tct ctc gtc ggc
48Met Ala Leu Pro Ala Tyr Lys Thr Ala Phe Leu Glu Ser Leu Val Gly 1
5 10 15 caa cgt gct gac ttt cgg cac ctt cac cct gaa gtc ggg tcg ccg
tac 96Gln Arg Ala Asp Phe Arg His Leu His Pro Glu Val Gly Ser Pro
Tyr 20 25 30 tcc ccc tac ttc ttc aac gcc ggc atc ttc aac acc gcc
tct ctc ctc 144Ser Pro Tyr Phe Phe Asn Ala Gly Ile Phe Asn Thr Ala
Ser Leu Leu 35 40 45 tcc gcc ctc tcc acc gca tac gcc cac acc atc
atc acc ttc ctc gct 192Ser Ala Leu Ser Thr Ala Tyr Ala His Thr Ile
Ile Thr Phe Leu Ala 50 55 60 gag aac cct tcc atc ccc aag ccc gac
gtc atc ttc ggc ccc gca tac 240Glu Asn Pro Ser Ile Pro Lys Pro Asp
Val Ile Phe Gly Pro Ala Tyr 65 70 75 80 aaa ggc atc ccc ctc gcg tgc
gcc acc ctc ctt gaa ctc aac cgc atc 288Lys Gly Ile Pro Leu Ala Cys
Ala Thr Leu Leu Glu Leu Asn Arg Ile 85 90 95 gac ccc gcc acc tgg
ggc agc gtg tcc tac agc tac aac cgc aaa gaa 336Asp Pro Ala Thr Trp
Gly Ser Val Ser Tyr Ser Tyr Asn Arg Lys Glu 100 105 110 gcc aag gat
cac ggc gaa ggc ggc aac att gtc ggc gcc gct ctg aag 384Ala Lys Asp
His Gly Glu Gly Gly Asn Ile Val Gly Ala Ala Leu Lys 115 120 125 ggc
aag acc gtg ctt gtg atc gac gat gtc atc acg gcc ggt acc gcc 432Gly
Lys Thr Val Leu Val Ile Asp Asp Val Ile Thr Ala Gly Thr Ala 130 135
140 atg cgt gag acc ctc aac ctg gtc gcc aag gag ggc ggc aag gtc gtc
480Met Arg Glu Thr Leu Asn Leu Val Ala Lys Glu Gly Gly Lys Val Val
145 150 155 160 gga ttc act gtt gct ctg gac cgc ttg gag aag atg ccc
gga ccc aag 528Gly Phe Thr Val Ala Leu Asp Arg Leu Glu Lys Met Pro
Gly Pro Lys 165 170 175 gac gag aac ggt gtc gag gac gat aag ccc aga
atg agt gct atg ggt 576Asp Glu Asn Gly Val Glu Asp Asp Lys Pro Arg
Met Ser Ala Met Gly 180 185 190 cag atc cgt aag gag tat ggt gtg ccc
acg acg agt att gtt act ctg 624Gln Ile Arg Lys Glu Tyr Gly Val Pro
Thr Thr Ser Ile Val Thr Leu 195 200 205 gat gat ttg atc aag ttg atg
cag gcg aag ggc aat gag gcc gat atg 672Asp Asp Leu Ile Lys Leu Met
Gln Ala Lys Gly Asn Glu Ala Asp Met 210 215 220 aag cgg ttg gag gag
tat agg gct aag tat cag gct agt gat 714Lys Arg Leu Glu Glu Tyr Arg
Ala Lys Tyr Gln Ala Ser Asp 225 230 235 14238PRTAspergillus niger
14Met Ala Leu Pro Ala Tyr Lys Thr Ala Phe Leu Glu Ser Leu Val Gly 1
5 10 15 Gln Arg Ala Asp Phe Arg His Leu His Pro Glu Val Gly Ser Pro
Tyr 20 25 30 Ser Pro Tyr Phe Phe Asn Ala Gly Ile Phe Asn Thr Ala
Ser Leu Leu 35 40 45 Ser Ala Leu Ser Thr Ala Tyr Ala His Thr Ile
Ile Thr Phe Leu Ala 50 55 60 Glu Asn Pro Ser Ile Pro Lys Pro Asp
Val Ile Phe Gly Pro Ala Tyr 65 70 75 80 Lys Gly Ile Pro Leu Ala Cys
Ala Thr Leu Leu Glu Leu Asn Arg Ile 85 90 95 Asp Pro Ala Thr Trp
Gly Ser Val Ser Tyr Ser Tyr Asn Arg Lys Glu 100 105 110 Ala Lys Asp
His Gly Glu Gly Gly Asn Ile Val Gly Ala Ala Leu Lys 115 120 125 Gly
Lys Thr Val Leu Val Ile Asp Asp Val Ile Thr Ala Gly Thr Ala 130 135
140 Met Arg Glu Thr Leu Asn Leu Val Ala Lys Glu Gly Gly Lys Val Val
145 150 155 160 Gly Phe Thr Val Ala Leu Asp Arg Leu Glu Lys Met Pro
Gly Pro Lys 165 170 175 Asp Glu Asn Gly Val Glu Asp Asp Lys Pro Arg
Met Ser Ala Met Gly 180 185 190 Gln Ile Arg Lys Glu Tyr Gly Val Pro
Thr Thr Ser Ile Val Thr Leu 195 200 205 Asp Asp Leu Ile Lys Leu Met
Gln Ala Lys Gly Asn Glu Ala Asp Met 210 215 220 Lys Arg Leu Glu Glu
Tyr Arg Ala Lys Tyr Gln Ala Ser Asp 225 230 235 15831DNAAspergillus
oryzaeCDS(1)..(831) 15atg tct tcc aag tcg caa ttg acc tac agc gca
cgc gct agc aag cac 48Met Ser Ser Lys Ser Gln Leu Thr Tyr Ser Ala
Arg Ala Ser Lys His 1 5 10 15 ccc aat gcg ctc gta aag aag ctc ttc
gag gtt gcc gag gcc aag aaa 96Pro Asn Ala Leu Val Lys Lys Leu Phe
Glu Val Ala Glu Ala Lys Lys 20 25 30 acc aat gtc acc gtt tcc gcc
gac gtg aca acc acc aaa gag ctg ctg 144Thr Asn Val Thr Val Ser Ala
Asp Val Thr Thr Thr Lys Glu Leu Leu 35 40 45 gat ttg gct gac cgg
ctc ggt ccg tac att gcc gtg atc aaa act cac 192Asp Leu Ala Asp Arg
Leu Gly Pro Tyr Ile Ala Val Ile Lys Thr His 50 55 60 atc gat atc
ctc tcc gat ttc agc gaa gaa acc atc acc ggt ctg aag 240Ile Asp Ile
Leu Ser Asp Phe Ser Glu Glu Thr Ile Thr Gly Leu Lys 65 70 75 80 gcc
ctt gca gag aag cac aat ttc ctc atc ttc gaa gat cgc aag ttc 288Ala
Leu Ala Glu Lys His Asn Phe Leu Ile Phe Glu Asp Arg Lys Phe 85 90
95 atc gat atc gga aac aca gtc caa aag cag tac cat ggc ggc act ctg
336Ile Asp Ile Gly Asn Thr Val Gln Lys Gln Tyr His Gly Gly Thr Leu
100 105 110 cgt atc tct gag tgg gcc cac atc atc aac tgc agt att ctg
ccc ggt 384Arg Ile Ser Glu Trp Ala His Ile Ile Asn Cys Ser Ile Leu
Pro Gly 115 120 125 gag ggt atc gtc gag gct ctg gcc cag act gct tcg
gcc gag gac ttc 432Glu Gly Ile Val Glu Ala Leu Ala Gln Thr Ala Ser
Ala Glu Asp Phe 130 135 140 ccc tac ggc tcc gag agg ggc ctt ttg atc
ctt gcg gag atg acc tcc 480Pro Tyr Gly Ser Glu Arg Gly Leu Leu Ile
Leu Ala Glu Met Thr Ser 145 150 155 160 aag gga tct ttg gct acc ggt
cag tat act act tct tct gtt gac tat 528Lys Gly Ser Leu Ala Thr Gly
Gln Tyr Thr Thr Ser Ser Val Asp Tyr 165 170 175 gct cgg aag tat aag
aag ttt gtg atg gga ttc gtc tcg aca cgt cac 576Ala Arg Lys Tyr Lys
Lys Phe Val Met Gly Phe Val Ser Thr Arg His 180 185 190 ctt ggc gag
gtt cag tct gaa gtt agc tcg cct tcg gag gag gaa gat 624Leu Gly Glu
Val Gln Ser Glu Val Ser Ser Pro Ser Glu Glu Glu Asp 195 200 205 ttt
gtc gtc ttc acg aca ggt gtc aac ctc tcc tcg aag ggt gac aag 672Phe
Val Val Phe Thr Thr Gly Val Asn Leu Ser Ser Lys Gly Asp Lys 210 215
220 ctg gga cag cag tac caa act cct gag tcg gct gtt gga cgc ggt gcc
720Leu Gly Gln Gln Tyr Gln Thr Pro Glu Ser Ala Val Gly Arg Gly Ala
225 230 235 240 gac ttt att att gct ggc cgt gga att tat gct gct cct
gat ccc gtg 768Asp Phe Ile Ile Ala Gly Arg Gly Ile Tyr Ala Ala Pro
Asp Pro Val 245 250 255 gag gcg gcg aac cag tac cag aag gag gga tgg
gat gca tac ctg aag 816Glu Ala Ala Asn Gln Tyr Gln Lys Glu Gly Trp
Asp Ala Tyr Leu Lys 260 265 270 cgt gtt ggt gcg caa 831Arg Val Gly
Ala Gln 275 16277PRTAspergillus oryzae 16Met Ser Ser Lys Ser Gln
Leu Thr Tyr Ser Ala Arg Ala Ser Lys His 1 5 10 15 Pro Asn Ala Leu
Val Lys Lys Leu Phe Glu Val Ala Glu Ala Lys Lys 20 25 30 Thr Asn
Val Thr Val Ser Ala Asp Val Thr Thr Thr Lys Glu Leu Leu 35 40 45
Asp Leu Ala Asp Arg Leu Gly Pro Tyr Ile Ala Val Ile Lys Thr His 50
55 60 Ile Asp Ile Leu Ser Asp Phe Ser Glu Glu Thr Ile Thr Gly Leu
Lys 65 70 75 80 Ala Leu Ala Glu Lys His Asn Phe Leu Ile Phe Glu Asp
Arg Lys Phe 85 90 95 Ile Asp Ile Gly Asn Thr Val Gln Lys Gln Tyr
His Gly Gly Thr Leu 100 105 110 Arg Ile Ser Glu Trp Ala His Ile Ile
Asn Cys Ser Ile Leu Pro Gly 115 120 125 Glu Gly Ile Val Glu Ala Leu
Ala Gln Thr Ala Ser Ala Glu Asp Phe 130 135 140 Pro Tyr Gly Ser Glu
Arg Gly Leu Leu Ile Leu Ala Glu Met Thr Ser 145 150 155 160 Lys Gly
Ser Leu Ala Thr Gly Gln Tyr Thr Thr Ser Ser Val Asp Tyr 165 170 175
Ala Arg Lys Tyr Lys Lys Phe Val Met Gly Phe Val Ser Thr Arg His 180
185 190 Leu Gly Glu Val Gln Ser Glu Val Ser Ser Pro Ser Glu Glu Glu
Asp 195 200 205 Phe Val Val Phe Thr Thr Gly Val Asn Leu Ser Ser Lys
Gly Asp Lys 210 215 220 Leu Gly Gln Gln Tyr Gln Thr Pro Glu Ser Ala
Val Gly Arg Gly Ala 225 230 235 240 Asp Phe Ile Ile Ala Gly Arg Gly
Ile Tyr Ala Ala Pro Asp Pro Val 245 250 255 Glu Ala Ala Asn Gln Tyr
Gln Lys Glu Gly Trp Asp Ala Tyr Leu Lys 260 265 270 Arg Val Gly Ala
Gln 275 17580DNAhumanmisc_feature(423)..(574)n is a, c, g, or t
17cgaacccccg cgccgccttt gcgagggtgg agttgcctta gggttagggt tagggttagg
60gttagggtta gggttagggt tagggttagg gttagggtta gggttagggt tagggttagg
120gttagggtta gggttagggt tagggtcagg gtcagggtag ggtcaggggt
agggtcaggg 180gtaggggtag ggtcagggtt agggttaggg ttagggttag
ggttagggtt agggttaggg 240tcagggttag ggttagggtt aggggtaggg
gtaggggtag ggttagggtt agggttaggg 300ttagggttag ggttagggtt
agggtcaggg tcagggtcag gggtagggta gggtagggtt 360aggggttagg
gttagggtta gggttagggt tagggttagg gttaagggtt aagggttaag
420ggnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnntgggaa 580
* * * * *
References