U.S. patent application number 14/608018 was filed with the patent office on 2015-10-22 for methods and compositions for improving sugar transport, mixed sugar fermentation, and production of biofuels.
The applicant listed for this patent is The Board of Trustees of the University of Illinois, BP Corporation North America Inc., The Regents of the University of California. Invention is credited to William T. BEESON, IV, Jin Ho CHOI, James H. DOUDNA CATE, Jing DU, Jonathan M. GALAZKA, N. Louise GLASS, Suk-Jin HA, Yong-Su JIN, Soo Rin KIM, Sijin LI, Chaoguang TIAN, Xiaomin YANG, Huimin ZHAO.
Application Number | 20150299755 14/608018 |
Document ID | / |
Family ID | 43497647 |
Filed Date | 2015-10-22 |
United States Patent
Application |
20150299755 |
Kind Code |
A1 |
GLASS; N. Louise ; et
al. |
October 22, 2015 |
METHODS AND COMPOSITIONS FOR IMPROVING SUGAR TRANSPORT, MIXED SUGAR
FERMENTATION, AND PRODUCTION OF BIOFUELS
Abstract
The present disclosure relates to host cells containing a
recombinant polynucleotide encoding a polypeptide where the
polypeptide transports cellodextrin into the cell. The present
disclosure further relates to methods of increasing transport of
cellodextrin into a cell, methods of increasing growth of a cell on
a medium containing cellodextrin, methods of co-fermenting
cellulose-derived and hemicellulose-derived sugars, and methods of
making hydrocarbons or hydrocarbon derivatives by providing a host
cell containing a recombinant polynucleotide encoding a polypeptide
where the polypeptide transports cellodextrin into the cell. The
present disclosure relates to host cells containing a recombinant
polynucleotide encoding a polypeptide where the polypeptide
transports a pentose into the cell, methods of increasing transport
of a pentose into a cell, methods of increasing growth of a cell on
a medium containing pentose sugars, and methods of making
hydrocarbons or hydrocarbon derivatives by providing a host cell
containing a recombinant polynucleotide encoding a polypeptide
where the polypeptide transports a pentose into the cell.
Inventors: |
GLASS; N. Louise; (Orinda,
CA) ; TIAN; Chaoguang; (Tianjin, CN) ; BEESON,
IV; William T.; (Indianapolis, IN) ; ZHAO;
Huimin; (Champaign, IL) ; DU; Jing;
(Champaign, IL) ; CHOI; Jin Ho; (Urbana, IL)
; DOUDNA CATE; James H.; (Berkeley, CA) ; GALAZKA;
Jonathan M.; (Berkeley, CA) ; HA; Suk-Jin;
(Savoy, IL) ; JIN; Yong-Su; (Champaign, IL)
; KIM; Soo Rin; (Savoy, IL) ; LI; Sijin;
(Urbana, IL) ; YANG; Xiaomin; (Albany,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California
The Board of Trustees of the University of Illinois
BP Corporation North America Inc. |
Oakland
Urbana
Houston |
CA
IL
TX |
US
US
US |
|
|
Family ID: |
43497647 |
Appl. No.: |
14/608018 |
Filed: |
January 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14289570 |
May 28, 2014 |
9012177 |
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14608018 |
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13802533 |
Mar 13, 2013 |
8765410 |
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14289570 |
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12843844 |
Jul 26, 2010 |
8431360 |
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13802533 |
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61285526 |
Dec 10, 2009 |
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61271833 |
Jul 24, 2009 |
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Current U.S.
Class: |
435/69.1 |
Current CPC
Class: |
C07K 14/705 20130101;
Y02E 50/17 20130101; C12N 15/81 20130101; Y02E 50/10 20130101; C12P
21/02 20130101; Y02E 50/16 20130101; C07K 14/37 20130101; C12N
15/80 20130101 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C07K 14/705 20060101 C07K014/705; C12N 15/80 20060101
C12N015/80 |
Claims
1.-17. (canceled)
18. A method of co-fermenting cellulose-derived and
hemicellulose-derived sugars, comprising: providing a host cell,
wherein the host cell comprises a first recombinant polynucleotide
encoding a cellodextrin transporter and a second recombinant
polynucleotide encoding a catalytic domain of a .beta.-glucosidase,
and culturing the host cell in a medium comprising a
cellulose-derived sugar and a hemicellulose-derived sugar, wherein
expression of the recombinant polynucleotides enables
co-fermentation of the cellulose-derived sugar and the
hemicellulose-derived sugar.
19. The method of claim 18, wherein the first recombinant
polynucleotide encodes a polypeptide comprising transmembrane
.alpha.-helix 1, transmembrane .alpha.-helix 2, transmembrane
.alpha.-helix 3, transmembrane .alpha.-helix 4, transmembrane
.alpha.-helix 5, transmembrane .alpha.-helix 6, transmembrane
.alpha.-helix 7, transmembrane .alpha.-helix 8, transmembrane
.alpha.-helix 9, transmembrane .alpha.-helix 10, transmembrane
.alpha.-helix 11, and transmembrane .alpha.-helix 12, and
transmembrane .alpha.-helix 1 comprises SEQ ID NO: 1.
20. The method of claim 18, wherein the first recombinant
polynucleotide encodes a polypeptide comprising transmembrane
.alpha.-helix 1, transmembrane .alpha.-helix 2, transmembrane
.alpha.-helix 3, transmembrane .alpha.-helix 4, transmembrane
.alpha.-helix 5, transmembrane .alpha.-helix 6, transmembrane
.alpha.-helix 7, transmembrane .alpha.-helix 8, transmembrane
.alpha.-helix 9, transmembrane .alpha.-helix 10, transmembrane
.alpha.-helix 11, and transmembrane .alpha.-helix 12, and
transmembrane .alpha.-helix 2 comprises SEQ ID NO: 2.
21. The method of claim 18, wherein the first recombinant
polynucleotide encodes a polypeptide comprising transmembrane
.alpha.-helix 1, transmembrane .alpha.-helix 2, transmembrane
.alpha.-helix 3, transmembrane .alpha.-helix 4, transmembrane
.alpha.-helix 5, transmembrane .alpha.-helix 6, transmembrane
.alpha.-helix 7, transmembrane .alpha.-helix 8, transmembrane
.alpha.-helix 9, transmembrane .alpha.-helix 10, transmembrane
.alpha.-helix 11, and transmembrane .alpha.-helix 12, and a loop
connecting transmembrane .alpha.-helix 2 and transmembrane
.alpha.-helix 3 comprises SEQ ID NO: 3.
22. The method of claim 18 wherein the first recombinant
polynucleotide encodes a polypeptide comprising transmembrane
.alpha.-helix 1, transmembrane .alpha.-helix 2, transmembrane
.alpha.-helix 3, transmembrane .alpha.-helix 4, transmembrane
.alpha.-helix 5, transmembrane .alpha.-helix 6, transmembrane
.alpha.-helix 7, transmembrane .alpha.-helix 8, transmembrane
.alpha.-helix 9, transmembrane .alpha.-helix 10, transmembrane
.alpha.-helix 11, and transmembrane .alpha.-helix 12, and
transmembrane .alpha.-helix 5 comprises SEQ ID NO: 4.
23. The method of claim 18, wherein the first recombinant
polynucleotide encodes a polypeptide comprising transmembrane
.alpha.-helix 1, transmembrane .alpha.-helix 2, transmembrane
.alpha.-helix 3, transmembrane .alpha.-helix 4, transmembrane
.alpha.-helix 5, transmembrane .alpha.-helix 6, transmembrane
.alpha.-helix 7, transmembrane .alpha.-helix 8, transmembrane
.alpha.-helix 9, transmembrane .alpha.-helix 10, transmembrane
.alpha.-helix 11, and transmembrane .alpha.-helix 12, and
transmembrane .alpha.-helix 6 comprises SEQ ID NO: 5.
24. The method of claim 18, wherein the first recombinant
polynucleotide encodes a polypeptide comprising transmembrane
.alpha.-helix 1, transmembrane .alpha.-helix 2, transmembrane
.alpha.-helix 3, transmembrane .alpha.-helix 4, transmembrane
.alpha.-helix 5, transmembrane .alpha.-helix 6, transmembrane
.alpha.-helix 7, transmembrane .alpha.-helix 8, transmembrane
.alpha.-helix 9, transmembrane .alpha.-helix 10, transmembrane
.alpha.-helix 11, and transmembrane .alpha.-helix 12, and the
sequence between transmembrane .alpha.-helix 6 and transmembrane
.alpha.-helix 7 comprises SEQ ID NO: 6.
25. The method of claim 18, wherein the first recombinant
polynucleotide encodes a polypeptide comprising transmembrane
.alpha.-helix 1, transmembrane .alpha.-helix 2, transmembrane
.alpha.-helix 3, transmembrane .alpha.-helix 4, transmembrane
.alpha.-helix 5, transmembrane .alpha.-helix 6, transmembrane
.alpha.-helix 7, transmembrane .alpha.-helix 8, transmembrane
.alpha.-helix 9, transmembrane .alpha.-helix 10, transmembrane
.alpha.-helix 11, and transmembrane .alpha.-helix 12, and
transmembrane .alpha.-helix 7 comprises SEQ ID NO: 7.
26. The method of claim 18, wherein the first recombinant
polynucleotide encodes a polypeptide comprising transmembrane
.alpha.-helix 1, transmembrane .alpha.-helix 2, transmembrane
.alpha.-helix 3, transmembrane .alpha.-helix 4, transmembrane
.alpha.-helix 5, transmembrane .alpha.-helix 6, transmembrane
.alpha.-helix 7, transmembrane .alpha.-helix 8, transmembrane
.alpha.-helix 9, transmembrane .alpha.-helix 10, transmembrane
.alpha.-helix 11, and transmembrane .alpha.-helix 12, and
transmembrane .alpha.-helix 10 and transmembrane .alpha.-helix 11
and the sequence between them comprise SEQ ID NO: 8.
27. The method of claim 18, wherein the cellodextrin transporter
has at least 29%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 99%, or at least 100% amino acid
identity to NCU00801 or NCU08114.
28. The method of claim 18, wherein the .beta.-glucosidase is from
Neurospora crassa.
29. The method of claim 18, wherein the .beta.-glucosidase is
NCU00130.
30. The method of claim 18, wherein the host cell further comprises
one or more recombinant polynucleotides encoding one or more
enzymes involved in pentose utilization.
31. The method of claim 30, wherein the one or more enzymes
involved in pentose utilization are selected from the group
consisting of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P
4 epimerase, xylose isomerase, xylulokinase, aldose reductase,
L-arabinitol 4-dehydrogenase, L-xylulose reductase, and xylitol
dehydrogenase.
32. The method of claim 18, wherein the host cell further comprises
a third recombinant polynucleotide encoding a pentose
transporter.
33. The method of claim 32, wherein the pentose transporter is
selected from the group consisting of NCU00821, NCU04963, NCU06138,
STL12/XUT6, SUT2, SUT3, XUT1, and XUT3.
34. The method of claim 18, wherein the cellulose-derived sugar is
selected from the group consisting of cellobiose, cellotriose, and
cellotetraose, and the hemicellulose-derived sugar is xylose.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/289,570, filed May 28, 2014, which is a divisional of U.S.
application Ser. No. 13/802,533, filed Mar. 13, 2013, now U.S. Pat.
No. 8,765,410, which is a divisional of U.S. application Ser. No.
12/843,844, filed Jul. 26, 2010, now U.S. Pat. No. 8,431,360, which
claims the benefit of U.S. Provisional Application No. 61/285,526,
filed Dec. 10, 2009, and U.S. Provisional Application No.
61/271,833, filed Jul. 24, 2009, all of which are hereby
incorporated by reference in their entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
677792000112SeqList.txt, date recorded: Jan. 26, 2015, size: 104
KB).
FIELD OF THE INVENTION
[0003] The present disclosure relates to methods and compositions
for increasing the transport of sugars into cells, for increasing
growth of cells, for increasing synthesis of hydrocarbons and
hydrocarbon derivatives, and for co-fermenting cellulose-derived
and hemicellulose-derived sugars.
BACKGROUND OF THE INVENTION
[0004] Biofuels are under intensive investigation due to the
increasing concerns about energy security, sustainability, and
global climate change (Lynd et al., 1991). Bioconversion of
plant-derived lignocellulosic materials into biofuels has been
regarded as an attractive alternative to chemical production of
fossil fuels (Lynd et al. 2008; Hahn-Hagerdal et al. 2006).
Lignocellulosic biomass is composed of cellulose, hemicellulose,
and lignin.
[0005] The engineering of microorganisms to perform the conversion
of lignocellulosic biomass to ethanol efficiently remains a major
goal of the biofuels field. Much research has been focused on
genetically manipulating microorganisms that naturally ferment
simple sugars to alcohol to express cellulases and other enzymes
that would allow them to degrade lignocellulosic biomass polymers
and generate ethanol within one cell. However, an area that has
been less well studied is that of sugar transporters. An
understanding of the regulation of sugar transport and the genetic
engineering of microorganisms to have improved sugar-uptake ability
will greatly improve efficiency (Stephanopoulos 2007). Furthermore,
other types of proteins involved in the regulation of cellulase
expression and activity remain to be fully explored.
[0006] Saccharomyces cerevisiae, also known as baker's yeast, has
been used for bioconversion of hexose sugars into ethanol for
thousands of years. It is also the most widely used microorganism
for large scale industrial fermentation of D-glucose into ethanol.
S. cerevisiae is a very suitable candidate for bioconversion of
lignocellulosic biomass into biofuels (van Maris et al., 2006). It
has a well-studied genetic and physiological background, ample
genetic tools, and high tolerance to high ethanol concentration and
inhibitors presented in lignocellulosic hydrolysates (Jeffries
2006). The low fermentation pH of S. cerevisiae can also prevent
bacterial contamination during fermentation.
[0007] Unfortunately, wild type S. cerevisiae cannot utilize
pentose sugars (Hector et al., 2008). To overcome this limitation,
pentose utilization pathways from pentose-assimilating organisms
have been introduced into S. cerevisiae, allowing fermentation of
D-xylose and L-arabinose (Hahn-Hagerdal et al., 2007; Brat et al.,
2009; Wisselink et al., 2007, 2009; Wiedemann and Boles 2008;
Karhumma et al., 2006). However, efficient conversion of pentose
sugars into biofuels is limited by multiple issues including
cellular redox imbalance, low influx of pentose phosphate pathway,
and lack of efficient pentose transport into the cell (Hector et
al., 2008).
[0008] In addition, both natural and engineered microorganisms show
reduced ethanol tolerance during xylose fermentation as compared to
glucose fermentation (Jeffries and Jin 2000). Combined with the
lower fermentation rate, the reduced ethanol tolerance during
xylose fermentation poses a significant problem in fermentation of
sugar mixtures containing the high concentrations of glucose
(.about.70-100 g/L) and xylose (.about.40-60 g/L) present in
cellulosic hydrolysates. Since microorganisms utilize glucose
preferentially, at the time of glucose depletion (when cells begin
to use xylose), the ethanol concentration is already high enough
(.about.35-45 g/L) to further reduce the xylose fermentation rate.
As a result, sequential utilization of xylose after glucose
depletion because of "glucose repression" is a significant
challenge to be overcome in order to successfully utilize mixed
sugars in cellulosic hydrolysates.
[0009] Thus, a need exists for the identification of additional
genes that are critical for the degradation of lignocellulose and
for their use in the engineering of microorganisms for improved
growth on lignocellulose and uptake of compounds resulting from
lignocellulose degradation. A further need exists for improved
methods of efficient conversion of pentose sugars into biofuels and
of mixed sugar fermentation for the production of biofuels.
BRIEF SUMMARY OF THE INVENTION
[0010] In order to meet these needs, the invention described herein
provides methods of increasing transport of cellodextrin into a
cell, methods of increasing growth of a cell on a medium containing
cellodextrin, methods of co-fermenting cellulose-derived and
hemicellulose-derived sugars, and methods of making hydrocarbons or
hydrocarbon derivatives by providing a host cell containing a
recombinant polynucleotide encoding a polypeptide where the
polypeptide transports cellodextrin into the cell. Further
described are host cells containing a recombinant polynucleotide
encoding a polypeptide where the polypeptide transports
cellodextrin into the cell. Further described herein are host cells
containing a recombinant polynucleotide encoding a polypeptide
where the polypeptide transports a pentose into the cell, methods
of increasing transport of a pentose into a cell, methods of
increasing growth of a cell on a medium containing pentose sugars,
and methods of making hydrocarbons or hydrocarbon derivatives by
providing a host cell containing a recombinant polynucleotide
encoding a polypeptide where the polypeptide transports a pentose
into the cell.
[0011] As used herein, cellodextrin refers to glucose polymers of
varying length and includes, without limitation, cellobiose (2
glucose monomers), cellotriose (3 glucose monomers), cellotetraose
(4 glucose monomers), cellopentaose (5 glucose monomers), and
cellohexaose (6 glucose monomers).
[0012] Thus one aspect includes methods of increasing transport of
cellodextrin into a cell, including providing a host cell, where
the host cell contains a recombinant polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and transmembrane
.alpha.-helix 1 contains SEQ ID NO: 1, and culturing the cell in a
medium such that the recombinant polynucleotide is expressed, where
expression of the recombinant polynucleotide results in increased
transport of cellodextrin into the cell compared with a cell that
does not contain the recombinant polynucleotide.
[0013] Another aspect includes methods of increasing transport of
cellodextrin into a cell, including providing a host cell, where
the host cell contains a recombinant polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and transmembrane
.alpha.-helix 2 contains SEQ ID NO: 2, and culturing the cell in a
medium such that the recombinant polynucleotide is expressed, where
expression of the recombinant polynucleotide results in increased
transport of cellodextrin into the cell compared with a cell that
does not contain the recombinant polynucleotide.
[0014] Another aspect includes methods of increasing transport of
cellodextrin into a cell, including providing a host cell, where
the host cell contains a recombinant polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and a loop connecting
transmembrane .alpha.-helix 2 and transmembrane .alpha.-helix 3
contains SEQ ID NO: 3, and culturing the cell in a medium such that
the recombinant polynucleotide is expressed, where expression of
the recombinant polynucleotide results in increased transport of
cellodextrin into the cell compared with a cell that does not
contain the recombinant polynucleotide.
[0015] Another aspect includes methods of increasing transport of
cellodextrin into a cell, including providing a host cell, where
the host cell contains a recombinant polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and transmembrane
.alpha.-helix 5 contains SEQ ID NO: 4, and culturing the cell in a
medium such that the recombinant polynucleotide is expressed, where
expression of the recombinant polynucleotide results in increased
transport of cellodextrin into the cell compared with a cell that
does not contain the recombinant polynucleotide.
[0016] Another aspect includes methods of increasing transport of
cellodextrin into a cell, including providing a host cell, where
the host cell contains a recombinant polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and transmembrane
.alpha.-helix 6 contains SEQ ID NO: 5, and culturing the cell in a
medium such that the recombinant polynucleotide is expressed, where
expression of the recombinant polynucleotide results in increased
transport of cellodextrin into the cell compared with a cell that
does not contain the recombinant polynucleotide.
[0017] Another aspect includes methods of increasing transport of
cellodextrin into a cell, including providing a host cell, where
the host cell contains a recombinant polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and sequence between
transmembrane .alpha.-helix 6 and transmembrane .alpha.-helix 7
contains SEQ ID NO: 6, and culturing the cell in a medium such that
the recombinant polynucleotide is expressed, where expression of
the recombinant polynucleotide results in increased transport of
cellodextrin into the cell compared with a cell that does not
contain the recombinant polynucleotide.
[0018] Another aspect includes methods of increasing transport of
cellodextrin into a cell, including providing a host cell, where
the host cell contains a recombinant polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and transmembrane
.alpha.-helix 7 contains SEQ ID NO: 7, and culturing the cell in a
medium such that the recombinant polynucleotide is expressed, where
expression of the recombinant polynucleotide results in increased
transport of cellodextrin into the cell compared with a cell that
does not contain the recombinant polynucleotide.
[0019] Another aspect includes methods of increasing transport of
cellodextrin into a cell, including providing a host cell, where
the host cell contains a recombinant polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and transmembrane
.alpha.-helix 10 and transmembrane .alpha.-helix 11 and the
sequence between them contains SEQ ID NO: 8, and culturing the cell
in a medium such that the recombinant polynucleotide is expressed,
where expression of the recombinant polynucleotide results in
increased transport of cellodextrin into the cell compared with a
cell that does not contain the recombinant polynucleotide.
[0020] In certain embodiments that may be combined with any of the
preceding aspects, the polypeptide has at least 29%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 99%,
or at least 100% amino acid identity to NCU00801 or NCU08114. In
certain embodiments that may be combined with any of the preceding
embodiments, the host cell contains a second recombinant
polynucleotide encoding at least a catalytic domain of a
.beta.-glucosidase. In certain embodiments that may be combined
with the preceding embodiments having a host cell containing a
second recombinant polynucleotide encoding at least a catalytic
domain of a .beta.-glucosidase, the .beta.-glucosidase is from
Neurospora crassa. In certain embodiments that may be combined with
the preceding embodiments having a host cell containing a second
recombinant polynucleotide encoding at least a catalytic domain of
a .beta.-glucosidase from Neurospora crassa, the .beta.-glucosidase
is encoded by NCU00130. In certain embodiments that may be combined
with any of the preceding embodiments, the host cell further
contains one or more recombinant polynucleotides where the one or
more polynucleotides encode one or more enzymes involved in pentose
utilization. In certain embodiments that may be combined with the
preceding embodiments having a host cell further containing one or
more recombinant polynucleotides where the one or more
polynucleotides encode one or more enzymes involved in pentose
utilization, the one or more enzymes are selected from one or more
of the group consisting of L-arabinose isomerase, L-ribulokinase,
L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose
reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and
xylitol dehydrogenase. In certain embodiments that may be combined
with any of the preceding embodiments, the host cell further
contains a third recombinant polynucleotide where the third
recombinant polynucleotide encodes a pentose transporter. In
certain embodiments that may be combined with the preceding
embodiments having the host cell further containing a third
recombinant polynucleotide where the third recombinant
polynucleotide encodes a pentose transporter, the pentose
transporter is selected from the group consisting of NCU00821,
NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, and XUT3.
[0021] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 1 contains
SEQ ID NO: 1, and the polypeptide is a cellodextrin transporter,
and culturing the host cell in a medium containing cellodextrin,
where the host cell grows at a faster rate in the medium than a
cell that does not contain the recombinant polynucleotide.
[0022] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 2 contains
SEQ ID NO: 2, and the polypeptide is a cellodextrin transporter,
and culturing the host cell in a medium containing cellodextrin,
where the host cell grows at a faster rate in the medium than a
cell that does not contain the recombinant polynucleotide.
[0023] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and a loop connecting transmembrane
.alpha.-helix 2 and transmembrane .alpha.-helix 3 contains SEQ ID
NO: 3, and the polypeptide is a cellodextrin transporter, and
culturing the host cell in a medium containing cellodextrin, where
the host cell grows at a faster rate in the medium than a cell that
does not contain the recombinant polynucleotide.
[0024] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 5 contains
SEQ ID NO: 4, and the polypeptide is a cellodextrin transporter,
and culturing the host cell in a medium containing cellodextrin,
where the host cell grows at a faster rate in the medium than a
cell that does not contain the recombinant polynucleotide.
[0025] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 6 contains
SEQ ID NO: 5, and the polypeptide is a cellodextrin transporter,
and culturing the host cell in a medium containing cellodextrin,
where the host cell grows at a faster rate in the medium than a
cell that does not contain the recombinant polynucleotide.
[0026] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and sequence between transmembrane
.alpha.-helix 6 and transmembrane .alpha.-helix 7 contains SEQ ID
NO: 6, and the polypeptide is a cellodextrin transporter, and
culturing the host cell in a medium containing cellodextrin, where
the host cell grows at a faster rate in the medium than a cell that
does not contain the recombinant polynucleotide.
[0027] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 7 contains
SEQ ID NO: 7, and the polypeptide is a cellodextrin transporter,
and culturing the host cell in a medium containing cellodextrin,
where the host cell grows at a faster rate in the medium than a
cell that does not contain the recombinant polynucleotide.
[0028] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 10 and
transmembrane .alpha.-helix 11 and the sequence between them
contain SEQ ID NO: 8, and the polypeptide is a cellodextrin
transporter, and culturing the host cell in a medium containing
cellodextrin, where the host cell grows at a faster rate in the
medium than a cell that does not contain the recombinant
polynucleotide.
[0029] In certain embodiments that may be combined with any of the
preceding aspects of increasing growth of cells, the polypeptide
has at least 29%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 99%, or at least 100% amino acid
identity to NCU00801 or NCU08114. In certain embodiments that may
be combined with any of the preceding embodiments, the host cell
contains an endogenous or a second recombinant polynucleotide where
the polynucleotide encodes at least a catalytic domain of a
.beta.-glucosidase. In certain embodiments that may be combined
with the preceding embodiments having a host cell containing an
endogenous or a second recombinant polynucleotide where the
polynucleotide encodes at least a catalytic domain of a
.beta.-glucosidase, the .beta.-glucosidase is from Neurospora
crassa. In certain embodiments that may be combined with the
preceding embodiments having a host cell containing an endogenous
or a second recombinant polynucleotide where the polynucleotide
encodes at least a catalytic domain of a .beta.-glucosidase from
Neurospora crassa, the .beta.-glucosidase is encoded by
NCU00130.
[0030] Another aspect includes methods of co-fermenting
cellulose-derived and hemicellulose-derived sugars, containing
providing a host cell, where the host cell contains a first
recombinant polynucleotide encoding a cellodextrin transporter and
a second recombinant polynucleotide encoding a catalytic domain of
a .beta.-glucosidase, and culturing the host cell in a medium
containing a cellulose-derived sugar and a hemicellulose-derived
sugar, where expression of the recombinant polynucleotides enables
co-fermentation of the cellulose-derived sugar and the
hemicellulose-derived sugar. In certain embodiments, the first
recombinant polynucleotide encodes a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 1 contains
SEQ ID NO: 1. In certain embodiments, the first recombinant
polynucleotide encodes a polypeptide containing transmembrane
.alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4,
.alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8,
.alpha.-helix 9, .alpha.-helix 10, .alpha.-helix 11, .alpha.-helix
12, and transmembrane .alpha.-helix 2 contains SEQ ID NO: 2. In
certain embodiments, the first recombinant polynucleotide encodes a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and a loop connecting
transmembrane .alpha.-helix 2 and transmembrane .alpha.-helix 3
contains SEQ ID NO: 3. In certain embodiments, the first
recombinant polynucleotide encodes a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 5 contains
SEQ ID NO: 4. In certain embodiments, the first recombinant
polynucleotide encodes a polypeptide containing transmembrane
.alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4,
.alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8,
.alpha.-helix 9, .alpha.-helix 10, .alpha.-helix 11, .alpha.-helix
12, and transmembrane .alpha.-helix 6 contains SEQ ID NO: 5. In
certain embodiments, the first recombinant polynucleotide encodes a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and sequence between
transmembrane .alpha.-helix 6 and transmembrane .alpha.-helix 7
contains SEQ ID NO: 6. In certain embodiments, the first
recombinant polynucleotide encodes a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 7 contains
SEQ ID NO: 7. In certain embodiments, the first recombinant
polynucleotide encodes a polypeptide containing transmembrane
.alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4,
.alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8,
.alpha.-helix 9, .alpha.-helix 10, .alpha.-helix 11, .alpha.-helix
12, and transmembrane .alpha.-helix 10 and transmembrane
.alpha.-helix 11 and the sequence between them contain SEQ ID NO:
8. In certain embodiments that may be combined with any of the
preceding embodiments, the polypeptide has at least 29%, at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
99%, or at least 100% amino acid identity to NCU00801 or NCU08114.
In certain embodiments that may be combined with any of the
preceding embodiments, the .beta.-glucosidase is from Neurospora
crassa. In certain embodiments that may be combined with the
preceding embodiments having a host cell containing a second
recombinant polynucleotide encoding a catalytic domain of a
.beta.-glucosidase from Neurospora crassa, the .beta.-glucosidase
is encoded by NCU00130. In certain embodiments that may be combined
with any of the preceding embodiments, the host cell further
contains one or more recombinant polynucleotides where the one or
more polynucleotides encode one or more enzymes involved in pentose
utilization. In certain embodiments that may be combined with the
preceding embodiments having a host cell further containing one or
more recombinant polynucleotides where the one or more
polynucleotides encode one or more enzymes involved in pentose
utilization, the one or more enzymes are selected from one or more
of the group consisting of L-arabinose isomerase, L-ribulokinase,
L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose
reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and
xylitol dehydrogenase. In certain embodiments that may be combined
with any of the preceding embodiments, the host cell further
contains a third recombinant polynucleotide where the third
recombinant polynucleotide encodes a pentose transporter. In
certain embodiments that may be combined with the preceding
embodiments having the host cell further containing a third
recombinant polynucleotide where the third recombinant
polynucleotide encodes a pentose transporter, the pentose
transporter is selected from the group consisting of NCU00821,
NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, and XUT3. In
certain embodiments that may be combined with any of the preceding
embodiments, the cellulose-derived sugar is selected from the group
consisting of cellobiose, cellotriose, and celltetraose, and the
hemicellulose-derived sugar is xylose.
[0031] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 1 contains
SEQ ID NO: 1, and the polypeptide transports cellodextrin into the
host cell for the synthesis of hydrocarbons or hydrocarbon
derivatives, and culturing the host cell in a medium containing
cellodextrin or a source of cellodextrin to increase the synthesis
of hydrocarbons or hydrocarbon derivatives by the host cell, where
transport of cellodextrin into the cell is increased upon
expression of the recombinant polynucleotide.
[0032] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
containing providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 2 contains
SEQ ID NO: 2, and the polypeptide transports cellodextrin into the
host cell for the synthesis of hydrocarbons or hydrocarbon
derivatives, and culturing the host cell in a medium containing
cellodextrin or a source of cellodextrin to increase the synthesis
of hydrocarbons or hydrocarbon derivatives by the host cell, where
transport of cellodextrin into the cell is increased upon
expression of the recombinant polynucleotide.
[0033] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and a loop connecting transmembrane
.alpha.-helix 2 and transmembrane .alpha.-helix 3 contains SEQ ID
NO: 3, and the polypeptide transports cellodextrin into the host
cell for the synthesis of hydrocarbons or hydrocarbon derivatives,
and culturing the host cell in a medium containing cellodextrin or
a source of cellodextrin to increase the synthesis of hydrocarbons
or hydrocarbon derivatives by the host cell, where transport of
cellodextrin into the cell is increased upon expression of the
recombinant polynucleotide.
[0034] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 5 contains
SEQ ID NO: 4, and the polypeptide transports cellodextrin into the
host cell for the synthesis of hydrocarbons or hydrocarbon
derivatives, and culturing the host cell in a medium containing
cellodextrin or a source of cellodextrin to increase the synthesis
of hydrocarbons or hydrocarbon derivatives by the host cell, where
transport of cellodextrin into the cell is increased upon
expression of the recombinant polynucleotide.
[0035] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 6 contains
SEQ ID NO: 5, and the polypeptide transports cellodextrin into the
host cell for the synthesis of hydrocarbons or hydrocarbon
derivatives, and culturing the host cell in a medium containing
cellodextrin or a source of cellodextrin to increase the synthesis
of hydrocarbons or hydrocarbon derivatives by the host cell, where
transport of cellodextrin into the cell is increased upon
expression of the recombinant polynucleotide.
[0036] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and sequence between transmembrane
.alpha.-helix 6 and transmembrane .alpha.-helix 7 contains SEQ ID
NO: 6, and the polypeptide transports cellodextrin into the host
cell for the synthesis of hydrocarbons or hydrocarbon derivatives,
and culturing the host cell in a medium containing cellodextrin or
a source of cellodextrin to increase the synthesis of hydrocarbons
or hydrocarbon derivatives by the host cell, where transport of
cellodextrin into the cell is increased upon expression of the
recombinant polynucleotide.
[0037] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 7 contains
SEQ ID NO: 7, and the polypeptide transports cellodextrin into the
host cell for the synthesis of hydrocarbons or hydrocarbon
derivatives, and culturing the host cell in a medium containing
cellodextrin or a source of cellodextrin to increase the synthesis
of hydrocarbons or hydrocarbon derivatives by the host cell, where
transport of cellodextrin into the cell is increased upon
expression of the recombinant polynucleotide.
[0038] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, .alpha.-helix 12, and transmembrane .alpha.-helix 10 and
transmembrane .alpha.-helix 11 and the sequence between them
contain SEQ ID NO: 8, and the polypeptide transports cellodextrin
into the host cell for the synthesis of hydrocarbons or hydrocarbon
derivatives, and culturing the host cell in a medium containing
cellodextrin or a source of cellodextrin to increase the synthesis
of hydrocarbons or hydrocarbon derivatives by the host cell, where
transport of cellodextrin into the cell is increased upon
expression of the recombinant polynucleotide.
[0039] In certain embodiments that may be combined with any of the
preceding aspects increasing the synthesis of hydrocarbons or
hydrocarbon derivatives, the polypeptide has at least 29%, at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
99%, or at least 100% amino acid identity to NCU00801 or NCU08114.
In certain embodiments that may be combined with any of the
preceding embodiments, the host cell further contains a second
recombinant polynucleotide where the polynucleotide encodes at
least a catalytic domain of a .beta.-glucosidase. In certain
embodiments that may be combined with preceding embodiments having
the host cell further containing a second recombinant
polynucleotide where the polynucleotide encodes at least a
catalytic domain of a .beta.-glucosidase, the .beta.-glucosidase is
from Neurospora crassa. In certain embodiments that may be combined
with preceding embodiments having the host cell further containing
a second recombinant polynucleotide where the polynucleotide
encodes at least a catalytic domain of a .beta.-glucosidase from
Neurospora crassa, the .beta.-glucosidase is encoded by NCU00130.
In certain embodiments that may be combined with any of the
preceding embodiments, the source of the cellodextrin contains
cellulose. In certain embodiments that may be combined with any of
the preceding embodiments, the hydrocarbons or hydrocarbon
derivatives can be used as fuel. In certain embodiments that may be
combined with the preceding embodiments having the hydrocarbons or
hydrocarbon derivatives used as fuel, the hydrocarbons or
hydrocarbon derivatives contain ethanol. In certain embodiments
that may be combined with the preceding embodiments having the
hydrocarbons or hydrocarbon derivatives used as fuel, the
hydrocarbons or hydrocarbon derivatives contain butanol.
[0040] In certain embodiments that may be combined with any of the
preceding aspects, the medium contains a cellulase-containing
enzyme mixture from an altered organism, where the
cellulase-containing mixture has reduced .beta.-glucosidase
activity compared to a cellulase-containing mixture from an
unaltered organism. In certain embodiments that may be combined
with any of the preceding aspects, the host cell is selected from
the group consisting of Saccharomyces sp., Saccharomyces
cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus,
Saccharomyces pastorianus, Saccharomyces carlsbergensis,
Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus,
Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis,
Sporotrichum thermophile, Candida shehatae, Candida tropicalis,
Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium
phytofermentans, Clostridium thermocellum, Clostridium
beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica,
Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium
saccharolyticum, and Bacillus subtilis. In certain embodiments that
may be combined with any of the preceding aspects, cellodextrin is
selected from one or more of the group consisting of cellobiose,
cellotriose, and cellotetraose.
[0041] Another aspect includes host cells containing a recombinant
polynucleotide encoding a polypeptide having transmembrane
.alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4,
.alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8,
.alpha.-helix 9, .alpha.-helix 10, .alpha.-helix 11, .alpha.-helix
12, an intracellular N-terminus, an intracellular C-terminus, and a
sequence selected from the group consisting of SEQ ID NO: 1 in
transmembrane .alpha.-helix 1, SEQ ID NO: 2 in transmembrane
.alpha.-helix 2, SEQ ID NO: 3 in a loop connecting transmembrane
.alpha.-helix 2 and transmembrane .alpha.-helix 3, SEQ ID NO: 4 in
transmembrane .alpha.-helix 5, SEQ ID NO: 5 in transmembrane
.alpha.-helix 6, SEQ ID NO: 6 in the sequence between transmembrane
.alpha.-helix 6 and transmembrane .alpha.-helix 7, SEQ ID NO: 7 in
transmembrane .alpha.-helix 7, and SEQ ID NO: 8 in transmembrane
.alpha.-helix 10 and transmembrane .alpha.-helix 11 and the
sequence between them, where the polypeptide is a cellodextrin
transporter. In certain embodiments, the polypeptide has at least
29%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 99%, or at least 100% amino acid identity to NCU00801
or NCU08114. In certain embodiments that may be combined with
either of the preceding embodiments, the host cell further contains
a second recombinant polynucleotide where the second recombinant
polynucleotide encodes a catalytic domain of a .beta.-glucosidase.
In certain embodiments that may be combined with preceding
embodiments having the host cell further containing a second
recombinant polynucleotide where the second recombinant
polynucleotide encodes a catalytic domain of a .beta.-glucosidase,
the .beta.-glucosidase is from Neurospora crassa. In certain
embodiments that may be combined with the preceding embodiments
having the host cell further containing a second recombinant
polynucleotide where the second recombinant polynucleotide encodes
a catalytic domain of a .beta.-glucosidase from Neurospora crassa,
the .beta.-glucosidase is encoded by NCU00130. In certain
embodiments that may be combined with any of the preceding
embodiments, the host cell further contains one or more recombinant
polynucleotides where the one or more polynucleotides encode one or
more enzymes involved in pentose utilization. In certain
embodiments that may be combined with the preceding embodiments
having the host cell further containing one or more recombinant
polynucleotides where the one or more polynucleotides encode one or
more enzymes involved in pentose utilization, the one or more
enzymes are selected from one or more of the group consisting of
L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase,
xylose isomerase, xylulokinase, aldose reductase, L-arabinitol
4-dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase.
In certain embodiments that may be combined with any of the
preceding embodiments, the host cell further contains a third
recombinant polynucleotide where the third recombinant
polynucleotide encodes a pentose transporter. In certain
embodiments that may be combined with the preceding embodiment
having the host cell further containing a third recombinant
polynucleotide where the third recombinant polynucleotide encodes a
pentose transporter, the pentose transporter is selected from the
group consisting of NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2,
SUT3, XUT1, and XUT3.
[0042] In certain embodiments that may be combined with any of the
preceding aspects, the host cell further contains one or more
inducible promoters operably linked to the one or more recombinant
polynucleotides.
[0043] Another aspect includes a host cell containing a recombinant
polynucleotide encoding a polypeptide selected from the group
consisting of NCU00821 and STL12/XUT6, where the polypeptide
transports xylose into the cell.
[0044] Another aspect includes a host cell containing a recombinant
polynucleotide encoding a XUT1 polypeptide, where the polypeptide
transports arabinose into the cell.
[0045] Another aspect includes a host cell containing a recombinant
polynucleotide encoding an NCU06138 polypeptide, where the
polypeptide transports arabinose and glucose into the cell.
[0046] Another aspect includes a host cell containing a recombinant
polynucleotide encoding a polypeptide selected from the group
consisting of SUT2, SUT3, and XUT3, where the polypeptide
transports xylose and glucose into the cell.
[0047] Another aspect includes a host cell containing a recombinant
polynucleotide encoding an NCU04963 polypeptide, where the
polypeptide transports xylose, arabinose, and glucose into the
cell.
[0048] In certain embodiments that may be combined with any of the
preceding aspects having a host cell containing a recombinant
polynucleotide encoding a pentose transporter, the host cell
further contains one or more recombinant polynucleotides where the
one or more polynucleotides encode one or more enzymes involved in
pentose utilization. In certain embodiments that may be combined
with the preceding embodiment having the host cell further
containing one or more recombinant polynucleotides where the one or
more polynucleotides encode one or more enzymes involved in pentose
utilization, the one or more enzymes are selected from one or more
of the group consisting of L-arabinose isomerase, L-ribulokinase,
L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose
reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and
xylitol dehydrogenase.
[0049] Another aspect includes methods of increasing transport of
xylose into a cell, including providing a host cell, where the host
cell contains a recombinant polynucleotide encoding a polypeptide
selected from the group consisting of NCU00821 and STL12/XUT6, and
culturing the cell such that the recombinant polynucleotide is
expressed, where expression of the recombinant polynucleotide
results in increased transport of xylose into the cell compared
with a cell that does not contain the recombinant
polynucleotide.
[0050] Another aspect includes methods of increasing transport of
arabinose into a cell, including providing a host cell, where the
host cell contains a recombinant polynucleotide encoding a XUT1
polypeptide, and culturing the cell such that the recombinant
polynucleotide is expressed, where expression of the recombinant
polynucleotide results in increased transport of arabinose into the
cell compared with a cell that does not contain the recombinant
polynucleotide.
[0051] Another aspect includes methods of increasing transport of
arabinose or glucose into a cell, including providing a host cell,
where the host cell contains a recombinant polynucleotide encoding
a NCU06138 polypeptide, and culturing the cell such that the
recombinant polynucleotide is expressed, where expression of the
recombinant polynucleotide results in increased transport of
arabinose or glucose into the cell compared with a cell that does
not contain the recombinant polynucleotide.
[0052] Another aspect includes methods of increasing transport of
xylose or glucose into a cell, including providing a host cell,
where the host cell contains a recombinant polynucleotide encoding
a polypeptide selected from the group consisting of SUT2, SUT3, and
XUT3, and culturing the cell such that the recombinant
polynucleotide is expressed, where expression of the recombinant
polynucleotide results in increased transport of xylose or glucose
into the cell compared with a cell that does not contain the
recombinant polynucleotide.
[0053] Another aspect includes methods of increasing transport of
xylose, arabinose, or glucose into a cell, including providing a
host cell, where the host cell contains a recombinant
polynucleotide encoding a NCU04963 polypeptide, and culturing the
cell such that the recombinant polynucleotide is expressed, where
expression of the recombinant polynucleotide results in increased
transport of xylose, arabinose, or glucose into the cell compared
with a cell that does not contain the recombinant
polynucleotide.
[0054] In certain embodiments that may be combined with any of the
preceding aspects of increasing transport of xylose, arabinose, or
glucose into cells, the method further includes one or more
recombinant polynucleotides where the one or more polynucleotides
encode one or more enzymes involved in pentose utilization. In
certain embodiments that may be combined with the preceding
embodiments having the method further including one or more
recombinant polynucleotides where the one or more polynucleotides
encode one or more enzymes involved in pentose utilization, the one
or more enzymes are selected from one or more of the group
consisting of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P
4 epimerase, xylose isomerase, xylulokinase, aldose reductase,
L-arabinitol 4-dehydrogenase, L-xylulose reductase, and xylitol
dehydrogenase.
[0055] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide where the polynucleotide encodes a
polypeptide selected from the group consisting of NCU00821 and
STL12/XUT6, and the polypeptide transports xylose, and culturing
the host cell in a medium containing xylose, where the host cell
grows at a faster rate in the medium than a cell that does not
contain the recombinant polynucleotide.
[0056] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide where the polynucleotide encodes a
XUT1 polypeptide, and the polypeptide transports arabinose, and
culturing the host cell in a medium containing arabinose, where the
host cell grows at a faster rate in the medium than a cell that
does not contain the recombinant polynucleotide.
[0057] Another aspect includes method of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide where the polynucleotide encodes an
NCU06138 polypeptide, and the polypeptide transports arabinose and
glucose, and culturing the host cell in a medium containing
arabinose or glucose, where the host cell grows at a faster rate in
the medium than a cell that does not contain the recombinant
polynucleotide.
[0058] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide where the polynucleotide encodes a
polypeptide selected from the group consisting of SUT2, SUT3, and
XUT3, and the polypeptide transports xylose and glucose, and
culturing the host cell in a medium including xylose or glucose,
where the host cell grows at a faster rate in the medium than a
cell that does not contain the recombinant polynucleotide.
[0059] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide where the polynucleotide encodes a
NCU04963 polypeptide, and the polypeptide transports xylose,
arabinose, and glucose, and culturing the host cell in a medium
containing xylose, arabinose, or glucose, where the host cell grows
at a faster rate in the medium than a cell that does not contain
the recombinant polynucleotide.
[0060] In certain embodiments that may be combined with the
preceding aspects of increasing growth of cells by culturing a host
cell containing a recombinant polynucleotide encoding a polypeptide
that transports xylose and/or arabinose and/or glucose, the host
cell further contains one or more endogenous or recombinant
polynucleotides encoding one or more enzymes involved in pentose
utilization. In certain embodiments that may be combined with the
preceding embodiments having the host cell further containing one
or more endogenous or recombinant polynucleotides encoding one or
more enzymes involved in pentose utilization, the one or more
enzymes are selected from one or more of the group consisting of
L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase,
xylose isomerase, xylulokinase, aldose reductase, L-arabinitol
4-dehydrogenase, L-xylulose reductase, and xylitol
dehydrogenase.
[0061] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a polypeptide selected from the
group consisting of NCU00821 and STL12/XUT6, where the polypeptide
transports xylose into the host cell for the synthesis of
hydrocarbons or hydrocarbon derivatives, and culturing the host
cell in a medium containing xylose or a source of xylose to
increase the synthesis of hydrocarbons or hydrocarbon derivatives
by the host cell, where transport of xylose into the cell is
increased upon expression of the recombinant polynucleotide.
[0062] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a XUT1 polypeptide, where the
polypeptide transports arabinose into the host cell for the
synthesis of hydrocarbons or hydrocarbon derivatives, and culturing
the host cell in a medium containing arabinose or a source of
arabinose to increase the synthesis of hydrocarbons or hydrocarbon
derivatives by the host cell, where transport of arabinose into the
cell is increased upon expression of the recombinant
polynucleotide.
[0063] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding an NCU06138 polypeptide, where
the polypeptide transports arabinose or glucose into the host cell
for the synthesis of hydrocarbons or hydrocarbon derivatives, and
culturing the host cell in a medium containing arabinose or glucose
or a source of arabinose or glucose to increase the synthesis of
hydrocarbons or hydrocarbon derivatives by the host cell, where
transport of arabinose or glucose into the cell is increased upon
expression of the recombinant polynucleotide.
[0064] Another aspect includes method of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding a polypeptide selected from the
group consisting of SUT2, SUT3, and XUT3, where the polypeptide
transports xylose or glucose into the host cell for the synthesis
of hydrocarbons or hydrocarbon derivatives, and culturing the host
cell in a medium containing xylose or glucose or a source of xylose
or glucose to increase the synthesis of hydrocarbons or hydrocarbon
derivatives by the host cell, where transport of xylose or glucose
into the cell is increased upon expression of the recombinant
polynucleotide.
[0065] Another aspect includes methods of increasing the synthesis
of hydrocarbons or hydrocarbon derivatives by a host cell,
including providing a host cell, where the host cell contains a
recombinant polynucleotide encoding an NCU04963 polypeptide, where
the polypeptide transports xylose, arabinose, or glucose into the
host cell for the synthesis of hydrocarbons or hydrocarbon
derivatives, and culturing the host cell in a medium containing
xylose, arabinose, or glucose or a source of xylose, arabinose, or
glucose to increase the synthesis of hydrocarbons or hydrocarbon
derivatives by the host cell, where transport of xylose, arabinose,
or glucose into the cell is increased upon expression of the
recombinant polynucleotide.
[0066] In certain embodiments that may combine any of the preceding
aspects of increasing the synthesis of hydrocarbons or hydrocarbon
derivatives by culturing a host cell containing a recombinant
polynucleotide encoding a polypeptide that transports glucose, the
source of glucose contains cellulose. In certain embodiments that
may combine any of the preceding embodiments, the source of xylose
or arabinose contains hemicellulose. In certain embodiments that
may combine any of the preceding embodiments, the hydrocarbons or
hydrocarbon derivatives can be used as fuel. In certain embodiments
that may combine the preceding embodiment having the hydrocarbons
or hydrocarbon derivatives used as fuel, the hydrocarbons or
hydrocarbon derivatives contain ethanol. In certain embodiments
that may combine the preceding embodiment having the hydrocarbons
or hydrocarbon derivatives used as fuel, the hydrocarbons or
hydrocarbon derivatives contain butanol.
[0067] In certain embodiments that may combine any of the preceding
embodiments, the host cell is selected from the group consisting of
Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces
monacensis, Saccharomyces bayanus, Saccharomyces pastorianus,
Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces
sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces
fragilis, Pichia stipitis, Sporotrichum thermophile, Candida
shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis,
Clostridium sp., Clostridium phytofermentans, Clostridium
thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum,
Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca,
Thermoanaerobacterium saccharolyticum, and Bacillus subtilis.
[0068] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide where the polynucleotide encodes a
NCU07705 polypeptide, and culturing the cell in a medium containing
cellulose, where the host cell grows at a faster rate in the medium
than a cell that does not contain the recombinant polynucleotide.
In certain embodiments, the host cell is selected from the group
consisting of Saccharomyces sp., Saccharomyces cerevisiae,
Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces
pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe,
Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis,
Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile,
Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas
mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium
thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum,
Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca,
Thermoanaerobacterium saccharolyticum, and Bacillus subtilis. In
certain embodiments, the host cell further contains an inducible
promoter operably linked to the recombinant polynucleotide. In
certain embodiments, expression of cellulases is increased in the
host cell upon expression of the recombinant polynucleotide.
[0069] Another aspect includes methods of increasing growth of a
cell on a biomass polymer, including providing a host cell, where
the host cell contains an endogenous polynucleotide where the
polynucleotide encodes an NCU05137 polypeptide, inhibiting
expression of the endogenous polynucleotide, and culturing the cell
in a medium containing the biomass polymer, where the host cell
grows at a faster rate in the medium than a cell in which
expression of the endogenous polynucleotide is not inhibited. In
certain embodiments, the host cell is selected from the group
consisting of Saccharomyces sp., Saccharomyces cerevisiae,
Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces
pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe,
Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis,
Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile,
Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas
mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium
thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum,
Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca,
Thermoanaerobacterium saccharolyticum, and Bacillus subtilis. In
certain embodiments, cellulase activity of the host cell is
increased upon inhibiting expression of the endogenous
polynucleotide. In certain embodiments, hemicellulase activity of
the host cell is increased upon inhibiting expression of the
endogenous polynucleotide. In certain embodiments, inhibiting
expression of the endogenous polynucleotide contains mutating or
deleting a gene containing the endogenous polynucleotide. In
certain embodiments, the biomass polymer is cellulose. In certain
embodiments, the biomass polymer is hemicellulose.
[0070] Another aspect includes methods of increasing growth of a
cell, including providing a host cell, where the host cell contains
a recombinant polynucleotide where the polynucleotide encodes a
polypeptide selected from the group consisting of NCU01517,
NCU09133, and NCU10040, and culturing the cell in a medium
containing hemicellulose, where the host cell grows at a faster
rate in the medium than a cell that does not contain the
recombinant polynucleotide. In certain embodiments, the host cell
is selected from the group consisting of Saccharomyces sp.,
Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces
bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis,
Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus,
Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis,
Sporotrichum thermophile, Candida shehatae, Candida tropicalis,
Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium
phytofermentans, Clostridium thermocellum, Clostridium
beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica,
Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium
saccharolyticum, and Bacillus subtilis. In certain embodiments, the
host cell further contains an inducible promoter operably linked to
the recombinant polynucleotide. In certain embodiments,
hemicellulase activity of the host cell is increased upon
expression of the recombinant polynucleotide.
[0071] Another aspect includes methods of degrading cellulose,
including providing a composition containing cellulose, and
contacting the composition with a cellulase-containing enzyme
mixture from an altered organism, where the cellulase-containing
mixture has reduced .beta.-glucosidase activity compared to a
cellulase-containing mixture from an unaltered organism, and where
the cellulose is degraded by the cellulase-containing mixture. In
certain embodiments, the organism is altered by mutation of a gene
encoding a .beta.-glucosidase. In certain embodiments, the organism
is altered by reducing the expression of a .beta.-glucosidase. In
certain embodiments that may be combined with any of the preceding
embodiments, the organism is selected from the group consisting of
a fungus and a bacterium. In certain embodiments that may be
combined with any of the preceding embodiments having the organism
selected from the group consisting of a fungus and a bacterium, the
organism is a filamentous fungus. In certain embodiments that may
be combined with any of the preceding embodiments, the cellulose is
from plant material. In certain embodiments that may be combined
with the preceding embodiments having the cellulose from plant
material, the plant material is selected from the group consisting
of switchgrass, Miscanthus, rice hulls, bagasse, flax, bamboo,
sisal, abaca, straw, leaves, grass clippings, corn stover, corn
cobs, distillers grains, legume plants, sorghum, sugar cane, sugar
beet pulp, wood chips, sawdust, and biomass crops.
[0072] Yet another aspect includes methods of increasing the
synthesis of hydrocarbons or hydrocarbon derivatives by a host cell
comprising providing a host cell, wherein the host cell comprises a
recombinant polynucleotide wherein the polynucleotide encodes a
polypeptide encoded by a sequence selected from the group
consisting of NCU00801, NCU00988, NCU01231, NCU04963, NCU05519,
NCU05853, NCU05897, NCU06138, NCU00809, NCU08114, NCU10021, and any
of the genes listed in Table 15 and culturing the host cell in a
medium comprising a source of a compound to increase the synthesis
of hydrocarbons or hydrocarbon derivatives by the host cell,
wherein the compound is a substrate for the synthesis of the
hydrocarbons or hydrocarbon derivatives, and wherein transport of
the compound into the cell is increased upon expression of the
recombinant polynucleotide. In certain embodiments, the host cell
is selected from the group consisting of Saccharomyces cerevisiae,
Escherichia coli, Zymomonas mobilis, Neurospora crassa, Candida
shehatae, Clostridium sp., Clostridium phytofermentans, Clostridium
thermocellum, Moorella thermocetica, Thermoanaerobacterium
saccharolyticum, Klebsiella oxytoca, and Pichia stipitis. In
certain embodiments, the host cell further comprises an inducible
promoter operably linked to the recombinant polynucleotide. In
certain embodiments, the recombinant polynucleotide encodes a
polypeptide having at least 50% amino acid identity to the
polypeptide encoded by a sequence selected from the group
consisting of NCU00801, NCU00988, NCU01231, NCU04963, NCU05519,
NCU05853, NCU05897, NCU06138, NCU00809, NCU08114, NCU10021, and any
of the genes listed in Table 15. In some embodiments, the
hydrocarbons or hydrocarbon derivatives can be used as fuel. In
certain embodiments, the medium comprises cellulose. In other
embodiments, the medium comprises hemicellulose. In certain
embodiments, the compound is a sugar. In certain embodiments that
may be combined with the preceding embodiments, the sugar is a
pentose. In certain embodiments that may be combined with the
preceding embodiments, the sugar is a hexose. In certain
embodiments that may be combined with the preceding embodiments,
the sugar is a disaccharide. In certain embodiments that may be
combined with the preceding embodiments, the sugar is an
oligosaccharide. In other embodiments, the compound is a plant
phenol. In certain embodiments that may be combined with the
preceding embodiments, the plant phenol is quinic acid. In certain
embodiments that may be combined with the preceding embodiments,
the plant phenol is nicotinamide. In other embodiments, the
compound is pyruvate or lactate.
[0073] Another aspect includes methods of increasing growth of a
cell on a biomass polymer comprising providing a host cell, wherein
the host cell comprises a recombinant polynucleotide wherein the
polynucleotide encodes a polypeptide encoded by any of the
Neurospora or Pichia stipitis genes listed in Table 10, in
Supplemental Data, Dataset S1, page 3 in Tian et al., PNAS, 2009,
vol. 106, no. 52, 22157-22162, the disclosure of which is hereby
incorporated by reference, in Table 15, or NCU01517, NCU09133, or
NCU10040 and culturing the cell in a medium comprising the biomass
polymer, wherein the host cell grows at a faster rate in the medium
than a cell that does not comprise the recombinant polynucleotide.
In certain embodiments, the polynucleotide encodes a polypeptide
encoded by any of the sequences NCU00130.2, NCU00248.2, NCU00326.2,
NCU00762.2, NCU00810.2, NCU00890.2, NCU03328.2, NCU03415.2,
NCU03731.2, NCU03753.2, NCU04197.2, NCU04249.2, NCU04287.2,
NCU04349.2, NCU04475.2, NCU04997.2, NCU05057.2, NCU05159.2,
NCU05493.2, NCU05751.2, NCU05770.2, NCU05932.2, NCU06009.2,
NCU06490.2, NCU07340.2, NCU07853.2, NCU07997.2, NCU08744.2,
NCU08746.2, NCU08760.2, NCU09108.2, NCU09495.2, NCU09680.2, or
NCU10045.2.
[0074] In certain embodiments, the polynucleotide encodes a
polypeptide encoded by NCU07705. In certain embodiments, the
recombinant polynucleotide encodes a polypeptide having at least
50% amino acid identity to the polypeptide encoded by any of the
Neurospora or Pichia stipitis genes listed in Table 10, in
Supplemental Data, Dataset S1, page 3 in Tian et al., 2009, or in
Table 15. In certain embodiments, the polynucleotide encodes a
polypeptide having at least 50% amino acid identity to the
polypeptide encoded by any of the sequences NCU00130.2, NCU00248.2,
NCU00326.2, NCU00762.2, NCU00810.2, NCU00890.2, NCU03328.2,
NCU03415.2, NCU03731.2, NCU03753.2, NCU04197.2, NCU04249.2,
NCU04287.2, NCU04349.2, NCU04475.2, NCU04997.2, NCU05057.2,
NCU05159.2, NCU05493.2, NCU05751.2, NCU05770.2, NCU05932.2,
NCU06009.2, NCU06490.2, NCU07340.2, NCU07853.2, NCU07997.2,
NCU08744.2, NCU08746.2, NCU08760.2, NCU09108.2, NCU09495.2,
NCU09680.2, or NCU10045.2. In certain embodiments, the recombinant
polynucleotide encodes a polypeptide having at least 50% amino acid
identity to the polypeptide encoded by NCU07705. In certain
embodiments, the biomass polymer is cellulose. In other
embodiments, the biomass polymer is hemicellulose. In certain
embodiments, the host cell is selected from the group consisting of
Saccharomyces cerevisiae, Escherichia coli, Zymomonas mobilis,
Neurospora crassa, Candida shehatae, Clostridium sp., Clostridium
phytofermentans, Clostridium thermocellum, Moorella thermocetica,
Thermoanaerobacterium saccharolyticum, Klebsiella oxytoca, and
Pichia stipitis. In certain embodiments, the host cell further
comprises an inducible promoter operably linked to the recombinant
polynucleotide. In certain embodiments, expression of cellulases is
increased in the host cell upon expression of the recombinant
polynucleotide. In other embodiments, expression of hemicellulases
is increased in the host cell upon expression of the recombinant
polynucleotide.
[0075] Yet another aspect includes methods of increasing growth of
a cell on a biomass polymer comprising providing a host cell,
wherein the host cell comprises an endogenous polynucleotide
wherein the polynucleotide encodes a polypeptide encoded by any of
the Neurospora or Pichia stipitis genes listed in Table 10, in
Supplemental Data, Dataset S1, page 3 in Tian et al., 2009, or in
Table 15, or, inhibiting expression of the endogenous
polynucleotide, and culturing the cell in a medium comprising the
biomass polymer, wherein the host cell grows at a faster rate in
the medium than a cell in which expression of the endogenous
polynucleotide is not inhibited. In certain embodiments, the
endogenous polynucleotide encodes a polypeptide encoded by any of
the sequences NCU00130.2, NCU00248.2, NCU00326.2, NCU00762.2,
NCU00810.2, NCU00890.2, NCU03328.2, NCU03415.2, NCU03731.2,
NCU03753.2, NCU04197.2, NCU04249.2, NCU04287.2, NCU04349.2,
NCU04475.2, NCU04997.2, NCU05057.2, NCU05159.2, NCU05493.2,
NCU05751.2, NCU05770.2, NCU05932.2, NCU06009.2, NCU06490.2,
NCU07340.2, NCU07853.2, NCU07997.2, NCU08744.2, NCU08746.2,
NCU08760.2, NCU09108.2, NCU09495.2, NCU09680.2, or NCU10045.2. In
certain embodiments, the endogenous polynucleotide encodes a
polypeptide encoded by NCU05137. In certain embodiments, the
endogenous polynucleotide encodes a polypeptide having at least 50%
amino acid identity to the polypeptide encoded by any of the
Neurospora or Pichia stipitis genes listed in Table 10, in
Supplemental Data, Dataset S1, page 3 in Tian et al., 2009, or in
Table 15. In certain embodiments, the endogenous polynucleotide
encodes a polypeptide having at least 50% amino acid identity to
the polypeptide encoded by any of the sequences NCU00130.2,
NCU00248.2, NCU00326.2, NCU00762.2, NCU00810.2, NCU00890.2,
NCU03328.2, NCU03415.2, NCU03731.2, NCU03753.2, NCU04197.2,
NCU04249.2, NCU04287.2, NCU04349.2, NCU04475.2, NCU04997.2,
NCU05057.2, NCU05159.2, NCU05493.2, NCU05751.2, NCU05770.2,
NCU05932.2, NCU06009.2, NCU06490.2, NCU07340.2, NCU07853.2,
NCU07997.2, NCU08744.2, NCU08746.2, NCU08760.2, NCU09108.2,
NCU09495.2, NCU09680.2, or NCU10045.2. In certain embodiments, the
endogenous polynucleotide encodes a polypeptide having at least 50%
amino acid identity to the polypeptide encoded by NCU05137. In
certain embodiments, the host cell is selected from the group
consisting of Saccharomyces cerevisiae, Escherichia coli, Zymomonas
mobilis, Neurospora crassa, Candida shehatae, Clostridium sp.,
Clostridium phytofermentans, Clostridium thermocellum, Moorella
thermocetica, Thermoanaerobacterium saccharolyticum, Klebsiella
oxytoca, and Pichia stipitis. In certain embodiments, the biomass
polymer is cellulose. In other embodiments, the biomass polymer is
hemicellulose. In certain embodiments, cellulase activity of the
host cell is increased upon inhibiting expression of the endogenous
polynucleotide. In other embodiments, hemicellulase activity of the
host cell is increased upon inhibiting expression of the endogenous
polynucleotide. In certain embodiments, inhibiting expression of
the endogenous polynucleotide comprises mutating or deleting a gene
comprising the endogenous polynucleotide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0076] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0077] FIG. 1 shows the domain structure of the polypeptide encoded
by NCU07705.
[0078] FIG. 2 shows the phylogenetic analysis of NCU05137. The
predicted orthologs of N. crassa NCU05137 were retrieved from NCBI
and JGI based on amino acid sequences showing significant
similarity by BLAST. All identified filamentous fungal orthologs
are shown; NCBI E values were 0.0 except for B. fuckeliana, which
was 9e-175. Homologs of NCU05137 were also identified in a number
of bacteria (E value.about.e-30). YP 981875 from Polaromonas
naphthalenivorans (a beta-proteobacterium) was used as an outgroup.
A. Aspergillus; N.=Neosartorya; P. chyrosogenum=Penicillium;
S.=Sclerotinia; B.=Botryotinia; P.=Pyrenophora; C.=Cochliobolus; N.
haematococca=Nectria; P. anserina=Podospora; N.=Neurospora. The
tree was made by MEGA3, NJ. Bar=0.2 substitutions per amino acid
site.
[0079] FIG. 3 shows an analysis of N. crassa FGSC2489 and T. reesei
QM9414 endoglucanase activity when grown on Miscanthus and Avicel
as a sole carbon source. Endoglucanase activity in culture
filtrates of N. crassa WT strain FGSC2489 and T. reesei QM9414. N.
crassa was grown on Vogel's minimal medium containing 2% of either
Avicel or Miscanthus powder as a sole carbon source at 25.degree.
C. T. reesei strain was inoculated in MA medium with either 1%
Avicel or Miscanthus powder as sole carbon source at 25.degree. C.
Both strains were inoculated with the same amount of conidia
(1.times.10.sup.6/mL in 100 mL culture). The endoglucanase activity
in the cultures at different time points were measured at pH 4.5
using Azo-CM-cellulose as a substrate according to the
manufacturer's instructions (Megazyme, Ireland).
[0080] FIGS. 4A-C show transcriptional profiling of N. crassa grown
on Miscanthus and Avicel. FIG. 4A shows hierarchical clustering
analysis of 769 genes showing expression differences in Miscanthus
culture. Dark shading indicates higher relative expression and
light shading indicates lower relative expression. Lane 1:
Expression profile of a 16 hr Vogel's minimal medium N. crassa
culture (Vogel 1956). Lane 2: Expression profile of a culture grown
on Miscanthus as a sole carbon source for 16 hrs. Lanes 3, 4, 5:
Expression profiles from cultures grown on Miscanthus for 40 hrs, 5
days, and 10 days. The three clusters are shown as C1, C2, and C3.
The cluster that showed increased expression levels of most of the
cellulase and hemicellulase genes is boxed (C3 cluster). FIG. 4B
shows analysis of the overlap in expression profiles between the N.
crassa Miscanthus versus Avicel grown cultures (Top). Analysis and
overlap of proteins detected in the culture filtrates of N. crassa
grown on Miscanthus and Avicel by tandem mass spectrometry
(Bottom). FIG. 4C shows functional category (FunCat) enrichment
analysis (Ruepp 2004) of the 231 genes that showed an increase in
relative expression levels in Miscanthus cultures. Functional
categories that showed significant enrichment (p<0.001),
including the unclassified group are shown.
[0081] FIGS. 5A-B show the relative expression levels of N. crassa
genes encoding cellulases (A) and hemicellulases (B) during growth
on minimal medium (MM) and during growth on Miscanthus for 16 hr,
40 hr, 5 days and 10 days. FIG. 5A shows cellulases. FIG. 5B shows
hemicellulases.
[0082] FIGS. 6A-C show the protein profile and enzymatic activity
of culture supernatants from strains containing deletions of genes
encoding secreted proteins identified by MS. FIG. 6A shows SDS-PAGE
of proteins present in the culture filtrates of 16 deletion strains
as compared to wild type when grown on Avicel for 7 days. Deletion
strains were chosen based on identification of the protein by mass
spectrometry in both Miscanthus and Avicel culture filtrates.
Strains are ordered based on gene NCU number, the wild-type strain
is FGSC 2489. Missing protein bands that correspond to the deleted
genes are marked with boxes. FIG. 6B shows the total secreted
protein, azo-CMCase, and .beta.-glucosidase activity assays (see
Example 5) performed on 16 deletion strains and the wild-type
parental strain (FGSC 2489) using the same sample from FIG. 6A.
Activities and protein concentrations were normalized compared to
wild type levels and represent the average of triplicate biological
measurements. FIG. 6C show cellulase activity of the culture
filtrates from the 16 deletion strains using the same samples as in
FIG. 6A. Culture filtrates were diluted 10 fold and mixed with 5
mg/mL Avicel (see Example 5) to assess Avicelase activity. Glucose
(black) and cellobiose (white) were measured after 8 hours of
incubation at 40.degree. C.
[0083] FIG. 7 shows the identity of N. crassa secreted proteins
based on mutant analysis from a culture grown on Avicel as a sole
carbon source. SDS-PAGE of secreted proteins from WT N. crassa
(FGSC 2489) grown on 2% Avicel in 100 mL shake flasks for 7 days at
25.degree. C. 15 .mu.L of unconcentrated culture filtrate was
loaded onto Criterion 4-15% 26-well gel. Proto Blue Safe
(Coomassie) from National Diagnostics was used to stain the gel.
The protein bands were identified in this study as shown in FIG. 6A
based on analysis of secreted proteins in deletion strains.
[0084] FIGS. 8A-B show the profile of secreted proteins and
expression of cbh-1 (NCU07340) and gh6-2 (NCU09680; CBHII) in
.DELTA.NCU04952 and .DELTA.NCU05137. FIG. 8A shows SDS-PAGE of
total secreted proteins in WT, .DELTA.NCU04952, and
.DELTA.NCU05137. Cultures were grown on Avicel from conidia, and
harvested at 30 hrs, two days (48 hrs) and three days (72 hrs) (see
Example 5). Lanes 1-3, 20.times. concentrated culture filtrates
after 30 hrs of growth on Avicel from WT, .DELTA.NCU04952, and
.DELTA.NCU05137 strains, respectively. Lanes 4-6, unconcentrated
culture filtrates after two days of growth from WT,
.DELTA.NCU04952, and .DELTA.NCU05137 strains, respectively. Lane
7-9, unconcentrated culture filtrates after three days of growth
from WT and .DELTA.NCU04952 and .DELTA.NCU05137 strains,
respectively. FIG. 8B shows RT-PCR of cbh-1 (NCU07340; CBHI) and
gh6-2 (NCU09680; CBHII) in the WT, .DELTA.NCU04952, and
.DELTA.NCU05137 strains during growth on Avicel. The WT and
deletion strains were grown on Avicel from conidia, and harvested
at 48 hrs and 72 hrs (see Example 5). The minimal medium (MM)
culture, with sucrose as a sole carbon source (Vogel 1956), was
grown for 16 hrs (similar developmental time point). The fold
induction of cbh-1 and gh6-2 were relative to the expression of
these genes under MM conditions, with actin gene expression used as
the control in all samples.
[0085] FIG. 9 shows a model of plant cell wall deconstruction in N.
crassa. Induction: Extracellular enzymes expressed at low levels
generate secondary metabolites that signal N. crassa to
dramatically increase the expression level of genes encoding plant
cell wall degrading enzymes, most of which are secreted.
Utilization: Extracellular enzymes and transporters specific for
translocation of cell wall degradation products enable N. crassa to
utilize plant cell material for growth. Some extracellular proteins
(NCU05137, NCU05057, and NCU04952) may generate metabolites that
modulate gene expression of cellulases and hemicellulase during the
utilization phase; double hexagon (cellobiose), double pentagon
(xylobiose), hexagon (glucose), and pentagon (xylose). The depicted
plant cell wall-degrading enzymes include CBH(I), CBH(II), EG2,
EG1, EG6, and xylanase. Additional cellulolytic enzymes are not
shown. Thickness of arrows indicates relative strength of
response.
[0086] FIG. 10-1, FIG. 10-2, FIG. 10-3, FIG. 10-4, FIG. 10-5, FIG.
10-6, FIG. 10-7, FIG. 10-8, FIG. 10-9, FIG. 10-10, FIG. 10-11, and
FIG. 10-12 show BLAST results from searching the sequences of N.
crassa putative transporters against a database of S. thermophile
protein sequences or from searching the sequences of S. thermophile
putative transporters against a database of N. crassa protein
sequences.
[0087] FIGS. 11A-B show the growth phenotype of a N. crassa strain
lacking NCU08114. FIG. 11A shows shaker flasks of WT (left) and
.DELTA.NCU08114 (right) N. crassa strains after 3 days of growth
with crystalline cellulose as a carbon source. FIG. 11B shows the
mean Alamar Blue.COPYRGT. fluorescence from N. crassa cultures
grown with either sucrose or crystalline cellulose as a carbon
source for 16 or 28 hours, respectively. Fluorescence was
normalized by setting WT to 100%. Error bars were the standard
deviation between measurements from three biological replicates. N.
crassa lacking NCU00801 did not have an obvious phenotype. N.
crassa secreted .beta.-glucosidases (Tian et al., 2009) that
hydrolyzed cellodextrins to glucose, which was subsequently taken
up by monosaccharide transporters (Scarborough 1973). This
alternate route of consumption led to an underestimate of the
cellodextrin transport defect in these deletion lines.
[0088] FIGS. 12A-D show (A) cellobiose consumption for S.
cerevisiae strains expressing NCU00801, NCU05853, or NCU08114 along
with NCU00130; (B) cellotriose consumption for S. cerevisiae
strains expressing NCU00801, NCU05853, or NCU08114 along with
NCU00130; (C) cellotetraose consumption for S. cerevisiae strains
expressing NCU00801, NCU05853, or NCU08114 along with NCU00130; and
(D) cellohexaose consumption for S. cerevisiae strains expressing
NCU00801, NCU05853, or NCU08114 along with NCU00130. FIG. 12A shows
cellobiose consumption. FIG. 12B shows cellotriose consumption.
FIG. 12C shows cellotetraose consumption. FIG. 12D shows
cellohexaose consumption.
[0089] FIG. 13 shows cellodextrin consumption by N. crassa strains
lacking NCU008114 or NCU00801. The indicated N. crassa strains were
incubated with 90 .mu.M of the respective sugars for 15 minutes.
Bars represent the mean concentration of sugars remaining in the
supernatant following the incubation from two independent
experiments. Error bars were the standard deviation between these
experiments.
[0090] FIG. 14 shows cellobiose transport by a S. cerevisiae strain
expressing NCU00801/cbt1. Shown is cellobiose transport by yeast
with (.largecircle.) or without ( ) CBT1. Both strains expressed
the intracellular .beta.-glucosidase, NCU00130. The initial
concentration of cellobiose was 50 .mu.M. All values were the mean
between two measurements, with error bars representing the standard
deviation between these measurements.
[0091] FIGS. 15A-B show localization and quantification of GFP
fused to CBT1 and CBT2. FIG. 15A shows images of S. cerevisiae
strains expressing cbt1 (left), or cbt2 (right), fused to GFP at
their C-terminus. FIG. 15B shows GFP fluorescence of yeast strains
without a cellobiose transporter, or expressing cbt1 or cbt2 fused
to GFP at their C-terminus. Values were the mean from three
biological replicates, and error bars represent the standard
deviation between these replicates.
[0092] FIGS. 16A-C show cellodextrin transport by N. crassa
transport systems expressed in S. cerevisiae. FIG. 16A shows
cellobiose-mediated growth of yeast strains expressing the gene
NCU00801 (named cbt1, .largecircle.), NCU08114 (named cbt2, ), or
no transporter ( ). All strains also expressed the intracellular
.beta.-glucosidase, NCU00130. A representative experiment is shown.
Growth rates from three independent experiments were as follows:
cbt1, 0.0341.+-.0.0010 hr.sup.-1; cbt2, 0.0131.+-.0.0008 hr.sup.-1;
no transporter, 0.0026.+-.0.0001 hr.sup.-1. FIG. 16B shows growth
of yeast strains on cellotriose and cellotetraose. Strains
expressing the intracellular .beta.-glucosidase, NCU00130, as well
as the transporters listed in the legend, were grown with 0.5%
(w/v) of cellotriose (G3) or cellotetraose (G4) serving as the sole
carbon source. A representative experiment is shown. Growth rates
from three independent experiments were as follows: cbt1
cellotriose, 0.0332.+-.0.0004 hr.sup.-1; cbt1 cellotetraose
0.0263.+-.0.0020 hr.sup.-1; no transporter cellotriose,
0.0043.+-.0.0015 hr.sup.-1; cbt2 cellotriose, 0.0178.+-.0.0005
hr.sup.-1; cbt2 cellotetraose 0.0041.+-.0.0003 hr.sup.-1; no
transporter cellotetraose, 0.0031.+-.0.0008 hr.sup.-1. FIG. 16C
shows glucose produced from cellobiose (G2), cellotriose (G3), and
cellotetraose (G4) hydrolysis by purified NCU00130. The mean and
standard deviation of three independent measurements are shown.
Residual glucose in incubations without enzyme (2 nmol) was
subtracted from the values shown.
[0093] FIG. 17 shows growth of S. cerevisiae strains expressing
cbt1 (.largecircle.), cbt2 (), or no transporter ( ) on glucose.
All strains expressed the .beta.-glucosidase, NCU00130. A
representative experiment is shown.
[0094] FIG. 18 shows cellobiose-mediated growth of S. cerevisiae
strains in 250 mL flasks. Values represent the mean OD between two
replicate cultures of yeast strains expressing the
.beta.-glucosidase, NCU00130, cbt1 or cbt2, or a strain expression
NCU00130, but lacking any transporters. Error bars represent the
standard deviation between replicates.
[0095] FIG. 19 shows kinetics of cellobiose transport by CBT1 and
CBT2. The rate of cellobiose transport was determined as a function
of cellobiose concentration by yeast strains expressing either cbt1
or cbt2. The transport rate was normalized for transporter
abundance.
[0096] FIG. 20 shows the ability of S. cerevisiae expressing the
combinations of Neurospora genes shown on the x-axis to grow on
cellobiose, cellotriose, or cellotetraose.
[0097] FIG. 21 shows competition by cellodextrins for cellobiose
transport in strains carrying cbt1 or cbt2. A 5-fold excess of the
respective unlabeled sugar was included during assays of
[.sup.3H]-cellobiose transport. Substrates of CBT1 or CBT2 would
decrease the [.sup.3H]-cellobiose transport rate by competing for
binding. Bars represent the mean from three replicates. Error bars
represent the standard deviation between these replicates. Values
were normalized by setting the rate of [.sup.3H]-cellobiose
transport without a competing sugar to 100.
[0098] FIG. 22 shows the SDS-PAGE gel of purified NCU00130. Lane 1,
Protein molecular weight standards, in kDa. Lane 2, NCU00130 after
purification over nickel-NTA resin. Molecular weights in kDa are
shown to the left.
[0099] FIG. 23 shows maximum likelihood phylogenetic analysis of
the cellobiose transporters NCU08114 and NCU00801. With the
exception of S. cerevisiae HXT1 and K. lactis LACP, all genes
encoding proteins shown are reported to increases in expression
level when the fungus comes into contact with plant cell wall
material or cellobiose (Tian et al., 2009; Noguchi et al., 2009;
Wymelenberg et al., 2010; Martin et al., 2010). S. cerevisiae HXT1,
a low affinity glucose transporter (Reifenberger et al., 1997), was
used as an outgroup.
[0100] FIGS. 24A-C show cellobiose fermentation, and simultaneous
saccharification and fermentation of cellulose, by S. cerevisiae
expressing the cellobiose transport system from N. crassa. FIG. 24A
shows cellobiose fermentation to ethanol. Ethanol produced by yeast
strains with CBT1 ( ), or without CBT1 (.largecircle.). Cellobiose
concentration during the fermentation reaction using yeast strains
with CBT1 (), or without CBT1 (A). FIG. 24B shows SSF using yeast
strains with and without CBT1. Cellobiose ( ) and glucose ()
concentrations in the presence of a strain with CBT1, and
cellobiose (.largecircle.) and glucose (.DELTA.) concentrations in
the presence of a strain lacking CBT1. Note, 0.1 mg/mL
cellobiose=292 .mu.M. FIG. 24C shows ethanol produced during SSF
using a strain with CBT1 ( ), or without CBT1 (.largecircle.). In
all panels, values are the mean of 3 biological replicates. Error
bars were the standard deviation between these replicates. All
strains also expressed the intracellular .beta.-glucosidase,
NCU00130.
[0101] FIG. 25 shows use of cellodextrin transport pathways from
filamentous fungi during simultaneous saccharification and
fermentation of cellulose by yeast. The cellodextrin (Cdex)
transport pathway (black) includes a cellodextrin transporter (CBT)
and intracellular .beta.-glucosidase (.beta.G). The sugar
catabolism pathway presented in standard yeast includes hexose
transporters (HXT). In SSF, both cellulases (GH) and extracellular
.beta.-glucosidase (.beta.G) could be used.
[0102] FIGS. 26A-C show residues in NCU00801 and NCU08114 that are
critical for function. FIG. 26A shows Ala-scan of cbt1/NCU00801.
FIG. 26B shows polypeptide sequence (important residues marked) of
cbt1/NCU00801. FIG. 26C shows polypeptide sequence (important
residues marked) of cbt2/NCU08114.
[0103] FIGS. 27A-C show a comparison of S. cerevisiae strains
expressing cellobiose transporters from P. stipitis. FIG. 27A shows
cell growth of S. cerevisiae strains expressing .beta.-glucosidase
and orthologs of cellobiose transporters NCU00801, NCU08114, and
NCU05853. FIG. 27B shows a comparison of cellobiose transporters
from P. stipitis: cell growth of S. cerevisiae strains expressing
.beta.-glucosidase and cellobiose transporters. FIG. 27C shows a
comparison of cellobiose transporters from P. stipitis: xylose
consumption and ethanol production by S. cerevisiae strains
expressing .beta.-glucosidase and cellobiose transporters.
[0104] FIGS. 28A-C show alignments of cellobiose transporter
orthologs. FIG. 28A shows alignment of cellobiose transporter
orthologs including ones that did not appear to have transporter
function under the conditions tested. FIG. 28B shows alignment of
cellobiose transporter orthologs that had transport function. FIG.
28C shows alignment of NCU00801 and NCU08114.
[0105] FIGS. 29A-B show functionally important motifs marked in
homology models of NCU00801 and NCU08114. FIG. 29A shows location
of cellobiose transporters motifs on NCU00801 homology model. Motif
[LIVM]-Y-[FL]-x(13)-[YF]-D (SEQ ID NO: 1) is shown in red. Motif
[YF]-x(2)-G-x(5)-[PVF]-x(6)-[DQ] (SEQ ID NO: 2) is shown in light
green. Motif G-R-[RK] (SEQ ID NO: 3) is shown in dark blue. Motif
R-x(6)-[YF]-N(SEQ ID NO: 4) is shown in yellow. Motif
WR-[IVLA]-P-x(3)-Q (SEQ ID NO: 5) is shown in magenta. Motif
P-E-S-P-R-x-L-x(8)-A-x(3)-L-x(2)-Y-H (SEQ ID NO: 6) is shown in
cyan. Motif F-[GST]-Q-x-S-G-N-x-[LIV] (SEQ ID NO: 7) is shown in
orange. Motif L-x(3)-[YIV]-x(2)-E-x-L-x(4)-R-[GA]-K-G (SEQ ID NO:
8) is shown in dark green. I. View of NCU00801 from the cytoplasmic
side looking into the putative cellobiose binding pore. Note that
in this image, some of the residues connecting transmembrane
helices 6 and 7 have been removed for clarity as they occlude the
pore. II. View of one side of NCU00801. III. View of the side
opposite to that shown in II. FIG. 29B shows location of cellobiose
transporters motifs on NCU08114 homology model. Motif
[LIVM]-Y-[FL]-x(13)-[YF]-D (SEQ ID NO: 1) is shown in red. Motif
[YF]-x(2)-G-x(5)-[PVF]-x(6)-[DQ] (SEQ ID NO: 2) is shown in light
green. Motif G-R-[RK] (SEQ ID NO: 3) is shown in dark blue. Motif
R-x(6)-[YF]-N (SEQ ID NO: 4) is shown in yellow. Motif
WR-[IVLA]-P-x(3)-Q (SEQ ID NO: 5) is shown in magenta. Motif
P-E-S-P-R-x-L-x(8)-A-x(3)-L-x(2)-Y-H (SEQ ID NO: 6) is shown in
cyan. Motif F-[GST]-Q-x-S-G-N-x-[LIV] (SEQ ID NO: 7) is shown in
oranges. Motif L-x(3)-[YIV]-x(2)-E-x-L-x(4)-R-[GA]-K-G (SEQ ID NO:
8) is shown in dark green. I. View of NCU08114 from the cytoplasmic
side looking into the putative cellobiose binding pore. Note that
in this image, some of the residues connecting transmembrane
helices 6 and 7 have been removed for clarity as they occlude the
pore. II. View of one side of NCU08114. III. View of the side
opposite to that shown in II.
[0106] FIGS. 30A-B show the cloning process used in the
construction of plasmid expressing: (A) putative transporters and
(B) transporter-GFP fusion proteins. FIG. 30A shows putative
transporters. FIG. 30B shows transporter-GFP fusion proteins.
[0107] FIG. 31 shows pentose transport activity of putative
transporters identified to have glucose-uptake activity.
[0108] FIG. 32 shows pentose transport activity of putative
transporters identified to not have glucose-uptake activity.
[0109] FIGS. 33A-B show pentose uptake of NCU00821 (AN25),
STL12/XUT6 (Xyp29), and XUT1 (Xyp32). FIG. 33A shows xylose uptake.
FIG. 33B shows arabinose uptake.
[0110] FIG. 34 shows .sup.14C-labeled sugar uptake by S. cerevisiae
expressing STL12/XUT6 (Xyp29).
[0111] FIG. 35 shows localizations of transporters expressed in S.
cerevisiae cells as monitored by GFP fluorescence. First row from
left to right: NCU00821-GFP fluorescence, NCU00821 nuclei; second
row from left to right: STL12/XUT6-GFP fluorescence, STL12/XUT6
nuclei.
[0112] FIGS. 36A-C show the effect on pH upon addition of maltose
to un-buffered cell suspension expressing: (a) NCU00821 (AN25), (b)
STL12/XUT6 (Xyp29), and (c) XUT1 (Xyp32). The black arrows indicate
the time points when maltose was added. FIG. 36A shows NCU00821
(AN25). FIG. 36B shows STL12/XUT6 (Xyp29). FIG. 36C shows XUT1
(Xyp32).
[0113] FIGS. 37A-F show results of a symporter assay of NCU00821,
STL12/XUT6, and XUT1. FIG. 37A shows NCU00821 for xylose. FIG. 37B
shows NCU00821 for arabinose. FIG. 37C shows XUT1 for arabinose.
FIG. 37D shows XUT1 for xylose. FIG. 37E shows STL12/XUT6 for
arabinose. FIG. 37F shows STL12/XUT6 for xylose. The black arrows
the time points when maltose was added.
[0114] FIGS. 38A-G show phenotypic analyses of transporter
overexpression. FIG. 38A shows OD. FIG. 38B shows xylose
concentration. FIG. 38C shows xylose consumption in 0.5%
xylose-containing media. FIG. 38D shows OD. FIG. 38E shows xylose
concentration. FIG. 38F shows xylose consumption in 5%
xylose-containing media. FIG. 38G shows the growth curve of S.
cerevisiae containing pentose transporters introduced on pRS424, a
multicopy plasmid.
[0115] FIG. 39 shows maps of the plasmids used for cloning of
heterologous transporters.
[0116] FIG. 40 shows results of the sugar-uptake assay by S.
cerevisiae strains expressing pentose transporter orthologs.
[0117] FIGS. 41A-1 to 41C-3 show sequence alignments of the pentose
transporter orthologs by Clustal W (1.81). FIGS. 41A-1 to 41A-2
show alignment of the xylose transporter orthologs. FIGS. 41B-1 to
41B-2 show alignment of the arabinose transporters. FIGS. 41C-1 to
41C-3 show alignment of xylose and arabinose transporters.
Consensus key: *--single, fully conserved residue; :--conservation
of strong groups; .--conservation of weak groups.
[0118] FIG. 42 describes the different S. cerevisiae strains
engineered to express xylose-utilizing enzymes.
[0119] FIG. 43 shows xylose metabolism (as monitored by xylose
consumption, ethanol production, etc.) of three S. cerevisiae
strains of different backgrounds expressing identical cassettes
containing xylose utilization pathway enzymes.
[0120] FIG. 44 shows xylose-uptake rates and metabolite yields of
three S. cerevisiae strains of different backgrounds expressing
identical cassettes containing xylose utilization pathway
enzymes.
[0121] FIGS. 45A-C show xylose fermentation by the S. cerevisiae
strain DA24 under various conditions. FIG. 45A shows 40 g/L xylose
in a shaker flask. FIG. 45B shows 80 g/L xylose in a shaker flask.
FIG. 45C shows 80 g/L xylose in a bioreactor. Symbols: xylose ( ),
ethanol (.diamond-solid.), and OD.sub.600 (1).
[0122] FIGS. 46A-B show a comparison of xylose consumption and
ethanol production between (a) S. cerevisiae DA24 and (b) P.
stipitis. Symbols: xylose ( ), ethanol (.diamond-solid.), and
OD.sub.600 ( ). FIG. 46A shows S. cerevisiae DA24. FIG. 46B shows
P. stipitis.
[0123] FIG. 47 describes the experimental design used to test the
effect of XYL2 over-expression levels on xylose metabolism in
engineered S. cerevisiae.
[0124] FIG. 48 shows the effect of additional XYL2 integration
(i.e. increased XYL2 expression level) into the genome of
engineered xylose-fermenting S. cerevisiae.
[0125] FIG. 49 shows the effect of additional simultaneous
over-expression of XYL2 and XYL3 on xylose fermentation by
engineered S. cerevisiae.
[0126] FIG. 50 describes S. cerevisiae strains expressing different
levels of xylose-fermenting enzymes.
[0127] FIG. 51 shows the effect of differential XYL1 expression of
fermentation by engineered S. cerevisiae.
[0128] FIG. 52 describes S. cerevisiae strains engineered to
over-express identical XYL2 and XYL3 but different reductases (XYL1
vs. GRE3).
[0129] FIG. 53 shows the effect of over-expressing XYL1 versus GRE3
on xylose fermentation by engineered S. cerevisiae grown in 40 g/L
xylose.
[0130] FIG. 54 shows the effect of over-expressing XYL1 versus GRE3
on xylose fermentation by engineered S. cerevisiae grown in 80 g/L
xylose.
[0131] FIGS. 55A-C show the thermal and pH-dependent properties of
different wild-type LAD enzymes: anLAD ( ), tlLAD
(.diamond-solid.), and pcLAD ( ). FIG. 55A shows
temperature-dependent catalytic activities. FIG. 55B shows thermal
inactivation at 50.degree. C. over time. FIG. 55C shows
pH-dependent catalytic activities. Error bars indicate standard
error of the mean (n=3).
[0132] FIG. 56 shows an alignment of XDH from N. crassa (ncXDH) and
P. stipitis (psXDH).
[0133] FIG. 57 show a comparison of pH rate profiles of N. crassa
LAD and XDH. Data taken from the characterization of LAD was
performed in universal buffer MES/Tris/glycine, and overlapped with
data for ncXDH (closed triangles) and ncLAD (closed circles)
performed in universal buffer acetic acid/MES/Tris for lower pH
values.
[0134] FIG. 58 shows ethanol production by S. cerevisiae strain
L2612 transformed with xylose isomerase enzyme from Bacteroids
stercoris (BtXI), Bifidobacterium longum (BfXI), and BtXIO coding
for codon-optimized BtXI. The XI gene was cloned into the pRs424TEF
vector.
[0135] FIG. 59 shows xylose consumption and ethanol production by
S. cerevisiae strain D452-2, which had BtXI integrated into its
genome by the vector pRS403TEF. Comparison is also made to
xylose-fermentation by S. cerevisiae strain L2612, which expresses
BtXI from a plasmid
[0136] FIG. 60 shows xylose fermentation by S. cerevisiae strain,
containing integrated BtXI and expressing XYL2 or XYL3 or XYL2 and
XYL3.
[0137] FIG. 61 shows the necessity of XYL3 expression in S.
cerevisiae engineered to over-express enzymes, such as GND1,
involved in the pentose phosphate pathway in order to efficiently
metabolize xylose.
[0138] FIGS. 62A-B show the effect of over-expression of NCU09705
homologs in E. coli, S. cerevisiae, and P. stipitis on fermentation
parameters. Over-expression of galM, GAL10-Sc, GAL10-Ps, YHR210C,
and YNR071C on (A) cellobiose consumption, growth, and ethanol
production; and on (B) ethanol yield and productivity. FIG. 62A
shows cellobiose consumption, growth, and ethanol production. FIG.
62B shows ethanol yield and productivity.
[0139] FIGS. 63A-C show the experimental design enabling
simultaneous co-fermentation of cellobiose and xylose without
glucose repression through integration of a cellodextrin
assimilation pathway from filamentous fungi (N. crassa) and
modified xylose metabolic pathway from the xylose-fermenting yeast
P. stipitis into S. cerevisiae. FIG. 63A shows a strain improvement
strategy to engineer yeast strain capable of fermenting two
non-metabolizable sugars (cellobiose and xylose). The cellodextrin
assimilation pathway consists of a cellodextrin transporter
(NCU00801) and an intracellular .beta.-glucosidase (NCU00130) from
N. crassa. The modified xylose metabolic pathway utilizes xylose
reductase isozymes (wild-type XR and mutant XR.sup.R276H), xylitol
dehydrogenase (XYL2), and xylulokinase (XKS1). FIG. 63B shows
fermentation profile of a sugar mixture containing glucose and
xylose by the engineered S. cerevisiae developed in this study.
Glucose fermentation repressed xylose fermentation completely so
that xylose fermentation begins only after glucose depletion. FIG.
63C shows fermentation profile of a sugar mixture containing
cellobiose and xylose by the engineered S. cerevisiae developed in
this study. Cellobiose and xylose are simultaneously utilized, as
neither carbon source repressed consumption of the other.
[0140] FIG. 64 shows the scheme for plasmid construction. The
pRS425 shuttle vector was linearized followed by assembly of the
cellobiose transporter and .beta.-glucosidase genes using the DNA
assembler method (Shao et al., 2009).
[0141] FIGS. 65A-G show the change in concentrations of cellobiose
(.box-solid.), glucose ( ), D-xylose (.tangle-solidup.), ethanol
(), and biomass (.quadrature.) during co-fermentation of 4%
cellobiose and 5% D-xylose by S. cerevisiae strains (a) SL01, (b)
SL04, (c) SL02, (d) SL05, (e) SL03, (f) SL06, and (g) SL00 as a
function of time. FIG. 65A shows SL01. FIG. 65B shows SL04. FIG.
65C shows SL02. FIG. 65D shows SL05. FIG. 65E shows SL03. FIG. 65F
shows SL06. FIG. 65G shows SL00.
[0142] FIGS. 66A-D show the change in concentrations of cellobiose
(.box-solid.), glucose ( ), D-xylose (.tangle-solidup.), ethanol
(), and biomass (.quadrature.) in S. cerevisiae strains SL01 (a, c)
and SL00 (b, d) grown in cellobiose-xylose mixtures in shake-flasks
(a, b) or bioreactors (c, d) plotted as a function of time. FIG.
66A shows S. cerevisiae strain SL01 grown in cellobiose-xylose
mixtures in shake-flasks. FIG. 66B shows S. cerevisiae strain SL00
grown in cellobiose-xylose mixtures in shake-flasks. FIG. 66C shows
S. cerevisiae strain SL01 grown in cellobiose-xylose mixtures in
bioreactors. FIG. 66D shows S. cerevisiae strain SL00 grown in
cellobiose-xylose mixtures in bioreactors.
[0143] FIGS. 67A-D show the change in concentrations of cellobiose
(.box-solid.), glucose ( ), D-xylose (.tangle-solidup.), ethanol
(), and biomass (.quadrature.) in S. cerevisiae strains SL01 (a, c)
and SL00 (b, d) grown in media containing 5 g/L glucose-40 g/L
cellobiose-50 g/L xylose mixture (a, b) or 10 g/L glucose-40 g/L
cellobiose-50 g/L xylose mixture (c, d) in bioreactors, plotted as
a function of time. FIG. 67A shows S. cerevisiae strain SL01 grown
in media containing 5 g/L glucose-40 g/L cellobiose-50 g/L xylose
mixture. FIG. 67B shows S. cerevisiae strains SL00 grown in media
containing 5 g/L glucose-40 g/L cellobiose-50 g/L xylose mixture.
FIG. 67C shows S. cerevisiae strain SL01 grown in media containing
10 g/L glucose-40 g/L cellobiose-50 g/L xylose mixture. FIG. 67D
shows S. cerevisiae strains SL00 grown in media containing 10 g/L
glucose-40 g/L cellobiose-50 g/L xylose mixture.
[0144] FIGS. 68A-C show a comparison of cellobiose utilizations by
.beta.-glucosidase (NCU00130)-containing S. cerevisiae strain
expressing (a) NCU00801, (b) NCU00809, and (c) NCU08114. Symbols:
cellobiose (.box-solid.), ethanol (.diamond-solid.), and OD.sub.600
( ). FIG. 68A shows NCU00801. FIG. 68B shows NCU00809. FIG. 68C
shows NCU08114.
[0145] FIGS. 69A-C show co-fermentation of cellobiose and xylose by
the S. cerevisiae strain DA24-16BT3 grown in mixtures containing
various concentrations of the two sugars: (a) 20 g/L (each) of
cellobiose and xylose, (b) 30 g/L (each) of cellobiose and xylose,
and (c) 40 g/L (each) of cellobiose and xylose. Symbols: cellobiose
(.tangle-solidup.), xylose (.box-solid.), ethanol
(.diamond-solid.), and OD.sub.600 ( ). FIG. 69A shows 20 g/L (each)
of cellobiose and xylose. FIG. 69B shows 30 g/L (each) of
cellobiose and xylose. FIG. 69C shows 40 g/L (each) of cellobiose
and xylose.
[0146] FIGS. 70A-C show the synergistic effects of co-fermentation
of cellobiose and xylose by the S. cerevisiae strain DA24-16BT3.
Symbols: cellobiose (.tangle-solidup.), xylose (.box-solid.),
ethanol (.diamond-solid.), and OD.sub.600 ( ). FIG. 70A shows 40
g/L cellobiose. FIG. 70B shows 40 g/L (each) of cellobiose and
xylose. FIG. 70C shows 40 g/L xylose.
[0147] FIGS. 71A-B show co-fermentation of glucose, cellobiose, and
xylose by the S. cerevisiae strain DA24-16BT3 and the wild-type P.
stipitis strain. Symbols: cellobiose (.tangle-solidup.), xylose
(.box-solid.), ethanol (.diamond-solid.), OD.sub.600 ( ), and
glucose (). FIG. 71A shows DA24-16BT3. FIG. 71B shows P.
stipitis.
[0148] FIG. 72 shows HPLC chromatograms from each time point,
suggesting cellotriose and cellotetraose accumulation during
c-fermentation of cellobiose and xylose by the S. cerevisiae strain
DA24-16BT3.
[0149] FIG. 73 shows HPAEC analysis demonstrating cellodextrin
accumulation in fermentation medium after 22 hours fermentation by
the S. cerevisiae strain DA24-16BT3 during co-fermentation of
cellobiose and xylose. (G1: glucose, G2: cellobiose, G3:
cellotriose, G4: cellotetraose, and G5: cellopentaose).
[0150] FIGS. 74A-B show a comparison of sugar utilization by S.
cerevisiae transformants expressing (a) an integrated copy of
NCU00801 and (b) NCU00801 on a multi-copy plasmid, during
co-fermentation of 40 g/L (each) of cellobiose and xylose. Symbols:
cellobiose (.tangle-solidup.), xylose (.box-solid.), ethanol
(.diamond-solid.), and OD.sub.600 ( ). FIG. 74A shows S. cerevisiae
transformants expressing an integrated copy of NCU00801. FIG. 74B
shows S. cerevisiae transformants expressing NCU00801 on a
multi-copy plasmid.
[0151] FIGS. 75A-B show ethanol production by cultivation of two
different yeast strains. FIG. 75A shows the two different S.
cerevisiae strains used in study: DA24-16 and D452BT. A xylose
molecule is shown as a pentagon and a cellobiose molecule is shown
as two hexagons. FIG. 75B shows mixed cultures of xylose-fermenting
strain and cellobiose-fermenting strain.
[0152] FIG. 76 shows a listing of 354 xylan-induced genes in N.
crassa.
[0153] FIG. 77 shows secreted protein levels, reducing sugar, and
azo-xylanase activity for various N. crassa knock-out strains.
Secreted protein levels were relatively constant for all
strains.
[0154] FIG. 78A shows total secreted protein and CMC-activity for
wild type, .DELTA.NCU05137, and .DELTA.NCU05137/.DELTA.NCU05137-GFP
Neurospora strains. FIG. 78B shows a Coomassie stain of total
protein in supernatants from cultures of the three different
strains.
[0155] FIG. 79 shows localization of NCU05137-GFP in conidia.
[0156] FIG. 80 shows localization of NCU05137-GFP in the hypha
tip.
DETAILED DESCRIPTION OF THE INVENTION
[0157] The present disclosure relates to host cells containing a
recombinant polynucleotide encoding a polypeptide containing
transmembrane .alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3,
.alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7,
.alpha.-helix 8, .alpha.-helix 9, .alpha.-helix 10, .alpha.-helix
11, and .alpha.-helix 12, where one or more of the following is
true: transmembrane .alpha.-helix 1 comprises SEQ ID NO: 1,
transmembrane .alpha.-helix 2 comprises SEQ ID NO: 2, the loop
connecting transmembrane .alpha.-helix 2 and transmembrane
.alpha.-helix 3 comprises SEQ ID NO: 3, transmembrane .alpha.-helix
5 comprises SEQ ID NO: 4, transmembrane .alpha.-helix 6 comprises
SEQ ID NO: 5, sequence between transmembrane .alpha.-helix 6 and
transmembrane .alpha.-helix 7 comprises SEQ ID NO: 6, transmembrane
.alpha.-helix 7 comprises SEQ ID NO: 7, and transmembrane
.alpha.-helix 10 and transmembrane .alpha.-helix 11 and the
sequence between them comprise SEQ ID NO: 8, and where the
polypeptide transports cellodextrin into the cell. Further
described herein are methods of increasing transport of
cellodextrin into a cell, methods of increasing growth of a cell on
a medium containing cellodextrin, methods of co-fermenting
cellulose-derived and hemicellulose-derived sugars, and methods of
making hydrocarbons or hydrocarbon derivatives using the host
cells. Further described herein are host cells containing a
recombinant polynucleotide encoding a polypeptide where the
polypeptide transports a pentose into the cell, methods of
increasing transport of a pentose into a cell, methods of
increasing growth of a cell on a medium containing pentose sugars,
and methods of making hydrocarbons or hydrocarbon derivatives by
providing a host cell containing a recombinant polynucleotide
encoding a polypeptide where the polypeptide transports a pentose
into the cell.
[0158] As used herein, cellodextrin refers to glucose polymers of
varying length and includes, without limitation, cellobiose (2
glucose monomers), cellotriose (3 glucose monomers), cellotetraose
(4 glucose monomers), cellopentaose (5 glucose monomers), and
cellohexaose (6 glucose monomers).
[0159] As used herein, sugar refers to monosaccharides (e.g.,
glucose, fructose, galactose, xylose, arabinose), disaccharides
(e.g., cellobiose, sucrose, lactose, maltose), and oligosaccharides
(typically containing 3 to 10 component monosaccharides).
[0160] Polynucleotides of the Invention
[0161] The invention herein relates to host cells and methods of
using such host cells where the host cells comprise recombinant
polynucleotides encoding polypeptides capable of transporting
various sugars.
[0162] As used herein, the terms "polynucleotide," "nucleic acid
sequence," "sequence of nucleic acids," and variations thereof
shall be generic to polydeoxyribonucleotides (containing
2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to
any other type of polynucleotide that is an N-glycoside of a purine
or pyrimidine base, and to other polymers containing
non-nucleotidic backbones, provided that the polymers contain
nucleobases in a configuration that allows for base pairing and
base stacking, as found in DNA and RNA. Thus, these terms include
known types of nucleic acid sequence modifications, for example,
substitution of one or more of the naturally occurring nucleotides
with an analog; inter-nucleotide modifications, such as, for
example, those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoramidates, carbamates, etc.), with
negatively charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), and with positively charged linkages
(e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters);
those containing pendant moieties, such as, for example, proteins
(including nucleases, toxins, antibodies, signal peptides,
poly-L-lysine, etc.); those with intercalators (e.g., acridine,
psoralen, etc.); and those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.). As used herein,
the symbols for nucleotides and polynucleotides are those
recommended by the IUPAC-IUB Commission of Biochemical Nomenclature
(Biochem. 9:4022, 1970).
[0163] As used herein, a "polypeptide" is an amino acid sequence
comprising a plurality of consecutive polymerized amino acid
residues (e.g., at least about 15 consecutive polymerized amino
acid residues, optionally at least about 30 consecutive polymerized
amino acid residues, at least about 50 consecutive polymerized
amino acid residues). In many instances, a polypeptide comprises a
polymerized amino acid residue sequence that is a transporter, a
transcription factor, a predicted protein of unknown function, or a
domain or portion or fragment thereof. A transporter is involved in
the movement of ions, small molecules, or macromolecules, such as a
carbohydrate, across a biological membrane. A transcription factor
can regulate gene expression and may increase or decrease gene
expression in a host cell. The polypeptide optionally comprises
modified amino acid residues, naturally occurring amino acid
residues not encoded by a codon, and non-naturally occurring amino
acid residues.
[0164] As used herein, "protein" refers to an amino acid sequence,
oligopeptide, peptide, polypeptide, or portions thereof whether
naturally occurring or synthetic.
[0165] Recombinant polynucleotides of the invention include any
polynucleotides that encode a polypeptide encoded by any of the
genes listed in Table 10, in Supplemental Data, Dataset S1, page 3
in Tian et al., 2009; in Tables 14, 15, 16, 29; or in FIG. 76. In
preferred embodiments, polynucleotides of the invention include any
polynucleotides that encode a polypeptide encoded by any of the
sequences NCU00801, NCU00809, NCU08114, NCU00130, NCU00821,
NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, XUT3, NCU07705,
NCU05137, NCU01517, NCU09133, or NCU10040.
[0166] Table 1 shows polynucleotides of the invention including
sequences listed below or sequences encoding polypeptides listed
below.
TABLE-US-00001 NCBI Reference Gene Alternate Sequence/GenBank
Name/Locus Name Accession Number Organism NCU00801 cbt1
XP_963801.1/EAA34565 N. crassa NCU00809 XP_964302.1/EAA35116.1 N.
crassa NCU00821 AN25 XP_964364.2/EAA35128.2 N. crassa NCU00988 Xy33
XP_963898.1/EAA34662.1 N. crassa NCU01231 XP_961597.2/EAA32361.2 N.
crassa NCU01494 AN49 XP_955927.2/EAA26691.2 N. crassa NCU02188
AN28-3 XP_959582.2/EAA30346.2 N. crassa NCU04537 Xy50
XP_955977.1/EAA26741.1 N. crassa NCU04963 AN29-2
XP_959411.2/EAA30175.2 N. crassa NCU05519 XP_960481.1/EAA31245.1 N.
crassa NCU05853 XP_959844.1/EAA30608.1 N. crassa NCU05897
XP_959888.1/EAA30652.1 N. crassa NCU06138 Xy31
XP_960000.1/EAA30764.1 N. crassa NCU08114 cbt2
XP_963873.1/EAA34637.1 N. crassa NCU09287 AN41
XP_958139.1/EAA28903.1 N. crassa NCU10021 XP_958069.2/EAA28833.2 N.
crassa XP_001387242 Ap26 XP_001387242 P. stipitis HGT3 Xyp30-1
XP_001386715.1/ P. stipitis ABN68686.1 STL1 Xyp30 XP_001383774.1/
P. stipitis ABN65745.1 STL12/XUT6 Xyp29 XP_001386589.1/ P. stipitis
ABN68560.1 SUT2 Ap31 XP_001384295.2/ P. stipitis ABN66266.2 SUT3
Xyp37 XP_001386019.2/ P. stipitis ABN67990.2 XUT1 Xyp32
XP_001385583.1/ P. stipitis ABN67554.1 XUT2 Xyp31 XP_001387242.1/
P. stipitis EAZ63219.2 XUT3 Xyp33 XP_001387138.1/ P. stipitis
EAZ63115.1 XUT7 Xyp28 XP_001387067.1/ P. stipitis EAZ63044.1
NCU07705 cdr-1 XP_962291.1/EAA33055 N. crassa NCU05137
XP_956635.1/EAA27399 N. crassa NCU01517 XP_956966.1/EAA27730 N.
crassa NCU09133 XP_958905.1/EAA29669 N. crassa NCU10040 N.
crassa
[0167] In certain embodiments, the recombinant polynucleotides of
the invention encode polypeptides having at least about 20%, or at
least about 29%, or at least about 30%, or at least about 40%, or
at least about 50%, or at least about 55%, or at least about 60%,
or at least about 65%, or at least about 70%, or at least about
75%, or at least about 80%, or at least about 85%, or at least
about 90%, or at least about 92%, or at least about 94%, or at
least about 96%, or at least about 98%, or at least about 99%, or
at least about 100% amino acid residue sequence identity to a
polypeptide encoded by any of the genes listed in genes listed in
Table 10, in Supplemental Data, Dataset S1, page 3 in Tian et al.,
2009; in Tables 14, 15, 16, 29; or in FIG. 76. In preferred
embodiments, the polynucleotides of the invention encode
polypeptides having at least about 20%, or at least about 29%, or
at least about 30%, or at least about 40%, or at least about 50%,
or at least about 55%, or at least about 60%, or at least about
65%, or at least about 70%, or at least about 75%, or at least
about 80%, or at least about 85%, or at least about 90%, or at
least about 92%, or at least about 94%, or at least about 96%, or
at least about 98%, or at least about 99%, or at least about 100%
amino acid residue sequence identity to a polypeptide encoded by
any of the sequences NCU00801, NCU00809, NCU08114, NCU00130,
NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, XUT3,
NCU07705, NCU05137, NCU01517, NCU09133, or NCU10040.
[0168] Polynucleotides of the invention further include
polynucleotides that encode conservatively modified variants of
polypeptides encoded by the genes listed above. "Conservatively
modified variants" as used herein include individual substitutions,
deletions or additions to a polypeptide sequence which result in
the substitution of an amino acid with a chemically similar amino
acid. Conservative substitution tables providing functionally
similar amino acids are well known in the art. Such conservatively
modified variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles of the disclosure. The
following eight groups contain amino acids that are conservative
substitutions for one another: 1) Alanine (A), Glycine (G); 2)
Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine
(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (); 6) Phenylalanine (F), Tyrosine (Y),
Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),
Methionine (M) (see, e.g., Creighton, Proteins (1984)).
[0169] Polynucleotides of the invention further include
polynucleotides that encode homologs or orthologs of polypeptides
encoded by any of the genes listed in Table 10, in Supplemental
Data, Dataset 51, page 3 in Tian et al., 2009; in Tables 14, 15,
16, 29; or in FIG. 76. "Homology" as used herein refers to sequence
similarity between a reference sequence and at least a fragment of
a second sequence. Homologs may be identified by any method known
in the art, preferably, by using the BLAST tool to compare a
reference sequence to a single second sequence or fragment of a
sequence or to a database of sequences. As described below, BLAST
will compare sequences based upon percent identity and similarity.
"Orthology" as used herein refers to genes in different species
that derive from a common ancestor gene.
[0170] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same. Two sequences
are "substantially identical" if two sequences have a specified
percentage of amino acid residues or nucleotides that are the same
(i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified
region, or, when not specified, over the entire sequence), when
compared and aligned for maximum correspondence over a comparison
window, or designated region as measured using one of the following
sequence comparison algorithms or by manual alignment and visual
inspection. Optionally, the identity exists over a region that is
at least about 50 nucleotides (or 10 amino acids) in length, or
more preferably over a region that is 100 to 500 or 1000 or more
nucleotides (or 20, 50, 200, or more amino acids) in length.
[0171] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters. When comparing two sequences for identity, it
is not necessary that the sequences be contiguous, but any gap
would carry with it a penalty that would reduce the overall percent
identity. For blastn, the default parameters are Gap opening
penalty=5 and Gap extension penalty=2. For blastp, the default
parameters are Gap opening penalty=11 and Gap extension
penalty=1.
[0172] A "comparison window," as used herein, includes reference to
a segment of any one of the number of contiguous positions
including, but not limited to from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1981), by the homology alignment algorithm of Needleman
and Wunsch (1970) J Mol Biol 48(3):443-453, by the search for
similarity method of Pearson and Lipman (1988) Proc Natl Acad Sci
USA 85(8):2444-2448, by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
[see, e.g., Brent et al., (2003) Current Protocols in Molecular
Biology, John Wiley & Sons, Inc. (Ringbou Ed)].
[0173] Two examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1997)
Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J.
Mol Biol 215(3)-403-410, respectively. Software for performing
BLAST analyses is publicly available through the National Center
for Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4, and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix [see Henikoff and Henikoff, (1992) Proc Natl Acad
Sci USA 89(22):10915-10919] alignments (B) of 50, expectation (E)
of 10, M=5, N=-4, and a comparison of both strands.
[0174] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul, (1993) Proc Natl Acad Sci USA 90(12):5873-5877). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0175] Other than percentage of sequence identity noted above,
another indication that two nucleic acid sequences or polypeptides
are substantially identical is that the polypeptide encoded by the
first nucleic acid is immunologically cross-reactive with the
antibodies raised against the polypeptide encoded by the second
nucleic acid, as described below. Thus, a polypeptide is typically
substantially identical to a second polypeptide, for example, where
the two peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules or their complements hybridize
to each other under stringent conditions, as described below. Yet
another indication that two nucleic acid sequences are
substantially identical is that the same primers can be used to
amplify the sequence.
[0176] As described herein, polynucleotides of the invention
include members of the Major Facilitator Superfamily sugar
transporter family, including NCU00988, NCU10021, NCU04963,
NCU06138, NCU00801, NCU08114, and NCU05853. Members of the Major
Facilitator Superfamily (MFS) (Transporter Classification #2.A.1)
of transporters almost always consist of 12 transmembrane
.alpha.-helices, with an intracellular N- and C-terminus (S. S.
Pao, I. T. Paulsen, M. H. Saier, Jr., Microbiol Mol Biol Rev 62, 1
(March, 1998)). While the primary sequence of MFS transporters
varies widely, all are thought to share the tertiary structure of
the E. coli lactose permease (LacY) (J. Abramson et al., Science
301, 610 (Aug. 1, 2003)), and the E. coli Pi/glycerol-3-phospate
(GlpT) (Y. Huang, M. J. Lemieux, J. Song, M. Auer, D. N. Wang,
Science 301, 616 (Aug. 1, 2003)). In these examples the six N- and
C-terminal helices form two distinct domains connected by a long
cytoplasmic loop between helices 6 and 7. This symmetry corresponds
to a duplication event thought to have given rise to the MFS.
Substrate binds within a hydrophilic cavity formed by helices 1, 2,
4, and 5 of the N-terminal domain, and helices 7, 8, 10, and 11 of
the C-terminal domain. This cavity is stabilized by helices 3, 6,
9, and 12.
[0177] The Sugar Transporter family of the MFS (Transporter
Classification #2.A.1.1) is defined by motifs found in
transmembrane helices 6 and 12 (PESPR (SEQ ID NO: 9)/PETK (SEQ ID
NO: 10)), and loops 2 and 8 (GRR/GRK) (M. C. Maiden, E. O. Davis,
S. A. Baldwin, D. C. Moore, P. J. Henderson, Nature 325, 641 (Feb.
12-18, 1987)). The entire Hidden Markov Model (HMM) for this family
can be viewed at pfam.janelia.org/family/PF00083#tabview=tab3.
PROSITE (N. Hulo et al., Nucleic Acids Res 34, D227 (Jan. 1, 2006))
uses two motifs to identify members of this family. The first is
[LIVMSTAG]-[LIVMFSAG]-{SH}-{RDE}-[LIVMSA]-[DE]-{TD}-[LIVMFYWA]-G-R-[RK]-x-
(4,6)-[GSTA] (SEQ ID NO: 11). The second is
[LIVMF]-x-G-[LIVMFA]-{V}-x-G-{KP}-x(7)-[LIFY]-x(2)-[EQ]-x(6)-[RK]
(SEQ ID NO: 12). As an example of how to read a PROSITE motif, the
following motif, [AC]-x-V-x(4)-{ED}, is translated as: [Ala or
Cys]-any-Val-any-any-any-any-{any but Glu or Asp} (SEQ ID NO:
13).
[0178] As described herein, NCU00801, NCU00809, NCU08114,
XP.sub.--001268541.1, and LAC2 were discovered to encode
polypeptides that transport cellodextrins. Further, alanine
scanning experiments and sequence analyses were used to determine
that a recombinant polypeptide containing 12 transmembrane
.alpha.-helices, and one or more of the sequences selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ
ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:
8 encodes a polypeptide that transports cellodextrin.
[0179] Thus, in one aspect, the invention provides a polynucleotide
encoding a polypeptide containing transmembrane .alpha.-helix 1,
.alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5,
.alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9,
.alpha.-helix 10, .alpha.-helix 11, .alpha.-helix 12, and
transmembrane .alpha.-helix 1 comprises SEQ ID NO: 1. In another
aspect, the invention provides a polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and transmembrane
.alpha.-helix 2 comprises SEQ ID NO: 2. In another aspect, the
invention provides a polynucleotide encoding a polypeptide
containing transmembrane .alpha.-helix 1, .alpha.-helix 2,
.alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6,
.alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and a loop connecting
transmembrane .alpha.-helix 2 and transmembrane .alpha.-helix 3
comprises SEQ ID NO: 3. In another aspect, the invention provides a
polynucleotide encoding a polypeptide containing transmembrane
.alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4,
.alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8,
.alpha.-helix 9, .alpha.-helix 10, .alpha.-helix 11, .alpha.-helix
12, and transmembrane .alpha.-helix 5 comprises SEQ ID NO: 4. In
another aspect, the invention provides a polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and transmembrane
.alpha.-helix 6 comprises SEQ ID NO: 5. In another aspect, the
invention provides a polynucleotide encoding a polypeptide
containing transmembrane .alpha.-helix 1, .alpha.-helix 2,
.alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6,
.alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and sequence between
transmembrane .alpha.-helix 6 and transmembrane .alpha.-helix 7
comprises SEQ ID NO: 6. In another aspect, the invention provides a
polynucleotide encoding a polypeptide containing transmembrane
.alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4,
.alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8,
.alpha.-helix 9, .alpha.-helix 10, .alpha.-helix 11, .alpha.-helix
12, and transmembrane .alpha.-helix 7 comprises SEQ ID NO: 7. In
another aspect, the invention provides a polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, and transmembrane
.alpha.-helix 10 and transmembrane .alpha.-helix 11 and the
sequence between them comprise SEQ ID NO: 8.
[0180] Each of the above described aspects may be combined in any
number of combinations. A polynucleotide according to any of these
aspects may encode a polypeptide containing 1, 2, 3, 4, 5, 6, or 7
of any of SEQ ID NOs: 1-8, or the polypeptide may contain all of
SEQ ID NOs: 1-8. For example, a polynucleotide may encode a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, where transmembrane
.alpha.-helix 1 comprises SEQ ID NO: 1, a loop connecting
transmembrane .alpha.-helix 2 and transmembrane .alpha.-helix 3
comprises SEQ ID NO: 3, and transmembrane .alpha.-helix 7 comprises
SEQ ID NO: 7. Or, in another example, a polynucleotide may encode a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, where transmembrane
.alpha.-helix 2 comprises SEQ ID NO: 2, transmembrane .alpha.-helix
3 comprises SEQ ID NO: 3, transmembrane .alpha.-helix 6 comprises
SEQ ID NO: 5, and transmembrane .alpha.-helix 10 and transmembrane
.alpha.-helix 11 and the sequence between them comprise SEQ ID NO:
8.
[0181] In certain embodiments of the above described aspects, the
polypeptide has at least 29%, at least 30%, at least 35%, at least
40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 99%, or at least 100% amino
acid identity to NCU00801 or NCU08114.
[0182] As further described herein, NCU08221 and STL12/XUT6 were
discovered to encode polypeptides that transport xylose. XUT1 was
discovered to encode a polypeptide that transports arabinose.
NCU06138 was discovered to encode a polypeptide that transports
arabinose or glucose. SUT2, SUT3, and XUT3 were discovered to
encode polypeptides that transport xylose or glucose. NCU04963 was
discovered to encode a polypeptide that transports xylose,
arabinose, or glucose. In preferred embodiments, polynucleotides of
the invention include recombinant polynucleotides encoding a
NCU08221 or STL12/XUT6 polypeptide, where the polypeptide
transports xylose. In other preferred embodiments, polynucleotides
of the invention include recombinant polynucleotides encoding a
XUT1 polypeptide, where the polypeptide transports arabinose. In
other preferred embodiments, polynucleotides of the invention
include recombinant polynucleotides encoding a NCU06138
polypeptide, where the polypeptide transports arabinose or glucose.
In other preferred embodiments, polynucleotides of the invention
include recombinant polynucleotides encoding a SUT2, SUT3, or XUT3
polypeptide, where the polypeptide transports xylose or glucose. In
other preferred embodiments, polynucleotides of the invention
include recombinant polynucleotides encoding a NCU04963
polypeptide, where the polypeptide transports xylose, arabinose, or
glucose.
[0183] The polynucleotides of the invention that encode
polypeptides encoded by NCU07705 are predicted by FunCat (Ruepp,
2004; webpage broad.mit.edu/annotation/genome/neurospora/Home.html)
to encode an unclassified protein. However, BLAST analysis of the
polypeptide encoded by NCU07705 revealed that the polypeptide has
high similarity to many C6 zinc finger domain containing
transcription factors (see FIG. 1; a list of exemplary homologs can
be found in FIG. 23 of related U.S. Appl. No. 61/271,833).
Polynucleotides of the invention include polynucleotides that
encode these homologs of the polypeptide encoded by NCU07705 or any
other homologs identified with any methods known in the art.
[0184] In another aspect of the invention, polynucleotides of the
invention include those polynucleotides that encode polypeptides
encoded by NCU05137. FunCat classifies the polypeptide encoded by
NCU05137 to be an unclassified protein. However, NCU05137 is highly
conserved in the genomes of a number of filamentous ascomycete
fungi (see FIG. 2). Polynucleotides of the invention include
polynucleotides that encode these homologs of the polypeptide
encoded by NCU05137 or any other homologs identified with any
methods known in the art.
[0185] In another aspect of the invention, polynucleotides of the
invention include those polynucleotides that encode polypeptides
encoded by NCU01517, NCU09133, or NCU10040. FunCat classifies the
polypeptide encoded by NCU01517 to be a glucoamylase precursor.
FunCat classifies the polypeptides encoded by NCU09133 and NCU10040
to be unclassified proteins. Polynucleotides of the invention
include polynucleotides that encode these homologs of the
polypeptide encoded by NCU01517, NCU09133, or NCU10040 or any other
homologs identified with any methods known in the art.
[0186] Predicted functions of these polypeptides can be confirmed
by performing functional analyses of the polynucleotide and its
encoded protein. These analyses may include, for example,
phenotypic analysis of strains containing deletions of the
polynucleotide, genetic complementation experiments, phenotypic
analysis of strains over expressing a wild-type copy of the
polynucleotide, expression and purification of a recombinant form
of the polypeptide, and subsequent characterization of the
biochemical properties and activity of the recombinant
polypeptide.
[0187] Sequences of the polynucleotides of the invention are
prepared by any suitable method known to those of ordinary skill in
the art, including, for example, direct chemical synthesis or
cloning. For direct chemical synthesis, formation of a polymer of
nucleic acids typically involves sequential addition of 3 `-blocked
and 5`-blocked nucleotide monomers to the terminal 5'-hydroxyl
group of a growing nucleotide chain, wherein each addition is
effected by nucleophilic attack of the terminal 5'-hydroxyl group
of the growing chain on the 3'-position of the added monomer, which
is typically a phosphorus derivative, such as a phosphotriester,
phosphoramidite, or the like. Such methodology is known to those of
ordinary skill in the art and is described in the pertinent texts
and literature [e.g., in Matteucci et al., (1980) Tetrahedron Lett
21:719-722; U.S. Pat. Nos. 4,500,707; 5,436,327; and 5,700,637]. In
addition, the desired sequences may be isolated from natural
sources by splitting DNA using appropriate restriction enzymes,
separating the fragments using gel electrophoresis, and thereafter,
recovering the desired nucleic acid sequence from the gel via
techniques known to those of ordinary skill in the art, such as
utilization of polymerase chain reactions (PCR; e.g., U.S. Pat. No.
4,683,195).
[0188] Each polynucleotide of the invention can be incorporated
into an expression vector. "Expression vector" or "vector" refers
to a compound and/or composition that transduces, transforms, or
infects a host cell, thereby causing the cell to express nucleic
acids and/or proteins other than those native to the cell, or in a
manner not native to the cell. An "expression vector" contains a
sequence of nucleic acids (ordinarily RNA or DNA) to be expressed
by the host cell. Optionally, the expression vector also comprises
materials to aid in achieving entry of the nucleic acid into the
host cell, such as a virus, liposome, protein coating, or the like.
The expression vectors contemplated for use in the present
invention include those into which a nucleic acid sequence can be
inserted, along with any preferred or required operational
elements. Further, the expression vector must be one that can be
transferred into a host cell and replicated therein. Preferred
expression vectors are plasmids, particularly those with
restriction sites that have been well documented and that contain
the operational elements preferred or required for transcription of
the nucleic acid sequence. Such plasmids, as well as other
expression vectors, are well known to those of ordinary skill in
the art.
[0189] Incorporation of the individual polynucleotides may be
accomplished through known methods that include, for example, the
use of restriction enzymes (such as BamHI, EcoRI, HhaI, XhoI, XmaI,
and so forth) to cleave specific sites in the expression vector,
e.g., plasmid. The restriction enzyme produces single stranded ends
that may be annealed to a polynucleotide having, or synthesized to
have, a terminus with a sequence complementary to the ends of the
cleaved expression vector. Annealing is performed using an
appropriate enzyme, e.g., DNA ligase. As will be appreciated by
those of ordinary skill in the art, both the expression vector and
the desired polynucleotide are often cleaved with the same
restriction enzyme, thereby assuring that the ends of the
expression vector and the ends of the polynucleotide are
complementary to each other. In addition, DNA linkers maybe used to
facilitate linking of nucleic acids sequences into an expression
vector.
[0190] A series of individual polynucleotides can also be combined
by utilizing methods that are known to those having ordinary skill
in the art (e.g., U.S. Pat. No. 4,683,195).
[0191] For example, each of the desired polynucleotides can be
initially generated in a separate PCR. Thereafter, specific primers
are designed such that the ends of the PCR products contain
complementary sequences. When the PCR products are mixed,
denatured, and reannealed, the strands having the matching
sequences at their 3' ends overlap and can act as primers for each
other. Extension of this overlap by DNA polymerase produces a
molecule in which the original sequences are "spliced" together. In
this way, a series of individual polynucleotides may be "spliced"
together and subsequently transduced into a host cell
simultaneously. Thus, expression of each of the plurality of
polynucleotides is affected.
[0192] Individual polynucleotides, or "spliced" polynucleotides,
are then incorporated into an expression vector. The invention is
not limited with respect to the process by which the polynucleotide
is incorporated into the expression vector. Those of ordinary skill
in the art are familiar with the necessary steps for incorporating
a polynucleotide into an expression vector. A typical expression
vector contains the desired polynucleotide preceded by one or more
regulatory regions, along with a ribosome binding site, e.g., a
nucleotide sequence that is 3-9 nucleotides in length and located
3-11 nucleotides upstream of the initiation codon in E. coli. See
Shine and Dalgarno (1975) Nature 254(5495):34-38 and Steitz (1979)
Biological Regulation and Development (ed. Goldberger, R. F.),
1:349-399 (Plenum, New York).
[0193] The term "operably linked" as used herein refers to a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of the DNA
sequence or polynucleotide such that the control sequence directs
the expression of a polypeptide.
[0194] Regulatory regions include, for example, those regions that
contain a promoter and an operator. A promoter is operably linked
to the desired polynucleotide, thereby initiating transcription of
the polynucleotide via an RNA polymerase enzyme. An operator is a
sequence of nucleic acids adjacent to the promoter, which contains
a protein-binding domain where a repressor protein can bind. In the
absence of a repressor protein, transcription initiates through the
promoter. When present, the repressor protein specific to the
protein-binding domain of the operator binds to the operator,
thereby inhibiting transcription. In this way, control of
transcription is accomplished, based upon the particular regulatory
regions used and the presence or absence of the corresponding
repressor protein. Examples include lactose promoters (Lad
repressor protein changes conformation when contacted with lactose,
thereby preventing the Lad repressor protein from binding to the
operator) and tryptophan promoters (when complexed with tryptophan,
TrpR repressor protein has a conformation that binds the operator;
in the absence of tryptophan, the TrpR repressor protein has a
conformation that does not bind to the operator). Another example
is the tac promoter (see de Boer et al., (1983) Proc Natl Acad Sci
USA 80(1):21-25). As will be appreciated by those of ordinary skill
in the art, these and other expression vectors may be used in the
present invention, and the invention is not limited in this
respect.
[0195] Although any suitable expression vector may be used to
incorporate the desired sequences, readily available expression
vectors include, without limitation: plasmids, such as pSC1O1,
pBR322, pBBR1MCS-3, pUR, pEX, pMR100, pCR4, pBAD24, pUC19;
bacteriophages, such as Ml 3 phage and .lamda. phage. Of course,
such expression vectors may only be suitable for particular host
cells. One of ordinary skill in the art, however, can readily
determine through routine experimentation whether any particular
expression vector is suited for any given host cell. For example,
the expression vector can be introduced into the host cell, which
is then monitored for viability and expression of the sequences
contained in the vector. In addition, reference may be made to the
relevant texts and literature, which describe expression vectors
and their suitability to any particular host cell.
[0196] Host Cells of the Invention
[0197] The invention herein relates to host cells containing
recombinant polynucleotides encoding polypeptides where the
polypeptides transport cellodextrin or a pentose into the cell.
Further described herein are methods of increasing transport of
cellodextrin into a host cell, methods of increasing growth of a
host cell on a medium containing cellodextrin, methods of
co-fermenting cellulose-derived and hemicellulose-derived sugars,
and methods of making hydrocarbons or hydrocarbon derivatives by
providing a host cell containing a recombinant polynucleotide
encoding a polypeptide where the polypeptide transports
cellodextrin into the cell. Further described herein are methods of
increasing transport of a pentose into a host cell, methods of
increasing growth of a host cell on a medium containing pentose
sugars, and methods of making hydrocarbons or hydrocarbon
derivatives by providing a host cell containing a recombinant
polynucleotide encoding a polypeptide where the polypeptide
transports a pentose into the cell.
[0198] "Host cell" and "host microorganism" are used
interchangeably herein to refer to a living biological cell that
can be transformed via insertion of recombinant DNA or RNA. Such
recombinant DNA or RNA can be in an expression vector. Thus, a host
organism or cell as described herein may be a prokaryotic organism
(e.g., an organism of the kingdom Eubacteria) or a eukaryotic cell.
As will be appreciated by one of ordinary skill in the art, a
prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic
cell has a membrane-bound nucleus.
[0199] Any prokaryotic or eukaryotic host cell may be used in the
present invention so long as it remains viable after being
transformed with a sequence of nucleic acids. Preferably, the host
cell is not adversely affected by the transduction of the necessary
nucleic acid sequences, the subsequent expression of the proteins
(e.g., transporters), or the resulting intermediates. Suitable
eukaryotic cells include, but are not limited to, fungal, plant,
insect or mammalian cells.
[0200] In preferred embodiments, the host is a fungal strain.
"Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota, and Zygomycota (as defined by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
Fungi, 8th edition, 1995, CAB International, University Press,
Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et
al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et
al., 1995, supra).
[0201] In particular embodiments, the fungal host is a yeast
strain. "Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Biology and Activities of
Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0202] In a more preferred embodiment, the yeast host is a Candida,
Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia strain.
[0203] In certain embodiments, the yeast host is a Saccharomyces
carlsbergensis (Todkar, 2010), Saccharomyces cerevisiae (Duarte et
al., 2009), Saccharomyces diastaticus, Saccharomyces douglasii,
Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces
monacensis (GB-Analysts Reports, 2008), Saccharomyces bayanus
(Kristen Publicover, 2010), Saccharomyces pastorianus (Nakao et
al., 2007), Saccharomyces pombe (Mousdale, 2008), or Saccharomyces
oviformis strain. In other preferred embodiments, the yeast host is
Kluyveromyces lactis (O. W. Merten, 2001), Kluyveromyces fragilis
(Pestal et al., 2006; Siso, 1996), Kluyveromyces marxiamus (K.
Kourkoutas et al., 2008), Pichia stipitis (Almeida et al., 2008),
Candida shehatae (Ayhan Demirbas, 2003), or Candida tropicalis
(Jamai et al., 2006). In other embodiments, the yeast host may be
Yarrowia lipolytica (Biryukova E. N., 2009), Brettanomyces
custersii (Spindler D. D. et al., 1992), or Zygosaccharomyces roux
(Chaabane et al., 2006).
[0204] In another particular embodiment, the fungal host is a
filamentous fungal strain. "Filamentous fungi" include all
filamentous forms of the subdivision Eumycota and Oomycota (as
defined by Hawksworth et al., 1995, supra). The filamentous fungi
are generally characterized by a mycelial wall composed of chitin,
cellulose, glucan, chitosan, mannan, and other complex
polysaccharides. Vegetative growth is by hyphal elongation and
carbon catabolism is obligately aerobic. In contrast, vegetative
growth by yeasts such as Saccharomyces cerevisiae is by budding of
a unicellular thallus and carbon catabolism may be
fermentative.
[0205] In preferred embodiments, the filamentous fungal host is,
but not limited to, an Acremonium, Aspergillus, Fusarium, Humicola,
Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium,
Thielavia, Tolypocladium, or Trichoderma strain.
[0206] In certain embodiments, the filamentous fungal host is an
Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,
Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae
strain. In other embodiments, the filamentous fungal host is a
Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,
Fusarium culmorum, Fusarium graminearum, Fusarium graminum,
Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,
Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,
Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium
sulphureum, Fusarium torulosum, Fusarium trichothecioides, or
Fusarium venenatum strain. In yet other preferred embodiments, the
filamentous fungal host is a Humicola insolens, Humicola
lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora
crassa, Penicillium purpurogenum, Scytalidium thermophilum,
Sporotrichum thermophile (Topakas et al., 2003), or Thielavia
terrestris strain. In a further embodiment, the filamentous fungal
host is a Trichoderma harzianum, Trichoderma koningii, Trichoderma
longibrachiatum, Trichoderma reesei, or Trichoderma viride
strain.
[0207] In other preferred embodiments, the host cell is
prokaryotic, and in certain embodiments, the prokaryotes are E.
coli (Dien, B. S. et al., 2003; Yomano, L. P. et al., 1998;
Moniruzzaman et al., 1996), Bacillus subtilis (Susana Romero et
al., 2007), Zymomonas mobilis (B. S. Dien et al, 2003; Weuster
Botz, 1993; Alterthum and Ingram, 1989), Clostridium sp. (Zeikus,
1980; Lynd et al., 2002; Demain et al., 2005), Clostridium
phytofermentans (Leschine S., 2010), Clostridium thermocellum (Lynd
et al., 2002), Clostridium beijerinckii (Giles Clark, 2008),
Clostridium acetobutylicum (Moorella thermoacetica) (Huang W. C. et
al., 2004; Dominik et al., 2007), Thermoanaerobacterium
saccharolyticum (Marietta Smith, 2009), or Klebsiella oxytoca
(Dien, B. S. et al., 2003; Zhou et al., 2001; Brooks and Ingram,
1995). In other embodiments, the prokaryotic host cells are
Carboxydocella sp. (Dominik et al., 2007), Corynebacterium
glutamicum (Masayuki Inui, et al., 2004), Enterobacteriaceae
(Ingram et al., 1995), Erwinia chrysanthemi (Zhou and Ingram, 2000;
Zhou et al., 2001), Lactobacillus sp. (McCaskey, T. A., et al.,
1994), Pediococcus acidilactici (Zhou, S. et al., 2003),
Rhodopseudomonas capsulata (X. Y. Shi et al., 2004), Streptococcus
lactis (J. C. Tang et al., 1988), Vibrio furnissii (L. P. Wackett,
2010), Vibrio furnissii M1 (Park et al, 2001), Caldicellulosiruptor
saccharolyticus (Z. Kadar et al., 2004), or Xanthomonas campestris
(S. T. Yang et al., 1987). In other embodiments, the host cells are
cyanobacteria. Additional examples of bacterial host cells include,
without limitation, those species assigned to the Escherichia,
Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas,
Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia,
Vitreoscilla, Synechococcus, Synechocystis, and Paracoccus
taxonomical classes.
[0208] In especially preferred embodiments of the invention, the
host cell is Saccharomyces sp., Saccharomyces cerevisiae,
Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces
pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe,
Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis,
Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile,
Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas
mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium
thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum,
Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca,
Thermoanaerobacterium saccharolyticum, or Bacillus subtilis.
Saccharomyces sp. may include Industrial Saccharomyces strains.
Argueso et al. discuss the genome structure of an Industrial
Saccharomyces strain commonly used in bioethanol production as well
as specific gene polymorphisms that are important for bioethanol
production (Genome Research, 19: 2258-2270, 2009).
[0209] The host cells of the present invention may be genetically
modified in that recombinant nucleic acids have been introduced
into the host cells, and as such the genetically modified host
cells do not occur in nature. The suitable host cell is one capable
of expressing one or more nucleic acid constructs encoding one or
more proteins for different functions.
[0210] "Recombinant nucleic acid" or "heterologous nucleic acid" or
"recombinant polynucleotide" as used herein refers to a polymer of
nucleic acids wherein at least one of the following is true: (a)
the sequence of nucleic acids is foreign to (i.e., not naturally
found in) a given host cell; (b) the sequence may be naturally
found in a given host cell, but in an unnatural (e.g., greater than
expected) amount; or (c) the sequence of nucleic acids comprises
two or more subsequences that are not found in the same
relationship to each other in nature. For example, regarding
instance (c), a recombinant nucleic acid sequence will have two or
more sequences from unrelated genes arranged to make a new
functional nucleic acid. Specifically, the present invention
describes the introduction of an expression vector into a host
cell, wherein the expression vector contains a nucleic acid
sequence coding for a protein that is not normally found in a host
cell or contains a nucleic acid coding for a protein that is
normally found in a cell but is under the control of different
regulatory sequences. With reference to the host cell's genome,
then, the nucleic acid sequence that codes for the protein is
recombinant.
[0211] In some embodiments, the host cell naturally produces any of
the proteins encoded by the polynucleotides of the invention. The
genes encoding the desired proteins may be heterologous to the host
cell or these genes may be endogenous to the host cell but are
operatively linked to heterologous promoters and/or control regions
which result in the higher expression of the gene(s) in the host
cell. In other embodiments, the host cell does not naturally
produce the desired proteins, and comprises heterologous nucleic
acid constructs capable of expressing one or more genes necessary
for producing those molecules.
[0212] "Endogenous" as used herein with reference to a nucleic acid
molecule or polypeptide and a particular cell or microorganism
refers to a nucleic acid sequence or peptide that is in the cell
and was not introduced into the cell using recombinant engineering
techniques; for example, a gene that was present in the cell when
the cell was originally isolated from nature.
[0213] "Genetically engineered" or "genetically modified" refer to
any recombinant DNA or RNA method used to create a prokaryotic or
eukaryotic host cell that expresses a protein at elevated levels,
at lowered levels, or in a mutated form. In other words, the host
cell has been transfected, transformed, or transduced with a
recombinant polynucleotide molecule, and thereby been altered so as
to cause the cell to alter expression of a desired protein. Methods
and vectors for genetically engineering host cells are well known
in the art; for example various techniques are illustrated in
Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley
& Sons, New York, 1988, and quarterly updates). Genetically
engineering techniques include but are not limited to expression
vectors, targeted homologous recombination and gene activation
(see, for example, U.S. Pat. No. 5,272,071 to Chappel) and
trans-activation by engineered transcription factors (see, for
example, Segal et al., (1999) Proc Natl Acad Sci USA
96(6):2758-2763).
[0214] Genetic modifications that result in an increase in gene
expression or function can be referred to as amplification,
overproduction, overexpression, activation, enhancement, addition,
or up-regulation of a gene. More specifically, reference to
increasing the action (or activity) of enzymes or other proteins
discussed herein generally refers to any genetic modification of
the host cell in question which results in increased expression
and/or functionality (biological activity) of the enzymes or
proteins and includes higher activity or action of the proteins
(e.g., specific activity or in vivo enzymatic activity), reduced
inhibition or degradation of the proteins, and overexpression of
the proteins. For example, gene copy number can be increased,
expression levels can be increased by use of a promoter that gives
higher levels of expression than that of the native promoter, or a
gene can be altered by genetic engineering or classical mutagenesis
to increase the biological activity of an enzyme or action of a
protein. Combinations of some of these modifications are also
possible.
[0215] Genetic modifications which result in a decrease in gene
expression, in the function of the gene, or in the function of the
gene product (i.e., the protein encoded by the gene) can be
referred to as inactivation (complete or partial), deletion,
interruption, blockage, silencing, or down-regulation, or
attenuation of expression of a gene. For example, a genetic
modification in a gene which results in a decrease in the function
of the protein encoded by such gene, can be the result of a
complete deletion of the gene (i.e., the gene does not exist, and
therefore the protein does not exist), a mutation in the gene which
results in incomplete or no translation of the protein (e.g., the
protein is not expressed), or a mutation in the gene which
decreases or abolishes the natural function of the protein (e.g., a
protein is expressed which has decreased or no enzymatic activity
or action). More specifically, reference to decreasing the action
of proteins discussed herein generally refers to any genetic
modification in the host cell in question, which results in
decreased expression and/or functionality (biological activity) of
the proteins and includes decreased activity of the proteins (e.g.,
decreased transport), increased inhibition or degradation of the
proteins as well as a reduction or elimination of expression of the
proteins. For example, the action or activity of a protein of the
present invention can be decreased by blocking or reducing the
production of the protein, reducing protein action, or inhibiting
the action of the protein. Combinations of some of these
modifications are also possible. Blocking or reducing the
production of a protein can include placing the gene encoding the
protein under the control of a promoter that requires the presence
of an inducing compound in the growth medium. By establishing
conditions such that the inducer becomes depleted from the medium,
the expression of the gene encoding the protein (and therefore, of
protein synthesis) could be turned off. Blocking or reducing the
action of a protein could also include using an excision technology
approach similar to that described in U.S. Pat. No. 4,743,546,
incorporated herein by reference. To use this approach, the gene
encoding the protein of interest is cloned between specific genetic
sequences that allow specific, controlled excision of the gene from
the genome. Excision could be prompted by, for example, a shift in
the cultivation temperature of the culture, as in U.S. Pat. No.
4,743,546, or by some other physical or nutritional signal.
[0216] In general, according to the present invention, an increase
or a decrease in a given characteristic of a mutant or modified
protein (e.g., enzyme activity, ability to transport compounds) is
made with reference to the same characteristic of a wild-type
(i.e., normal, not modified) protein that is derived from the same
organism (from the same source or parent sequence), which is
measured or established under the same or equivalent conditions.
Similarly, an increase or decrease in a characteristic of a
genetically modified host cell (e.g., expression and/or biological
activity of a protein, or production of a product) is made with
reference to the same characteristic of a wild-type host cell of
the same species, and preferably the same strain, under the same or
equivalent conditions. Such conditions include the assay or culture
conditions (e.g., medium components, temperature, pH, etc.) under
which the activity of the protein (e.g., expression or biological
activity) or other characteristic of the host cell is measured, as
well as the type of assay used, the host cell that is evaluated,
etc. As discussed above, equivalent conditions are conditions
(e.g., culture conditions) which are similar, but not necessarily
identical (e.g., some conservative changes in conditions can be
tolerated), and which do not substantially change the effect on
cell growth or enzyme expression or biological activity as compared
to a comparison made under the same conditions.
[0217] Preferably, a genetically modified host cell that has a
genetic modification that increases or decreases the activity of a
given protein (e.g., a transporter, an enzyme) has an increase or
decrease, respectively, in the activity or action (e.g.,
expression, production and/or biological activity) of the protein,
as compared to the activity of the wild-type protein in a wild-type
host cell, of at least about 5%, and more preferably at least about
10%, and more preferably at least about 15%, and more preferably at
least about 20%, and more preferably at least about 25%, and more
preferably at least about 30%, and more preferably at least about
35%, and more preferably at least about 40%, and more preferably at
least about 45%, and more preferably at least about 50%, and more
preferably at least about 55%, and more preferably at least about
60%, and more preferably at least about 65%, and more preferably at
least about 70%, and more preferably at least about 75%, and more
preferably at least about 80%, and more preferably at least about
85%, and more preferably at least about 90%, and more preferably at
least about 95%, or any percentage, in whole integers between 5%
and 100% (e.g., 6%, 7%, 8%, etc.). The same differences are
preferred when comparing an isolated modified nucleic acid molecule
or protein directly to the isolated wild-type nucleic acid molecule
or protein (e.g., if the comparison is done in vitro as compared to
in vivo).
[0218] In another aspect of the invention, a genetically modified
host cell that has a genetic modification that increases or
decreases the activity of a given protein (e.g., a transporter, an
enzyme) has an increase or decrease, respectively, in the activity
or action (e.g., expression, production and/or biological activity)
of the protein, as compared to the activity of the wild-type
protein in a wild-type host cell, of at least about 2-fold, and
more preferably at least about 5-fold, and more preferably at least
about 10-fold, and more preferably about 20-fold, and more
preferably at least about 30-fold, and more preferably at least
about 40-fold, and more preferably at least about 50-fold, and more
preferably at least about 75-fold, and more preferably at least
about 100-fold, and more preferably at least about 125-fold, and
more preferably at least about 150-fold, or any whole integer
increment starting from at least about 2-fold (e.g., 3-fold,
4-fold, 5-fold, 6-fold, etc.).
[0219] Host Cell Components
[0220] In one aspect, host cells of the invention contain a
polynucleotide encoding a polypeptide containing transmembrane
.alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4,
.alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8,
.alpha.-helix 9, .alpha.-helix 10, .alpha.-helix 11, .alpha.-helix
12, where transmembrane .alpha.-helix 1 comprises SEQ ID NO: 1. In
another aspect, host cells of the invention contain a
polynucleotide encoding a polypeptide containing transmembrane
.alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4,
.alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8,
.alpha.-helix 9, .alpha.-helix 10, .alpha.-helix 11, .alpha.-helix
12, where transmembrane .alpha.-helix 2 comprises SEQ ID NO: 2. In
another aspect, host cells of the invention contain a
polynucleotide encoding a polypeptide containing transmembrane
.alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4,
.alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8,
.alpha.-helix 9, .alpha.-helix 10, .alpha.-helix 11, .alpha.-helix
12, where the loop connecting transmembrane .alpha.-helix 2 and
transmembrane .alpha.-helix 3 comprises SEQ ID NO: 3. In another
aspect, host cells of the invention contain a polynucleotide
encoding a polypeptide containing transmembrane .alpha.-helix 1,
.alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5,
.alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9,
.alpha.-helix 10, .alpha.-helix 11, .alpha.-helix 12, where
transmembrane .alpha.-helix 5 comprises SEQ ID NO: 4. In another
aspect, host cells of the invention contain a polynucleotide
encoding a polypeptide containing transmembrane .alpha.-helix 1,
.alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5,
.alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9,
.alpha.-helix 10, .alpha.-helix 11, .alpha.-helix 12, where
transmembrane .alpha.-helix 6 comprises SEQ ID NO: 5. In another
aspect, host cells of the invention contain a polynucleotide
encoding a polypeptide containing transmembrane .alpha.-helix 1,
.alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5,
.alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9,
.alpha.-helix 10, .alpha.-helix 11, .alpha.-helix 12, where
sequence between transmembrane .alpha.-helix 6 and transmembrane
.alpha.-helix 7 comprises SEQ ID NO: 6. In another aspect, host
cells of the invention contain a polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, where transmembrane
.alpha.-helix 7 comprises SEQ ID NO: 7. In another aspect, host
cells of the invention contain a polynucleotide encoding a
polypeptide containing transmembrane .alpha.-helix 1, .alpha.-helix
2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix
6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, where transmembrane
.alpha.-helix 10 and transmembrane .alpha.-helix 11 and the
sequence between them comprise SEQ ID NO: 8.
[0221] Each of the above described aspects may be combined in any
number of combinations. A host cell may contain a polynucleotide
encoding a polypeptide containing 1, 2, 3, 4, 5, 6, or 7 of any of
SEQ ID NOs: 1-8, or the polypeptide may contain all of SEQ ID NOs:
1-8. For example, a host cell may contain a polynucleotide encoding
a polypeptide containing transmembrane .alpha.-helix 1,
.alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5,
.alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9,
.alpha.-helix 10, .alpha.-helix 11, .alpha.-helix 12, where
transmembrane .alpha.-helix 1 comprises SEQ ID NO: 1, a loop
connecting transmembrane .alpha.-helix 2 and transmembrane
.alpha.-helix 3 comprises SEQ ID NO: 3, and transmembrane
.alpha.-helix 7 comprises SEQ ID NO: 7. Or, in another example, a
host cell may contain a polynucleotide encoding a polypeptide
containing transmembrane .alpha.-helix 1, .alpha.-helix 2,
.alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6,
.alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, .alpha.-helix 12, where transmembrane
.alpha.-helix 2 comprises SEQ ID NO: 2, transmembrane .alpha.-helix
3 comprises SEQ ID NO: 3, transmembrane .alpha.-helix 6 comprises
SEQ ID NO: 5, and transmembrane .alpha.-helix 10 and transmembrane
.alpha.-helix 11 and the sequence between them comprise SEQ ID NO:
8.
[0222] In certain embodiments of the above described aspects, the
polypeptide has at least 29%, at least 30%, at least 35%, at least
40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 99%, or at least 100% amino
acid identity to NCU00801 or NCU08114.
[0223] In preferred embodiments, the host cells further contain a
polynucleotide, where the polynucleotide encodes a catalytic domain
of a .beta.-glucosidase. As used herein, .beta.-glucosidase refers
to a .beta.-D-glucoside glucohydrolase (E.C. 3.2.1.21), which
catalyzes the hydrolysis of terminal non-reducing .beta.-D-glucose
residues with the release of .beta.-D-glucose. A catalytic domain
of .beta.-glucosidase has .beta.-glucosidase activity as
determined, for example, according to the basic procedure described
by Venturi et al., 2002. A catalytic domain of a .beta.-glucosidase
is any domain that catalyzes the hydrolysis of terminal
non-reducing residues in .beta.-D-glucosides with release of
glucose. In preferred embodiments, the .beta.-glucosidase is a
glycosyl hydrolase family 1 member. Members of this group can be
identified by the motif,
[LIVMFSTC]-[LIVFYS]-[LIV]-[LIVMST]-E-N-G-[LIVMFAR]-[CSAGN] (SEQ ID
NO: 14).
[0224] Here, E is the catalytic glutamate (webpage
expasy.org/cgi-bin/prosite-search-ac?PD0000495). In certain
embodiments, the polynucleotide encoding a catalytic domain of
.beta.-glucosidase is heterologous to the host cell. In preferred
embodiments, the catalytic domain of .beta.-glucosidase is located
intracellularly in the host cell. In preferred embodiments, the
.beta.-glucosidase is from N. crassa, and in particularly preferred
embodiments, the .beta.-glucosidase is NCU00130. In certain
embodiments, the .beta.-glucosidase may be an ortholog of NCU00130.
Examples of orthologs of NCU00130 include, without limitation, T.
melanosporum, CAZ82985.1; A. oryzae, BAE57671.1; P. placenta,
EED81359.1; P. chrysosporium, BAE87009.1; Kluyveromyces lactis,
CAG99696.1; Laccaria bicolor, EDR09330; Clavispora lusitaniae,
EEQ37997.1; and Pichia stipitis, ABN67130.1. Other
.beta.-glucosidases could be used include those from the glycosyl
hydrolase family 3. These .beta.-glucosidases can be identified by
the following motif according to PROSITE:
[LIVM](2)-[KR]-x-[EQKRD]-x(4)-G-[LIVMFTC]-[LIVT]-[LIVMF]-[ST]-D--
x(2)-[SGADNIT] (SEQ ID NO: 15). Here D is the catalytic aspartate.
Typically, any .beta.-glucosidase may be used that contains the
conserved domain of
.beta.-glucosidase/6-phospho-.beta.-glucosidase/.beta.-galactos-
idase found in NCBI sequence COG2723. Catalytic domains from
specific .beta.-glucosidases may be preferred depending on the
cellodextrin transporter contained in the host cell.
[0225] In certain embodiments, the host cell contains one or more
polynucleotides, where the one or more polynucleotides encode one
or more enzymes involved in pentose utilization. The one or more
polynucleotides may be endogenous or heterologous to the host cell.
Pentose, as used herein, refers to any monosaccharide with five
carbon atoms. Examples of pentoses include, without limitation,
xylose, arabinose, mannose, galactose, and rhamnose. The one or
more enzymes involved in pentose utilization may include, for
example, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4
epimerase, xylose isomerase, xylulokinase, aldose reductase,
L-arabitinol 4-dehydrogenase, L-xylulose reductase, and xylitol
dehydrogenase in any combination. These enzymes may come from any
organism that naturally metabolizes pentose sugars. Examples of
such organisms include, for example, Kluyveromyces sp., Zymomonas
sp., E. coli, Clostridium sp., and Pichia sp.
[0226] Examples 12-15 describe ways in which the pentose
utilization pathway in the host cell may be improved or made to be
more efficient. Strain background of a host cell can affect the
efficiency of its pentose utilization pathway. In embodiments of
the invention where the host cell is a Saccharomyces sp., preferred
pentose utilizing strains include DA24-16 (see Example 13) and
L2612 (see Example 16). Other host cells containing polynucleotides
encoding enzymes involved in pentose utilization include a DuPont
Zymomonas strain (WO 2009/058927) and a Saccharomyces strain (U.S.
Pat. No. 5,789,210).
[0227] In certain embodiments of the invention, the host cell
contains a recombinant polynucleotide encoding a pentose
transporter. In certain embodiments, pentose transporters include
those transporters discovered and described herein, including
NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, and
XUT3 (see Example 11). In other embodiments, pentose transporters
may include Gxs1 from C. intermedia, Aut1 from P. stipitis, Xylhp
from D. hansenii (Nobre et al., 1999), xylose transporter from K.
marxianus (Stambuk et al., 2003), LAT1 and LAT2 from Ambrosiozyma
monospora (EMBL AY923868 and AY923869, respectively, R. Verho et
al.), ART1 from C. arabinofermentans (Fonseca et al., 2007), KmLAT1
from K. marxiamus (Knoshaug et al., 2007), PgLAT2 from P.
guilliermondii (Knoshaug et al., 2007), and araT from P. stipitis
(Boles & Keller, 2008).
[0228] Methods of Producing and Culturing Host Cells of the
Invention
[0229] The invention herein relates to host cells containing
recombinant polynucleotides encoding polypeptides where the
polypeptide transports cellodextrin or a pentose into the cell.
Further described herein are methods of increasing transport of
cellodextrin into a host cell, methods of increasing growth of a
host cell on a medium containing cellodextrin, methods of
co-fermenting cellulose-derived and hemicellulose-derived sugars,
and methods of making hydrocarbons or hydrocarbon derivatives by
providing a host cell containing a recombinant polynucleotide
encoding a polypeptide where the polypeptide transports
cellodextrin into the cell. Further described herein are methods of
increasing transport of a pentose into a host cell, methods of
increasing growth of a host cell on a medium containing pentose
sugars, and methods of making hydrocarbons or hydrocarbon
derivatives by providing a host cell containing a recombinant
polynucleotide encoding a polypeptide where the polypeptide
transports a pentose into the cell.
[0230] Methods of producing and culturing host cells of the
invention may include the introduction or transfer of expression
vectors containing the recombinant polynucleotides of the invention
into the host cell. Such methods for transferring expression
vectors into host cells are well known to those of ordinary skill
in the art. For example, one method for transforming E. coli with
an expression vector involves a calcium chloride treatment wherein
the expression vector is introduced via a calcium precipitate.
Other salts, e.g., calcium phosphate, may also be used following a
similar procedure. In addition, electroporation (i.e., the
application of current to increase the permeability of cells to
nucleic acid sequences) may be used to transfect the host cell.
Also, microinjection of the nucleic acid sequences provides the
ability to transfect host cells. Other means, such as lipid
complexes, liposomes, and dendrimers, may also be employed. Those
of ordinary skill in the art can transfect a host cell with a
desired sequence using these or other methods.
[0231] The vector may be an autonomously replicating vector, i.e.,
a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the host, is integrated into the genome
and replicated together with the chromosome(s) into which it has
been integrated. Furthermore, a single vector or plasmid or two or
more vectors or plasmids which together contain the total DNA to be
introduced into the genome of the host, or a transposon may be
used.
[0232] The vectors preferably contain one or more selectable
markers which permit easy selection of transformed hosts. A
selectable marker is a gene the product of which provides, for
example, biocide or viral resistance, resistance to heavy metals,
prototrophy to auxotrophs, and the like. Selection of bacterial
cells may be based upon antimicrobial resistance that has been
conferred by genes such as the amp, gpt, neo, and hyg genes.
[0233] Suitable markers for yeast hosts are, for example, ADE2,
HIS3, LEU2, LYS2, METS, TRP1, and URA3. Selectable markers for use
in a filamentous fungal host include, but are not limited to, amdS
(acetamidase), argB (ornithine carbamoyltransferase), bar
(phosphinothricin acetyltransferase), hph (hygromycin
phosphotransferase), niaD (nitrate reductase), pyrG
(orotidine-5'-phosphate decarboxylase), sC (sulfate
adenyltransferase), and trpC (anthranilate synthase), as well as
equivalents thereof. Preferred for use in Aspergillus are the amdS
and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and
the bar gene of Streptomyces hygroscopicus. Preferred for use in
Trichoderma are bar and amdS.
[0234] The vectors preferably contain an element(s) that permits
integration of the vector into the host's genome or autonomous
replication of the vector in the cell independent of the
genome.
[0235] For integration into the host genome, the vector may rely on
the gene's sequence or any other element of the vector for
integration of the vector into the genome by homologous or
nonhomologous recombination. Alternatively, the vector may contain
additional nucleotide sequences for directing integration by
homologous recombination into the genome of the host. The
additional nucleotide sequences enable the vector to be integrated
into the host genome at a precise location(s) in the chromosome(s).
To increase the likelihood of integration at a precise location,
the integrational elements should preferably contain a sufficient
number of nucleic acids, such as 100 to 10,000 base pairs,
preferably 400 to 10,000 base pairs, and most preferably 800 to
10,000 base pairs, which are highly homologous with the
corresponding target sequence to enhance the probability of
homologous recombination. The integrational elements may be any
sequence that is homologous with the target sequence in the genome
of the host. Furthermore, the integrational elements may be
non-encoding or encoding nucleotide sequences. On the other hand,
the vector may be integrated into the genome of the host by
non-homologous recombination.
[0236] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host in question. The origin of replication may
be any plasmid replicator mediating autonomous replication which
functions in a cell. The term "origin of replication" or "plasmid
replicator" is defined herein as a sequence that enables a plasmid
or vector to replicate in vivo. Examples of origins of replication
for use in a yeast host are the 2 micron origin of replication,
ARS1, ARS4, the combination of ARS1 and CEN3, and the combination
of ARS4 and CEN6. Examples of origins of replication useful in a
filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991;
Cullen et al., 1987; WO 00/24883). Isolation of the AMA1 gene and
construction of plasmids or vectors comprising the gene can be
accomplished according to the methods disclosed in WO 00/24883.
[0237] For other hosts, transformation procedures may be found, for
example, in Jeremiah D. Read, et al., Applied and Environmental
Microbiology, August 2007, p. 5088-5096, for Kluyveromyces, in
Osvaldo Delgado, et al., FEMS Microbiology Letters 132, 1995,
23-26, for Zymomonas, in U.S. Pat. No. 7,501,275 for Pichia
stipitis, and in WO 2008/040387 for Clostridium,
[0238] More than one copy of a gene may be inserted into the host
to increase production of the gene product. An increase in the copy
number of the gene can be obtained by integrating at least one
additional copy of the gene into the host genome or by including an
amplifiable selectable marker gene with the nucleotide sequence
where cells containing amplified copies of the selectable marker
gene, and thereby additional copies of the gene, can be selected
for by cultivating the cells in the presence of the appropriate
selectable agent.
[0239] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
[0240] The host cell is transformed with at least one expression
vector. When only a single expression vector is used (without the
addition of an intermediate), the vector will contain all of the
nucleic acid sequences necessary.
[0241] Once the host cell has been transformed with the expression
vector, the host cell is allowed to grow. Methods of the invention
may include culturing the host cell such that recombinant nucleic
acids in the cell are expressed. For microbial hosts, this process
entails culturing the cells in a suitable medium. Typically cells
are grown at 35.degree. C. in appropriate media. Preferred growth
media in the present invention include, for example, common
commercially prepared media such as Luria Bertani (LB) broth,
Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other
defined or synthetic growth media may also be used and the
appropriate medium for growth of the particular host cell will be
known by someone skilled in the art of microbiology or fermentation
science. Temperature ranges and other conditions suitable for
growth are known in the art (see, e.g., Bailey and Ollis 1986).
[0242] According to some aspects of the invention, the culture
media contains a carbon source for the host cell. Such a "carbon
source" generally refers to a substrate or compound suitable to be
used as a source of carbon for prokaryotic or simple eukaryotic
cell growth. Carbon sources can be in various forms, including, but
not limited to polymers, carbohydrates, acids, alcohols, aldehydes,
ketones, amino acids, peptides, etc. These include, for example,
various monosaccharides such as glucose, oligosaccharides,
polysaccharides, a biomass polymer such as cellulose or
hemicellulose, xylose, arabinose, disaccharides, such as sucrose,
saturated or unsaturated fatty acids, succinate, lactate, acetate,
ethanol, etc., or mixtures thereof. The carbon source can
additionally be a product of photosynthesis, including, but not
limited to glucose.
[0243] In preferred embodiments, the carbon source is a biomass
polymer such as cellulose or hemicellulose. "A biomass polymer" as
described herein is any polymer contained in biological material.
The biological material may be living or dead. A biomass polymer
includes, for example, cellulose, xylan, xylose, hemicellulose,
lignin, mannan, and other materials commonly found in biomass.
Non-limiting examples of sources of a biomass polymer include
grasses (e.g., switchgrass, Miscanthus), rice hulls, bagasse,
cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, leaves,
grass clippings, corn stover, corn cobs, distillers grains, legume
plants, sorghum, sugar cane, sugar beet pulp, wood chips, sawdust,
and biomass crops (e.g., Crambe).
[0244] In addition to an appropriate carbon source, media must
contain suitable minerals, salts, cofactors, buffers and other
components, known to those skilled in the art, suitable for the
growth of the cultures and promotion of the enzymatic pathways
necessary for the fermentation of various sugars and the production
of hydrocarbons and hydrocarbon derivatives. Reactions may be
performed under aerobic or anaerobic conditions where aerobic,
anoxic, or anaerobic conditions are preferred based on the
requirements of the microorganism. As the host cell grows and/or
multiplies, expression of the enzymes, transporters, or other
proteins necessary for growth on various sugars or biomass
polymers, sugar fermentation, or synthesis of hydrocarbons or
hydrocarbon derivatives is affected.
[0245] Methods of Increasing Transport of a Sugar into a Cell
[0246] The present invention provides methods of increasing
transport of a sugar into a cell. In one aspect, the invention
provides a method of transporting cellodextrin into a cell,
including a first step of providing a host cell, where the host
cell contains a recombinant polynucleotide encoding a polypeptide
containing transmembrane .alpha.-helix 1, .alpha.-helix 2,
.alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5, .alpha.-helix 6,
.alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9, .alpha.-helix
10, .alpha.-helix 11, and .alpha.-helix 12, where one or more of
the following is true: transmembrane .alpha.-helix 1 comprises SEQ
ID NO: 1, transmembrane .alpha.-helix 2 comprises SEQ ID NO: 2, the
loop connecting transmembrane .alpha.-helix 2 and transmembrane
.alpha.-helix 3 comprises SEQ ID NO: 3, transmembrane .alpha.-helix
5 comprises SEQ ID NO: 4, transmembrane .alpha.-helix 6 comprises
SEQ ID NO: 5, sequence between transmembrane .alpha.-helix 6 and
transmembrane .alpha.-helix 7 comprises SEQ ID NO: 6, transmembrane
.alpha.-helix 7 comprises SEQ ID NO: 7, and transmembrane
.alpha.-helix 10 and transmembrane .alpha.-helix 11 and the
sequence between them comprise SEQ ID NO: 8. The method includes a
second step of culturing the cell such that the recombinant
polynucleotide is expressed, where expression of the recombinant
polynucleotide results in increased transport of cellodextrin into
the cell compared with a cell that does not contain the recombinant
polynucleotide. Transport of cellodextrin into a cell may be
measured by any method known to one of skill in the art, including
those methods described in Example 9 such as measuring uptake of
[.sup.3H]-cellobiose into cells or measuring the ability of an S.
cerevisiae host cell to grow when cellobiose is the sole carbon
source. Typically, the host cell containing the recombinant
polynucleotide and the host cell that does not contain the
recombinant polynucleotide will otherwise be identical in genetic
background.
[0247] In certain embodiments, the polypeptide has at least 29%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99%, or at least 100% amino acid identity to NCU00801 or
NCU08114. In certain embodiments, the host cell also contains a
recombinant polynucleotide encoding a catalytic domain of a
.beta.-glucosidase. Such embodiments are useful for host cells
lacking the endogenous ability to utilize cellodextrins.
Preferably, the catalytic domain of the .beta.-glucosidase is
intracellular. In preferred embodiments, the .beta.-glucosidase is
from Neurospora crassa. In particularly preferred embodiments, the
.beta.-glucosidase is encoded by NCU00130.
[0248] In methods of increasing transport of cellodextrin into a
cell, the cell may be cultured in a medium containing a
cellulase-containing enzyme mixture from an altered organism, where
the mixture has reduced .beta.-glucosidase activity compared to a
cellulase-containing mixture from an unaltered organism. The
organism may be altered to reduce the expression of
.beta.-glucosidase, such as by mutation of a gene encoding
.beta.-glucosidase or by targeted RNA interference or the like.
[0249] In another aspect, the invention provides a method of
increasing transport of xylose into a cell, including the steps of
providing a host cell, where the host cell contains a recombinant
polynucleotide encoding a NCU00821 or STL12/XUT6 polypeptide, and
culturing the cell such that the recombinant polynucleotide is
expressed, where expression of the recombinant polynucleotide
results in increased transport of xylose into the cell compared
with a cell that does not contain the recombinant polynucleotide.
In another aspect, the invention provides a method of increasing
transport of arabinose into a cell, including the steps of
providing a host cell, where the host cell contains a recombinant
polynucleotide encoding a XUT1 polypeptide, and culturing the cell
such that the recombinant polynucleotide is expressed, where
expression of the recombinant polynucleotide results in increased
transport of arabinose into the cell compared with a cell that does
not contain the recombinant polynucleotide. In yet another aspect,
the invention provides a method of increasing transport of
arabinose or glucose into a cell, including the steps of providing
a host cell, where the host cell contains a recombinant
polynucleotide encoding a NCU06138 polypeptide, and culturing the
cell such that the recombinant polynucleotide is expressed, where
expression of the recombinant polynucleotide results in increased
transport of arabinose or glucose into the cell compared with a
cell that does not contain the recombinant polynucleotide. In yet
another aspect the invention provides a method of increasing
transport of xylose or glucose into a cell, including the steps of
providing a host cell, where the host cell comprises a recombinant
polynucleotide encoding a SUT2, SUT3, or XUT3 polypeptide, and
culturing the cell such that the recombinant polynucleotide is
expressed, where expression of the recombinant polynucleotide
results in increased transport of xylose or glucose into the cell
compared with a cell that does not contain the recombinant
polynucleotide. In another aspect, the invention provides a method
of increasing transport of xylose, arabinose, or glucose into a
cell, including the steps of providing a host cell, where the host
cell contains a recombinant polynucleotide encoding a NCU04963
polypeptide, and culturing the cell such that the recombinant
polynucleotide is expressed, where expression of the recombinant
polynucleotide results in increased transport of xylose, arabinose,
or glucose into the cell compared with a cell that does not contain
the recombinant polynucleotide.
[0250] Transport of xylose, arabinose, or glucose into a cell may
be measured by any method known to one of skill in the art,
including those methods described in Example 11. These methods
include, for example, measuring D-xylose or L-arabinose transport
by extracting accumulated D-xylose and xylitol or L-arabinose and
arabinitol from the host cell using osmosis and analyzing it using
high performance liquid chromatography and measuring glucose
transport by using host cells lacking the ability to grow on
glucose as a sole carbon source. Typically, the host cell
containing the recombinant polynucleotide and the host cell that
does not contain the recombinant polynucleotide will otherwise be
identical in genetic background.
[0251] In certain embodiments, the host cell also contains one or
more recombinant polynucleotides where the one or more
polynucleotides encode one or more enzymes involved in pentose
utilization. The one or more enzymes may be, for example,
L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase,
xylose isomerase, xylulokinase, aldose reductase, L-arabitinol
4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, or
any other pentose utilization enzymes known in the art.
[0252] Methods of Increasing Growth of a Cell
[0253] The present invention further provides methods of increasing
the growth of a cell. In one aspect the invention provides methods
of increasing growth of a cell, including a first step of providing
a host cell, where the host cell contains a recombinant
polynucleotide encoding a polypeptide containing transmembrane
.alpha.-helix 1, .alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4,
.alpha.-helix 5, .alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8,
.alpha.-helix 9, .alpha.-helix 10, .alpha.-helix 11, and
.alpha.-helix 12, where one or more of the following is true:
transmembrane .alpha.-helix 1 comprises SEQ ID NO: 1, transmembrane
.alpha.-helix 2 comprises SEQ ID NO: 2, the loop connecting
transmembrane .alpha.-helix 2 and transmembrane .alpha.-helix 3
comprises SEQ ID NO: 3, transmembrane .alpha.-helix 5 comprises SEQ
ID NO: 4, transmembrane .alpha.-helix 6 comprises SEQ ID NO: 5,
sequence between transmembrane .alpha.-helix 6 and transmembrane
.alpha.-helix 7 comprises SEQ ID NO: 6, transmembrane .alpha.-helix
7 comprises SEQ ID NO: 7, and transmembrane .alpha.-helix 10 and
transmembrane .alpha.-helix 11 and the sequence between them
comprise SEQ ID NO: 8, and the polypeptide transports cellodextrin.
The method includes a second step of culturing the host cell in a
medium containing cellodextrin, where the host cell grows at a
faster rate in the medium than a cell that does not contain the
recombinant polynucleotide. The growth rate of a host cell may be
measured by any method known to one of skill in the art. Typically,
growth rate of a cell will be measured by evaluating cell
concentration in suspension by optical density. Preferably, the
host cell containing the recombinant polynucleotide and the host
cell that does not contain the recombinant polynucleotide will
otherwise be identical in genetic background. Media containing
cellodextrins may have resulted from enzymatic treatment of biomass
polymers such as cellulose.
[0254] In certain embodiments, the polypeptide has at least 29%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99%, or at least 100% amino acid identity to NCU00801 or
NCU08114. In certain embodiments, the host cell also contains a
recombinant polynucleotide encoding a catalytic domain of a
.beta.-glucosidase. Such embodiments are useful for host cells
lacking the endogenous ability to utilize cellodextrins.
Preferably, the catalytic domain of the .beta.-glucosidase is
intracellular. In preferred embodiments, the .beta.-glucosidase is
from Neurospora crassa. In particularly preferred embodiments, the
.beta.-glucosidase is encoded by NCU00130.
[0255] In methods of increasing growth of a cell, the culturing
medium may contain a cellulase-containing enzyme mixture from an
altered organism, where the mixture has reduced .beta.-glucosidase
activity compared to a cellulase-containing mixture from an
unaltered organism. The organism may be altered to reduce the
expression of .beta.-glucosidase, such as by mutation of a gene
encoding .beta.-glucosidase or by targeted RNA interference or the
like.
[0256] In another aspect, the invention provides a method of
increasing growth of a cell, including the steps of providing a
host cell, where the host cell contains a recombinant
polynucleotide where the polynucleotide encodes a NCU00821 or
STL12/XUT6 polypeptide, and the polypeptide transports xylose, and
culturing the host cell in a medium containing xylose, where the
host cell grows at a faster rate in the medium than a cell that
does not contain the recombinant polynucleotide. In another aspect
the invention provides a method of increasing growth of a cell,
including the steps of providing a host cell, where the host cell
contains a recombinant polynucleotide where the polynucleotide
encodes a XUT1 polypeptide, and the polypeptide transports
arabinose, and culturing the host cell in a medium containing
arabinose, where the host cell grows at a faster rate in the medium
than a cell that does not contain the recombinant polynucleotide.
In yet another aspect, the invention provides a method of
increasing growth of a cell, including the steps of providing a
host cell, where the host cell contains a recombinant
polynucleotide where the polynucleotide encodes a NCU06138
polypeptide, and the polypeptide transports arabinose and glucose,
and culturing the host cell in a medium containing arabinose or
glucose, where the host cell grows at a faster rate in the medium
than a cell that does not contain the recombinant polynucleotide.
In another aspect, the invention provides a method of increasing
growth of a cell, including the steps of providing a host cell,
where the host cell contains a recombinant polynucleotide where the
polynucleotide encodes a SUT2, SUT3, or XUT3 polypeptide, and the
polypeptide transports xylose and glucose, and culturing the host
cell in a medium containing xylose or glucose, where the host cell
grows at a faster rate in the medium than a cell that does not
contain the recombinant polynucleotide. In yet another aspect, the
invention provides a method of increasing growth of a cell,
including the steps of providing a host cell, where the host cell
contains a recombinant polynucleotide where the polynucleotide
encodes a NCU04963 polypeptide, and the polypeptide transports
xylose, arabinose, and glucose, and culturing the host cell in a
medium containing xylose, arabinose, or glucose, where the host
cell grows at a faster rate in the medium than a cell that does not
contain the recombinant polynucleotide.
[0257] The growth rate of a host cell may be measured by any method
known to one of skill in the art. Typically, growth rate of a cell
will be measured by evaluating cell concentration in suspension by
optical density. Preferably, the host cell containing the
recombinant polynucleotide and the host cell that does not contain
the recombinant polynucleotide will otherwise be identical in
genetic background. Media containing xylose or arabinose may have
resulted from acid treatment of biomass polymers such as
hemicellulose. Media containing glucose may have resulted from
enzymatic treatment of biomass polymers such as cellulose.
[0258] In certain embodiments, the host cell also contains one or
more recombinant polynucleotides where the one or more
polynucleotides encode one or more enzymes involved in pentose
utilization. The one or more enzymes may be, for example,
L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase,
xylose isomerase, xylulokinase, aldose reductase, L-arabitinol
4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, or
any other pentose utilization enzymes known in the art.
[0259] In one aspect, the invention provides methods of increasing
growth of a cell on a biomass polymer. In preferred embodiments,
the biomass polymer is cellulose. In other preferred embodiments,
the biomass polymer is hemicellulose. According to one aspect of
the invention, the method includes providing a host cell comprising
a recombinant polynucleotide that encodes a NCU07705 polypeptide.
According to another aspect of the invention, the method includes
culturing the cell in a medium comprising the biomass polymer
wherein the host cell grows at a faster rate in the medium than a
cell that does not comprise the recombinant polynucleotide.
[0260] In another aspect of the invention, the invention provides a
method of increasing growth of a cell, including the steps of
providing a host cell, where the host cell contains a recombinant
polynucleotide where the polynucleotide encodes a NCU01517,
NCU09133, or NCU10040 polypeptide, and culturing the cell in a
medium containing hemicellulose, where the host cell grows at a
faster rate in the medium than a cell that does not contain the
recombinant polynucleotide.
[0261] According to another aspect of the invention, the method
includes providing a host cell comprising an endogenous
polynucleotide that encodes a NCU05137 polypeptide. According to
another aspect of the invention, the method includes inhibiting
expression of the endogenous polynucleotide and culturing the cell
in a medium comprising a biomass polymer wherein the host cell
grows at a faster rate in the medium than a cell in which
expression of the endogenous polynucleotide is not inhibited.
[0262] Methods of the invention may include culturing the host cell
such that recombinant nucleic acids in the cell are expressed. For
microbial hosts, this process entails culturing the cells in a
suitable medium. Typically cells are grown at 35.degree. C. in
appropriate media. Preferred growth media in the present invention
include, for example, common commercially prepared media such as
Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast
medium (YM) broth. Other defined or synthetic growth media may also
be used and the appropriate medium for growth of the particular
host cell will be known by someone skilled in the art of
microbiology or fermentation science. Temperature ranges and other
conditions suitable for growth are known in the art (see, e.g.,
Bailey and Ollis 1986).
[0263] The source of the biomass polymer in the medium may include,
for example, grasses (e.g., switchgrass, Miscanthus), rice hulls,
bagasse, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw,
leaves, grass clippings, corn stover, corn cobs, distillers grains,
legume plants, sorghum, sugar cane, sugar beet pulp, wood chips,
sawdust, and biomass crops (e.g., Crambe). In addition to a biomass
polymer, the medium must contain suitable minerals, salts,
cofactors, buffers and other components, known to those skilled in
the art, suitable for the growth of the cultures. The rate of
growth of the host cell may be measured by any methods known to one
of skill in the art.
[0264] In certain embodiments of the invention, the expression of
cellulases is increased in the host cell upon expression of a
recombinant polynucleotide. "Cellulase" as used herein refers to a
category of enzymes capable of hydrolyzing cellulose polymers to
shorter cello-oligosaccharide oligomers, cellobiose, and/or
glucose. Cellulases include, without limitation, exoglucanases,
exocellobiohydrolases, endoglucanases, and glucosidases. Expression
of cellulases may be measured by RT-PCR or other methods known in
the art.
[0265] In certain embodiments of the invention, the expression of
hemicellulases is increased in the host cell upon expression of a
recombinant polynucleotide. "Hemicellulase" as used herein refers
to a category of enzymes capable of hydrolyzing hemicellulose
polymers. Hemicellulases include, without limitation, xylanases,
mannanases, arabinases (both endo and exo kinds) and their
corresponding glycosidases. Expression of hemicellulases may be
measured by RT-PCR or other methods known in the art.
[0266] Inhibition of expression of the endogenous polynucleotide
may be achieved, for example, by genetic modifications which result
in a decrease in gene expression, in the function of the gene, or
in the function of the gene product (i.e., the protein encoded by
the gene) and can be referred to as inactivation (complete or
partial), deletion, interruption, blockage, silencing, or
down-regulation, or attenuation of expression of a gene. For
example, a genetic modification in a gene which results in a
decrease in the function of the protein encoded by such a gene can
be the result of a complete deletion of the gene (i.e., the gene
does not exist, and therefore the protein does not exist), a
mutation in the gene which results in incomplete or no translation
of the protein (e.g., the protein is not expressed), or a mutation
in the gene which decreases or abolishes the natural function of
the protein (e.g., a protein is expressed which has decreased or no
enzymatic activity or action). More specifically, reference to
decreasing the action of proteins discussed herein generally refers
to any genetic modification in the host cell in question which
results in decreased expression and/or functionality (biological
activity) of the proteins and includes decreased activity of the
proteins (e.g., decreased transport), increased inhibition or
degradation of the proteins, as well as a reduction or elimination
of expression of the proteins. For example, the action or activity
of a protein of the present invention can be decreased by blocking
or reducing the production of the protein, reducing protein action,
or inhibiting the action of the protein. Combinations of some of
these modifications are also possible. Blocking or reducing the
production of a protein can include placing the gene encoding the
protein under the control of a promoter that requires the presence
of an inducing compound in the growth medium. By establishing
conditions such that the inducer becomes depleted from the medium,
the expression of the gene encoding the protein (and therefore, of
protein synthesis) could be turned off. Blocking or reducing the
action of a protein could also include using an excision technology
approach similar to that described in U.S. Pat. No. 4,743,546. To
use this approach, the gene encoding the protein of interest is
cloned between specific genetic sequences that allow specific,
controlled excision of the gene from the genome. Excision could be
prompted by, for example, a shift in the cultivation temperature of
the culture, as in U.S. Pat. No. 4,743,546, or by some other
physical or nutritional signal.
[0267] In certain embodiments of the invention, cellulase activity
of the host cell is increased upon inhibiting expression of an
endogenous polynucleotide. Cellulase activity may be measured as
described in Example 5 and by any other methods known in the
art.
[0268] In certain embodiments of the invention, hemicellulase
activity of the host cell is increased upon inhibiting expression
of an endogenous polynucleotide. Hemicellulase activity may be
measured as described in Example 17 and by any other methods known
in the art.
[0269] Methods of Co-Fermentation
[0270] One aspect of the present invention provides methods of
co-fermenting cellulose-derived and hemicellulose-derived sugars.
As used herein, co-fermentation refers to simultaneous utilization
by a host cell of more than one sugar in the same vessel. The
method includes the steps of providing a host cell, where the host
cell contains a first recombinant polynucleotide encoding a
cellodextrin transporter and a second recombinant polynucleotide
encoding a catalytic domain of a .beta.-glucosidase, and culturing
the host cell in a medium containing a cellulose-derived sugar and
a hemicellulose-derived sugar, where expression of the recombinant
polynucleotides enables co-fermentation of the cellulose-derived
sugar and the hemicellulose-derived sugar.
[0271] The first recombinant polynucleotide may encode any
polypeptide that is capable of transporting cellodextrin into a
cell. Cellodextrin transport may be measured by any method known to
one of skill in the art, including the methods discussed in Example
9. In preferred embodiments, the first recombinant polynucleotide
encodes a polypeptide containing transmembrane .alpha.-helix 1,
.alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5,
.alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9,
.alpha.-helix 10, .alpha.-helix 11, and .alpha.-helix 12, where one
or more of the following is true: transmembrane .alpha.-helix 1
comprises SEQ ID NO: 1, transmembrane .alpha.-helix 2 comprises SEQ
ID NO: 2, the loop connecting transmembrane .alpha.-helix 2 and
transmembrane .alpha.-helix 3 comprises SEQ ID NO: 3, transmembrane
.alpha.-helix 5 comprises SEQ ID NO: 4, transmembrane .alpha.-helix
6 comprises SEQ ID NO: 5, sequence between transmembrane
.alpha.-helix 6 and transmembrane .alpha.-helix 7 comprises SEQ ID
NO: 6, transmembrane .alpha.-helix 7 comprises SEQ ID NO: 7, and
transmembrane .alpha.-helix 10 and transmembrane .alpha.-helix 11
and the sequence between them comprise SEQ ID NO: 8. Examples of
such polypeptides include NCU00801, NCU00809, NCU08114,
XP.sub.--001268541.1, and LAC2. In preferred embodiments, the first
recombinant polypeptide encodes NCU00801.
[0272] The second recombinant polynucleotide may encode any
catalytic domain capable of catalyzing the hydrolysis of terminal
non-reducing residues in .beta.-D-glucosides with release of
glucose. Preferably, the .beta.-glucosidase catalytic domain is
located intracellularly in the host cell. In certain embodiments
the source of the .beta.-glucosidase domain is a N. crassa
.beta.-glucosidase. In preferred embodiments the source of the
.beta.-glucosidase domain is NCU00130. Catalytic domains from
different sources may work best with different cellodextrin
transporters.
[0273] In certain embodiments, the host cell also contains one or
more recombinant polynucleotides where the one or more
polynucleotides encode one or more enzymes involved in pentose
utilization. Alternatively, one or more polynucleotides encoding
one or more enzymes involved in pentose utilization may be
endogenous to the host cell. The one or more enzymes may include,
for example, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P
4 epimerase, xylose isomerase, xylulokinase, aldose reductase,
L-arabitinol 4-dehydrogenase, L-xylulose reductase, xylitol
dehydrogenase, or any other pentose-utilizing enzymes known to one
of skill in the art.
[0274] In certain embodiments, the host cell contains a third
recombinant polynucleotide where the polynucleotide encodes a
pentose transporter. Alternatively, the host cell may contain an
endogenous polynucleotide encoding a pentose transporter. In
preferred embodiments, the pentose transporter transports xylose
and/or arabinose into the cell. In certain embodiments, the third
recombinant polynucleotide encodes a polypeptide such as NCU00821,
NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, or XUT3. The
expression of a pentose transporter in the host cell may enhance
the efficiency of co-fermentation if glucose is present along with
a pentose sugar is the growth medium.
[0275] In methods of co-fermentation as described herein,
cellulose-derived sugars preferably include cellobiose,
cellotriose, and celltetraose, and hemicellulose-derived sugars
preferably include xylose and arabinose. Typically, in order to
prepare the cellulose-derived sugars and hemicellulose-derived
sugars for co-fermentation by a host cell, lignocellulosic biomass
is first pretreated to alter its structure and allow for better
enzymatic hydrolysis of cellulose. Pretreatment may include
physical or chemical methods, including, for example, ammonia
fiber/freeze explosion, the lime method based on calcium or sodium
hydroxide, and steam explosion with or without an acid catalyst.
Acid treatment will release xylose and arabinose from the
hemicellulose component of the lignocellulosic biomass. Next,
preferably, the cellulose component of the pretreated biomass is
hydrolyzed by a mixture of cellulases. Examples of commercially
available cellulase mixtures include Celluclast 1.5L.RTM.
(Novozymes), Spezyme CP.RTM. (Genencor) (Scott W. Pryor, 2010, Appl
Biochem Biotechnol), and Cellulyve 50L (Lyven).
[0276] Cellulase mixtures typically contain endoglucanases,
exoglucanases, and .beta.-glucosidases. In methods of
co-fermentation as described herein, the amount of
.beta.-glucosidase activity in the cellulase mixture should be
minimized as much as possible. For example, the culturing medium
may contain a cellulase-containing enzyme mixture from an altered
organism, where the mixture has reduced .beta.-glucosidase activity
compared to a cellulase-containing mixture from an unaltered
organism. The organism may be altered to reduce the expression of
.beta.-glucosidase, such as by mutation of a gene encoding
.beta.-glucosidase or by targeted RNA interference or the like.
[0277] Surprisingly, as described in Example 17, co-fermentation of
cellobiose and xylose by the methods of the invention resulted in a
synergistic effect on sugar consumption and ethanol production by
the host cell.
[0278] Methods of Synthesis of Hydrocarbons or Hydrocarbon
Derivatives
[0279] One aspect of the present invention provides methods for
increasing the synthesis of hydrocarbons or hydrocarbon derivatives
by a host cell.
[0280] "Hydrocarbons" as used herein are organic compounds
consisting entirely of hydrogen and carbon. Hydrocarbons include,
without limitation, methane, ethane, ethene, ethyne, propane,
propene, propyne, cyclopropane, allene, butane, isobutene, butene,
butyne, cyclobutane, methylcyclopropane, butadiene, pentane,
isopentane, neopentane, pentene, pentyne, cyclopentane,
methylcyclobutane, ethylcyclopropane, pentadiene, isoprene, hexane,
hexene, hexyne, cyclohexane, methylcyclopentane, ethylcyclobutane,
propylcyclopropane, hexadiene, heptane, heptene, heptyne,
cycloheptane, methylcyclohexane. heptadiene, octane, octene,
octyne, cyclooctane, octadiene, nonane, nonene, nonyne,
cyclononane, nonadiene, decane, decene, decyne, cyclodecane, and
decadiene.
[0281] "Hydrocarbon derivatives" as used herein are organic
compounds of carbon and at least one other element that is not
hydrogen. Hydrocarbon derivatives include, without limitation,
alcohols (e.g., arabinitol, butanol, ethanol, glycerol, methanol,
1,3-propanediol, sorbitol, and xylitol); organic acids (e.g.,
acetic acid, adipic acid, ascorbic acid, citric acid,
2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric
acid, gluconic acid, glucuronic acid, glutaric acid,
3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,
malonic acid, oxalic acid, propionic acid, succinic acid, and
xylonic acid); esters; ketones (e.g., acetone); aldehydes (e.g.,
furfural); amino acids (e.g., aspartic acid, glutamic acid,
glycine, lysine, serine, and threonine); and gases (e.g., carbon
dioxide and carbon monoxide).
[0282] In preferred embodiments, the hydrocarbon or hydrocarbon
derivative can be used as fuel. In particularly preferred
embodiments, the hydrocarbon or hydrocarbon derivative is ethanol
or butanol.
[0283] According to one aspect of the invention, a method of
increasing the synthesis of hydrocarbons or hydrocarbon derivatives
by a host cell includes a first step of providing a host cell,
where the host cell contains a recombinant polynucleotide encoding
a polypeptide containing transmembrane .alpha.-helix 1,
.alpha.-helix 2, .alpha.-helix 3, .alpha.-helix 4, .alpha.-helix 5,
.alpha.-helix 6, .alpha.-helix 7, .alpha.-helix 8, .alpha.-helix 9,
.alpha.-helix 10, .alpha.-helix 11, and .alpha.-helix 12, where one
or more of the following is true: transmembrane .alpha.-helix 1
comprises SEQ ID NO: 1, transmembrane .alpha.-helix 2 comprises SEQ
ID NO: 2, the loop connecting transmembrane .alpha.-helix 2 and
transmembrane .alpha.-helix 3 comprises SEQ ID NO: 3, transmembrane
.alpha.-helix 5 comprises SEQ ID NO: 4, transmembrane .alpha.-helix
6 comprises SEQ ID NO: 5, sequence between transmembrane
.alpha.-helix 6 and transmembrane .alpha.-helix 7 comprises SEQ ID
NO: 6, transmembrane .alpha.-helix 7 comprises SEQ ID NO: 7, and
transmembrane .alpha.-helix 10 and transmembrane .alpha.-helix 11
and the sequence between them comprise SEQ ID NO: 8, and where the
polypeptide transports cellodextrin into the host cell for the
synthesis of hydrocarbons or hydrocarbon derivatives. The method
includes a second step of culturing the host cell in a medium
containing cellodextrin or a source of cellodextrin to increase the
synthesis of hydrocarbons or hydrocarbon derivatives by the host
cell, where transport of cellodextrin into the cell is increased
upon expression of the recombinant polynucleotide. In certain
embodiments, the polypeptide has at least 29%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 99%, or at
least 100% amino acid identity to NCU00801 or NCU08114. In certain
embodiments, the host cell also contains a recombinant
polynucleotide encoding a catalytic domain of a .beta.-glucosidase.
Such embodiments are useful for host cells lacking the endogenous
ability to utilize cellodextrins. Preferably, the catalytic domain
of the .beta.-glucosidase is intracellular. In preferred
embodiments, the .beta.-glucosidase is from Neurospora crassa. In
particularly preferred embodiments, the .beta.-glucosidase is
encoded by NCU00130. Transport of cellodextrin into the cell may be
measured by any methods known to one of skill in the art, including
the methods described in Example 9. Typically, the source of the
cellodextrin is cellulose.
[0284] The culturing medium may contain a cellulase-containing
enzyme mixture from an altered organism, where the mixture has
reduced .beta.-glucosidase activity compared to a
cellulase-containing mixture from an unaltered organism. The
organism may be altered to reduce the expression of
.beta.-glucosidase, such as by mutation of a gene encoding
.beta.-glucosidase or by targeted RNA interference or the like.
[0285] According to another aspect of the invention, a method of
increasing the synthesis of hydrocarbons or hydrocarbon derivatives
by a host cell includes the steps of providing a host cell, where
the host cell contains a recombinant polynucleotide encoding a
NCU00821 or STL12/XUT6 polypeptide, where the polypeptide
transports xylose into the host cell for the synthesis of
hydrocarbons or hydrocarbon derivatives, and culturing the host
cell in a medium containing xylose or a source of xylose to
increase the synthesis of hydrocarbons or hydrocarbon derivatives
by the host cell, where transport of xylose into the cell is
increased upon expression of the recombinant polynucleotide.
[0286] According to another aspect, a method of increasing the
synthesis of hydrocarbons or hydrocarbon derivatives by a host cell
includes the steps of providing a host cell, where the host cell
contains a recombinant polynucleotide encoding a XUT1 polypeptide,
where the polypeptide transports arabinose into the host cell for
the synthesis of hydrocarbons or hydrocarbon derivatives, and
culturing the host cell in a medium containing arabinose or a
source of arabinose to increase the synthesis of hydrocarbons or
hydrocarbon derivatives by the host cell, where transport of
arabinose into the cell is increased upon expression of the
recombinant polynucleotide.
[0287] According to yet another aspect, a method of increasing the
synthesis of hydrocarbons or hydrocarbon derivatives by a host cell
includes the steps of providing a host cell, where the host cell
contains a recombinant polynucleotide encoding a NCU06138
polypeptide, where the polypeptide transports arabinose or glucose
into the host cell for the synthesis of hydrocarbons or hydrocarbon
derivatives, and culturing the host cell in a medium comprising
arabinose or glucose or a source of arabinose or glucose to
increase the synthesis of hydrocarbons or hydrocarbon derivatives
by the host cell, where transport of arabinose or glucose into the
cell is increased upon expression of the recombinant
polynucleotide.
[0288] According to yet another aspect, a method of increasing the
synthesis of hydrocarbons or hydrocarbon derivatives by a host cell
includes the steps of providing a host cell, where the host cell
contains a recombinant polynucleotide encoding a SUT2, SUT3, or
XUT3 polypeptide, where the polypeptide transports xylose or
glucose into the host cell for the synthesis of hydrocarbons or
hydrocarbon derivatives, and culturing the host cell in a medium
containing xylose or glucose or a source of xylose or glucose to
increase the synthesis of hydrocarbons or hydrocarbon derivatives
by the host cell, where transport of xylose or glucose into the
cell is increased upon expression of the recombinant
polynucleotide.
[0289] According to another aspect, a method of increasing the
synthesis of hydrocarbons or hydrocarbon derivatives by a host cell
includes the steps of providing a host cell, where the host cell
contains a recombinant polynucleotide encoding a NCU04963
polypeptide, where the polypeptide transports xylose, arabinose, or
glucose into the host cell for the synthesis of hydrocarbons or
hydrocarbon derivatives, and culturing the host cell in a medium
comprising xylose, arabinose, or glucose or a source of xylose,
arabinose, or glucose to increase the synthesis of hydrocarbons or
hydrocarbon derivatives by the host cell, where transport of
xylose, arabinose, or glucose into the cell is increased upon
expression of the recombinant polynucleotide.
[0290] Transport of xylose, arabinose, or glucose into the cell may
by measured by any methods known to one of skill in the art,
including the methods described in Example 11. Typically, the
source of glucose is cellulose, and the source of xylose and
arabinose is hemicellulose.
[0291] Methods of Degrading Cellulose
[0292] One aspect of the present invention provides methods of
degrading cellulose. The methods include a first step of providing
a composition comprising cellulose. The cellulose is preferably
from plant material, such as switchgrass, Miscanthus, rice hulls,
bagasse, flax, bamboo, sisal, abaca, straw, leaves, grass
clippings, corn stover, corn cobs, distillers grains, legume
plants, sorghum, sugar cane, sugar beet pulp, wood chips, sawdust,
and biomass crops.
[0293] The methods include a second step of contacting the
composition with a cellulase-containing enzyme mixture from an
altered organism, where the cellulase-containing mixture has
reduced .beta.-glucosidase activity compared to a
cellulase-containing mixture from an unaltered organism. The
cellulose is degraded by the cellulase-containing mixture. The
organism may be altered by mutation of a gene encoding a
.beta.-glucosidase or by reducing the expression of a
.beta.-glucosidase with a technique such as RNA interference. The
organism may be a fungus or a bacterium. In preferred embodiments,
the organism is a filamentous fungus such as T. reesei.
[0294] Alternatively, the methods include a second step of
contacting the composition with a cellulase-containing enzyme
mixture that has been altered to reduce its .beta.-glucosidase
activity. For example, the cellulase-containing enzyme mixture may
be altered by affinity chromatography where .beta.-glucosidase
enzymes are captured during the chromatography, and thus removed
from the mixture. In another example, the cellulase-containing
enzyme mixture is altered by inactivation of .beta.-glucosidase
enzymes in the mixtures with an inhibitor. Examples of commercially
available cellulase mixtures include Celluclast 1.5L.RTM.
(Novozymes), Spezyme CP.RTM. (Genencor) (Scott W. Pryor, 2010, Appl
Biochem Biotechnol), and Cellulite 50L (Lyven).
[0295] It is to be understood that, while the invention has been
described in conjunction with the preferred specific embodiments
thereof, the foregoing description is intended to illustrate and
not limit the scope of the invention. Other aspects, advantages,
and modifications within the scope of the invention will be
apparent to those skilled in the art to which the invention
pertains.
[0296] The invention having been described, the following examples
are offered to illustrate the subject invention by way of
illustration, not by way of limitation.
EXAMPLES
Example 1
Transcriptome Analysis of N. crassa Grown on Miscanthus and
Avicel
[0297] In this example, the expression profile of the N. crassa
genome was examined during growth on Miscanthus or Avicel. Growth
and cellulase activity of N. crassa (FGSC 2489) cultured on Vogel's
minimal media with crystalline cellulose (Avicel) as the sole
carbon source was similar to that of T. reesei (QM9414) (FIG. 3);
N. crassa completely degraded Avicel in approximately 4 days. N.
crassa also grew rapidly on ground Miscanthus stems, suggesting
functional cellulase and hemicellulase degradative capacity. To
determine the transcriptome associated with plant cell wall
deconstruction in N. crassa, we used full genome microarrays
(Kasuga and Glass 2008; Tian et al., 2007; Kasuga et al., 2005) to
monitor gene expression profiles during growth of N. crassa on
ground Miscanthus stems. RNA was sampled after 16 hrs of growth on
sucrose and compared to RNA isolated from N. crassa grown on
Miscanthus medium at 16 and 40 hrs, 5 days and 10 days (FIGS. 4A-B;
also see Supplemental Data, Dataset S1, page 1 in Tian et al.,
2009; data can also be found at
bioinfo.townsend.yale.edu/browse.jsp, Experiment IDs 52 and
53).
[0298] A total of 769 N. crassa genes showed a statistically
significant difference in relative expression level among the four
Miscanthus samples as compared to the sucrose sample (see
Supplemental Data, Dataset S1, page 3 in Tian et al., 2009).
Hierarchical clustering showed that these genes fell into three
main clusters (FIG. 4A). The first cluster of genes (Cl; 300 genes)
showed the highest expression levels in minimal medium with sucrose
as a carbon source. Functional category (FunCat) analysis (Ruepp
2004) of these genes showed an enrichment for ribosomal proteins
and other functional categories associated with primary metabolism,
such as respiration, electron transport and amino acid metabolism
(see Supplemental Data, Dataset S1, page 4 in Tian et al., 2009).
The second cluster (C2) included 327 genes that showed the highest
expression levels in Miscanthus cultures at later time points (40
hrs to 10 days; FIG. 4A). Within this group were 89 genes that
showed a high relative expression level in Miscanthus cultures at
all time points. For further analyses, these 89 genes were added to
the cluster of genes that showed the highest expression levels from
the 16 hr Miscanthus cultures (C3 cluster, see below). FunCat
analysis (Ruepp 2004) of the remaining 238 genes showed one
functional category (C-compound and carbohydrate metabolism) was
slightly enriched (see Supplemental Data, Dataset S1, page 5 in
Tian et al., 2009).
[0299] A third cluster of 142 genes showed the highest relative
expression level after 16 hrs of growth of N. crassa on Miscanthus
(C3, FIG. 4A). FunCat analysis (Ruepp 2004) of these 142 genes plus
the 89 genes that showed high expression levels in Miscanthus
cultures at all time points (C3+ cluster; total 231 genes) showed
an enrichment for proteins involved with carbon metabolism,
including predicted cellulases and hemicellulases (FIG. 4C; also
see Supplemental Data, Dataset S1, page 6 in Tian et al., 2009). Of
the 23 predicted cellulase genes in the N. crassa genome, 18 showed
significant increases in expression levels during growth on
Miscanthus (see Table 1 in Tian et al., 2009), particularly at the
16 hr time point (FIG. 5A). Five genes showed an increase in
expression level over 200-fold (cbh-1 (CBH(I); NCU07340, gh6-2
(CBH(II)-like gene; NCU09680), gh6-3 (NCU07190) and two GH61 genes
(gh61-4; NCU01050 and NCU07898))).
[0300] Plant cell walls are complex structures composed of
cellulose microfibrils, hemicellulose, lignin, pectin, cutin, and
protein. Thus, we compared expression profiles of N. crassa grown
on Miscanthus to expression profiles of N. crassa grown on Avicel,
a pure form of crystalline cellulose (see Tian et al., 2009,
Supplemental Data, Dataset S1, page 2; data can also be found at
bioinfo.townsend.yale.edu/browse.jsp, Experiment IDs 52 and 53).
Over 187 genes showed a significant increase in relative expression
level during growth of N. crassa on Avicel. Of these genes, 114
overlapped with the 231 genes in the C3+ cluster (FIG. 4B). FunCat
analysis of the 114-overlap gene set showed a clear enrichment for
genes predicted to be involved in carbon metabolism (see
Supplemental Data, Dataset S1, page 6 in Tian et al., PNAS, 2009).
Within this gene set, there was a further enrichment for secreted
proteins; 53 of the 114 gene products were predicted to be
secreted. Of these 53 genes, 32 encode predicted proteins that have
annotation suggesting a role in plant cell wall degradation, while
16 encode putative or hypothetical proteins that lack any
functional prediction. The remaining 61 genes encode predicted
intracellular proteins, including ten predicted major facilitator
superfamily transporters (NCU00801, NCU00988, NCU01231, NCU04963,
NCU05519, NCU05853, NCU05897, NCU06138, NCU08114 and NCU10021) and
23 putative or hypothetical proteins.
[0301] Of the 117 genes within the Miscanthus-specific cluster
(FIG. 4B), 37 encode proteins predicted to be secreted. Nine
predicted hemicellulases or enzymes related to the degradation of
hemicellulose were identified (NCU00710, NCU04265, NCU04870,
NCU05751, NCU05965, NCU09170, NCU09775, NCU09923 and NCU09976)
(Tian et al., 2009-Table 2). The remaining 80 Miscanthus-specific
genes encode predicted intracellular proteins, including genes
involved in the metabolism of pentose sugars (for example,
NCU00891, xylitol dehydrogenase and NCU00643, a predicted
arabinitol dehydrogenase), a predicted sugar transporter
(NCU01132), and 48 proteins of unknown function.
Example 2
Secretome Analysis of N. crassa Grown on Miscanthus and Avicel
[0302] Lignocellulose degradation by fungi takes place
extracellularly and requires the secretion of proteins associated
with depolymerization of cell wall constituents (Lynd et al.,
2002). To compare with transcriptional profiling data, which showed
that genes encoding predicted cellulases, hemicellulases, and other
secreted proteins increased in expression levels when N. crassa was
grown on Miscanthus or Avicel, we analyzed the secretome of N.
crassa using a shotgun proteomics approach (FIG. 4B). Supernatants
from seven day old Miscanthus and Avicel cultures were digested
with trypsin and analyzed by liquid chromatography
nano-electrospray ionization tandem mass spectrometry (MS; see
Example 5). Secreted proteins that bound to phosphoric acid swollen
cellulose (PASC) were enriched and also analyzed by MS.
[0303] A total of 50 proteins were identified with confidence by
tandem MS (Tables 2 and 3). There were 34 proteins detected in the
Miscanthus grown N. crassa cultures, while 38 proteins were
identified from Avicel grown cultures; twenty-two proteins were
detected in both samples. Of these 22 proteins, 21 were predicted
to be secreted based on computational analyses and 19 showed
increased expression levels in both the Miscanthus and Avicel grown
cultures (Table 2). The overlap dataset included eight of the 23
predicted cellulases in N. crassa (Table 3). There were also five
predicted hemicellulases, a predicted .beta.-glucosidase (gh3-4;
NCU04952), five proteins with predicted activity on carbohydrates,
and two proteins with unknown function (NCU07143 and NCU05137)
(Table 4-5).
[0304] For Table 2, the annotation was generated by the Broad
Institute at webpage
broad.mit.edu/annotation/genome/neurospora/Home.html. The "sample
detected" was the sample in which peptides were detected for a
particular protein. Peptides were validated by manual inspection. A
protein was determined to be present if at least 1 peptide was
detected in each biological repeat. "TOTAL" refers to peptides
detected from a tryptic digest of all extracellular proteins. "PASC
BOUND" refers to peptides detected after enrichment for proteins
that bind to phosphoric acid swollen cellulose. "UNBOUND" refers to
proteins remaining in solution after removal of PASC bound
proteins.
TABLE-US-00002 TABLE 2 Proteins identified by LC-MS/MS In the
culture filtrates of Avicel grown Neurospora crassa GENE ID
ANNOTATION SAMPLE DETECTED NCU00206 Neurospora crassa hypothetical
protein similar to cellobiose dehydrogenase 830 nt TOTAL NCU00762
Neurospora crassa endoglucanase 3 precursor 391 nt TOTAL NCU01050
Neurospora crassa hypothetical protein similar to endoglucanase II
239 nt TOTAL NCU02343 Neurospora crassa hypothetical protein
similar to alpha L arabinofuranosidase A TOTAL 668 nt NCU04870
Neurospora crassa hypothetical protein similar to acetyl xylan
esterase 313 nt TOTAL NCU04952 Neurospora crassa hypothetical
protein similar to beta D glucoside glucohydrolase TOTAL 736 nt
NCU05137 Neurospora crassa conserved hypothetical protein 692 nt
TOTAL NCU05159 Neurospora crassa acetylxylan esterase precursor 301
nt TOTAL NCU05924 Neurospora crassa endo 1 4 beta xylanase 330 nt
TOTAL NCU07143 Neurospora crassa predicted protein 391 nt TOTAL
NCU07190 Neurospora crassa conserved hypothetical protein 384 nt
TOTAL NCU07225 Neurospora crassa endo 1 4 beta xylanase 2 precursor
255 nt TOTAL NCU07326 Neurospora crassa predicted protein 327 nt
TOTAL NCU07340 Neurospora crassa exoglucanase 1 precursor 522 nt
TOTAL NCU07898 Neurospora crassa predicted protein 242 nt TOTAL
NCU08189 Neurospora crassa hypothetical protein similar to endo 1 4
beta xylanase 385 nt TOTAL NCU08398 Neurospora crassa conserved
hypothetical protein 391 nt TOTAL NCU08760 Neurospora crassa
predicted protein 343 nt TOTAL NCU08785 Neurospora crassa conserved
hypothetical protein 291 nt TOTAL NCU09491 Neurospora crassa
feruloyl esterase B precursor 293 nt TOTAL NCU09680 Neurospora
crassa exoglucanase 2 precursor 485 nt TOTAL NCU09923 Neurospora
crassa hypothetical protein similar to beta xylosidase 775 nt TOTAL
NCU00206 Neurospora crassa hypothetical protein similar to
cellobiose dehydrogenase 830 nt PASC BOUND NCU00762 Neurospora
crassa endoglucanase 3 precursor 391 nt PASC BOUND NCU05159
Neurospora crassa acetylxylan esterase precursor 301 nt PASC BOUND
NCU05955 Neurospora crassa hypothetical protein similar to Cel74a
862 nt PASC BOUND NCU07225 Neurospora crassa endo 1 4 beta xylanase
2 precursor 255 nt PASC BOUND NCU07340 Neurospora crassa
exoglucanase 1 precursor 522 nt PASC BOUND NCU08760 Neurospora
crassa predicted protein 343 nt PASC BOUND NCU09680 Neurospora
crassa exoglucanase 2 precursor 485 nt PASC BOUND NCU09708
Neurospora crassa conserved hypothetical protein 465 nt PASC BOUND
NCU00762 Neurospora crassa endoglucanase 3 precursor 391 nt UNBOUND
NCU01651 Neurospora crassa conserved hypothetical protein 783 nt
UNBOUND NCU02343 Neurospora crassa hypothetical protein similar to
alpha L arabinofuranosidase A UNBOUND 668 nt NCU04202 Neurospora
crassa nucleoside diphosphate kinase 1 153 nt UNBOUND NCU04870
Neurospora crassa hypothetical protein similar to acetyl xylan
esterase 313 nt UNBOUND NCU04952 Neurospora crassa hypothetical
protein similar to beta D glucoside glucohydrolase UNBOUND 736 nt
NCU05057 Neurospora crassa endoglucanase EG 1 precursor 439 nt
UNBOUND NCU05137 Neurospora crassa conserved hypothetical protein
692 nt UNBOUND NCU05751 Neurospora crassa conserved hypothetical
protein 242 nt UNBOUND NCU05924 Neurospora crassa endo 1 4 beta
xylanase 330 nt UNBOUND NCU06239 Neurospora crassa conserved
hypothetical protein 514 nt UNBOUND NCU07143 Neurospora crassa
predicted protein 391 nt UNBOUND NCU07190 Neurospora crassa
conserved hypothetical protein 384 nt UNBOUND NCU07225 Neurospora
crassa endo 1 4 beta xylanase 2 precursor 255 nt UNBOUND NCU07326
Neurospora crassa predicted protein 327 nt UNBOUND NCU07898
Neurospora crassa predicted protein 242 nt UNBOUND NCU08189
Neurospora crassa hypothetical protein similar to endo 1 4 beta
xylanase 385 nt UNBOUND NCU08398 Neurospora crassa conserved
hypothetical protein 391 nt UNBOUND NCU08412 Neurospora crassa
conserved hypothetical protein 401 nt UNBOUND NCU08760 Neurospora
crassa predicted protein 343 nt UNBOUND NCU08785 Neurospora crassa
conserved hypothetical protein 291 nt UNBOUND NCU09024 Neurospora
crassa conserved hypothetical protein 625 nt UNBOUND NCU09175
Neurospora crassa conserved hypothetical protein 411 nt UNBOUND
NCU09267 Neurospora crassa conserved hypothetical protein 1048 nt
UNBOUND NCU09491 Neurospora crassa feruloyl esterase B precursor
293 nt UNBOUND NCU09775 Neurospora crassa hypothetical protein
similar to alpha L arabinofuranosidase 343 UNBOUND nt NCU09923
Neurospora crassa hypothetical protein similar to beta xylosidase
775 nt UNBOUND
TABLE-US-00003 TABLE 3 22 secreted proteins detected in both
Miscanthus and Avicel cultures Gene name Gene annotation Profiling
kos CBM1 Signal P NCU00206.2 CBDH both heter yes yes NCU00762.2
probable cellulase precursor both 16747 yes yes NCU01050.2 related
to cel1 protein precursor both 16543 no yes NCU04952.2 probable
beta-D-glucoside glucohydrolase both 13732 no yes NCU05057.2
probable endo-1,4-beta-glucanase both 13343 no yes NCU05137.2
conserved hypothetical protein both 11682 no yes NCU05924.2
probable endo-beta-1,4-D-xylanase both 15626 no yes NCU05955.2
probable endoglucanase C both 13535 yes yes NCU07143.2 hypothetical
both No no yes NCU07190.2 CBHII homolog both 19315 no yes
NCU07225.2 probable endo-1,4-beta-xylanase B both heter yes yes
precursor NCU07326.2 related to putative arabinase both 19534 no
yes NCU07340.2 CBHI both 15630 yes yes NCU07898.2 related to cel1
protein precursor both 19600 no yes NCU08189.2 similar to
endo-1,4-beta xylanase both 19861 no yes NCU08398.2 related to
aldose 1-epimerase both 20310 no yes NCU08412.2 hypothetical
protein 401 nt none No no no NCU08760.2 related to family 61
endoglucanase both 15664 yes yes NCU09024.2 hypothetical protein
625 nt none No no yes NCU09175.2 glucan 1,3-beta-glucosidase
precursor both 11750 no yes NCU09491.2 feruloyl esterase B
precursor mis No no yes NCU09680.2 CBHII both 15633 yes yes
[0305] Table 4 shows predicted cellulase genes in Neurospora
crassa
TABLE-US-00004 GH.sup.1 EL.sup.5 EL.sup.5 Gene Family CBM1.sup.2
SP.sup.3 MS.sup.4 Miscanthus Avicel NCU00762 5 yes yes both 29.6
31.5 NCU03996 6 no no ND6 ND ND NCU07190 6 no yes both 526.0 119
NCU09680 6 yes yes both 230.9 251.3 NCU04854 7 no yes ND 32.9 10.8
NCU05057 7 no yes both 8.7 7.4 NCU05104 7 no yes ND 11.6 NC7
NCU07340 7 yes yes both 426.4 382.2 NCU05121 45 yes yes avi 8.6
17.2 NCU00836 61 yes yes ND 91.2 31 NCU01050 61 no yes both 206.7
382.1 NCU01867 61 yes yes ND 2.2 NC NCU02240 61 yes yes avi 193.5
84 NCU02344 61 no yes ND 8.1 4.1 NCU02916 61 yes yes ND 85.2 17.7
NCU03000 61 no yes ND NC ND NCU03328 61 no yes ND 26.4 23.8
NCU05969 61 no yes ND ND 12.7 NCU07520 61 no yes ND ND ND NCU07760
61 yes yes ND 3.7 NC NCU07898 61 no yes both 376.3 230 NCU07974 61
no yes ND NC NC NCU08760 61 yes yes both 107.5 44.7 .sup.1Glucoside
Hydrolase; .sup.2CBM1, carbohydrate binding module; .sup.3Signal
peptide prediction (signalP = webpage
cbs.dtu.dk/services/SignalP/); .sup.4MS, mass spectrometry
analysis; .sup.5EL, relative expression level; 6ND, not detected;
7NC, no change in expression level versus minimal media.
TABLE-US-00005 TABLE 5 Cellulases and Hemicellulases identified by
LC-MS/MS Predicted cellulases in the genome of Neurospora crassa GH
MIS Gene ID Family AV MS MIS MS AV ARRAY ARRAY NCU00762 5 + + 31.5
29.6 NCU00836 61 - - 31 91.2 NCU01050 61 + + 382.1 206.7 NCU01867
61 - - 1 1 NCU02240 61 + - 84 193.5 NCU02344 61 - - 4.1 8.1
NCU02916 61 - - 17.7 85.2 NCU03000 61 - - 1 1 NCU03328 61 - - 23.8
26.4 NCU03996 6 - - 2.5 6.3 NCU04854 7 - - 10.8 32.9 NCU05057 7 + +
7.4 8.7 NCU05104 7 - - 1 1 NCU05121 45 + - 17.2 8.6 NCU05969 61 - -
12.7 12.3 NCU07190 6 + + 119 526 NCU07340 7 + + 382.2 426.4
NCU07520 61 - - 1 1 NCU07760 61 - - 1 3.4 NCU07898 61 + + 230.5
376.3 NCU07974 61 - - 1 1 NCU08760 61 + + 44.7 107.5 NCU09680 6 + +
251.3 230.9 Predicted hemicellulases in the genome of Neurospora
crassa GH Gene ID Family AV MS MIS MS AV Array MIS Array NCU00852
43 - - 1 1 NCU00972 53 - - 9.03 15.6 NCU01900 43 - - 10.03 26
NCU02343 51 - + 6.63 174.6 NCU02855 11 + - 10.2 364 NCU04997 10 - -
1 25.6 NCU05924 10 + + 55.9 149.3 NCU05955 74 + + 19.9 50.5
NCU05965 43 - - 1 5.4 NCU06861 43 - - 1 1 NCU07130 10 - - 1 1
NCU07225 11 + + 11.43 33.5 NCU07326 43 + + 104.5 426.6 NCU07351 67
- - 1 1 NCU08087 26 - - 1 1 NCU08189 10 + + 39.8 94.4 NCU09170 43 -
- 1 16.7 NCU09652 43 - - 12.2 95.4 NCU09775 54 - + 1 48.3 GH
Family--Glycosyl Hydrolase Family; AV MS--Protein detected by
LC-MS/MS in the culture filtrates of Avicel grown Neurospora
crassa. (+) detected, (-) not detected; MIS MS--Protein detected by
LC-MS/MS in the culture filtrates of Miscanthus grown Neurospora
crassa. (+) detected, (-) not detected; AV ARRAY--Fold upregulation
after 30 hours of growth on Avicel relative to 16 hours of growth
on sucrose from profiling data; MIS ARRAY--Fold upregulation after
16 hours of growth on Miscanthus relative to 16 hours of growth on
sucrose from profiling data, peptides detected only in Miscanthus
culture filtrates.
[0306] There were 16 proteins identified with confidence only in
the Avicel culture and 14 of these were predicted to be secreted
(Table 6) including two predicted cellulases (gh61-1; NCU02240 and
gh45-1; NCU05121), one xylanase (gh11-1; NCU02855), one predicted
protease (NCU04205), three other proteins with predicted activity
on carbohydrates (NCU08909, NCU05974 and gh30-1 (NCU04395)), three
Neurospora-specific proteins of unknown function, and four
conserved hypothetical proteins, including one protein with a
cellulose binding domain (NCU09764). Twelve proteins were specific
for culture filtrates of Miscanthus cultures and seven of these
were predicted to be secreted (Table 3). Three of the five
predicted intracellular proteins were conserved hypothetical
proteins. The remaining two included a predicted glyoxal oxidase
(NCU09267, identified from the N. crassa Miscanthus transcriptome)
and a nucleoside diphosphate kinase (ndk-1; NCU04202, not
identified in the N. crassa transcriptome). The seven proteins
predicted to be secreted included three predicted esterases
(NCU04870, NCU05159, and NCU08785), two predicted xylanases (GH51;
NCU02343 and GH54; NCU09775), a predicted
.quadrature..beta.-xylosidase (gh3-7; NCU09923) and a conserved
hypothetical protein (NCU05751).
TABLE-US-00006 TABLE 6 Proteins identified by LC-MS/MS In the
culture filtrates of Avicel grown Neurospora crassa GENE ID
ANNOTATION SAMPLE DETECTED NCU00206 Neurospora crassa hypothetical
protein similar to cellobiose dehydrogenase 830 nt TOTAL NCU00762
Neurospora crassa endoglucanase 3 precursor 391 nt TOTAL NCU00798
Neurospora crassa predicted protein 313 nt TOTAL NCU01050
Neurospora crassa hypothetical protein similar to endoglucanase II
239 nt TOTAL NCU01595 Neurospora crassa protein SOF1 446 nt TOTAL
NCU02240 Neurospora crassa hypothetical protein similar to
endoglucanase II 323 nt TOTAL NCU02696 Neurospora crassa
hypothetical protein similar to DEAD DEAH box RNA helicase 1195
TOTAL nt NCU02855 Neurospora crassa endo 1 4 beta xylanase A
precursor 221 nt TOTAL NCU04952 Neurospora crassa hypothetical
protein similar to beta D glucoside glucohydrolase 736 TOTAL nt
NCU05057 Neurospora crassa endoglucanase EG 1 precursor 439 nt
TOTAL NCU05137 Neurospora crassa conserved hypothetical protein 692
nt TOTAL NCU05924 Neurospora crassa endo 1 4 beta xylanase 330 nt
TOTAL NCU05955 Neurospora crassa hypothetical protein similar to
Cel74a 862 nt TOTAL NCU07143 Neurospora crassa predicted protein
391 nt TOTAL NCU07190 Neurospora crassa conserved hypothetical
protein 384 nt TOTAL NCU07225 Neurospora crassa endo 1 4 beta
xylanase 2 precursor 255 nt TOTAL NCU07326 Neurospora crassa
predicted protein 327 nt TOTAL NCU07340 Neurospora crassa
exoglucanase 1 precursor 522 nt TOTAL NCU07898 Neurospora crassa
predicted protein 242 nt TOTAL NCU08171 Neurospora crassa predicted
protein 382 nt TOTAL NCU08412 Neurospora crassa conserved
hypothetical protein 401 nt TOTAL NCU08760 Neurospora crassa
predicted protein 343 nt TOTAL NCU09491 Neurospora crassa feruloyl
esterase B precursor 293 nt TOTAL NCU09680 Neurospora crassa
exoglucanase 2 precursor 485 nt TOTAL NCU09764 Neurospora crassa
conserved hypothetical protein 406 nt TOTAL NCU00206 Neurospora
crassa hypothetical protein similar to cellobiose dehydrogenase 830
nt PASC BOUND NCU00762 Neurospora crassa endoglucanase 3 precursor
391 nt PASC BOUND NCU05121 Neurospora crassa endoglucanase V 294 nt
PASC BOUND NCU05955 Neurospora crassa hypothetical protein similar
to Cel74a 862 nt PASC BOUND NCU07225 Neurospora crassa endo 1 4
beta xylanase 2 precursor 255 nt PASC BOUND NCU07340 Neurospora
crassa exoglucanase 1 precursor 522 nt PASC BOUND NCU08760
Neurospora crassa predicted protein 343 nt PASC BOUND NCU09680
Neurospora crassa exoglucanase 2 precursor 485 nt PASC BOUND
NCU00206 Neurospora crassa hypothetical protein similar to
cellobiose dehydrogenase 830 nt UNBOUND NCU00762 Neurospora crassa
endoglucanase 3 precursor 391 nt UNBOUND NCU00798 Neurospora crassa
predicted protein 313 nt UNBOUND NCU01050 Neurospora crassa
hypothetical protein similar to endoglucanase II 239 nt UNBOUND
NCU04205 Neurospora crassa predicted protein 346 nt UNBOUND
NCU04395 Neurospora crassa endo 1 6 beta D glucanase precursor 481
nt UNBOUND NCU04952 Neurospora crassa hypothetical protein similar
to beta D glucoside glucohydrolase 736 UNBOUND nt NCU05057
Neurospora crassa endoglucanase EG 1 precursor 439 nt UNBOUND
NCU05134 Neurospora crassa hypothetical protein 124 nt UNBOUND
NCU05137 Neurospora crassa conserved hypothetical protein 692 nt
UNBOUND NCU05852 Neurospora crassa conserved hypothetical protein
254 nt UNBOUND NCU05924 Neurospora crassa endo 1 4 beta xylanase
330 nt UNBOUND NCU05974 Neurospora crassa hypothetical protein
similar to cell wall glucanosyltransferase Mwg1 UNBOUND 365 nt
NCU07143 Neurospora crassa predicted protein 391 nt UNBOUND
NCU07190 Neurospora crassa conserved hypothetical protein 384 nt
UNBOUND NCU07225 Neurospora crassa endo 1 4 beta xylanase 2
precursor 255 nt UNBOUND NCU07326 Neurospora crassa predicted
protein 327 nt UNBOUND NCU07340 Neurospora crassa exoglucanase 1
precursor 522 nt UNBOUND NCU07898 Neurospora crassa predicted
protein 242 nt UNBOUND NCU08171 Neurospora crassa predicted protein
382 nt UNBOUND NCU08189 Neurospora crassa hypothetical protein
similar to endo 1 4 beta xylanase 385 nt UNBOUND NCU08398
Neurospora crassa conserved hypothetical protein 391 nt UNBOUND
NCU08412 Neurospora crassa conserved hypothetical protein 401 nt
UNBOUND NCU08760 Neurospora crassa predicted protein 343 nt UNBOUND
NCU08909 Neurospora crassa hypothetical protein similar to beta 1 3
glucanosyltransferase 543 nt UNBOUND NCU08936 Neurospora crassa
clock controlled gene 15 412 nt UNBOUND NCU09024 Neurospora crassa
conserved hypothetical protein 625 nt UNBOUND NCU09046 Neurospora
crassa predicted protein 187 nt UNBOUND NCU09175 Neurospora crassa
conserved hypothetical protein 411 nt UNBOUND NCU09491 Neurospora
crassa feruloyl esterase B precursor 293 nt UNBOUND
ANNOTATION--Generated by the Broad Institute (webpage at
broad.mit.edu/annotation/genome/neurospora/Home.html); SAMPLE
DETECTED--Sample in which peptides were detected for a particular
protein. Peptides were validated by manual inspection. A protein
was determined to be present if at least 1 peptide was detected in
each biological repeat. TOTAL, peptides detected from a tryptic
digest of all extracellular proteins; PASC BOUND, peptides detected
after enrichment for proteins that bind to phosphoric acid swollen
cellulose; UNBOUND, proteins remaining in solution after removal of
PASC bound proteins.
[0307] Many plant cell wall degrading enzymes contain a
cellulose-binding module (CBM), which aids in attachment of the
enzyme to the substrate (Linder and Teeri 1996). Within the N.
crassa genome, proteins encoded by 19 genes are predicted to
contain a CBM1 domain (Cantarel et al., 2009). Of these 19 genes,
16 showed an increase in relative gene expression in
Miscanthus-grown cultures (Table 7).
TABLE-US-00007 TABLE 7 Effect of Miscanthus and Avicel on N. crassa
gene expression Gene CBM Avicel name prediction Annotation Mis
Array array MS NCU00206 cazy and mips probable cellobiose 164 12
both dehydrogenase NCU00710 cazy and mips acetylxylan esterase 30
no detect none NCU00762 cazy and mips EG2 29 31 both NCU00836 cazy
and mips EG, GH61 91 30 none NCU01867 cazy and mips EG, GH61
2.2-d10 no none difference NCU02240 cazy and mips EG, GH61 193 84
avi NCU02916 cazy and mips EG, GH61 85 17 none NCU04500 cazy and
mips similar to chitinase 4 no detect no detect none NCU04997 cazy
and mips similar to xylanase no detect no detect none NCU05121 cazy
and mips EG, GH45 8.5 17 avi NCU05159 cazy and mips acetylxylan
esterase 34 10 mis precursor NCU05955 cazy and mips GH74 50 19 both
NCU07225 cazy and mips xylanase 33 11 both NCU07340 cazy and mips
CBH1 426 382 both NCU07760 cazy and mips EG, GH61 3.7 no none
difference NCU08760 cazy and mips EG, GH61 107 44 both NCU09416
cazy and mips hypothetical no detect 27 none NCU09680 cazy and mips
CBH2 230 251 both NCU09764 cazy and mips hypothetical 18 16.6
avi
[0308] From the 50 proteins identified by MS, 11 contained a CBM1
domain. PASC was used to enrich for proteins that bind to cellulose
(see Example 4 for methods). Nine proteins were identified by MS
that bound to PASC from the supernatant of Miscanthus-grown N.
crassa cultures, while eight proteins from the Avicel supernatants
were identified; seven cellulose binding proteins were identified
in both (Tables 2, 3, 8). These included NCU00206, a predicted
cellobiose dehydrogenase; gh5-1 (NCU00762), a predicted
endoglucanase; NCU05955, a predicted GH74 xyloglucanase; gh11-2
(NCU07225), a predicted endoxylanase; cbh-1 (NCU07340); gh61-5
(NCU08760), a predicted endoglucanase; and gh6-2 (NCU09680), a
predicted cellobiohydrolase 2 precursor.
TABLE-US-00008 TABLE 8 Proteins identified by LC-MS/MS in the
culture filtrates of Avicel-grown Neurospora crassa GENE ID
ANNOTATION CULTURE NCU00206 Neurospora crassa hypothetical protein
similar to cellobiose dehydrogenase 830 nt BOTH NCU00762 Neurospora
crassa endoglucanase 3 precursor 391 nt BOTH NCU01050 Neurospora
crassa hypothetical protein similar to endoglucanase II 239 nt BOTH
NCU04952 Neurospora crassa hypothetical protein similar to beta D
glucoside glucohydrolase BOTH 736 nt NCU05057 Neurospora crassa
endoglucanase EG 1 precursor 439 nt BOTH NCU05137 Neurospora crassa
conserved hypothetical protein 692 nt BOTH NCU05924 Neurospora
crassa endo 1 4 beta xylanase 330 nt BOTH NCU05955 Neurospora
crassa hypothetical protein similar to Cel74a 862 nt BOTH NCU07143
Neurospora crassa predicted protein 391 nt BOTH NCU07190 Neurospora
crassa conserved hypothetical protein 384 nt BOTH NCU07225
Neurospora crassa endo 1 4 beta xylanase 2 precursor 255 nt BOTH
NCU07326 Neurospora crassa predicted protein 327 nt BOTH NCU07340
Neurospora crassa exoglucanase 1 precursor 522 nt BOTH NCU07898
Neurospora crassa predicted protein 242 nt BOTH NCU08189 Neurospora
crassa hypothetical protein similar to endo 1 4 beta xylanase 385
nt BOTH NCU08398 Neurospora crassa conserved hypothetical protein
391 nt BOTH NCU08412 Neurospora crassa conserved hypothetical
protein 401 nt BOTH NCU08760 Neurospora crassa predicted protein
343 nt BOTH NCU09024 Neurospora crassa conserved hypothetical
protein 625 nt BOTH NCU09175 Neurospora crassa conserved
hypothetical protein 411 nt BOTH NCU09491 Neurospora crassa
feruloyl esterase B precursor 293 nt BOTH NCU09680 Neurospora
crassa exoglucanase 2 precursor 485 nt BOTH NCU00798 Neurospora
crassa predicted protein 313 nt AV NCU01595 Neurospora crassa
protein SOF1 446 nt AV NCU02240 Neurospora crassa hypothetical
protein similar to endoglucanase II 323 nt AV NCU02696 Neurospora
crassa hypothetical protein similar to DEAD DEAH box RNA helicase
AV 1195 nt NCU02855 Neurospora crassa endo 1 4 beta xylanase A
precursor 221 nt AV NCU04205 Neurospora crassa predicted protein
346 nt AV NCU04395 Neurospora crassa endo 1 6 beta D glucanase
precursor 481 nt AV NCU05121 Neurospora crassa endoglucanase V 294
nt AV NCU05134 Neurospora crassa hypothetical protein 124 nt AV
NCU05852 Neurospora crassa conserved hypothetical protein 254 nt AV
NCU05974 Neurospora crassa hypothetical protein similar to cell
wall glucanosyltransferase AV Mwg1 365 nt NCU08171 Neurospora
crassa predicted protein 382 nt AV NCU08909 Neurospora crassa
hypothetical protein similar to beta 1 3 glucanosyltransferase 543
AV nt NCU08936 Neurospora crassa clock controlled gene 15 412 nt AV
NCU09046 Neurospora crassa predicted protein 187 nt AV NCU09764
Neurospora crassa conserved hypothetical protein 406 nt AV NCU01651
Neurospora crassa conserved hypothetical protein 783 nt MIS
NCU02343 Neurospora crassa hypothetical protein similar to alpha L
arabinofuranosidase A 668 MIS nt NCU04202 Neurospora crassa
nucleoside diphosphate kinase 1 153 nt MIS NCU04870 Neurospora
crassa hypothetical protein similar to acetyl xylan esterase 313 nt
MIS NCU05159 Neurospora crassa acetylxylan esterase precursor 301
nt MIS NCU05751 Neurospora crassa conserved hypothetical protein
242 nt MIS NCU06239 Neurospora crassa conserved hypothetical
protein 514 nt MIS NCU08785 Neurospora crassa conserved
hypothetical protein 291 nt MIS NCU09267 Neurospora crassa
conserved hypothetical protein 1048 nt MIS NCU09708 Neurospora
crassa conserved hypothetical protein 465 nt MIS NCU09775
Neurospora crassa hypothetical protein similar to alpha L
arabinofuranosidase 343 nt MIS NCU09923 Neurospora crassa
hypothetical protein similar to beta xylosidase 775 nt MIS
ANNOTATION--Generated by the Broad Institute (webpage
broad.mit.edu/annotation/genome/neurospora/Home.html);
CULTURE--Culture in which peptides were detected for a particular
protein. BOTH, peptides detected in both Avicel and Miscanthus
culture filtrates; AV, peptides detected only in Avicel culture
filtrates; MIS, peptides detected only in Miscanthus culture
filtrates.
Example 3
Characterization of Extracellular Proteins and Cellulase Activity
in Strains Containing Deletions in Genes Identified in the Overlap
of the Transcriptome/Secretome Datasets
[0309] Of the 22 extracellular proteins detected in both the
Miscanthus and Avicel grown cultures, homokaryotic strains
containing deletions in genes encoding 16 of these extracellular
proteins were available to the public (Dunlap et al., 2007). None
of these 16 deletion strains had been previously characterized with
respect to their influence on plant cell wall or cellulose
degradation in N. crassa. The 16 deletion strains were grown both
on media containing sucrose or Avicel as a preferred carbon source.
All strains showed a wild type growth phenotype on sucrose. On
medium containing Avicel, the bulk growth of the 16 deletion
strains was monitored for a 7-day period. After seven days, the
total secreted protein, endoglucanase activity, .beta.-glucosidase
activity, and aggregate Avicelase activity of the culture filtrates
was measured and compared with the wild-type strain from which all
the mutants were derived (FIGS. 6A-C). SDS-PAGE was also done on
unconcentrated culture supernatants to investigate the relative
abundance of secreted proteins.
[0310] There were growth deficiencies on Avicel for strains
containing deletions of two predicted exoglucanases (cbh-1;
NCU07340 and gh6-2; NCU09680) and a predicted .beta.-glucosidase
(gh3-4; NCU04952). The cbh-1 mutant was the most severe; after
seven days much of the Avicel remained, while in the wild-type
strain all of the Avicel was degraded by this time. For 10 of the
16 deletion strains, SDS-PAGE analysis of the secreted proteins
showed an altered extracellular protein profile where a single band
disappeared, thus allowing assignment of a particular protein band
to a predicted gene (FIG. 6A, boxes; FIG. 7). These included
NCU00762 (gh5-1), NCU04952 (gh3-4), NCU05057 (gh7-1), NCU05137,
NCU05924 (gh10-1), NCU05955, NCU07190 (gh6-3), NCU07326, NCU07340
(cbh-1), and NCU09680 (gh6-2).
[0311] For the majority of the deletion strains, the total secreted
protein, endoglucanase, .beta.-glucosidase, and Avicelase
activities of the culture supernatants were similar to wild type
(FIG. 6B, C and Table 9).
TABLE-US-00009 TABLE 9 Enzyme Activity of Deletion Strains
[Secreted Azo- Gene Growth Protein] CMCase Bgl [CB] [GLC] Name on
Avicel (% of WT) (% of WT) (% of WT) (mM) (mM) NCU00762 * * * 113
.+-. 8 33 .+-. 2 102 .+-. 2 0.9 .+-. 0.0 2.6 .+-. 0.1 NCU01050 * *
* 98 .+-. 12 92 .+-. 8 88 .+-. 5 0.8 .+-. 0.2 2.9 .+-. 0.3 NCU04952
* * * 146 .+-. 6 124 .+-. 5 1 .+-. 0.3 2.24 .+-. 0.2 0.6 .+-. 0.0
NCU05057 * * * 143 .+-. 10 98 .+-. 3 100 .+-. 10 1.7 .+-. 0.1 3.6
.+-. 0.1 NCU05137 * * * 154 .+-. 12 156 .+-. 10 178 .+-. 3 1.0 .+-.
0.0 3.8 .+-. 0.1 NCU05924 * * * 108 .+-. 3 108 .+-. 5 101 .+-. 4
1.1 .+-. 0.1 2.6 .+-. 0.2 NCU05955 * * * 92 .+-. 10 94 .+-. 8 98
.+-. 7 0.9 .+-. 0.1 2.3 .+-. 0.1 NCU07190 * * * 111 .+-. 7 136 .+-.
6 92 .+-. 1 1.1 .+-. 0.0 2.6 .+-. 0.0 NCU07326 * * * 105 .+-. 4 114
.+-. 17 85 .+-. 11 1.0 .+-. 0.0 2.3 .+-. 0.0 NCU07340 * 41 .+-. 2.2
43 .+-. 9 56 .+-. 9 0.1 .+-. 0.0 0.5 .+-. 0.1 NCU07898 * * * 84
.+-. 7 .sup. 86 .+-. 1.5 59 .+-. 15 0.5 .+-. 0.3 2.3 .+-. 0.5
NCU08189 * * * 83 .+-. 12 80 .+-. 8 69 .+-. 15 0.5 .+-. 0.1 2.3
.+-. 0.4 NCU08398 * * * 95 .+-. 11 107 .+-. 7 97 .+-. 3 0.6 .+-.
0.1 1.8 .+-. 0.0 NCU08760 * * * 115 .+-. 3 126 .+-. 6 115 .+-. 8
0.9 .+-. 0.1 2.6 .+-. 0.1 NCU09175 * * * 96 .+-. 7 115 .+-. 0 101
.+-. 8 0.7 .+-. 0.0 1.9 .+-. 0.1 NCU09680 * * 118 .+-. 7 165 .+-. 7
150 .+-. 1 0.23 .+-. 0.1 1.7 .+-. 0.1 WT * * * 100 .+-. 7 100 .+-.
12 100 .+-. 6 0.97 .+-. 0.0 2.4 .+-. 0.1
[0312] Deviations from this trend were seen with the .DELTA.gh5-1
(NCU00762), .DELTA.gh3-4 (NCU04952), .DELTA.NCU05137, .DELTA.cbh-1
(NCU07340), and .DELTA.gh6-2 (NCU09680) mutants. In .DELTA.gh5-1
(NCU00762), .DELTA.gh3-4 (NCU04952), and .DELTA.cbh-1 (NCU07340),
Avicelase, endoglucanase or A-glucosidase activities were lower
than the corresponding wild-type activities. In particular, the
deletion of NCU04952 eliminated all .beta.-glucosidase activity
from the culture supernatant, as evidenced by PNPGase activity and
by higher levels of cellobiose and lower levels of glucose in the
Avicelase enzyme assays (FIG. 6B, C). Despite lowering
endoglucanase activity, the culture filtrate from .DELTA.gh5-1
(NCU00762) showed no significant deficiency in Avicelase activity
relative to the wild-type strain (FIG. 6C). As expected, mutations
in cbh-1 (NCU07340) resulted in lower endoglucanase and Avicelase
activity, due to poor growth. A strain containing a deletion of
NCU09680, encoding a CBH(II)-like protein (gh6-2), also showed
reduced cellobiose accumulation, as observed with .DELTA.cbh-1
mutant (FIG. 6C).
[0313] Mutations in three strains resulted in an increased level of
secreted proteins, especially CBH(I) (FIG. 6A); gh3-4 (NCU04952),
gh7-1 (NCU05057) and a hypothetical protein gene (NCU05137). In
addition to increased levels of secreted proteins, the
.DELTA.NCU05137 mutant showed increased endoglucanase,
.beta.-glucosidase, and Avicelase activity (FIG. 6B, C). NCU05137
is highly conserved in the genomes of a number of filamentous
ascomycete fungi, including other cellulolytic fungi, but notably
does not have an ortholog in T. reesei (FIG. 2). It is possible
that the increase in CBH(I) levels observed in .DELTA.gh3-4,
.DELTA.gh7-1, and .DELTA.NCU05137 could be due to either increased
secretion, protein stability or, alternatively, feedback that
results in an increase in expression of cbh-1. To differentiate
these possibilities, the profile of extracellular proteins produced
by .DELTA.NCU05137 and .DELTA.gh3-4 (NCU04952) was compared with
gene expression levels of cbh-1 (NCU07340) and gh6-2 (CBH(II);
NCU09680) as assayed by quantitative RT-PCR (FIG. 8). The strains
.DELTA.NCU05137 and .DELTA.gh3-4 showed a higher level of CBH(I)
protein as early as two days in an Avicel-grown culture.
Quantitative RT-PCR of cbh-1 and gh6-2 from Avicel-grown cultures
showed that both genes exhibited high expression levels in wild
type and the .DELTA.NCU05137 and .DELTA.gh3-4 mutants after two
days of growth. However, although expression of both of these genes
decreased significantly on day three in the wild-type strain, both
cbh-1 and gh6-2 expression levels increased in the .DELTA.NCU05137
mutant, and decreased less than wild type in .DELTA.gh3-4 (FIG. 8).
Sustained expression of cbh-1 and gh6-2 genes in the
.DELTA.NCU05137 and .DELTA.gh3-4 mutants could be responsible for
the observed increase in CBH(I) and CBH(II) protein levels.
Example 4
Materials and Methods for Transcriptome and Secretome Studies
Strains
[0314] All Neurospora crassa strains were obtained from the Fungal
Genetics Stock Center (FGSC; webpage fgsc.net) (Supplemental Data,
Dataset S1, page 1 in Tian et al., 2009). Gene deletion strains
were from the N. crassa functional genomics project (Dunlap et al.,
2007). Trichoderma reesei QM9414 was a gift from Dr. Monika Schmoll
(Vienna University of Technology). Strains were grown on Vogel's
salts (Vogel 1956) with 2% (w/v) carbon source (Miscanthus, sucrose
or Avicel (Sigma)). Miscanthus.times.giganteus (milled stem to -0.1
mm) was a gift from the University of Illinois.
[0315] Enzyme Activity Measurements
[0316] Total extracellular protein content was determined using a
Bio-Rad DC Protein Assay kit (Bio-Rad). Endoglucanase activity in
culture supernatants was measured with an azo-CMC kit (Megazyme
SCMCL). .beta.-glucosidase activity was measured by mixing 10-fold
diluted culture supernatant with 500 .mu.M 4-nitrophenyl
.beta.-D-glucopyranoside in 50 mM sodium acetate buffer, pH 5.0,
for 10 minutes at 40.degree. C. The reaction was quenched with 5%
w/v sodium carbonate, and the absorbance at 400 nm was measured.
Avicelase activity was measured by mixing 2-fold diluted culture
supernatant with 50 mM sodium acetate, pH 5.0, and 5 mg/mL Avicel
at 40.degree. C. Supernatants were analyzed for glucose content
using a coupled enzyme assay with glucose oxidase/peroxidase. Fifty
.mu.L of the avicelase reaction was transferred to 150 .mu.L of
glucose detection reagent containing 100 mM sodium acetate pH 5.0,
10 U/mL horseradish peroxidase, 10 U/mL glucose oxidase, and 1 mM
o-dianisidine. After 30 minutes absorption was measured at 540 nm.
Cellobiose concentrations were determined using a coupled enzyme
assay with cellobiose dehydrogenase (CDH) from Sporotrichum
thermophile. CDH was isolated from S. thermophile similar to
previous reports (Canevascini 1988). Fifty .mu.L of the avicelase
reaction was transferred to 250 .mu.L of cellobiose detection
reagent containing 125 mM sodium acetate pH 5.0, 250 .mu.M
dichlorophenol indophenol, and 0.03 mg/mL CDH. After 10 minutes
absorption was measured at 530 nm.
[0317] RNA Isolation, Microarray Analysis, and Signal Peptide
Predictions
[0318] Mycelia were harvested by filtration and flash frozen in
liquid nitrogen. Total RNA was isolated using trizol (Tian et al.,
2007; Kasuga et al., 2005). Microarray hybridization and data
analysis were as previously described (Tian et al., 2007).
Normalized expression values were analyzed using BAGEL (Bayesian
analysis of gene expression levels) (Townsend 2004; Townsend and
Hartl 2002), which infers relative gene expression levels and
credible intervals for each gene at each experimental time point.
Signal peptides were predicted using the N-terminal 70 amino acid
region of each predicted protein with the signalP3 program (webpage
cbs.dtu.dk/services/SignalP-3.0/). Original profiling data is
obtainable at (webpage yale.edu/townsend/Links/ffdatabase/).
[0319] Protein Gel Electrophoresis
[0320] Except where otherwise noted, unconcentrated culture
supernatants were treated with 5.times.SDS loading dye and boiled
for 5 minutes before loading onto Criterion 4-15% Tris-HCl
polyacrylamide gels. Coomassie dye was used for staining.
[0321] Preparation of Tryptic Peptides for Secretome Analysis
[0322] Culture supernatants were concentrated with 10 kDa MWCO PES
spin concentrators.
[0323] Cellulose binding proteins were isolated from the culture
supernatant by addition of phosphoric acid swollen cellulose
(PASC). Five mL of a suspension of 10 mg/mL PASC was added to 10 mL
of culture supernatant. After incubation at 4.degree. C. for 5
minutes, the mixture was centrifuged and the pelleted PASC was then
washed with 20 pellet volumes of 100 mM sodium acetate pH 5.0. The
supernatant after treatment with PASC was saved as the unbound
fraction and concentrated. 36 mg of urea, 5 .mu.L of 1M Tris PH
8.5, and 5 .mu.L of 100 mM DTT were then added to 100 .mu.L of
concentrated culture supernatant or protein-bound PASC and the
mixture was heated at 60.degree. C. for 1 hour. After heating 700
.mu.L of 25 mM ammonium bicarbonate and 140 .mu.L of methanol were
added to the solution followed by treatment with 50 .mu.L of 100
.mu.g/mL trypsin in 50 mM sodium acetate pH 5.0. For the PASC bound
proteins, the PASC was removed by centrifugation after heating, and
the supernatant was then treated with trypsin. The trypsin was left
to react overnight at 37.degree. C. After digestion the volume was
reduced by speedvac and washed with MilliQ water three times.
Residual salts in the sample were removed by using OMIX
microextraction pipette tips according to the manufacturer's
instructions.
[0324] Liquid Chromatography of Tryptic Peptides
[0325] Trypsin-digested proteins were analyzed using a tandem mass
spectrometer that was connected in-line with ultraperformance
liquid chromatography (UPLC). Peptides were separated using a
nanoAcquity UPLC (Waters, Milford, Mass.) equipped with C18
trapping (180 .mu.m.times.20 mm) and analytical (100
.mu.m.times.100 mm) columns and a 10 .mu.L sample loop. Solvent A
was 0.1% formic acid/99.9% water and solvent B was 0.1% formic
acid/99.9% acetonitrile (v/v). Sample solutions contained in 0.3 mL
polypropylene snap-top vials sealed with septa caps (Wheaton
Science, Millville, N.J.) were loaded into the nanoAcquity
autosampler prior to analysis. Following sample injection (2 .mu.L,
partial loop), trapping was performed for 5 min with 100% A at a
flow rate of 3 .mu.L/min. The injection needle was washed with 750
.mu.L each of solvents A and B after injection to avoid
cross-contamination between samples. The elution program consisted
of a linear gradient from 25% to 30% B over 55 min, a linear
gradient to 40% B over 20 min, a linear gradient to 95% B over 0.33
min, isocratic conditions at 95% B for 11.67 min, a linear gradient
to 1% B over 0.33 min, and isocratic conditions at 1% B for 11.67
min, at a flow rate of 500 nL/min. The analytical column and sample
compartment were maintained at 35.degree. C. and 8.degree. C.,
respectively.
[0326] Mass Spectrometry
[0327] The column was connected to a NanoEase nanoelectrospray
ionization (nanoESI) emitter mounted in the nanoflow ion source of
a quadrupole time-of-flight mass spectrometer (Q-Tof Premier,
Waters). The nanoESI source parameters were as follows: nanoESI
capillary voltage 2.3 kV, nebulizing gas (nitrogen) pressure 0.15
mbar, sample cone voltage 30 V, extraction cone voltage 5 V, ion
guide voltage 3 V, and source block temperature 80.degree. C. No
cone gas was used. The collision cell contained argon gas at a
pressure of 8.times.10-3 mbar. The Tof analyzer was operated in "V"
mode. Under these conditions, a mass resolving power1 of
1.0.times.104 (measured at m/z=771) was routinely achieved, which
is sufficient to resolve the isotopic distributions of the singly
and multiply charged peptide ions measured in this study. Thus, an
ion's mass and charge could be determined independently, i.e., the
ion charge was determined from the reciprocal of the spacing
between adjacent isotope peaks in the m/z spectrum. External mass
calibration was performed immediately prior to analysis, using
solutions of sodium formate. Survey scans were acquired in the
positive ion mode over the range m/z=450-1800 using a 0.95 s scan
integration and a 0.05 s interscan delay. In the data-dependent
mode, up to five precursor ions exceeding an intensity threshold of
35 counts/second (cps) were selected from each survey scan for
tandem mass spectrometry (MS/MS) analysis. Real-time deisotoping
and charge state recognition were used to select 2+, 3+, 4+, 5+,
and 6+ charge state precursor ions for MS/MS. Collision energies
for collisionally activated dissociation (CAD) were automatically
selected based on the mass and charge state of a given precursor
ion. MS/MS spectra were acquired over the range m/z=50-2500 using a
0.95 s scan integration and a 0.05 s interscan delay. Ions were
fragmented to achieve a minimum total ion current (TIC) of 30,000
cps in the cumulative MS/MS spectrum for a maximum of 3 s. To avoid
the occurrence of redundant MS/MS measurements, real time exclusion
was used to preclude re-selection of previously analyzed precursor
ions over an exclusion width of .+-.0.25 m/z unit for a period of
180 s.
[0328] Mass Spectrometry Data Analysis
[0329] The data resulting from LC-MS/MS analysis of
trypsin-digested proteins were processed using ProteinLynx Global
Server software (version 2.3, Waters), which performed background
subtraction (threshold 35% and fifth order polynomial), smoothing
(Savitzky-Golay2 10 times, over three channels), and centroiding
(top 80% of each peak and minimum peak width at half height four
channels) of the mass spectra and MS/MS spectra. The processed data
were searched against the N. crassa database (Broad Institute)
using the following criteria: tryptic fragments with up to five
missed cleavages, precursor ion mass tolerance 50 ppm, fragment ion
mass tolerance 0.1 Da, and the following variable
post-translational modifications: carbamylation of N-terminus and
Lys side chains, Met oxidation, and Ser/Thr dehydration. The
identification of at least three consecutive fragment ions from the
same series, i.e., b or y-type fragment ions, was required for
assignment of a peptide to an MS/MS spectrum. The MS/MS spectra
were manually inspected to verify the presence of the fragment ions
that uniquely identify the peptides.
[0330] Quantitative RT-PCR
[0331] The RT-PCR was performed in an ABI7300 with reagents from
Qiagen (SYBR-green RT-PCR kit (Cat No. 204243)). The primers for
CBHI (NCU07340) were: forward 5'-ATCTGGGAAGCGAACAAAG-3' (SEQ ID NO:
16) and reverse 5'-TAGCGGTCGTCGGAATAG-3' (SEQ ID NO: 17). The
primers for CBHII (NCU09680) were: forward 5'-CCCATCACCACTACTACC-3'
(SEQ ID NO: 18) and reverse 5'-CCAGCCCTGAACACCAAG-3' (SEQ ID NO:
19). Actin was used as a control for normalization. The primers for
actin were: forward 5'-TGA TCT TAC CGA CTA CCT-3' (SEQ ID NO: 20)
and reverse 5'-CAG AGC TTC TCC TTG ATG-3' (SEQ ID NO: 21).
Quantitative RT-PCR was performed according to Dementhon et al.,
(2006).
Example 5
Discussion of Transcriptome and Secretome Studies
[0332] Degradation of plant biomass requires the production of many
different enzymatic activities, which are regulated by the type and
complexity of the available plant material (FIG. 9) (Bouws et al.,
2008). The first systematic analyses of plant cell wall degradation
by a cellulolytic fungus are described here, which include
transcriptome, secretome, and mutant analyses. Profiling data
showed that N. crassa coordinately expresses a host of
extracellular and intracellular proteins when challenged by growth
on Miscanthus or Avicel (FIG. 9). Many of the most highly expressed
genes during growth on cellulosic substrates encode proteins
predicted to be involved in the metabolism of plant cell wall
polysaccharides, many of which were identified by MS analyses.
Genome comparisons of filamentous fungi show a large number of
glycosyl hydrolases (.about.200) with varying numbers of predicted
cellulases, from 10 in T. reesei (Martinez et al., 2008) to 60 in
Podospora anserina (Espagne et al., 2008), a dung-degrading species
closely related to N. crassa. A comparison between these results
and a recent transcriptome/secretome study on the white rot
basidiomycete fungus, Phanerochaete chrysosporium, (Wymelenberg et
al., 2009) showed little overlap in regulated genes (18 genes) and
secreted proteins (2 proteins) when both species were grown on pure
cellulose. These data suggest that different fungi may utilize
different gene sets for plant cell wall degradation. However, one
aspect that both studies had in common was the high number of
uncharacterized genes/proteins associated with cellulose
degradation. Other cellulolytic fungi, including P. chrysosporium,
do not have the genetic and molecular tools that are readily
available with N. crassa. Using the functional genomic tools
available with N. crassa, both the function and redundancy of plant
cell wall degrading enzyme systems can be addressed to create
optimal enzyme mixtures for industrial production of liquid fuels
from lignocellulose biomass.
[0333] In this study, it was found that cellobiohydrolase(I) (CBHI)
in N. crassa is the most highly produced extracellular protein
during growth on Avicel or Miscanthus, and deletion of this gene
caused the most severe growth deficiencies on cellulosic
substrates. These results are similar to those reported in T.
reesei (Suominen et al., 1993, Seiboth et al., 1997). Deletion of
cellobiohydrolase(II) also caused growth deficiencies on cellulosic
substrates, but to a much lesser extent than CBH(I), suggesting
that exoglucanase activity in N. crassa is predominantly from
CBH(I) and that cellulases and other CBHs do not compensate for the
loss of CBH(I). Here, it was shown that the three most highly
produced endoglucanases during growth on cellulosic substrates are
the proteins encoded by NCU05057, NCU00762, and NCU07190. These
proteins have homology to endoglucanases EG1, EG2, and EG6,
respectively. Deletion of these genes did not affect growth on
Avicel, although differences in the secreted protein levels and
endoglucanase activity were observed. Unexpectedly, in the
.DELTA.NCU05057 strain, extracellular protein levels were much
higher, especially CBH(I), suggesting that to maintain the
wild-type growth phenotype on crystalline cellulose the mutant was
forced to increase production of other cellulases or that the
products of NCU05057 catalysis may repress cellulase production. It
was concluded that no one endoglucanase in N. crassa is required
for growth on crystalline cellulose and that the different
endoglucanases have overlapping substrate specificities.
[0334] The glycoside hydrolase family 61 enzymes are greatly
expanded in N. crassa compared to T. reesei (Martinez et al.,
2008). These enzymes have poorly defined biological function, but
their general conservation and abundance in cellulolytic fungi
suggests an important role in plant cell wall metabolism. Here,
genes for 10 of the 14 GH61 enzymes were identified in the N.
crassa transcriptome, suggesting that these enzymes are utilized
during growth on cellulosic biomass. The four GH61 deletion strains
tested showed only small differences compared to wild type in the
secreted protein levels, endoglucanase, and total cellulase
activities. However, analyses of additional GH61 mutants and the
capacity to create strains containing multiple mutations in N.
crassa via sexual crosses will address redundancy and expedite
functional analysis of this family.
[0335] In addition to predicted cellulase genes, genes encoding
hemicellulases, carbohydrate esterases, .beta.-glucosidases,
.beta.-xylosidases, and other proteins predicted to have activity
on carbohydrates were identified in the N. crassa transcriptome
from both Miscanthus and Avicel. The fact that Avicel contains no
hemicellulose components suggests that cellulose is probably the
primary inducer of genes encoding plant cell wall degrading enzymes
in N. crassa. However, genes encoding some hemicellulases and
carbohydrate esterases were only expressed during growth on
Miscanthus. Similarly, in other cellulolytic fungi such as T.
reesei and Aspergillus niger, genes encoding some cellulases and
hemicellulases are coordinately regulated, while others are
differentially regulated (Stricker et al., 2008). As expected,
deletions of non-cellulase genes had little effect on growth on
Avicel or cellulase activity, with the exception of NCU05137 and
gh3-4. The .DELTA.NCU05137 strain secreted more protein, had higher
cellulase activity, and showed higher expression of cbh-1 (CBH(I))
and gh6-2 (CBH(II)) than wild type. NCU05137 encodes a secreted
hypothetical protein that has no homology to proteins of known
function, but is highly conserved in other cellulolytic fungi (FIG.
2; E value 0.0). NCU05137 also has more distant homologs, but also
of unknown function, in a number of bacterial species. The protein
product of NCU05137 may interfere with signaling processes
associated with induction of cellulase gene expression N. crassa
(FIG. 9). Similarly, mutations in gh3-4 (NCU04952) also increased
CBH(I) activity. Deletion of this gene completely removed PNPGase
activity and cellobiose accumulated in in vitro cellulase assays
using .DELTA.gh3-4 culture filtrates. All the data together
suggested that NCU04952 encodes the primary extracellular
.beta.-glucosidase in N. crassa. These data were consistent with
catabolite repression of cellulase production by glucose.
[0336] Extracellular degradation of cellulose and hemicellulose
results in the formation of soluble carbohydrates that are
subsequently transported into the cell (FIG. 9). In this study, 10
genes encoding permeases/transporters were identified which showed
significantly increased expression when N. crassa was grown on
Miscanthus or Avicel, suggesting their involvement in transport of
plant cell wall degradation products into the cell. The major
degradation products by cellulases and hemicellulases in vitro are
cellobiose, glucose, xylobiose, and xylose. Some of these
transporters may be functionally redundant or capable of
transporting oligosaccharides. The function of these putative
transporters was further explored (see Examples 7-9). Construction
of downstream processing strains capable of transporting
oligosaccharides by heterologous expression of N. crassa
transporters may improve industrial fermentation of biomass
hydrolysis products. None of these transporters or what they may
transport has been characterized at the molecular or functional
level in any filamentous fungi.
[0337] Many genes that showed increased expression levels during
growth on Miscanthus and Avicel encode proteins of unknown function
that are conserved in other cellulolytic fungi. By assessing the
phenotype of only 16 strains, a mutant in a gene encoding a protein
of unknown function that significantly affects cellulase activity
was identified. The well-understood genetics and availability of
functional genomic resources in N. crassa make it an ideal model
organism to determine the biological function of these proteins, as
well as regulatory aspects of cellulase and hemicellulase
production, and to dissect redundancies and synergies between
extracellular enzymes involved in the degradation of plant cell
walls.
Example 6
Screening of Mutants of Genes Upregulated During Growth on
Miscanthus
[0338] In order to analyze additional genes identified in the
transcriptional profiling experiment, the phenotypes of mutants of
188 genes that were upregulated in Neurospora grown on Miscanthus
for 16 hours were analyzed (see Example 1). A knockout mutant of
each gene was grown on minimal Vogel's medium for 10-14 days.
Conidia were harvested with 2 mL ddH.sub.2O and inoculated into 100
mL media in 250 mL flasks at a concentration of 10.sup.6 conidia
per mL. One of three different carbon sources was added to each
flask: 2% sucrose, 2% Avicel, or 2% Miscanthus (1 mm particles from
Calvin Laboratory, University of California, Berkeley, Calif.).
Cultures were grown at 25.degree. C. with 220 rpm of shaking for 4
days.
[0339] Table 10 lists the phenotypes of the mutants that showed a
significant difference in cellulase activity and growth on Avicel
or Miscanthus compared to wild-type. Growth on Avicel or Miscanthus
was evaluated by eye with a "+" scoring system. Wild-type growth
was set at "++". Total protein in the culture supernatant was
measured by Bradford assay (100 .mu.l supernatant to 900 .mu.l
Bradford dye). Endoglucanase activity was measured with the Azo-CMC
kit from Megazyme and indicated in Table 10 as the percentage of
endoglucanase activity in the mutant compared to wild type. Total
cellulase activity was measured by detecting cellobiose levels in
the supernatant as described in Example 4. Results are indicated in
Table 10 as a percentage of wild-type.
Table 10 shows mutant screening data
TABLE-US-00010 Broad Growth % WT FGSC Annotation Up- (Avi, Bradford
NCU# # (Domains) Pfam* Regulation Mis) (Avi) NCU00130.2 FGSC beta-
Glycosyl 394.6 ++, ++ 203.2477947 11823 glucosidase Hydrolase 1
(GH1) (2.5e-196) NCU00248.2 FGSC Predicted no significant 9.74 +,
++ 86.96013289 12214 Protein hit NCU00326.2 FGSC Conserved SMP-30/
7.7 +, ++ 33.02879291 15868 Hypothetical Gluconolaconase/ (SMP-30/
LRE-like region gluconolactonase) (3.5e-82) NCU00762.2 FGSC
Endoglucanase- Cellulase 29.6 ++, ++ 104.3504411 16747 3 precursor
(1.4e-69), (GH5, CBD1) Fungal cellulose binding domain (9.2e-14)
NCU00810.2 FGSC Similar to Glycosyl 5.3 ++, ++ 163.805047 11285
Glycosyl hydrolases Hydrolase family 2 (GH2, beta- (1.7e-145)
galactosidase) NCU00890.2 FGSC Similar Glycosyl 20.45 +, +
47.57417803 16749 to beta- hydrolases manosidase family 2 (GH2)
(4.1e-06) NCU03328.2 FGSC Conserved Glycosyl 26.4 ++, ++
100.1752848 16589 Hypothetical hydrolase (GH61) family 61 (2.3e-10)
NCU03415.2 FGSC Aldehyde Aldehyde 9.8 ++, ++ 104.2278204 12922
Dehydrogenase dehydrogenase family (2.5e-267) NCU03731.2 FGSC
Similar haloacid 2.7 ++, ++ 131.3691128 18653 to HAD dehalogenase-
Superfamily like hydrolase Hydrolase (9.2e-21) NCU03753.2 FGSC
ccg-1 (clock no significant 10.5 ++, ++ 107.6792892 16379
controlled hit gene) NCU04197.2 FGSC Conserved no significant 5.04
++, ++ 103.0668127 17499 Hypothetical hit NCU04249.2 FGSC
Hypothetical no significant 5.3 ++, ++ 93.29682366 18628 Protein
hit NCU04287.2 FGSC Predicted no significant 4.7 ++, ++ 115.5157859
14573 Protein hit NCU04349.2 FGSC Similar to BCDHK_A 2.9 ++, ++
87.87776465 18634 mitochondrial dom3 pyruvate (4.7e-78),
dehydrogenase Histidine kinase ATPase_c (6.9e-14) NCU04475.2 FGSC
Predicted no significant 76.7 +++, ++ 98.10205352 15386 Protein hit
NCU04997.2 FGSC Similar to Glycosyl 25.6 ++, ++ 105.3520176 15623
xylanase hydrolase (GH10, CBD1) family 10 (3.3e-148), Fungal
cellulose binding domain (2.1e-16) NCU05057.2 FGSC Endoglucanase
Glycosyl 8.7 ++, ++ 137.5316563 13342 EG-1 hydrolase precursor
family 7 (GH7) (3.3e-189) NCU05159.2 FGSC acetylxylan Cutinase 34.8
+++, ++ 86.18543871 13439 esterase (3.4e-110), precursor Fungal
cellulose (Cutinase, binding domain CBD1) (7.4e-14) NCU05493.2 FGSC
Predicted no significant 4.5 +, ++ 73.25266013 14625 Protein hit
NCU05519.2 FGSC Similar Major 2.8 ++ ,++ .sup. 85.31191321 19924 to
Tna1 Facilitator (MFS Superfamily transporter) (3.7e-40) NCU05751.2
FGSC Conserved GDSL-like 3.9 +, ++ 97.01648237 15757 Hypothetical
Lipase/ (GDSL- Acylhydrolase like lipase) (1.3e-11) NCU05770.2 FGSC
Peroxidase/ Peroxidase 11.9 ++, ++ 109.8630989 11532 Catalase 2
(9.4e-195) NCU05853 FGSC Sugar Sugar 130.7 + 40.27924687 13771
Transporter Transporter NCU05897.2 FGSC Similar to Major 20.9 +, ++
33.78464142 13717 l-fucose Facilitator permease (MFS Superfamily
transporter) (3.8e-16) NCU05932.2 FGSC Predicted no significant
38.2 ++, ++ 70.89826428 19952 Protein hit NCU06009.2 FGSC Similar
to Aldo/keto 6.9 +, ++ 148.6633726 14922 aldo/keto reductase
reductase family (4.8e-63) NCU06490.2 FGSC Conserved no significant
13.8 +, ++ 77.46104143 15539 Hypothetical hit NCU07340.2 FGSC
Exoglucanase- Glycosyl 426.4 +, ++ 21.09634551 15630 1 precursor,
hydrolase CBH1 (GH7) family 7 (1e-999), Fungal cellulose binding
domain (4.9e-18) NCU07853.2 FGSC Uricase Uricase 4.3 +++, ++ n/a
19036 (1.7e-119) NCU07997.2 FGSC Predicted no significant 4.5 ++,
++ n/a 18273 Protein hit NCU08114.2 FGSC Similar to Sugar 6.7 +, ++
81.69263905 17869 MFS hexose (and other) transporter transporter
(MFS (5.1e-88), transporter) Major Facilitator Superfamily
(3.8e-24) NCU08744.2 FGSC Predicted no significant 2.3 ++, ++ n/a
11387 Protein, hit possible TF (basic region leucine zipper)
NCU08746.2 FGSC Conserved Starch 6 ++, ++ 98.69504624 18358
Hypothetical binding domain (starch (5.36-54) binding domain)
NCU08760.2 FGSC Predicted Fungal cellulose 107.5 ++, ++ 158.1395349
15664 Protein binding domain (CBD1) (1.9e-11), Glycosyl hydrolase
family 61 (1.3e-9) NCU09108.2 FGSC Conserved no significant 4.1 ++,
++ n/a 19207 Hypothetical hit NCU09495.2 FGSC set-6, histone SET
domain 26.2 ++, ++ 109.3300111 12411 methyltransferase (6.9e-5)
NCU09680.2 FGSC Exoglucanase- Glycosyl 230.9 +, ++ 102.7131783
15633 2 precursor, hydrolases CBH2 (GH6, family 6 CBD1) (1.1e-152),
Fungal cellulose binding domain (1.2e-13) NCU10045.2 FGSC
pectinesterase Pectinesterase 10.9 +, ++ 105.3085012 18480
precursor (4.4e-22) % WT % WT % WT % WT % WT Bradford endo endo
cellobiose cellobiose NCU# (Mis) (Avi) (Mis) (Avi) (Mis) NCU00130.2
118.3987972 152.2858578 129.3547494 n/a n/a NCU00248.2 86.04471858
30.39187506 156.5050144 93.05143946 89.27698219 NCU00326.2
144.1210486 39.91568458 227.0366809 89.76872415 79.05154639
NCU00762.2 84.57056944 26.92790756 39.26890058 n/a n/a NCU00810.2
123.5564757 161.2908993 159.4983744 102.2745211 91.73345664
NCU00890.2 101.5974441 43.25546345 164.0819718 n/a n/a NCU03328.2
109.9667248 142.6962073 167.0075481 n/a n/a NCU03415.2 96.61435373
96.4633125 63.45523329 76.96643943 103.1273983 NCU03731.2
110.5801446 145.0235135 134.627995 230.1450412 100.4172375
NCU03753.2 111.3481086 74.42402278 129.2196777 n/a n/a NCU04197.2
99.08305414 108.9737808 89.86128625 75.17285531 96.05075054
NCU04249.2 106.1012167 79.0053469 84.16141236 64.07989522
100.124185 NCU04287.2 102.2361065 125.5086234 127.9282577
202.516129 183.8679245 NCU04349.2 89.36205196 71.41381803
145.2415813 208.4329349 101.2993763 NCU04475.2 122.2034851
156.3643221 127.0676692 n/a n/a NCU04997.2 114.5840184 123.3295466
231.6983895 136.4189483 102.5403983 NCU05057.2 95.69220651
133.5226686 174.2679356 182.023775 97.81330657 NCU05159.2
39.51658235 92.2873845 67.11779449 n/a n/a NCU05493.2 104.4102564
102.3841739 116.8954593 70.37185126 99.42837929 NCU05519.2
101.0666667 118.8447721 87.77719113 51.6886931 97.87501655
NCU05751.2 111.4051282 114.7202911 136.3780359 87.71492649
105.5920583 NCU05770.2 86.73412029 69.1872525 146.2155388 n/a n/a
NCU05853 24.41259790 n/a n/a n/a n/a NCU05897.2 34.72754541
26.3266891 86.25954198 n/a n/a NCU05932.2 76.87132044 80.78910753
117.9596823 58.07431478 96.7108463 NCU06009.2 74.06784413
120.602266 99.48075748 70.89513625 97.00573241 NCU06490.2
80.26352677 76.95289207 79.38301772 59.91109168 99.99371385
NCU07340.2 95.21973786 35.54661301 96.99134496 93.62619808
78.44902553 NCU07853.2 n/a 120.9286562 168.2340648 65.7599456
99.14659177 NCU07997.2 n/a 148.127436 98.11912226 60.65548063
93.78704271 NCU08114.2 79.22624054 85.18187239 92.97495418
58.83068556 93.1432252 NCU08744.2 n/a 168.8527368 110.7628004
136.2451567 97.44134197 NCU08746.2 79.11410149 111.0713576
120.2504582 447.2796518 100.5753667 NCU08760.2 86.17964534
208.2590783 81.00013738 97.32646961 84.34251774 NCU09108.2 n/a
93.22148788 111.8077325 60.89420655 97.24517906 NCU09495.2
122.5327679 129.9223915 130.8971013 152.7495439 92.25216554
NCU09680.2 95.20046261 89.54680464 102.6789394 94.61873756
83.87661343 NCU10045.2 101.5138772 109.8886901 132.5290165
83.25906421 103.6151641 *Note: All sequences were searched against
Pfam ls models and hits were accepted with an e-value <.0001
Example 7
Further Analyses of Transporter Genes
[0340] As described in Example 1, ten genes encoding predicted
sugar transporter proteins showed increased expression levels when
Neurospora was grown on Miscanthus and Avicel: NCU00801, NCU00988,
NCU01231, NCU04963, NCU05519, NCU05853, NCU05897, NCU06138,
NCU08114 and NCU10021. Deletion strains for nine of these genes
were available from the Fungal Genetics Stock Center. A deletion
strain of NCU10021 was not available.
[0341] Deletion mutations of NCU05853, NCU05897, or NCU08114
resulted in strains that showed a growth defect on Miscanthus or
Avicel and/or had a cellulase enzyme defect (see Example 6; Table
10). .DELTA.NCU05853 showed reduced growth on Avicel and reduced
endoglucanase activity compared to wild-type. .DELTA.NCU05897
showed reduced growth on Avicel and reduced endoglucanase activity
compared to wild-type, and .DELTA.NCU08114 showed reduced growth on
Avicel and reduced cellobiose levels compared to wild-type.
Notably, in a comparison with expression analysis of Sporotrichum
thermophile, another filamentous fungus, the homologs of NCU05853
(ST8454) and NCU08114 (ST5194) were also upregulated when S.
thermophile was grown on Avicel compared to glucose (see Example 8,
Table 11), further indicating their importance in cellulose
utilization.
Table 11 shows S. thermophile expression data
TABLE-US-00011 Like Gene Gene Name NCU# Length Glu Avi Cot Glu_norm
Avi_norm Cot_norm Avi/Glu Cot/Glu jgi|Spoth1|108 NCU00988 1937 322
370 293 42.97830583 60.2250594 48.07756207 1.149068 0.9099379
890|estExt_fge nesh1_pg.C_6 0848 jgi|Spoth1|-484 NCU01132 1539 113
59 56 15.08244894 9.60345542 9.188885583 0.522124 0.4955752
39|e_gw1.3.33 67.1 jgi|Spoth1|790 NCU01231 1776 1171 1206 469
156.2968824 196.30114 76.95691676 1.029889 0.4005124 30|estExt_Gen
ewise1Plus.C.sub.-- 31624 jgi|Spoth1|116 NCU05519 1680 103 78 54
13.74771895 12.6960936 8.860711098 0.757282 0.5242718
270|estExt_fge nesh1_pm.C_5 0266 jgi|Spoth1|841 NCU05853 1706 2703
20760 14284 360.7775176 3379.11414 2343.822173 7.680355 5.2844987
64|estExt_Gen ewise1Plus.C.sub.-- 62100 jgi|Spoth1|102 NCU05897
1446 1510 546 322 201.5442292 88.8726553 52.8360921 0.361589
0.213245 977|fgenesh1.sub.-- pm.5_#_763 jgi|Spoth1|843 NCU06138
1605 1131 1330 2376 150.9579624 216.484673 389.8712883 1.17595
2.1007958 05|estExt_Gen ewise1Plus.C.sub.-- 70023 jgi|Spoth1|114
NCU08114 1945 2246 22423 10779 299.7803568 3649.80137 1768.696388
9.983526 4.7991986 107|estExt_fge nesh1_pm.C_2 0669 jgi|Spoth1|112
NCU10021 2026 6204 5287 5619 828.0664888 860.567268 922.006216
0.852192 0.905706 305|estExt_fge nesh1_kg.C_6 0263 jgi|Spoth1|439
NCU00801 1614 41 71 159 5.472392979 11.5567006 26.08987157 1.731707
3.8780488 41|e_gw1.2.42 09.1 jgi|Spoth1|625 NCU04963 2204 799 1548
641 106.6449266 251.968627 105.1799225 1.937422 0.8022528
21|estExt_Gen ewise1.C_217 57
[0342] In order to narrow down the identity of each predicted
transporter's substrate, strains containing deletion mutations of
NCU05853 or NCU08114 were cultured on glucose, xylose, cellobiose,
xylan and Avicel (Table 12). The culturing medium contained Vogel's
medium plus 2% of the carbon source. Both mutants showed greatly
reduced growth on Avicel but not on xylan, glucose, xylose, or
cellobiose.
Table 12 shows growth of deletion mutants on different sugars
TABLE-US-00012 Gene Growth on Growth on Growth on Growth on Growth
on Growth on Growth on Name Sucrose Avicel Mis Xylan Glucose Xylose
Cellobiose NCU00801 * * * * * * * * * NCU00988 * * * * * * * * *
NCU01231 * * * * * * * * * NCU04963 * * * * * * * * * NCU05519 * *
* * * * * * * NCU05853 * * * * * * * * * * * * * * * * * * NCU05897
* * * * * * NCU06138 * * * * * * * * * NCU08114 * * * * * * * * * *
* * * * * * * * NCU10021 No deletion strain wt * * * * * * * * * *
* * * * * * * * * * *
[0343] To investigate the role of these transporters in utilization
of hemicellulose, the expression of the ten transporter genes was
examined when Neurospora was grown on xylan. Methods were used as
described in Example 4, except that strains were grown on Vogel's
salts with 2% (w/v) xylan. Expression of all ten transporters was
upregulated during growth on xylan (Table 13), suggesting that they
can transport sugars derived from hemicellulose degradation (e.g.,
xylobiose, xylose, arabinose, xylo-oligosaccharides) as well as
from cellulose degradation (e.g., cellobiose, glucose,
cello-oligosaccharides). The mutant growth results and expression
analyses suggested that at least two of the predicted transporters,
NCU05853 and NCU08114, can transport disaccharides (cellobiose,
xylobiose) and/or oligosaccharides (cellodextrins).
Table 13 shows expression analysis of transporter genes
TABLE-US-00013 Gene Name wt-Xylan 4 h Fold change in St-Avicel-4
h/Glucose-4 h NCU00801 ~6 10 NCU00988.2 31.1 NO CHANGE NCU01231.2
732.1 NO CHANGE NCU04963.2 96.5 NO DETECT NCU05519.2 3.9 NO CHANGE
NCU05853.2 71.2 8.5 NCU05897.2 122.3 NO CHANGE NCU06138.2 141.0 NO
CHANGE NCU08114.2 10.0 11 NCU10021.2 44.7 NO CHANGE
Example 8
Expression Analysis of Sporotrichum thermophile Homologs of N.
crassa Transporters During Growth on Various Carbon Sources
[0344] In order to compare the expression of homologous genes from
a different filamentous fungus, the expression profile of
Sporotrichum thermophile was analyzed from cultures grown on
glucose, Avicel, or cotton. cDNA was isolated from cultures grown
on minimal media with a carbon source of glucose, Avicel, or cotton
for 16-30 hours.
[0345] First, in order to identify homologs of Neurospora
transporter proteins in the S. thermophile genome, each Neurospora
sequence was compared against a database of S. thermophile proteins
with BLAST. The sequences of S. thermophile proteins found by this
method were then compared to a database of Neurospora proteins with
BLAST. These results are listed in FIG. 10. The amino acid
sequences for all of the S. thermophile homologs of putative
Neurospora transporters that were identified can be found in SEQ ID
NOs: 22-32.
[0346] Next, the expression profile of the S. thermophile homologs
was examined. The data is presented in Table 11. The first column
contains the S. thermophile gene name from the Joint Genome
Institute S. thermophile assembly. The second column contains the
NCU number for the most closely related putative transporter in
Neurospora. The third column contains the gene length of the S.
thermophile gene in nucleotides. The fourth to sixth columns
contain the expression level (number of reads, comparable to
absolute expression level) during growth on Vogel's minimal media
supplemented with 2% of glucose, Avicel, or cotton balls as the
carbon source. The seventh to ninth columns contain the normalized
expression data (the # of reads divided by the total reads in the
dataset). The final two columns contain the relative expression
level data for each gene as a ratio of Avicel/glucose or
cotton/glucose. Homologs of NCU5853, NCU8114, and NCU0801 were
upregulated when grown on both Avicel and cotton. The homolog of
NCU6138 was upregulated when grown on cotton, and the homolog of
NCU4963 was upregulated when grown on Avicel. These data provided
further support that putative transporters NCU5853, NCU8114,
NCU0801, NCU6138, and NCU4963 are important for the utilization of
cellulose.
Example 9
Identification and Analysis of Cellodextrin Transporters
[0347] When grown on pure cellulose, N. crassa was shown to
increase transcription of seven Major Facilitator Superfamily sugar
transporters as well as an intracellular .beta.-glucosidase (Ex. 1;
also see Supplemental Data, Dataset S1, page 6 in Tian et al.,
PNAS, 2009). Notably, knockout strains lacking individual
transporters from this set grew more slowly on crystalline
cellulose, suggesting that they may play a direct role in
cello-oligosaccharide uptake under cellulolytic conditions (Ex. 7;
Tables 10, 12). For example, deletion of NCU08114 resulted in
severely retarded N. crassa growth (FIGS. 11A-B), and reduced N.
crassa consumption of cellobiose (FIGS. 12A-D and 13). In this
example, transporter genes NCU00801/cbt1 and NCU08114/cbt2 were
further analyzed and identified to encode transporters of
cellodextrin.
[0348] To assay the function of each transporter individually, the
fact that cellobiose is not catabolized by S. cerevisiae and is not
accumulated in its cytoplasm was exploited (FIG. 14). It was
reasoned that expression of a functional cellobiose transporter in
conjunction with an intracellular .beta.-glucosidase would allow S.
cerevisiae to grow when cellobiose is presented as the sole carbon
source. Yeast strains were engineered to express the transporters
NCU00801 or NCU08114 fused to Green Fluorescent Protein (GFP), and
the putative intracellular .beta.-glucosidase, NCU00130. Both
transporters were expressed and localized correctly to the plasma
membrane (FIGS. 15A-B). The strains expressing NCU00801 or NCU08114
allowed yeast to grow with specific growth rates of 0.0341
hr.sup.-1 and 0.0131 hr.sup.-1, respectively (FIG. 16A). These
growth rates correspond to 30% and 12% of the growth rate on
glucose, respectively (FIG. 17). Growth could not be explained by
the extracellular hydrolysis of cellobiose to glucose followed by
transport, as a strain expressing only the putative intracellular
.beta.-glucosidase grew at a rate of 0.0026 hr.sup.-1 (FIG. 16A),
and did not grow in large-scale cultures (FIG. 18). Based on these
observations, NCU00801 and NCU08114, which were named CBT1 and
CBT2, were determined to function as cellobiose transporters.
[0349] To directly assay transporter function, the uptake of
[.sup.3H]-cellobiose into yeast cells was measured. Both CBT1 and
CBT2 were found to be high-affinity cellobiose transporters, with
K.sub.m values of 4.0.+-.0.3 .mu.M and 3.2.+-.0.2 .mu.M,
respectively (FIG. 19). The expression-normalized V.sub.max of CBT1
was 2.2 times that of CBT2, a fact that explained differences seen
in the yeast growth assays. Notably, cellodextrin molecules longer
than cellobiose supported the growth of yeast expressing cbt1 and
cbt2 (FIG. 20; FIG. 16B), suggesting that cellodextrin molecules
are transported by CBT1 and CBT2. In agreement, cellobiose
transport by CBT1 and CBT2 was inhibited by excess cellotriose, and
CBT1 activity was also inhibited by cellotetraose (FIG. 21).
Furthermore, upon purification, the .beta.-glucosidase, NCU00130
(FIG. 22), was found to hydrolyze cellobiose, cellotriose, and
cellotetraose (FIG. 16C).
[0350] Orthologs of cbt1 and cbt2 were identified and found to be
widely distributed in the fungal kingdom (FIG. 23). Recent
expression data shows their importance to various interactions
between fungi and plants. For example, when the ascomycete, Tuber
melanosporum, or the basidiomycete, Laccaria bicolor, interacts
symbiotically with root tips to form ectomycorrihzas, the ortholog
of cbt1 is upregulated in both (Martin et al., 2010). Likewise, the
saprophytes, Aspergillus oryzae (Noguchi et al., 2009), Postia
placenta (Vanden Wymelenberg et al., 2010), and Phanerochaete
chrysosporium (Vanden Wymelenberg et al., 2010), upregulate
orthologs of cbt2 when in contact with plant wall material. Certain
yeasts, such as Kluveromyces lactis and Pichia stipitis grow on
cellobiose (Freer, 1991; Preez et al., 1986), and cellobiose
transport has been reported in Clavispora lusitaniae (Freer and
Greene 1990). It was determined in this study that all of these
yeasts contain orthologs of cbt1, cbt2, or both (see below for
methods). Cellobiose transport has been observed in Hypocrea
jecorina (Trichoderma reesei), but since the transporter was not
identified, it is not clear if this activity can be ascribed to
orthologs of cbt1 or cbt2 (Kubicek et al., 1993).
[0351] The use of cellobiose transporters by cellulolytic fungi
suggests that they are essential for their optimal growth on
cellulose. To test whether cellobiose catabolism could improve
yeast ethanol production, the yeast strains constructed above were
grown under fermentation conditions. With little optimization,
yeast with a complete cellobiose catabolism pathway ported from N.
crassa were shown to ferment cellobiose to ethanol efficiently
(FIG. 24A), with an ethanol yield of 0.47, 86% of the theoretical
value (Bai et al., 2008). This was comparable to industrial yields
from glucose of 90-93% (Basso et al., 2008). The high affinity of
CBT1 and CBT2 for cellobiose compared to the hexose transporters of
S. cerevisiae (Reifenberger et al., 1997), and reported
extracellular .beta.-glucosidases (Chauve et al., 2010), suggested
that a cellobiose/cellodextrin transport system would be
particularly useful during SSF. For example, cellobiose/celldextrin
transport would lower the requirement for full hydrolysis of
cellulose to glucose, decrease cellobiose-mediated inhibition of
cellulolytic enzymes, and reduce the risk of contamination by
glucose-dependent organisms. Indeed, yeasts expressing a
cellobiose/cellodextrin transport system markedly improved the
efficiency of SSF reactions by reducing the steady state
concentration of both cellobiose and glucose, and increasing the
ethanol production rate (FIG. 24B, C).
[0352] Biofuel production from cellulose requires efficient and
economical depolymerization of plant biomass to sugars coordinated
with fuel production by improved host strains (Kumar et al., 2008).
Here it was shown that cellulolytic fungi use cello-oligosaccharide
transport pathways for optimal growth on plant biomass.
Furthermore, reconstitution of these pathways in yeast revealed
that they can be ported in a modular fashion to improve cellobiose
catabolism, with a minimal pathway composed of a transporter and an
intracellular cello-oligosaccharide hydrolase (FIG. 25). The use of
cellodextrin transport in biofuel-producing strains of yeast and
other organisms is critical for making cellulosic biofuel processes
more economically viable.
[0353] Transporter and .beta.-glucosidase orthologs
[0354] GenBank accession numbers or Joint Genome Institute (JGI)
protein ID (PID) numbers for cellodextrin transporters are as
follows: Tuber melanosporum, CAZ81962.1; Pichia stipitis,
ABN65648.2; Laccaria bicolor, EDR07962; Aspergillus oryzae,
BAE58341.1; Phanerochaete chrysosporium, PID 136620 (JGI) (Martinez
et al., 2004); Postia placenta, PID 115604 (JGI) (Martinez et al.,
2009). The GenBank accession number for Saccharomyces cerevisiae
HXT1 and Kluyveromyces lactis LACP are DAA06789.1 and CAA30053.1,
respectively. The P. chrysosporium and P. placenta genomes can be
accessed at genome.jgi-psf.org/Phchr1/Phchr1.home.html and
genome.jgi-psf.org/Pospl1/Pospl1.home.html, respectively.
[0355] GenBank accession numbers for cellodextrin hydrolases that
are orthologs of NCU00130 are as follows: T. melanosporum,
CAZ82985.1; A. oryzae, BAE57671.1; P. placenta, EED81359.1; and P.
chrysosporium, BAE87009.1. The other organisms that contain
cellodextrin transporter orthologs contain genes in the GH3 family
predicted to be intracellular .beta.-glucosidases (Bendtsen et al.,
2004; Cantarel et al., 2009), as follows: Kluyveromyces lactis,
CAG99696.1; Laccaria bicolor, EDR09330; Clavispora lusitaniae,
EEQ37997.1; and Pichia stipitis, ABN67130.1.
[0356] Strains and Media
[0357] The yeast strain used in this study was YPH499 (Sikorski et
al., 1989), which has the genotype: MATa ura3-52 lys2-801_amber
ade2-101_ochre trp1-.DELTA.63 his3-.DELTA.200 leu2-.DELTA.1. It was
grown in YPD media supplemented to 100 mg/L adenine hemisulfate.
Transformed strains (Becker et al., 2001) were grown in the
appropriate complete minimal dropout media, supplemented to 100
mg/L adenine hemisulfate. Neurospora crassa stains used in this
study were obtained from the Fungal Genetics Stock Center
(McCluskey 2004) and include WT (FGSC 2489) and two cellobiose
transporter deletion strains (FGSC 16575, .DELTA.NCU00801.2 and
FGSC 17868, .DELTA.NCU08114.2 (Colot et al., 2006)).
[0358] Plasmids and Cloning
[0359] Transporters were cloned into the 2.mu. plasmid, pRS426,
which was modified to include the S. cerevisiae PGK1 promoter
inserted between SacI and SpeI using the primers,
ATATATGAGCTCGTGAGTAAGGAAAGAGTGAGGAACTATC (SEQ ID NO: 53) and
ATATATACTAGTTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCC (SEQ ID NO: 54).
(In all primers above and below, restriction sites are underlined).
NCU00801 with a C-terminal Myc-tag and optimized Kozak sequence
(Miyasaka 1999) was then inserted between BamHI and EcoRI using the
primers, ATGGATCCAAAAATGTCGTCTCACGGCTCC (SEQ ID NO: 55) and
ATGAATTCCTACAAATCTTCTTCAGAAATCAATTTTTGTTCAGCAACGATAGCTTCGGAC (SEQ
ID NO: 56), and NCU08114 with a C-terminal Myc-tag and optimized
Kozak sequence was inserted between SpeI and ClaI using the
primers, ATACTAGTAAAAATGGGCATCTTCAACAAGAAGC (SEQ ID NO: 57) and
GCATATCGATCTACAAATCTTCTTCAGAAATCAATTTTTGTTCAGCAACAGACTTGCCCTCAT G
(SEQ ID NO: 58). To make GFP fusions, superfolder GFP (Pedelacq et
al., 2006) with an N-terminal linker of Gly-Ser-Gly-Ser was first
inserted between the ClaI and SalI sited of the PGK1
promoter-containing pRS426 plasmid with the primers,
TATTAAATCGATGGTAGTGGTAGTGTGAGCAAGGGCGAGGAG (SEQ ID NO: 59) and
TATTAAGTCGACCTACTTGTACAGCTCGTCCATGCC (SEQ ID NO: 60). Transporters
were then fused to GFP as follows: NCU00801 was inserted between
BamHI and EcoRI using the primers, GCATGGATCCATGTCGTCTCACGGCTCC
(SEQ ID NO: 61) and TATAATGAATTCAGCAACGATAGCTTCGGAC (SEQ ID NO:
62), and NCU08114 was inserted between SpeI and EcoRI using the
primers, TATTAAACTAGTATGGGCATCTTCAACAAGAAGC (SEQ ID NO: 63) and
TTATAAGAATTCAGCAACAGACTTGCCCTCATG (SEQ ID NO: 64).
[0360] The .beta.-glucosidase, NCU00130, was cloned into the 2.mu.
plasmid, pRS425, modified to include the PGK1 promoter described
above. NCU00130 with an optimized Kozak sequence and a C-terminal
6.times.His tag was inserted between SpeI and PstI using the
primers, GCATACTAGTAAAAATGTCTCTTCCTAAGGATTTCCTCT (SEQ ID NO: 65)
and ATACTGCAGTTAATGATGATGATGATGATGGTCCTTCTTGATCAAAGAGTCAAAG (SEQ ID
NO: 66). All constructs included the Cyc transcriptional terminator
between XhoI and KpnI. All N. crassa genes were amplified by PCR
from cDNA synthesized from mRNA isolated from N. crassa (FGSC 2489)
cultured on minimal media with pure cellulose (Avicel) as the sole
carbon source.
[0361] Yeast Growth Assays
[0362] To monitor growth on cello-oligosaccharides, engineered
strains were grown in 5 mL of complete minimal media with
appropriate dropouts overnight. These starter cultures were washed
three times with 25 mL of ddH.sub.2O, and resuspended to an OD (at
600 nm) of 0.1 in Yeast Nitrogen Base (YNB) plus the appropriate
Complete Supplemental Media (CSM) and 1% (w/v) of cellobiose, or
0.5% (w/v) of either cellotriose or cellotetraose. Assays were
performed in a Bioscreen C.TM. with constant shaking at maximum
amplitude at 30.degree. C. and a final assay volume of 0.4 mL. The
change in OD was measured either at 600 nm or using a wideband
filter from 450-580 nm. Growth rates were taken from the linear
portion of each growth curve, and are reported as the mean of three
independent experiments.+-.the standard deviation between these
experiments. Cellotriose and cellotetraose were obtained from
Seikagaku Biobusiness Corporation (Tokyo, Japan).
[0363] Purification of NCU00130 and Assay of its Activity
[0364] A 1 L culture of S. cerevisiae expressing cbt1 and NCU00130
was grown to an OD of 2.0 in complete minimal media. Cells were
harvested by centrifugation and resuspended in 30 mL of lysis
buffer (50 mM NaH.sub.2PO.sub.4 [pH 8.0], 300 mM NaCl, 10 mM
imidazole, 2 mM .beta.-ME, Complete.TM. Mini, EDTA free protease
inhibitor cocktail). Cells were lysed by sonication, and the lysate
was cleared by centrifugation at 15,000 g for 30 minutes. The
lysate was bound to 1 mL of nickel-NTA resin by gravity flow, and
washed three times with 25 mL wash buffer (identical to lysis
buffer but with 20 mM imidazole). NCU00130 was eluted with 5 mL of
elution buffer (identical to lysis buffer but with 250 mM
imidazole), and the appropriate fractions were pooled, exchanged
into storage buffer (Phosphate Buffered Saline (PBS), 2 mM DTT, 10%
glycerol), aliquoted, frozen in liquid nitrogen, and stored at
-80.degree. C. Purity was determined by SDS-PAGE (FIG. 22), and
protein concentration was determined from the absorbance at 280 nm,
using an extinction coefficient of 108,750 M.sup.-1 cm.sup.-1.
[0365] Purified NCU00130 was assayed from hydrolysis activity with
different cellodextrin substrates. Activity was measured by
incubating 5 pmol of enzyme with 500 .mu.M of each sugar in 150
.mu.L PBS plus 3 mM DTT. Reactions proceeded for 40 minutes at
30.degree. C. before 100 .mu.L was removed and quenched in 400
.mu.L of 0.1 M NaOH. The results were analyzed by ion
chromatography with a Dionex ICS-3000, with CarboPac PA200 column.
Peaks were detected with an electrochemical detector.
[0366] Phylogenetic Analysis of Transporter Orthologs
[0367] Amino acid sequences of orthologs of CBT1 and CBT2 were
obtained from online databases. Multiple sequence alignments were
performed using T-Coffee (Notredame et al., 2000). A maximum
likelihood phylogeny was determined using PhyML version 3.0
(Guindon and Gascuel 2003) with 100 Bootstraps. Both programs were
accessed through Phylogeny.fr (webpage phylogeny.fr/). The
resulting tree was visualized with FigTree v.1.2.1 (webpage
tree.bio.ed.ac.uk/).
[0368] Fermentation and SSF
[0369] In fermentation and SSF experiments, comparisons were made
between yeast expressing NCU00130 and either Myc-tagged cbt1, or no
transporter. These strains were grown aerobically overnight in
complete minimal media, washed three times with 25 mL water, and
resuspended to a final OD of 2.0 in 50 mL YNB plus the appropriate
CSM, and either 2% (w/v) cellobiose or 3% (w/v) pure cellulose
(Avicel), in sealed serum flasks. The SSF reactions also included
50 Filter Paper Units/g cellulose of filter-sterilized Celluclast
(Sigma C2730), without .beta.-glucosidase supplementation.
Reactions were carried out anaerobically at 30.degree. C. with
shaking. At indicated time points, 1 mL samples were removed and
filtered through a 0.2 .mu.m syringe filter. The ethanol, glucose,
and cellobiose concentration in the filtrate was determined by HPLC
with an Aminex HPX-87H column and refractive index detection.
[0370] N. crassa Growth and Alamar Blue.RTM. Assays
[0371] WT N. crassa (FGSC 2489), and the homokaryotic NCU08114
(FGSC 17868) (Colot et al., 2006) were acquired from the Fungal
Genetics Research Center (McCluskey 2003), and grown at 25.degree.
C. in 50 mL of Vogel's salts plus 2% of either sucrose or pure
cellulose (Avicel) in a 250 mL unbaffled flask. After 16 or 28
hours, respectively, 100 .mu.L of Alamar Blue.RTM. was added, and
cultures were incubated at room temperature for 20 minutes. At this
time, 1 mL samples were removed, debris pelleted, and the
fluorescence of 100 .mu.L of the supernatant determined with
excitation/emission wavelengths of 535/595 nm in a Beckman Coulter
Paradigm plate reader.
[0372] N. crassa Cellobiose Transport Assays
[0373] WT N. crassa (FGSC 2489), and homokaryotic deletion lines
(Colot et al., 2006) of NCU00801 (FGSC 16575) and NCU08114 (FGSC
17868) were acquired from the Fungal Genetics Stock Center
(McCluskey 2003), and grown for 16 hours in 50 mL of Vogel's salts
plus 2% (w/v) sucrose at 25.degree. C., starting with an inoculum
of 10.sup.6 conidia/mL. Mycelia were harvested by centrifugation,
washed three times with Vogel's salts, and transferred to Vogel's
salts plus 0.5% (w/v) pure cellulose (Avicel) for 4 hours to induce
the transporter expression. Ten mL of the culture was harvested by
centrifugation, washed three times with Vogel's salts, and
resuspended in 1 mL ddH.sub.2O plus cycloheximide (100 .mu.g/mL)
and 90 .mu.M of the respective cellodextrin (cellobiose,
cellotriose, or cellotetraose). To measure cellodextrin
consumption, 100 .mu.L was removed after 15 minutes, clarified by
centrifugation, and transferred into 900 .mu.L of 0.1 M NaOH. The
amount of sugar remaining in the supernatant was determined by HPLC
with a Dionex ICS-3000, using a CarboPac PA200 column. Peaks were
detected with an electrochemical detector.
[0374] GFP Fluorescence and Confocal Fluorescence Microscopy
[0375] Bulk-cell GFP fluorescence measurements were made in a
Beckman Coulter Paradigm plate reader with excitation/emission
wavelengths of 485/535 nm. Confocal fluorescence microscopy was
performed with cells at an OD (at 600 nm) of 0.8-1.2, using a
100.times.1.4 NA oil immersion objective on a Leica SD6000
microscope attached to a Yokogawa CSU-X1 spinning disc head with a
488 nm laser and controlled by Metamorph software. Z series were
recorded with a 200 nm step size and analyzed using ImageJ.
[0376] [.sup.3H] Cellobiose Transport Assays and Kinetic
Parameters
[0377] Transport assays were performed using a modification of the
oil-stop method (Arendt et al., 2007). Yeast strains expressing
either cbt1 or cbt2 fused to GFP were grown to an OD (at 600 nm of
1.5-3.0 in selective media, washed three times with ice cold assay
buffer (30 mM MES-NaOH [pH 5.6] and 50 mM ethanol), and resuspended
to an OD of 20. To start transport reactions, 50 .mu.L of cells
were added to 50 .mu.L of [.sup.3H] cellobiose layered over 100
.mu.L of silicone oil (Sigma 85419). Reactions were stopped by
spinning cells through oil for 1 minute at 17,000 g, tubes were
frozen in ethanol/dry ice, and tube-bottoms containing the
cell-pellets were clipped off into 1 mL of 0.5 M NaOH. The pellets
were solubilized overnight, 5 mL of Ultima Gold scintillation fluid
added, and CPM determined in a Tri-Carb 2900TR scintillation
counter. [.sup.3H] cellobiose was purchased from Moravek
Biochemicals, Inc. and had a specific activity of 4 Ci/mmol and a
purity of >99%. Kinetic parameters were determined by measuring
the linear rate of [.sup.3H] cellobiose uptake over 3 minutes for a
range of cellobiose concentrations. V.sub.max and K.sub.m values
were determined by fitting a single rectangular, 2-parameter
hyperbolic function to a plot of rates vs. cellobiose concentration
by non-linear regression in SigmaPlot.RTM.. V.sub.max values were
normalized for differences in transporter abundance by measuring
the GFP fluorescence from 100 .mu.L of cells at OD 20 immediately
before beginning transport assays. Kinetic parameters reported in
the text are mean.+-.the standard deviation from three separate
experiments. Competition assays were performed by measuring
transport of 50 .mu.M [.sup.3H]-cellobiose over 20 seconds in the
percent of 250 .mu.M of the respective competitors.
[0378] Large Scale Yeast Growth
[0379] To monitor growth on different carbon sources, engineered
strains were grown in 5 mL of complete minimal media with
appropriate dropouts overnight. These starter cultures were washed
three times with 25 mL of ddH2O and resuspended to an OD (at 600
nm) of 0.1 in 50 mL Yeast Nitrogen Base (YNB) plus the appropriate
Complete Supplemental Media (CSM) and 2% (w/v) cellobiose. Cultures
were grown in 250 mL unbaffled flasks at 30.degree. C., with
shaking at 200 rpm. The change in OD (at 600 nm) was monitored by
periodically removing samples.
Example 10
Identification of Critical Residues for Cellodextrin Transporter
Function
[0380] In this example, sequence analysis and mutagenesis studies
were used to identify conserved and functionally important residues
in the cellodextrin transporters. In addition, additional
cellodextrin transporters were identified.
[0381] The growth rates of yeast strains expressing various mutants
of the cellodextrin transporter NCU00801 (cbt1) or NCU08114 (cbt2)
and the wild-type .beta.-glucosidase NCU00130 were grown with
cellobiose as the sole carbon source. Amino acid residues at 96
positions of NCU00801 and at 96 positions of NCU08114 were
individually mutated to alanine using QuickChange.RTM. II
Site-directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) as per
the manufacturer's instructions. Strains were grown in synthetic
defined media-ura-leu 100 mg/L adenine with 2% cellobiose. Cultures
were started from two independent colonies.
[0382] As the results shown in FIG. 26 (a, b) indicate, mutant
strains that expressed NCU00801 with substitutions at W66, L73,
Y74, N87, Y89, D90, Q104, F107, G113, F120, Y123, D139, G142, K144,
M147, G150, Q169, F170, G173, R174, G178, G180, P189, Y191, E194,
P198, R201, Y208, W235, R236, Q242, .sup.257PESPRF.sup.262 (SEQ ID
NO: 67), Y279, G283, E296, D307, K308, W310, D312, R325, G336,
Y345, N369, D385, F462, P468, E476, T480, or G486 showed at least a
25% growth defect compared to wild-type strain.
[0383] The alanine scanning experiment on NCU08114 indicated the
following residues as being functionally important: L38, Y39, G54,
D56, F73, G91, P100, D104, G107, R108, M118, R139, F144, Q150,
P154, E159, P163, H165, R166, Y173, N174, W199, Q214,
.sup.222PESP.sup.225 (SEQ ID NO: 68), Y244, H245, D249, E258, E268,
Q302, W303, 5304, N306, Y312, F359, L360, F402, Y403, 5404, Y414,
E417, P420, Y421, K426, N442, N446, P447, W459, K460, E482, T483,
L488, E489, E490, D496, and G497 (FIG. 26b).
[0384] In particular, the motifs .sup.73LYF.sup.75,
.sup.257PESP.sup.260 (SEQ ID NO: 69), and .sup.278KYH.sup.2''
(residue numbering of NCU00801) appeared to be functionally
important in both transporters (residues .sup.257PESP.sup.260 (SEQ
ID NO: 69) of NCU00801 and residues .sup.222PESP.sup.225 (SEQ ID
NO: 68) of NCU08114), which have an amino acid sequence identity of
29% (FIG. 26b, c). Several residues that are conserved in
transporters in general (italicized in FIG. 26b, c), or in
.beta.-linked transporters in particular (double-underlined), were
experimentally shown to be important for transporter function
(underlined), e.g., D90 (NCU00801) and D56 (NCU08114), and L73
(NCU00801) and L38 (NCU08114). Results of the mutagenesis
experiment also implicated residues conserved in the
NCU00801/NCU08114 clade (capped) as being functionally important,
e.g., Q168 (NCU00801) and Q214 (NCU08114). Moreover, multiple
residues determined to be functionally important in this experiment
were previously shown to be conserved in the S. cerevisiae sugar
transporters (Hxt1/Hxt3), e.g., L73 (NCU00801) and L38
(NCU08114).
[0385] Orthologs of N. crassa cellodextrin transporters from
different organisms were also studied (FIGS. 27A-C). Representative
orthologs were synthesized by Genescript and cloned into the
expression vector, pRS426 containing the Cup1 promoter using the
sites BamHI and HindIII. These constructs were transformed into the
yeast strain, YPH499 along with the intracellular
.beta.-glucosidase, NCU00130. Transporter activity was determined
by measuring the growth rates of these strains when cellobiose was
present as the sole carbon source.
[0386] Alternatively, different fungal strains containing putative
orthologs were cultivated in rich media supplemented with
cellobiose. Total RNA was isolated and reverse transcribed into
cDNA. Polymerase chain reaction (PCR) was used to amplify the
putative transporter genes directly from cDNA. However, because the
regulation mechanism and expression pattern were unknown for
cellodextrin transporters in fungal species, cDNAs encoding the
putative transporters were not always obtainable despite alteration
of cultivation condition. In this case, primers were designed
according to the corresponding cDNA sequences from GenBank and used
to amplify the exons using genomic DNA as a template.
Overlap-extension PCR was then used to assemble the exons into the
full-length genes. The resulting PCR products were cloned into the
pRS424 shuttle vector containing a HXT7 promoter and a HXT7
terminator using the DNA assembler method. Yeast plasmids isolated
from transformants were retransformed into E. coli DH5.alpha., and
isolated E. coli plasmids were first checked by diagnostic PCR
using the primers used to amplify the original transporter genes.
The entire open reading frames were submitted for sequencing to
confirm the correct construction of the plasmids. In the orthologs
LAC2, LAC3, HXT2.1, and HXT2.6 from P. stipitis, one or more
alternative codons (CUG) substitute Ser for Leu. Most of the
cloning work was carried out using the yeast homologous
recombination mediated DNA assembler method. pRS424-HXT7-GFP
plasmid was used for cloning of putative cellodextrin transporters.
In this plasmid, the HXT7 promoter, the GFP gene flanked with the
EcoRI sites at both ends, and the HXT7 terminator were assembled
into the pRS424 shuttle vector (New England Biolabs) linearized by
ClaI and BamHI. PCR products of the putative transporters flanked
with DNA fragments sharing sequence identity to the HXT7 promoter
and terminator were co-transferred into CEN.PK2-1C with EcoRI
digested pRS424-HXT7-GFP using the standard lithium acetate method.
The resulting transformation mixture was plated on SC-Trp plates
supplemented with 2% D-glucose to recover transformants. Yeast
expressing putative cellodextrin transporter orthologs and NCU00130
were tested for growth on cellobiose as the sole carbon source.
[0387] A listing of the putative cellodextrin transporter orthologs
and results obtained from the study are shown in Table 14.
TABLE-US-00014 TABLE 14 Listing of putative cellodextrin
transporter orthologs and summary of results. NCBI Reference
Sequence/ NCBI GI Aver. N. crassa Number/JGI Growth Growth ortholog
Species number Rate Rate error Seq results* NCU00809 Chaetomium
globusom XP_001220480 -- -- OK CBS148.51 NCU00809 Podospora
anserina XP_001912722 -- -- -- NCU00809 Nectria haematococca
EEU41662 -- -- -- mpVI77-13-4 NCU00809 Aspergillus nidulans
XP_660803 -- -- 1 intron and FGSC A4 50 bp insertion NCU00809
Aspergillus terreus XP_001218592 -- -- -- NIH2624 NCU00809
Talaromyces stipitatus XP_002341594 -- -- -- ATCC 10500 NCU00809
Aspergillus niger XP_001395979 -- -- Ala > Val NCU00809
Aspergillus fumigatus XP_747891 -- -- -- Af293 NCU00809 Aspergillus
terreus XP_00120996 -- -- -- NIH2624 NCU00809 Aspergillus oryzae
RIB40 XP_001817400 -- -- OK NCU08114 Podospora anserina
XP_001908539 -- -- N/A NCU08114 Penicillium chrysogenum
XP_002568019 -- -- N/A Wisconsin 54-1255 NCU08114 Aspergillus
terreus XP_001209810 -- -- Wrong NIH2624 NCU08114 Aspergillus
oryzae RIB40 XP_001820343 -- -- OK NCU08114 Aspergillus terreus
XP_001210859 -- -- N/A NIH2624 NCU08114 Neurospora crassa
XP_001728155 -- -- N/A OR74A NCU08114 Aspergillus oryzae RIB40
XP_001826848 -- -- N/A NCU08114 Aspergillus nidulans XP_657617 --
-- OK FGSC A4 NCU08114 Talaromyces stipitatus XP_002487579 -- --
N/A ATCC 10500 NCU08114 Chaetomium globosum XP_001227497 -- --
Wrong CBS 148.51 NCU08114 Trichoderma atroviridae 215408
0.000836364 0.00064871 I, D NCU08114 Chaetomium globosum
XP_001220290.1 0.004036364 0.00047168 OK NCU08114 Aspergillus
nidulans ANID_08347 0.011109091 0.000072727 Other NCU08114
Pleurotus ostreatus 51322 0.00390303 0.00018212 -- NCU08114
Sporotrichum 114107 0.009569697 0.00216366 -- thermophile NCU00801
Aspergillus nidulans XP_660418.1 0.000860606 0.000438 P NCU00801
Magnaporthe grisea XP_364883.1 005090909 0.00138313 OK NCU00801
Aspergillus fumigatus XP_753099.1 0.003975758 0.00211951 OK
NCU00801 Trichoderma atroviridae 211304 0.002678788 0.00031193 D
NCU00801 Chaetomium globosum XP_001220469.1 0.005890909 0.00010285
OK NCU00801 Tremella mesenterica 63529 0.004381818 0.00115751 D
NCU00801 Heterobasidion. annosum 105952 0.002751515 0.00068763 D
NCU00801 Cryphonectria parasitica 252427 0.02250303 0.00021692 D
NCU00801 Trichoderma ressei 67752 0.003672727 0.00066233 D NCU00801
Aspergillus clavatus XP_001268541.1 0.014381818 0.00059613 OK
NCU00801 Neurospora discreta 77429 0.007060606 0.00110566 D
NCU00801 Trichoderma reesei 3405 0.003264646 0.001033998 D NCU00801
Sporotrichum 43941 0.013654545 0.00431534 -- thermophile NCU00801
Neurospora crassa XP_963801.1 0.048754872 0.00354017 -- NCU05853
Chaetomium globosum XP_001226269.1 0.003593939 0.00062306 OK
NCU05853 Trichoderma reesei 46819 0.002042424 0.000085924 D
NCU05853 Mycosphaerella 68287 0.00290101 0.00060123 D graminicola
NCU05853 Aspergillus flavus AFLA_000820A 0.003078788 0.00209132 --
-- None -- 0.0026 0.0001 -- NCU00809 Pichia stipitis CBS6054
XP_001383110.1/ FIGS. -- -- (LAC1) GI: 126133170 27A-C NCU00809
Pichia stipitis CBS6054 XP_001387231.1/ FIGS. -- -- (LAC2) GI:
126276337 27A-C NCU00809 Pichia stipitis CBS6054 XP_001383677.2/
FIGS. -- -- (LAC3) GI: 150864727 27A-C NCU08114 Pichia stipitis
CBS6054 XP_001386873.1/ FIGS. -- -- (HXT2.1) GI: 126275571 27A-C
NCU05853 Pichia stipitis CBS6054 XP_001382754.1/ FIGS. -- --
(HXT2.3) GI: 126132458 27A-C NCU08114 Pichia stipitis CBS6054
XP_001387757.1/ FIGS. -- -- (HXT2.4) GI: 126273939 27A-C NCU08114
Pichia stipitis CBS6054 XP_001385684.1/ FIGS. -- -- (HXT2.5) GI:
126138322 27A-C NCU08114 Pichia stipitis CBS6054 XP_001384653.2/
FIGS. -- -- (HXT2.6) GI: 15086543 27A-C *Wrong = difference between
tested sequence and sequence in NCBI or JGI databases; I =
insertion in tested sequence; D = deletion in tested sequence; P =
point mutation in tested sequence; OK = no difference between
tested sequence and sequence deposited in NCBI or JGI databases;
Other = other problems in sequencing, excluding insertion,
deletion, and point mutations in tested sequence; "--" = results
not yet available (study in progress). When accession numbers were
not available, the JGI number was used. The JGI number allows
access to the gene sequence via the JGI genome portal for this
organism (accessible from the following page:
genome.jgi-psf.org/programs/fungi/index.jsf). The A. flavus and A.
nidulans identifiers allow access to the genes through their genome
portals at webpage cadre-genomes.org.uk/ and webpage
broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html,
respectively.
[0388] In certain cases, the sequences of the cloned orthologs were
determined to be correct, and the yeast expressing those clones
were able to utilize cellobiose. Thus, these clones, LAC2 from
Pichia stipitis and XP.sub.--001268541.1 from Aspergillus clavatus
were confirmed to be functional cellobiose transporters. Testing of
the cellobiose transporting function of other clones is still in
progress. Cloned orthologs with sequences different from the
published sequences in databases (e.g., ones with insertions,
deletions, etc.) (Table 14) will be re-cloned, re-sequences, and
similarly tested for cellobiose transport activity by expressing
them in S. cerevisiae and monitoring growth rates.
[0389] An alignment of NCU00801, NUC08114, and functional orthologs
of these transporters is shown in FIGS. 28A-C. The alignment in
FIG. 28a includes both putative and confirmed cellodextrin
transporters, whereas the alignment in FIG. 28b includes only
confirmed cellodextrin transporters. In addition, FIG. 28c shows an
alignment of NCU00801 and NCU08114. The two transporters share 29%
amino acid sequence identity.
[0390] Motifs critical for cellodextrin transporter function were
identified by visual inspection of multiple sequence alignments
between sugar transporters. Specifically, motifs common to
cellodextrin transporters were identified from multiple sequence
alignments produced in T-COFFEE between putative cellodextrin
transporter orthologs and confirmed cellodextrin transporters. To
ensure that these motifs were largely unique to cellodextrin
transporters, their absence was confirmed from a multiple sequence
alignment between the hexose transporters of S. cerevisiae, the
human glucose transporter, Glutl, and two N. crassa monosaccharide
transporters produced in T-COFFEE.
[0391] The identified motifs are described below. In the motifs,
residues that were found to be critical to the function of NCU00801
are underlined. The residues that were critical for the function of
NCU08114 are marked with the superscript ".dagger.". The residues
that were critical to the function of both transporters are marked
with the superscript "*". All motifs were defined using PROSITE
notation. As an example of how to read a PROSITE motif, the
following motif, [AC]-x-V-x(4)-{ED}, is translated as: [Ala or
Cys]-any-Val-any-any-any-any-{any but Glu or Asp} (SEQ ID NO:
13)
[0392] Cellodextrin transporters, like all sugar transporters, have
12 transmembrane .alpha.-helices. The N- and C-terminus of
cellodextrin transporters are both intracellular.
The sequence before transmembrane helix 1 had no distinguishing
features. Transmembrane helix 1 contained the motif,
[L*IVM]-Y*-[FL]-x(13)-[YF]-D* (SEQ ID NO: 1). Transmembrane helix 2
contained the motif, [YF]-x(2)-G.sup..dagger.-x(5)-[PVF]-x(6)-[DQ]*
(SEQ ID NO: 2). The loop connecting transmembrane helix 2 and
transmembrane helix 3 contained the motif, G*-R.sup..dagger.-[RK]*
(SEQ ID NO: 3). Transmembrane helix 3 had no distinguishing
features. Transmembrane helix 4 had no distinguishing features.
Transmembrane helix 5 contained the motif,
R*-x(6)-[YF]*-N.sup..dagger. (SEQ ID NO: 4). Transmembrane helix 6
contained the motif, W*R-[IVLA]-P-x(3)-Q (SEQ ID NO: 5). The
sequence between transmembrane helix 6 and transmembrane helix 7
contained the motif,
P*-P*-R-x-L-x(8)-A-x(3)-L-x(2)-Y*-H.sup..dagger. (SEQ ID NO: 6).
Transmembrane helix 7 contained the motif,
F.sup..dagger.-[GST]-Q*-x-S.sup..dagger.-G-N.sup..dagger.-x-[LIV]
(SEQ ID NO: 7). Transmembrane helix 8 had no distinguishing
features. Transmembrane helix 9 had no distinguishing features.
Transmembrane helix 10 and transmembrane helix 11 and the sequence
between them contained the motif,
L-x(3)-[YIV].sup..dagger.-x(2)-E*-x-L-x(4)-R-[GA]-K.sup..dagger.-G
(SEQ ID NO: 8). Transmembrane helix 12 had no distinguishing
features. The sequence after transmembrane helix 12 had no
distinguishing features.
[0393] Homology models of NCU00801 and NCU08114 were produced from
the primary amino acid sequences of NCU00801 and NCU08114 using the
I-TASSER server at: zhanglab.ccmb.med.umich.edu/I-TASSER/ (Roy et
al., 2010). The top structural models produced by I-TASSER were
visualized in PYMOL (webpage pymol.org/). Mapping of the motifs was
also performed in PYMOL. The homology models of NCU00801 and
NCU08114 with the cellodextrin transporter motifs marked are shown
in FIG. 29 (a, b).
Example 11
Characterization of Novel Pentose-Specific Transporters from
Neurospora crassa and Pichia stipitis in Saccharomyces
cerevisiae
[0394] In this example, a bioinformatics approach was taken to
identify novel pentose-specific transporters in N. crassa and P.
stipitis.
Genome Mining of Pentose-Specific Transporters
[0395] Bioinformatics Study
[0396] To discover novel D-xylose-specific transporters, the genes
encoding the D-glucose/D-xylose symporter Gxs1 from C. intermedia
(Leandro et al., 2006) and the uncharacterized putative
L-arabinose-proton symporter Aut1 from P. stipitis (locus tag
PICST.sub.--87108) were used as probes in BLAST searches (webpage
ncbi.nlm.nih.gov/) against the sequenced genomes of two efficient
xylose-utilizing species, N. crassa and P. stipitis (Galagan et
al., 2003; Jeffries et al., 2007). Any proteins with known
D-glucose transport activity or activity other than sugar transport
were eliminated from the analyses. Using a cut-off of 25% minimal
sequence identity, 17 putative pentose transporter genes were
identified (Table 15), in addition to AUT1 from P. stipitis. These
putative pentose transporter genes shared 25-50% identity with
either GXS1 from C. intermedia or AUT1 from P. stipitis. All 17
putative pentose transporters were annotated as either
sugar-transport proteins or hypothetical proteins with unknown
activity. The D-glucose transporter genes SUT1 and SUT2 from P.
stipitis were also cloned for comparison. Table 15 shows the
putative pentose transporters obtained from BLAST using (a) AUT1
from P. stipitis as a probe and (b) GSX1 from C. intermedia as a
probe.
TABLE-US-00015 a. BLAST search results using AUT1 as a probe. %
identity with Length Name Origin AUT1 Annotation from NCBT (cDNA)
Locus Tag Ap31/SUT2 P. stipitis 31 sugar uptake (tentative) 1653
ABN66266 Ap26/XP_001387242 P. stipitis 26 sugar transporter 1404 XP
001387242 AN49/NCU01494 N. crassa 49 hypothetical protein 2025
EAA2669I NCU01494, similar to MFS sugar transporter AN41/NCU09287
N. crassa 41 hypothetical protein 1968 EAA28903 NCU09287, similar
to galactose-proton symporter AN29- N. crassa 29 hypothetical
protein 1584 EAA30175 2/NCU04963 NCU04963, similar to MFS
monosaccharide transporter AN28- N. crassa 28 hypothetical protein
1458 EAA30346 3/NCU02188 NCU02188, conserved hypothetical protein
AN25/NCU00821 N. crassa 25 sugar transporter 1689 EAA35128
TABLE-US-00016 b. BLAST search results using GSX1 as a probe. %
identity with Length Name Origin GSX1 Annotation from NCBI (cDNA)
Locus Tag Xy50/NCU04537 N. crassa 50 hypothetical protein NCU04537
1626 EAA26741 similar to monosaccharide transporter Xy31/NCU06138
N. crassa 31 hypothetical protein NCU06138, 1757 EAA30764 similar
to MFS monosaccharide transporter Xy33/NCU00988 N. crassa 33
hypothetical protein NCU00988, 1614 EAA34662 similar to MFS quinate
transporter Xyp37/SUT3 P. stipitis 37 sugar uptake (tentative) 1653
ABN67990 Xyp33/XUT3 P. stipitis 33 sugar transporter, putative
xylose 1656 EAZ63115 uptake (tentative); predicted transporter
(major facilitator superfamily) Xyp32/XUT1 P. stipitis 32 sugar
transporter, high affinity, 1701 ABN67554 putative; xylose uptake
(tentative) Xyp30/STLl P. stipitis 30 sugar transporter, strongly
1590 ABN65745 conserved Xyp31/XUT2 P. stipitis 31 sugar
transporter, xylose 1407 AAVQOIOOOO02 transporter (tentative)
similarly to GXSI (STLl) Xyp29/STLl2/ P. stipitis 29 sugar
transporter, putative 1641 ABN68560 XUT6 (STLl2); .xylose uptake
(tentative) Xyp30- P. stipitis 30 high affinity xylose transporter
1587 ABN68686 1/HGT3 (putative), xylose uptake (tentative)
Xyp28/XUT7 P. stipitis 28 xylose transporter, high affinity, 1257
EAZ63044 putative similarity to STLl3, high affinity sugar
transporters
[0397] Cloning of Putative Pentose Transporters
[0398] N. crassa and P. stipitis were cultivated in rich media
supplemented with either D-xylose or L-arabinose as carbon sources.
Total RNA was isolated and reverse transcribed into cDNA.
Polymerase chain reaction (PCR) was used to amplify the putative
transporter genes directly from cDNA. However, because the
regulatory mechanism and expression patterns of pentose
transporters in fungal species were unknown, cDNAs encoding the
putative pentose transporters were not always obtainable despite
alteration of cultivation conditions. In those cases, primers were
designed according to the corresponding cDNA sequences from GenBank
and used to amplify the exons with genomic DNA as templates.
Overlap-extension PCR was then used to assemble the exons into full
length genes. The resulting PCR products were cloned into the
pRS424-HXT7-GFP shuttle vector using the yeast homologous
recombination-mediated DNA assembler method (Shao et al., 2009). In
this plasmid, an HXT7 promoter, a GFP gene flanked with the EcoRI
sites at both ends, and an HXT7 terminator were assembled into the
pRS424 shuttle vector (New England Biolabs) linearized by ClaI and
BamHI. PCR products of the putative pentose transporters flanked
with DNA fragments, sharing sequence identity with the HXT7
promoter and terminator (FIG. 30a) were co-transferred into S.
cerevisiae CEN.PK2-1C strain (MAT.alpha. leu2-3,112 ura3-52,
trp1-289, his3-.DELTA.1 MAL2-8c) purchased from Euroscarf
(Frankfurt, Germany) with EcoRI digested pRS424-HXT7-GFP using the
standard lithium acetate method. The resulting transformation
mixture was plated on SC-Trp plates supplemented with 2%
D-glucose.
[0399] Yeast plasmids isolated from transformants using Zymoprep
Yeast Plasmid Miniprep II (Zymo Research, Orange, Calif.) were
re-transferred into Escherichia coli DH5.alpha. cells (Cell Media
Facility, University of Illinois at Urbana-Champaign, Urbana,
Ill.). The plasmids were isolated using the QIAprep Spin Miniprep
Kit (QIAGEN, Valencia, Calif.) and then checked by diagnostic PCR
with the primers used to amplify the original transporter genes.
The entire open reading frames were also submitted for DNA
sequencing to confirm correct construction (Core Sequencing
Facility, University of Illinois at Urbana-Champaign, Urbana,
Ill.). The DNA sequencing results were compared to gene sequences
in databases using Sequencher 4.7 (Gene Codes Corporation, Ann
Arbor, Mich.). All sequences of cloned putative transporters are
listed in SEQ ID NOs: 33-52.
[0400] Yeast strains were cultivated in synthetic dropout media to
maintain plasmids (0.17% Difco yeast nitrogen base without amino
acids and ammonium sulfate, 0.5% ammonium sulfate, 0.05% amino acid
drop out mix). YPA media supplemented with 2% of sugar was used to
grow yeast strains harboring no plasmids (1% yeast extract, 2%
peptone, 0.01% adenine hemisulfate). S. cerevisiae strains were
cultured at 30.degree. C. and 250 rpm for aerobic growth and at
30.degree. C. and 100 rpm for oxygen-limited conditions. Yeast
strains were grown under aerobic conditions for cell manipulation
unless specified otherwise. E. coli strains were cultured at
37.degree. C. and 250 rpm in Luria broth (LB) (Fisher Scientific,
Pittsburgh, Pa.). All restriction enzymes were purchased from New
England Biolabs (Ipswich, Mass.). All chemicals were purchased from
Sigma Aldrich (St. Louis, Mo.) or Fisher Scientific.
Transporter Activity Assay for Cloned Putative Transporters
[0401] Intracellular Accumulation of Pentose Sugars
[0402] The cloned putative pentose transporters were over-expressed
in an S. cerevisiae sugar transporter deletion strain, and uptake
of pentose sugars was measured. The D-xylose-uptake ability of
putative pentose transporters was determined by summation of
intracellular D-xylose and xylitol concentrations. D-xylose
accumulated within S. cerevisiae cells can be partially converted
to xylitol due to the presence of endogenous aldose reductase. Both
D-xylose and xylitol were extracted using osmosis and analyzed
using high performance liquid chromatography (HPLC).
[0403] The sugar transporter knock-out S. cerevisiae strain
EBY.VW4000 (CEN.PK2-1c .DELTA.hxt1-17, .DELTA.stl1, .DELTA.agt1,
.DELTA.yd1247w, .DELTA.yjr160c, .DELTA.gal2), which was a gift from
Professor E. Boles' laboratory (Institut fur Mikrobiologie,
Heinrich-Heine-Universitat, Universitasstr. 1, Geb. 26.12.01,
D-40225 Dusseldorf, Germany), had concurrent knock-outs of more
than 20 sugar transporters and sensors including HXT1-17 and GAL2.
Growth on D-glucose as the sole carbon source was completely
abolished in this strain, whereas uptake of maltose through a
different sugar transport system was retained. The EBY.VW4000
strain also exhibited minimal pentose-uptake under HPLC assay
conditions, which made it a suitable host for testing recombinant
D-xylose uptake. Plasmids over-expressing the cloned putative
pentose transporter genes were transferred into the EBY.VW4000
strain using the standard lithium acetate method, and single
colonies were used for measuring sugar uptake activity.
[0404] Cells were first cultured in 2 mL SC-Trp medium supplemented
with 2% maltose. Seed culture was then used to inoculate a 50 mL
culture in a 250 mL flask. The cells were harvested by
centrifugation after 24 hours of growth and re-suspended in YPA
medium supplemented with 2% D-xylose or L-arabinose to a final
OD.sub.600 of 10. At 30 min, 60 min, 120 min, and 24 hours, 5 mL
cultures were taken for measuring intracellular sugar
concentrations. Culture samples were washed twice with ice-cold
water and re-suspended in 3 mL of deionized water. Cell suspensions
were incubated at 37.degree. C. with 250 rpm agitation for 2 days
to extract intracellular sugars. The resulting cell suspension was
filtered through a 0.22 .mu.m PES filter (Corning, Lowell, Mass.)
before HPLC analysis. The concentrations of sugar and corresponding
sugar alcohol (discussed below) were determined using Shimadzu HPLC
equipped with a BioRad HPX-87C column (BioRad Laboratories,
Hercules, Calif.) and Shimadzu ELSD-LTII low
temperature-evaporative light scattering detector (Shimadzu)
following the manufacturer's protocol. The sugar-uptake activity
was calculated as mg of sugar extracted through osmosis per mL of
cell culture at OD.about.10.
[0405] Several putative pentose transporters were identified to be
active in uptake of D-glucose or D-xylose or both. Since D-glucose
can be metabolized once inside yeast, the D-glucose transport
activity could not be determined by measuring intracellular
D-glucose concentration. However, because the EBY.VW4000 strain
normally cannot grow on media containing D-glucose as the sole
carbon source, growth of the strain transformed with a putative
pentose transporter on D-glucose indicated that the putative
transporter has D-glucose transport activity.
[0406] Introduction of SUT3 (Xyp37), XUT3 (Xyp33), SUT2 (Ap31),
NCU04963 (An29-2), and NCU06138 (Xy31) restored growth of the
EBY.VW4000 strain on D-glucose and, thus, enabled glucose transport
activity. SUT3, XUT3, SUT2, and NCU04963 also had xylose transport
activity, whereas NCU04963 and NCU06138 showed arabinose transport
activity (FIG. 31). The rest of the putative transporters failed to
enable growth on D-glucose, and most of them also did not show any
pentose transport activity. However, NCU00821 and STL12/XUT6 showed
xylose transport activity, and XUT1 exhibited arabinose transport
activity, indicating they may be sugar transporters specific for
pentoses (FIG. 32).
[0407] To further confirm that STL12/XUT6 and XUT1 from P. stipitis
and NCU00821 from N. crassa were actually pentose-specific
transporters with no D-glucose-uptake activity, the sugar-uptake
assay was performed using .sup.14C-labeled D-glucose, D-xylose, and
L-arabinose as substrates. It was found that D-glucose- and
L-arabinose-uptake activities of the EBY.VW4000 strain
over-expressing only STL12/XUT6 and NCU00821 were too low to be
measured under assay conditions used to determine D-xylose-uptake
kinetics of both transporters.
[0408] .sup.14C-labeled D-glucose, L-arabinose, and D-xylose were
purchased from American Radiolabeled Chemicals (St. Louis, Mo.) as
solutions in 90% ethanol. Radiolabeled sugars were first dried in a
chemical hood and then re-suspended in water. Sugar solutions at
concentrations of 1.33 M and 1 M with specific radioactivity of
approximately 40,000 dpm/.mu.L, and at concentrations of 500 mM,
350 mM, 250 mM, 100 mM, and 50 mM with specific radioactivity of
about 20,000 dpm/.mu.L were used for the sugar-uptake assay. Cell
culture at the exponential phase was harvested and washed twice
with ice-cold water and re-suspended to about 60 mg dry cell weight
(DCW) per mL in 100 mM Tris-citrate buffer at pH 5. Three aliquots
of 160 .mu.L cell suspension were dried at 65.degree. C. for 24
hours to determine the DCW. The rest of the cell suspension was
kept on ice before use. For the sugar-uptake assay, cell suspension
was equilibrated at 30.degree. C. for 5 min before the assay. In a
50 mL conical tube, 160 .mu.L of cell suspension was mixed with 40
.mu.L of radio-labeled sugar solution for 40 or 60 seconds
(accurately timed). The reaction was stopped by adding 10 mL of
ice-cold water delivered by a syringe. The zero-time-point sample
was obtained by adding ice-cold water and cell suspension
simultaneously in a culture tube containing the radio-labeled
solution. The mixture was then filtered immediately through a
Whatman GF/C filter (Whatman, Florham Park, N.J.) pre-soaked in 40%
sugar solution and washed with 15 mL of ice-cold water. The filter
was placed in 3 mL of Econo I scintillation cocktail (Fisher
Scientific) and counted using a Beckman LS6500 scintillation
counter (Beckman Coulter, Brea, Calif.) for 1 min. All data points
were measured in three independent experiments. The sugar-uptake
rate was calculated as mmol sugar transported per hour per gram of
dry cell weight.
[0409] Intracellular accumulation of both D-xylose and L-arabinose
in EBY.VW4000 strains over-expressing STL12/XUT6, NCU00821, or XUT1
was also measured using HPLC. Cell cultures incubated with pentose
sugars for 30 min, 60 min, 120 min, and 24 hours were analyzed by
HPLC. The EBY.VW4000 strains over-expressing STL12/XUT6 or NCU00821
exhibited D-xylose uptake activity, whereas the strain
over-expressing XUT1 exhibited L-arabinose-uptake activity after a
24-hour incubation (FIGS. 33A-B).
[0410] The .sup.14C-labeled sugar uptake assay together with HPLC
analysis of intracellular sugar accumulations confirmed that among
the three most abundant monosaccharides in lignocellulosic
hydrolysates, D-glucose, D-xylose, and L-arabinose, STL12/XUT6 and
NCU00821 were responsible for D-xylose uptake and XUT1 was
responsible for L-arabinose uptake. Of note, most sugar
transporters studied in yeast for D-xylose uptake have higher
uptake activity towards D-glucose than towards D-xylose. Only
Trxlt1 from Trichoderma reesei after adaptive evolution exhibited
D-xylose-specific uptake activity (Saloheimo et al., 2007). This
data indicated that STL12/XUT6 from P. stipitis, NCU00821 from N.
crassa are the first two experimentally confirmed
naturally-occurring D-xylose-specific transporters introduced into
S. cerevisiae. Similarly, XUT1 from P. stipitis is the first
experimentally confirmed naturally-occurring L-arabinose-specific
transporter introduced into S. cerevisiae.
[0411] Kinetic Parameters
[0412] Using the .sup.14C-labeled sugar-uptake assay, kinetic
parameters of D-xylose transport through NCU00921, STL12/XUT6, and
XUT1 were determined. It was observed that under the assay
conditions, sugar uptake was within a linear range for the first 60
seconds (FIG. 34). The EBY.VW4000 strains over-expressing NCU00821,
STL12/XUT6, or XUT1 were incubated with labeled D-xylose or
L-arabinose for 40 or 60 seconds followed by addition of ice-cold
water to stop further sugar uptake. The reaction mixture was then
filtered and washed before measurement using a liquid scintillation
counter. The sugar-uptake rates and substrate concentrations were
fitted into a Michaelis-Menten equation by non-linear regression
using the Origin software (OriginLab Corporation, Northampton,
Mass.). The K.sub.m values for D-xylose uptake by the EBY.VW4000
strain harboring only NCU00821 or STL12/XUT6 were 175.7.+-.21.4 mM
and 56.0.+-.9.4 mM, respectively. The corresponding V.sub.max
values were 36.7.+-.2.9 and 41.5.+-.2.3 .mu.mol/h/gram DCW,
respectively. Similarly, the K.sub.m and V.sub.max values for
L-arabinose uptake by the EBY.VW4000 strain harboring XUT1 were
48.0.+-.13.2 mM and 5.6.+-.1.6 .mu.mol/h/gram DCW respectively.
[0413] In naturally-occurring D-xylose-assimilating fungal species,
both the high affinity D-xylose-proton symport system and the low
affinity D-xylose facilitated diffusion system are present. The
K.sub.m values of these two systems were determined to be 0.4-4 mM
for the symport system and around 140 mM for the facilitated
diffusion system (Leandro et al., 2006; Stambuk et al., 2003).
These values are close to the affinity of the D-glucose-uptake
system in S. cerevisiae, which has a K.sub.m of 1.5 mM for the high
affinity system and 20 mM for the low affinity system (Lang and
Cirillo 1987; Ramos et al., 1988). Unfortunately, the D-xylose
uptake affinity of wild-type S. cerevisiae is two orders of
magnitude lower than its affinity for D-glucose. The K.sub.m values
for D-xylose uptake in S. cerevisiae are only 190 mM for the high
affinity system and 1.5 M for the low affinity system (Kotter and
Ciriacy, 1993). The affinities of the newly discovered
D-xylose-specific transporters were lower when compared to the high
affinity D-xylose-uptake system in naturally occurring
D-xylose-assimilating yeasts. However, compared to the
D-xylose-uptake system in wild-type S. cerevisiae, NCU00821 and
STL12/XUT6 showed higher affinity towards D-xylose. In particular,
the K.sub.m of D-xylose uptake by STL12/XUT6 and XUT1 were only
one-fourth of the K.sub.m of xylose uptake by the transporter in
wild-type S. cerevisiae. The K.sub.m values of the
D-xylose-specific transporters were also close to those of Gxfl
(K.sub.m 88 mM) and Sut1 (K.sub.m 145 mM), which have been shown to
improve D-xylose fermentation in recombinant S. cerevisiae
(Runquist et al., 2009; Katahira et al., 2008). Thus, D-xylose
fermentation may be improved by introducing these newly discovered
D-xylose-specific transporters into S. cerevisiae.
[0414] Cellular Localization of Sugar Transporters
[0415] Sugar transporters are transmembrane proteins, and correct
folding and localization in the cell membrane is required for them
to be functional. Since no signal peptide was specifically added
when the putative pentose transporters were cloned, it was
important to ensure that the D-xylose-specific transporters were
correctly localized to the cell membrane. This was particularly
true for putative pentose transporters like NCU00821 cloned from
the filamentous fungi N. crassa, which exhibits a very different
physiology compared to S. cerevisiae. To study the cellular
localization of D-xylose-specific transporters in S. cerevisiae,
NCU00821, STL12/XUT6, and XUT1 were fused with Green Fluorescent
Protein (GFP) at the C-termini via linkers, and their localization
was monitored by fluorescent imaging.
[0416] The fusion proteins of the pentose-specific transporters
with the GFP at the C-terminus were constructed for the transporter
localization study. A GS-linker
(Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 70)) was
introduced between the transporter and the GFP. The GS-linker was
added to the N-terminus of the GFP open reading frame by a PCR
primer, resulting in a PCR product of GS-linker-GFP flanked with
nucleotide sequence homologous to the transporters at the 5'-end
and the HXT7 terminator at the 3'-end. Transporter genes were
amplified from the original pRS424-HXT7-transporter constructs to
generate DNA fragments of the transporters flanked with nucleotide
sequence identical to the HXT7 promoter at the 5'-end and
GS-linker-GFP at the 3'-end. These two fragments were then
co-transferred into the S. cerevisiae strain CEN.PK2-1C with
pRS424-HXT7-GFP digested with EcoRI (FIG. 30b). The resulting
transformation mixture was plated on SC-Trp plates supplemented
with 2% D-glucose.
[0417] Single colonies were inoculated into 2 mL of SC-Trp liquid
medium supplemented with 2% maltose. Cell culture was harvested at
the exponential phase. In a centrifuge tube, 250 .mu.L of cell
culture was stained with 10 .mu.L Hoechst 33342 nuclei dye
(Invitrogen, Carlsbad, Calif.) for 10 minutes at room temperature.
A small droplet of cell culture was then transferred onto a piece
of cover glass and fluorescent images were taken using an Andor
Technology Revolution System Spinning Disk Confocal Microscope
(Core facilities, Institute for Genomic Biology, University of
Illinois at Urbana-Champaign, Urbana, Ill.). Images were processed
using Imaris image analysis and visualization software (Bitplane,
Saint Paul, Minn.).
[0418] Yeast strains over-expressing pentose-specific transporters
showed a distinctive fluorescent halo at the cell periphery (FIG.
35). For NCU00821 and XUT1, almost all the GFP fluorescence
appeared in the cell membrane, while a large portion of
fluorescence in STL12/XUT6-over-expressing cells remained in the
cytoplasm. This could indicate inefficient export of the STL12/XUT6
transporter due to elevated expression of the membrane protein. It
was also noticed that not all the cells showed fluorescence,
indicating that expression of the transporter was not optimal.
Further improvements of transporter expression can be achieved
through altering the expression level and/or integrating the
transporter genes into the genome of recombinant S. cerevisiae.
Determination of the Type of Pentose Transporters
[0419] There are two types of sugar transporters in S. cerevisiae,
symporters and facilitators. For symporters, sugar uptake is
coupled to proton uptake. Sugar symporters usually exhibit high
affinity towards sugar. Meanwhile, sugar uptake through
facilitators is not coupled to proton transport, and facilitators
usually exhibit low sugar-uptake affinities (Leandro et al., 2006).
Symporter assays were performed for NCU00821, STL12/XUT6, and XUT1
expressed in the EBY.VW4000 strain.
[0420] To determine the type of transporters, pH change of the
EBY.VW4000 over-expressing pentose-specific transporters was
measured in un-buffered cell suspension containing D-xylose,
L-arabinose, or maltose using a Seven Multi pH meter equipped with
an USB communication module and Direct pH software (Mettler Toledo,
Columbus, Ohio). Plasmids encoding pentose-specific transporters
were transferred into EBY.VW4000 strain followed by plating on the
SC-Trp plates supplemented with 2% maltose. Single colonies were
inoculated in 2 mL SC-Trp medium supplemented with 2% maltose. Seed
culture was then used to inoculate a 400 mL culture in 2 L flasks.
The culture was harvested at OD.about.1 and washed twice with
ice-cold water. Cell pellets were re-suspended in 4 mL of water and
kept on ice before use. For the symporter assay, the pH electrode
was immersed in a water-jacketed beaker of 50 mL capacity kept at
25.degree. C. and provided with magnetic stirring. To the beaker,
23 mL of deionized water and 1 mL of cell suspension equilibrated
at 25.degree. C. was added. The pH was adjusted to 5, and a base
line was obtained. The pH change was recorded with addition of 1 mL
of 50% sugar solution at pH 5.
[0421] FIGS. 36A-C show pH changes in un-buffered cell suspension
after the addition of maltose. As was reported, pH in un-buffered
S. cerevisiae cell suspension went up with the addition of maltose.
One mL of 50% maltose solution was added to the un-buffered cell
suspension to ensure that the pH recording system was functional.
The pH elevations observed in all samples indicated that the pH
recording system could monitor transient pH changes in the
experimental setting.
[0422] No elevation of pH in un-buffered cell suspensions was
observed for any of the pentose-specific transporters, indicating
that pentose uptake through these transporters is not coupled with
proton transport (FIGS. 37A-F). Thus, NCU00821, STL12/XUT6, and
XUT1 were determined to be pentose facilitators.
[0423] This result was consistent with the fact that the kinetic
parameters of NCU00821 and STL12/XUT6 were similar to those of the
low affinity D-xylose facilitated diffusion system in
naturally-occurring D-xylose-assimilating yeasts. Despite the fact
that symporters have higher affinities towards D-xylose,
over-expression of symporters may not always facilitate sugar
utilization by D-xylose-assimilating strains due to the ATP
requirement to create the proton gradient. In fact, most of the
transporters shown to be beneficial for D-xylose fermentation are
facilitators (Runquist et al., 2009; Katahira et al., 2008).
Heterologous Over-Expression of D-Xylose-Specific Transporters
[0424] The over-expression of active heterologous D-xylose-specific
transporters in S. cerevisiae strains containing the D-xylose
utilization pathway was also investigated to determine whether
their over-expression could improve xylose utilization. Xylose
utilization was studied using a shake-flask under aerobic
conditions. Plasmids expressing the xylose transporters NCU00821,
NCU04963, XUT1, STL12/XUT6, and Hxt7 were introduced into strain
HZE63 (CEN.PK2 ura3::xylose utilization pathway). This strain had a
xylose utilization pathway integrated into the URA3 site onto the
chromosome. It was constructed using a plasmid from previous work
that contained xylulose reductase (XR) and xylitol dehydrogenase
(XDH) from N. crassa and xylulokinase (XKS) from P. stipitis. This
plasmid was digested with ApaI and transformed into yeast strain
CEN.PK2 to yield the strain HZE63.
[0425] The HZE63 strain transformed with the xylose
transporter-encoding plasmids was selected by plating on SC-Ura
plates supplemented with 2% glucose. The transformed strain was
pre-cultured in SC-Trp-Ura with 2% glucose and then inoculated into
SC-Trp-Ura supplemented with 0.5% or 5% of xylose to an initial
OD.sub.600=1.0. Cell cultures were grown in a 125 mL shake-flask
containing 50 mL of culture at 30.degree. C. and 250 rpm (FIGS.
38A-G).
[0426] Yeast plasmids of transformants were transformed into E.
coli DH5.alpha. cells. The plasmids were then isolated and checked
by diagnostic PCR and submitted for sequencing to confirm correct
construction. Plasmid maps can be found in FIG. 39.
[0427] Unfortunately, the advantage of pentose-specific transporter
over-expression could not be observed despite alteration of
expression strategies, cultivation conditions, and choice of the
D-xylose utilization pathway. There are several possible reasons.
Firstly, the over-expression of membrane proteins, such as sugar
transporters, could affect the integrity of the cell membrane and
consequently hamper cell growth (Wagner et al., 2006). It was
observed that transporter over-expression strains displayed a
slower growth rate even when D-glucose was used as a carbon source.
The final OD of 2-day cultures of strains carrying transporters
grown in glucose-containing SC-ura media was only 4, whereas the OD
of the negative control was around 6. Secondly, the D-xylose-uptake
activity of the wild-type S. cerevisiae through hexose transporters
is much higher than the D-xylose-uptake activity of a certain
D-xylose transporter over-expressed in a hexose transporter
knockout strain. The low sugar transport activity of newly
discovered D-xylose-specific transporters may make it hard to
observe the improvement of sugar uptake ability. Thirdly, even if
the introduction of new D-xylose-specific transporters could
improve the uptake of D-xylose into S. cerevisiae cells, the
benefit of D-xylose utilization can only be observed when the
D-xylose utilization pathway is efficient enough to make
sugar-uptake the limiting step. It was shown that the effect of
over-expression of sugar transporters depends on the strain
background and cultivation conditions (Runquist et al., 2010).
Examples 12-15 below describe the optimization of the xylose
utilization pathway in yeast.
[0428] Cloning of Additional Pentose-Specific Transporters
[0429] Orthologs of NCU00821, STL12/XUT6, and XUT1 were cloned and
tested for pentose uptake. Different fungal strains were cultivated
in rich media supplemented with glucose or pentoses. Total RNA was
isolated and reverse transcribed into cDNA. Polymerase chain
reaction (PCR) was used to amplify the putative transporter genes
directly from cDNA. However, because the regulation mechanism and
expression pattern were unknown for pentose transporters in fungal
species, cDNAs encoding the putative pentose transporters were not
always obtainable despite alteration of cultivation condition. In
this case, primers were designed according to the corresponding
cDNA sequences from GenBank and used to amplify the exons using
genomic DNA as a template. Overlap-extension PCR was then used to
assemble the exons into the full-length genes. The resulting PCR
products were cloned into the pRS424 shuttle vector containing a
HXT7 promoter and a HXT7 terminator using the DNA assembler method.
Yeast plasmids isolated from transformants were retransformed into
E. coli DH5.alpha., and isolated E. coli plasmids were first
checked by diagnostic PCR using the primers used to amplify the
original transporter genes. The entire open reading frames were
submitted for sequencing to confirm the correct construction of the
plasmids.
[0430] Most of the cloning work was carried out using the yeast
homologous recombination mediated DNA assembler method.
pRS424-HXT7-GFP plasmid was used for cloning of putative pentose
transporters. In this plasmid, the HXT7 promoter, the GFP gene
flanked with the EcoRI sites at both ends, and the HXT7 terminator
were assembled into the pRS424 shuttle vector (New England Biolabs)
linearized by ClaI and BamHI. PCR products of the putative pentose
transporters flanked with DNA fragments sharing sequence identity
to the HXT7 promoter and terminator were co-transferred into
CEN.PK2-1C with EcoRI digested pRS424-HXT7-GFP using the standard
lithium acetate method. The resulting transformation mixture was
plated on SC-Trp plates supplemented with 2% D-glucose.
Transformants were then tested for pentose transport activity.
[0431] The results are shown below in FIG. 40 and Table 16. Among
the eight putative pentose specific transporters [XP.sub.--960000
(NC52), CAG88709 (DH48), XP.sub.--457508 (DH61), XP.sub.--681669
(32-10), XP.sub.--001487429 (29-6), XP.sub.--001727326 (29-9),
XP.sub.--657854 (32-8), XP.sub.--720384 (29-4)], only NC52 enabled
cell growth on a glucose plate, which suggested that the other
seven transporters may be pentose-specific or inactive. Using the
HPLC-based pentose uptake assay, four xylose-specific transporters
were found, including XP.sub.--457508 (DH61), XP.sub.--001727326
(29-9), XP.sub.--720384 (29-4), and XP.sub.--681669 (32-10). In
addition, one arabinose-specific transporter, XP.sub.--657854
(32-8) was identified (FIG. 40; Top). Five additional putative
pentose specific transporters (XP.sub.--002488227, AB070824.1,
XP.sub.--001389300, XP.sub.--002488227, EEQ43601.1) were also
tested, none of which enabled cell growth in a glucose plate.
Further pentose uptake assays indicated that XP.sub.--002488227 and
AB070824.1 were xylose specific transporters (FIG. 40; Bottom). The
summary of these results are shown in Table 16D.
TABLE-US-00017 TABLE 16A Cloning of xylose-specific transporter
NCU00821 orthologs NCBI Reference Sequence Uptake Sequence Origin
Results* Assay Status XP_002488227 Talaromyces stipitatus Correct
Yes Cloned XP_001400900 Aspergillus niger Correct Yes Cloned
XP_001220481 Chaetomium globosum CBS148.51 No No Sequenced, one
intron XP_001912725 Podospora anserina No No OE-PCR, no PCR product
XP_660079 Aspergillus nidulans FGSCA4 Correct Yes Cloned AAL89823
Aspergillus niger Correct Yes Cloned XP_002382573 Aspergillus
flavus Wrong Yes Cloned NRRL3357 XP_459386 Debaryomyces hansenii No
No Genomic DNA, no CBS767 PCR product XP_001825132 Aspergillus
oryzae RIB40 Correct Yes Cloned XP_001389300 Aspergillus niger
Correct Yes Cloned *"Correct" = Sequence of clone matched sequence
in database(s); "Wrong" = Sequence of clone did not match sequence
in database(s); "No" = Results not available (work in progress)
TABLE-US-00018 TABLE 16B Cloning of xylose-specific transporter
STL12/XUT6 orthologs NCBI Reference Sequence Uptake Sequence Origin
Results* Assay Status XP_457508 Debaryomyces Correct No Cloned
(DH61) hansenii CBS767 XP_002551364 Candida tropicalis Wrong No No
MYA-3404 XP_001523322 Lodderomyces Wrong No No elongisporus NRRL
XP_720384 (29-4) Candida albicans Correct No Cloned SC5314
XP_456868 Debaryomyces Wrong No No hansenii CBS767 XP_001487429
Pichia Wrong No Cloned (29-6) guilliermondii ATCC 6260 XP_961039
Neurospora crassa Wrong No No CAG88709 (DH48) Debaryomyces Correct
No Cloned hansenii CBS767 XP_001727326 Aspergillus oryzae Correct
No Cloned (29-9) XP_001816757 Aspergillus oryzae Correct No Cloned
*"Correct" = Sequence of clone matched sequence in database(s);
"Wrong" = Sequence of clone did not match sequence in database(s);
"No" = Results not available (work in progress)
TABLE-US-00019 TABLE 16C Cloning of arabinose-specific transporter
XUT1 orthologs NCBI Reference Sequence Uptake Sequence Origin
Results* Assay Status XP_002545773 Candida tropicalis Correct Yes
Cloned MYA-3404 EEQ43601 Candida albicans Correct Yes Cloned WO-1
XP_001818631 Aspergillus oryzae No No No PCR RIB40 product
XP_002558275 Penicillium Wrong Yes Cloned chrysogenum Wisconsin
54-1255 XP_001390883 Aspergillus niger No No No PCR product
XP_750103 Aspergillus fumigatus Wrong No No Af293 XP_960000
Neurospora crassa Wrong No Cloned (NC52) OR74A XP_657854 (32-
Aspergillus nidulans Correct No Cloned 8) FGSC A4 XP_001825068
Aspergillus oryzae Correct No Cloned RIB40 XP_681669 (32-
Aspergillus nidulans Correct No Cloned 10) FGSC *"Correct" =
Sequence of clone matched sequence in database(s); "Wrong" =
Sequence of clone did not match sequence in database(s) (e.g.,
because of mutation in clone) "No" = Results not available (work in
progress)
TABLE-US-00020 TABLE 16D Listing of new xylose-specific
transporters and one arabinose-specific transporter. NCBI Reference
Xylose- Arabinose- Sequence Origin specific specific XP_457508
Debaryomyces hansenii Yes (DH61) CBS767 XP_001727326 Aspergillus
oryzae Yes (29-9) XP_720384 (29-4) Candida albicans SC5314 Yes
XP_681669 (32- Aspergillus nidulans FGSC Yes 10) A4 XP_657854
(32-8) Aspergillus nidulans FGSC Yes A4 XP_002488227 Talaromyces
stipitatus Yes AB070824.1 Aspergillus oryzae Yes
[0432] The orthologs with sequences inconsistent with the sequences
in databases (e.g., ones with mutations) will be re-cloned,
sequenced, expressed in yeast strains, and tested for sugar uptake
function. Similarly, the orthologs for which there is no sequencing
results will also be tested for transporter function.
[0433] Sequence alignments of the pentose transporter orthologs
were analyzed to identify conserved residues, which could have
potential roles in transporter function. Alignments of a sample of
xylose transporters (NCU0821, STL12/XUT6, XP.sub.--002488227.1, and
XP.sub.--002382573.1) and arabinose transporters (XUT1 and
EEQ43601.1) are shown in FIG. 41A-1 to A-2 and FIG. 41B-1 to B-2
respectively. Several residues are specifically conserved in xylose
transporters whereas others are specifically conserved in the
arabinose transporters. These residues may have critical roles in
transporting the specific pentose. An overall comparison of the
sequences of the xylose and arabinose transporters (FIG. 41C-1 to
C-3) shows that there are also residues that are conserved in both
types of pentose transporters, indicating functional roles in
uptake of pentoses in general.
[0434] Examples 12-15 relate to optimization of the xylose
utilization pathway in yeast.
Example 12
Engineering Pentose-Utilizing S. cerevisiae Strain
[0435] An efficient xylose metabolic pathway was reconstituted by
exploiting the concept of isoenzymes. Isoenzymes catalyze the same
chemical reaction with different kinetic or regulatory properties,
and are known to confer fine-tuned control of metabolic fluxes in
response to dynamic changes in the cytosolic environment. However,
no prior metabolic engineering approaches had employed isoenzymes
to increase fluxes of interest. This study demonstrated that
simultaneous expression of both wild-type and mutant xylulose
reductase (XR) isozymes could decrease xylitol accumulation and
increase the overall xylose fermentation rate.
[0436] Inspired by the prevalence of isoenzymes in living systems,
wild type XR and mutant XR (R276H) were co-expressed in S.
cerevisiae along with xylitol dehydrogenase (XDH) and xylulokinase
(XK) in order to construct a functional xylose metabolic pathway in
S. cerevisiae. The XR mutant had been reported to exhibit much
lower preference for NADPH over NADH whereas wild type XR showed
116 two-fold higher preference for NADPH over NADH (Watanabe et
al., 2007).
[0437] The xylose-metabolizing genes (wild-type XYL1, 2, and 3 and
mutant XYL1) from P. stipitis were PCR-amplified and placed under
the control of constitutive promoters (PGK1 and TDH3) to construct
expression cassettes. These integration cassettes were integrated
into the genome of the D452-2 strain.
[0438] Transformation of expression cassettes for constructing
xylose metabolic pathways was performed using the yeast
EZ-Transformation kit (BIO 101, Vista, Calif.). To select
transformants using an amino acid auxotrophic marker, yeast
synthetic complete (YSC) medium was used, which contained 6.7
g/liter yeast nitrogen base plus 20 g/liter glucose, 20 g/liter
agar, and CSM-Leu-Trp-Ura (BIO 101), which supplied appropriate
nucleotides and amino acids. Yeast strains were routinely
cultivated at 30.degree. C. in YP medium 234 (10 g/liter yeast
extract, 20 g/liter Bacto peptone) with 20 g/liter glucose.
[0439] The effect of S. cerevisiae strain background on
xylose-metabolizing efficiency was also tested by expressing
identical constructs containing optimized xylose utilization
pathway enzymes in several different yeast strains. The three
laboratory strains used were D452-2 (MAT.alpha., leu2, his3, ura3,
can1), L2612 (MAT.alpha., leu2-3, leu2-112, ura3-52, trp1-298,
can1, cyn1, gal+), and CEN.PK. Production of xylitol, acetate, and
ethanol was monitored together with use of xylose and OD.sub.600.
The results indicated that the D452-2 strain was the best amongst
the three tested strains (FIG. 42-44). S. cerevisiae D452-2 was
used for engineering of the xylose-metabolizing enzymes in yeast.
Strains and plasmids used in this study are described in Table
17.
TABLE-US-00021 TABLE 17 Strain and plasmids used in study Strain or
plasmid Description Reference Strain D452-2 MATa, leu2, his3, ura3,
can1 Hosaka et al., (1992) D801-130 D452-2 expressing
.beta.-glucosidase In this study (NCU00130) and cbt1 (NCU00801)
D809-130 D452-2 expressing .beta.-glucosidase In this study
(NCU00130) and NCU00809 D8114-130 D452-2 expressing
.beta.-glucosidase In this study (NCU00130) and cbt2 (NCU08114)
DA24 D452-2 expressing XYL1, mXYL1, In this study XYL2, and XKS1
(Isogenic of D452-2 except for leu2::TDH3P-XYL1-TDH3T,
ura3::URA3-PGKP-mXYL1-PGKT- PGKP-XYL2-PGKT, Ty3::neo-TDHP-
XKS1-TDHT) DA24-16 Evolved strain of DA24 in xylose In this study
containing media DA24-16BT3 DA24-16 expressing .beta.-glucosidase
In this study (NCU00130) in a multi-copy plasmid and cbt1
(NCU00801) though single- copy integration DA24-16BT-M DA24-16
expressing .beta.-glucosidase In this study (NCU00130) and cbt1
(NCU00801) in multi-copy plasmids Plasmid pRS425 LEU2, a multi copy
plasmid Christianson et al., (1992) pRS426 URA3, a multi copy
plasmid Christianson et al., (1992) pRS403 HIS3, an integrative
plasmid Sikorski et al., (1989) pRS405 URA3, an integrative plasmid
Sikorski et al., (1989) pRS425-.beta.- .beta.-glucosidase
(NCU00130) under the Submitted glucosidase control of PGK promoter
in pRS425 pRS426-cbt1 cbt1 under the control of PGK promoter
Submitted in pRS426 pRS426-cbt2 cbt2 under the control of PGK
promoter Submitted in pRS426 pRS426-NCU00809 NCU00809 under the
control of PGK Submitted promoter in pRS426 pRS403-cbt1 cbt1 under
the control of PGK promoter In this study in pRS403
[0440] The engineered xylose-fermenting S. cerevisiae strain (DA24)
consumed xylose and produced ethanol with negligible amounts of
xylitol accumulation. When 40 and 80 g/L of xylose were used as a
sole carbon source, the DA24 strain produced ethanol with
consistent yields (Y.sub.Ethanol/xylose=0.31.about.0.32 g/g) in
both shaker-flask and bioreactor fermentation experiments (FIGS.
45A-C). However, the DA24 strain consumed xylose slower than the
naturally existing xylose-fermenting yeast, P. stipitis. Xylose
fermentation capability of DA24 was further improved using an
evolutionary engineering approach (Sauer 2001). One of the strains
(DA24-16) isolated after repeated sub-cultures of the DA24 on
xylose-containing medium showed much faster xylose fermentation
rates as compared to the parental strain under various culture
conditions (Table 18).
Table 18 shows the comparison of fermentation parameters of the two
S. cerevisiae strains DA24 and DA24-16 under different sugar
conditions.
TABLE-US-00022 Sugar Produced consumption Carbon Ethanol rate Yield
Productivity source Strains (g/L) (g/L/h) (g/g) (g/L h) Xylose DA24
24 1.16 0.34 0.40 (80 g/L) DA24-16 28 1.32 0.35 0.47 Glucose DA24
34 1.45 0.39 0.74 (70 g/L) and DA24-16 45 1.78 0.42 0.96 xylose (40
g/L)
[0441] Interestingly, the DA24-16 strain consumed xylose as fast as
P. stipitis, the fastest xylose-fermenting yeast known. However,
ethanol yield by DA24-16 was slightly lower than that by P.
stipitis (FIGS. 46A-B).
[0442] A screen was set up using S. cerevisiae strain L2612
expressing the xylose-utilizing enzymes (strain YSX3) transformed
with a genomic library. Transformation was followed by serial
culture transfer in 40 g/L xylose under oxygen-limiting conditions
to enrich for strains that are efficient in utilizing xylose.
Fermentations were performed in 50 mL YPX media under
oxygen-limited conditions and 0.1% (50 .mu.L) of a fully grown cell
culture was transferred to the next serial culture when
OD.sub.600=10 was reached. After 10 serial cultures, cells were
spread with serial dilution on YPX (40 g/L) agar media. Through
fermentation experiments using 5 mL of YPX media, colonies were
screened for low xylitol and high ethanol formation. DNA sequencing
revealed that the two most efficient strains contained integrated
copies of XYL2, which was then cloned into a multi-copy plasmid
through homologous recombination and transformed into YSX3
cells.
[0443] The XYL2 gene was placed in integration vectors under the
control of promoters of different strength, e.g., TDHp or PGKp, and
transformed into YSX3 cells (FIG. 47). Studies were conducted to
monitor the effect of these plasmids on xylitol and ethanol
formation in the transformed yeast cells. The results indicated
that the YSX3 cells expressing higher levels of XYL2 (under the
PGKp) were more efficient at ethanol production and in addition,
produced lower amounts of xylitol (FIG. 48). When additional XYL3
was expressed in these cells (termed SR1 strain), the amount of
xylitol produced was further decreased in the resulting strain
SRu-23 (FIG. 49). Therefore, it appeared that XYL2 expression level
in engineered S. cerevisiae strains is a key factor for
implementing xylose fermentation, and when expression is under a
strong promoter, the strain has less xylitol accumulation as well
as high ethanol yield. Simultaneous over-expression of XYL2 and
XYL3 can further decrease the amount of xylitol accumulation.
However, when XYL1 was further over-expressed in a strain
over-expressing XYL2 and XYL3, there was considerable xylitol
accumulation and consequently decreased xylose fermentation (FIGS.
50-51). Therefore, it appeared that there was an optimal level of
XYL1 for efficient xylose fermentation.
[0444] Experiments were also carried out to test if over-expression
of endogenous GRE3 in S. cerevisiae expressing XYL2 and XYL3 could
facilitate xylose fermentation. For the construction of
pRS403-GRE3, GRE3 gene was amplified from S. cerevisiae D452-2 and
inserted into pR403 vector with TDH3 promoter and CYC terminator.
After linearization of pRS403-GRE3, it was integrated into the
genome of D452-2. The xylose-utilizing genes were introduced into
the yeast strain D452-2 (FIG. 52), and xylose fermentation
parameters were monitored. The results indicated that
over-expression of GRE3 was as effective as the over-expression of
XYL1 in ethanol production and xylitol accumulation, particularly
when cells were grown in 80 g/L of xylose at high OD inoculations
(FIGS. 53-54).
Example 13
Engineering LAD and XDH
[0445] L-arabinitol and xylitol accumulation, thought to be caused
by cofactor imbalance between NADPH-dependent XR and
NAD.sup.+-dependent XDH and LAD, has been regarded as a major
bottleneck during xylose fermentation in engineered S. cerevisiae
expressing the pentose-utilizing enzymes. While the imbalance
between XR and XDH has been corrected by engineering enzymes with
reversed cofactor preferences (Watanabe et al., 2007; Matsushika et
al., 2008; Bengtsson et al., 2009), this approach resulted in
reduced flux, as the modified enzymes had reduced specific
activities. The P. stipitis XR mutant had been reported to exhibit
much lower preference for NADPH over NADH whereas wild type psXR
showed two-fold higher preference for NADPH (Watanabe et al.,
2007).
[0446] In this study, similar studies were done on L-arabinitol
4-dehydrogenase (LAD) and XDH from N. crassa to alter cofactor
specificity and hence improve xylose fermentation in engineered S.
cerevisiae.
[0447] Identification of Putative LAD-Encoding Genes
[0448] Methods of identifying putative LAD-encoding genes and of
cloning LAD-encoding and putative LAD-encoding genes are
described.
[0449] Identification of Putative LAD-Encoding Genes
[0450] From a protein BLAST search using ncLAD (EAA36547.1) as a
probe, two putative genes were identified in P. chrysogenum
(XP.sub.--002569286.1) and P. guilliermondii (EDK37120.2),
respectively. The amino acid sequence identities of these two
proteins with ncLAD were 71% and 46%, respectively.
[0451] Cloning LAD-Encoding and Putative LAD-Encoding Genes
[0452] A. niger (NRRL 326), P. guilliermondii (NRRL Y2075), and P.
chrysogenum (NRRL 807) were obtained from the United States
Department of Agriculture Agricultural Research Service Culture
Collection (Peoria, Ill.). T. longibrachiatum (T. reesei, YSM 768)
was obtained from the German Resource Centre for Biological
Material (DSMZ).
[0453] A. niger, T. longibrachiatum, P. chrysogenum, and P.
guilliermondii were grown in liquid media or on agar plates
containing 1% yeast extract, 2% peptone, and 2% L-arabinose. Cells
were frozen in liquid nitrogen for the isolation of total RNA or
genomic DNA. Reverse transcription-PCR (RT-PCR) was performed on
mRNAs isolated from T. longibrachiatum, P. chrysogenum, and P.
guilliermondii to obtain cDNA, and PCR was used to obtain the genes
encoding (putative) LADs. For A. niger, the putative LAD gene could
not be amplified from cDNA due to unknown reasons. Thus, overlap
extension-PCR (OE-PCR) was used to clone this intron-containing
gene from the isolated genomic DNA. Note that all primer sequences
used to clone these genes are listed in Table 19.
TABLE-US-00023 TABLE 19 Primers used for the cloning of wild type
LADs. Restriction enzyme sites are in bold and italicized.
Restriction Enzyme Primer Sequence anLAD NdeI Fwd-fragment1.sup.a
5'-GACATCGATGA GCTACCGCAAC-3' SEQ ID NO: 71 Rev-fragment1
5'-GTGCACGTCGGACCCGCAGATTCC-3' SEQ ID NO: 72 BamHI
Fwd-fragment2.sup.b 5'-GGAATCTGCGGGTCCGACGTGCAC-3' SEQ ID NO: 73
Rev-fragment2 5'-CAGAAGATTTAA TGAACGTAGA-3' SEQ ID NO: 74 tlLAD
NdeI Fwd 5'-GACATCAGTGA TCGCCTTCC-3' SEQ ID NO: 75 BamHI Rev
5'-CCTGGATTGA TGAACGTATA-3' SEQ ID NO: 76 pcLAD NdeI For
5'-GACATCGATGA GCTTCCGCAAC-3' SEQ ID NO: 77 EcoRI Rev
5'-CCAGAAGTATTGA TGAACGTAGA-3' SEQ ID NO: 78 pgLAD NdeI Fwd
5'-GACATCGATGA GCGACTCTGC-3' SEQ ID NO: 79 BamHI Rev
5'-GGATACAGAATGA TGAACGTAGA-3' SEQ ID NO: 80 .sup.a,bFragment 1 and
2 indicate the upstream and downstream exons flanking the intron.
.sup.cSequences in bold (italicized) indicate restriction enzyme
sites.
[0454] PCR products were subcloned into pET-28a vector and the
constructs were used to transform into two E. coli strains, DH5c
and BL21 (DE3), by electroporation for cloning and expression,
respectively. NdeI/BamHI restriction sites were used for the
subcloning of the predicted genes from A. niger, T.
longibrachiatum, and P. guilliermondii, and NdeI/EcoRI sites were
used for P. chrysogenum. The constructs encoded (putative) LADs as
N-terminal His.sub.6-tagged fusions. Plasmids were sequenced using
BIGDYE.TM. Terminator sequencing method and analyzed with
3730.times.L Genetic Analyzer (Applied Biosystems, Foster City,
Calif.) at the Biotechnology Center at the University of Illinois
at Urbana-Champaign (Urbana, Ill.).
[0455] Protein Expression and Purification
[0456] Genes encoding pcLAD (XP.sub.--002569286.1), pgLAD
(EDK37120.2), anLAD (CAH69383.1), and tlLAD (AAL08944.1) were
cloned into the pET-28a vector and expressed in E. coli BL21 (DE3).
E. coli BL21 (DE3) containing the LAD genes were grown overnight at
30.degree. C. on a rotary shaker at 250 rpm. Overnight culture (50
.mu.L) was used to inoculate a fresh culture (5 mL), which was
grown at 30.degree. C. with shaking at 250 rpm until the optical
density at 600 nm (OD.sub.600) reached 0.6-1.0. The cultures were
then induced with 0.3 mM IPTG at 30.degree. C. for 3-4 hrs or at
18.degree. C. for 20 hrs.
[0457] The induced cells (1 mL) were lysed by re-suspending them in
1 mL of 50 mM potassium phosphate buffer (pH 7.0) with 1 mg/mL
lysozyme and shaking at 30.degree. C. and 250 rpm for 30 min. Cells
were kept at -80.degree. C. overnight and thawed at room
temperature. The resulting cell lysates were centrifuged at 13,200
rpm for 15 min, and the supernatant and precipitate were analyzed
for protein expression by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE).
[0458] For protein purification, the induced cells (400 mL) were
treated with 15 mL of Buffer A (20 mM Tris, 0.5 M NaCl, 20%
glycerol, pH 7.6) with 1 mg/mL lysozyme and shaken at 30.degree. C.
and 250 rpm for 30 min. After a freeze-thaw cycle, the resulting
product was further lysed by sonication followed by centrifugation
for 20 min at 12,000 rpm to remove cell debris. The supernatants
were applied to a column packed with Co.sup.2+-immobilized metal
affinity chromatography resin to purify His.sub.6-tagged proteins
following the manufacturer's instructions. The purified proteins
were desalted by ultrafiltration (Amicon Ultra, Millipore,
Billerica, Mass.) and washed with HEPES buffer (pH 7.0) containing
150 mM NaCl and 15% glycerol and kept at -20.degree. C. Protein
concentrations were determined by the Bradford method (Bradford
1976) according to the manufacturer's protocol.
[0459] Characterization of LAD Proteins
[0460] The steady-state kinetics, molecular weight, quaternary
structure, temperature dependence, pH dependence, L-arabinitol
dehydrogenase activity, and metal content of LAD enzymes were
analyzed.
[0461] L-Arabinitol Dehydrogenase Activity
[0462] Lysates were prepared from host cells expressing LAD from P.
chrysogenum, P. guilliermondii, A. niger, and T. longibrachiatum.
Ten microliters of cell lysate were used for an activity assay with
200 mM L-arabinitol and 2 mM NAD.sup.+ as the substrates in 50 mM
potassium phosphate buffer (pH 7.0). NADH production was monitored
by measuring absorbance at 340 nm (E=6.22 mM.sup.-1 cm.sup.-1)
using a Cary 300 Bio UV-vis spectrophotometer (Varian, Cary,
N.C.).
[0463] Steady-State Kinetics
[0464] Kinetic parameters of different LAD enzymes were determined.
Initial rates were determined by measuring the absorbance change at
340 nm using a UV-vis spectrophotomer at room temperature in 50 mM
potassium phosphate buffer (pH 7.0). Initial rates were measured at
various concentrations of the substrate (L-arabinitol) and
cofactors (NAD.sup.+/NADP.sup.+) (5 to 320 mM for L-arabinitol, 0.5
to 3.2 mM for cofactors). Enzyme kinetics for the substrate and
cofactors were analyzed using Michaelis-Menten kinetics, and
kinetic parameters were determined by fitting data to the
Lineweaver-Burk plot. The parameters for substrate were determined
by measuring initial rates at saturated cofactor concentrations
(3.2 mM) and those for cofactors were determined at saturated
substrate concentrations (320 mM). Assays were performed in
triplicate.
[0465] The cloned LADs showed different binding affinities and
catalytic activities for L-arabinitol: K.sub.m differed by two fold
and k.sub.cat by about three fold amongst the LADs. For
L-arabinitol, the K.sub.m values of anLAD, tlLAD, and pcLAD were
25.+-.1, 18.+-.1, and 37.+-.2 mM, and the k.sub.cat values were
507.+-.22, 346.+-.41, and 1085.+-.71 min.sup.-1, respectively
(Table 20). The tlLAD enzyme had the lowest K.sub.m while pcLAD
showed the highest catalytic activity (k.sub.cat) and efficiency
(k.sub.cat/K.sub.m) despite having the highest K.sub.m (Table 20).
For cofactor NAD.sup.+ kinetics, the cloned LADs showed K.sub.m
values in the range of 0.2-0.3 mM and catalytic efficiencies in the
range of 2526 to 3460 mM.sup.-1min.sup.-1 (Table 21). All cloned
LADs showed minimal activities toward NADP.sup.+ (Tables 20, 21).
The initial rates were not saturated at highest substrate and
cofactor concentration (320 mM for L-arabinitol and 3.2 mM for
NADP.sup.+) due to the large K.sub.m. Therefore, only the catalytic
efficiency of the enzyme was determined using 0.1 or 0.2 mM for
NADP and 10 or 20 mM for L-arabinitol (K.sub.m>>[5]) (Tables
20, 21).
TABLE-US-00024 TABLE 20 Kinetic parameters of LADs for L-arabinitol
at saturated cofactor concentrations. Specific activity (U/mg
K.sub.m k.sub.cat k.sub.cat/K.sub.m protein) (mM) (min.sup.-1)
(mM.sup.-1 min.sup.-1) anLAD NAD.sup.+ .sup. 11.7 .+-. 0.3.sup.a 25
.+-. 1 507 .+-. 22 20.0 .+-. 0.8 NADP.sup.+ --.sup.b -- -- 0.04
.+-. 0.01 tlLAD NAD.sup.+ 8.7 .+-. 0.1 18 .+-. 1 346 .+-. 41 19.0
.+-. 0.8 NADP.sup.+ -- -- -- 0.13 .+-. 0.02 pcLAD NAD.sup.+ 25.3
.+-. 1.4 37 .+-. 2 1085 .+-. 71 29 .+-. 1 NADP.sup.+ -- -- -- 0.04
.+-. 0.02 .sup.aError indicates standard deviation from the mean, n
= 3 .sup.bDash indicates not determined due to high K.sub.m for
indicated cofactor
TABLE-US-00025 TABLE 21 Kinetic parameters of LADs for NAD.sup.+
and NADP.sup.+ at saturated L-arabinitol concentration. K.sub.m
k.sub.cat k.sub.cat/K.sub.m (mM) (min.sup.-1) (mM.sup.-1
min.sup.-1) anLAD NAD.sup.+ .sup. 0.20 .+-. 0.01.sup.a 494 .+-. 11
2526 .+-. 83 NADP.sup.+ --.sup.b -- 20 .+-. 9 tlLAD NAD.sup.+ 0.2
.+-. 0.1 436 .+-. 96 2689 .+-. 646 NADP.sup.+ -- -- 17 .+-. 9 pcLAD
NAD.sup.+ 0.3 .+-. 0.1 1039 .+-. 165 3460 .+-. 505 NADP.sup.+ -- --
15 .+-. 4 .sup.aError indicates standard deviation from the mean, n
= 3 .sup.bDash indicates not determined due to high K.sub.m for
indicated cofactor
[0466] Molecular Weight and Quaternary Structure
[0467] Calculated molecular weights of the subunits of the four
proteins were 43 kDa (anLAD), 41 kDa (tlLAD), 42 kDa (pcLAD), and
42 kDa (pgLAD). The molecular weights of the proteins were
determined using a Bio-Sil SEC-250 column (300.times.7.8 mm,
Bio-Rad, Hercules, Calif.) on a Shimadzu HPLC system (Shimadzu,
Kyoto, Japan). The mobile phase consisted of 50 mM
Na.sub.2HPO.sub.4, 50 mM NaH.sub.2PO.sub.4, 150 mM NaCl, and 10 mM
NaN.sub.3 (pH 6.8) and the flow rate was 1.0 mL/min. The molecular
weights were calculated by comparing the retention times with those
of protein molecular weight standard.
[0468] The quaternary structures were determined based on the
molecular weights observed by HPLC and the molecular weights of
monomeric subunits which were determined by SDS-PAGE analysis.
Molecular weights of an-, tl-, and pcLAD were determined to be 178,
194, and 173 kDa, respectively. Comparing to the molecular weights
of the subunits determined by SDS-PAGE, results suggested that the
LADs were non-covalently linked tetramers in their native
forms.
[0469] Temperature and pH Dependence
[0470] The optimal temperatures of the proteins were determined by
assaying enzyme activities at temperatures ranging from 10 to
70.degree. C. Thermal inactivation was determined by measuring
enzyme activity after various incubation times at 50.degree. C. in
phosphate buffer. Enzyme activity was measured with 2 mM NAD.sup.+
and 200 mM L-arabinitol. Half-life of enzyme activity was
determined using a first-order exponential decay function.
Temperature was controlled by a Cary temperature controller
connected to the UV-vis spectrophotometer (Varian, Cary, N.C.).
pH-dependent enzyme activity was determined by measuring activity
at pH between 5.0 and 11.0 at saturated concentrations of NAD.sup.+
(2 mM) and L-arabinitol (200 mM) in a universal buffer (50 mM
morpholineethanesulfonic acid/50 mM Tris/50 mM glycine) (Ellis and
Morrison 1982).
[0471] The optimal temperatures of anLAD and pcLAD were between 40
and 50.degree. C., whereas tlLAD showed higher optimal temperature
between 55 and 65.degree. C. (FIG. 55a). Catalytic activities of
the LADs exponentially decreased with the length of incubation time
at 50.degree. C. and were almost completely deactivated after 100
min (FIG. 55b). tlLAD was the most thermally stable with a
half-life of 20 min at 50.degree. C., and anLAD was least stable
with a half-life of less than 5 min at 50.degree. C. All
characterized LADs showed activity in the pH range of 7 to 11 with
maximum activity around pH 9.4 (FIG. 55c). In the pH range outside
of 9 to 10, activity was significantly reduced and approximately
20% of activity remained at pH 7.0 (FIG. 55c). No activity was
detected at or below pH 5.0.
[0472] Metal Analysis
[0473] Duplicate samples for metal analysis were prepared in
phosphate buffered saline (PBS) by buffer exchange and
lyophilization. Each sample contained 1-2 mg of protein in 1 mL
buffer solution. The identity and content of the metal were
analyzed by inductively coupled plasma atomic emission spectrometry
(OES Optima 2000 DV, Perkin Elmer, Boston, Mass.) in the
Microanalytical Laboratory at the University of Illinois at
Urbana-Champaign (Urbana, Ill.).
[0474] Measured weight percentages of Zn.sup.2+ were close to those
calculated based on the 1:1 molar ratio (Table 22).
TABLE-US-00026 TABLE 22 Calculated and measured Zn.sup.2+ contents.
Calculated Weight.sup.a (%) Measured weight (%) anLAD 0.027 .sup.
0.027 .+-. 0.003.sup.b tlLAD 0.047 0.048 .+-. 0.003 pcLAD 0.048
0.061 .+-. 0.013 .sup.aCalculated molecular weights were determined
based on the buffer composition, protein concentration, and 1:1
molar ratio of LAD monomer subunit and Zn.sup.2+. Buffer solution
(1 L) contained NaCl (8 g), KCl (0.2 g), Na.sub.2HPO.sub.4 (1.44
g), and KH.sub.2PO.sub.4 (0.24 g). .sup.bAll samples were analyzed
in duplicate and errors were standard deviations.
[0475] Engineering of LAD Enzymes with Altered Cofactor
Specificity
[0476] Methods of altering the cofactor specificity of LADs were
determined, and mutated LADs were analyzed for altered cofactor
specificity and other characteristics.
[0477] Development of LADs with Altered Cofactor Specificity
[0478] Site-directed mutagenesis was performed to alter the
cofactor specificity of anLAD, tlLAD, and pcLAD from NAD.sup.+ to
NADP.sup.+. Amino acid numbers 224, 225, and 362 of naturally
occurring tlLAD were substituted with serine, arginine, and
threonine, respectively, to generate the tlLAD with altered
cofactor specificity. The amino acid sequences of cloned anLAD and
pcLAD were aligned with the T. longibrachiatum LAD (tlLAD)
sequence, and the amino acids that correspond to tlLAD amino acid
numbers 224, 225, and 362 were mutated. For all of the LADs with
altered cofactor specificity, two amino acid residues within the
.beta.-.alpha.-.beta. motif of the coenzyme binding domain were
replaced with serine and arginine, respectively: D213 and I214 for
anLAD, D224 and 1225 for tlLAD, and D212 and I1213 for pcLAD
(Korkhin et al., 1998; Pauly et al., 2003; Watanabe et al., 2005),
and the third mutation was introduced at A359 for anLAD, A362 for
tlLAD, and 5358 for pcLAD and replaced with threonine (For primer
sequences, see Table 23). Megaprimer PCR method was used to
introduce site-specific mutations using wild type LAD constructs as
the templates (Sarkar and Sommer 1990). Correct mutations were
confirmed by DNA sequence analysis.
TABLE-US-00027 TABLE 23 Primers used for site directed mutagenesis
by the megaprimer PCR method..sup.a Fwd-T7-pro
5'-TAATACGACTCACTATAGGG-3' SEQ ID NO: 81 Rev-T7-term
5'-GCTAGTTATTGCTCAGCGG-3' SEQ ID NO: 82 anLAD Fwd-D213S/I214R
5'-CCTATCGTCATTACCTCACGT.sup.bGACGAGGGGCGGCTG-3' SEQ ID NO: 83
Rev-D213S/I214R 5'-CAGCCGCCCCTCGTCACGTGAGGTAATGACGATAGG-3' SEQ ID
NO: 84 Fwd-A359T 5'-CCT TCGAAACGGCTACAAACCCCAAGACG-3 SEQ ID NO: 85
tlLAD Fwd-D214S/I215R 5'-GCTTGTCATCACATCACGTTCAGAGAGCCGTCTG-3' SEQ
ID NO: 86 Rev-D214S/I215R 5'-CAGACGGCTCTCTGAACGTGATGTGATGACAAGC-3'
SEQ ID NO: 87 Fwd-S362T 5'-GCATTTGAGACGTCAACAGATCCCAAGAGC-3' SEQ ID
NO: 88 pcLAD Fwd-D212S/I213R
5'-CCTATTGTCATCACTTCACGTGACGAGGGCCGCTTG-3' SEQ ID NO: 89
Rev-D212S/I213R 5'-CAAGCGGCCCTCGTCACGTGAAGTGATGACAATAGG-3' SEQ ID
NO: 90 Fwd-S358T 5'-CCTTTGAGACTGCCACAAACCCTAAGACCGGTG-3' SEQ ID NO:
91 .sup.aTo create mutant LADs, fragments 1 and 2 were amplified
using Fwd-T7-pro and Rev-D213S/I214R and Fwd-A359T and Rev-T7-term
primers, respectively. Fragment 3 was amplified using
Fwd-D123S/I214R and fragment 2 (Rev megaprimer). Full mutant genes
were amplified by overlap extension of fragment 1 and 3. Template
DNA was pET-28a plasmid. .sup.bSequences underlined were the
mutation sites.
[0479] Kinetic Analysis of LADs with Altered Cofactor
Specificity
[0480] In this example, "tlLAD mutant" is defined as tlLAD with the
mutations D224S/I225R/A362T; "anLAD mutant" is defined as anLAD
with the mutations D213S/1214R/A359T; and "pcLAD mutant" is defined
as pcLAD with the mutations D212S/1213R/S358T. The tlLAD mutant
showed significantly altered cofactor specificity from NAD.sup.+ to
NADP.sup.+. It also demonstrated the highest catalytic activity.
The K.sub.m and k.sub.cat of the tlLAD mutant for L-arabinitol with
NADP.sup.+ were 46.+-.4 mM and 170.+-.9 min.sup.-1, respectively
(Table 24). In all assays including the tlLAD mutant with saturated
NAD.sup.+, a plateau of reaction rate was not observed in the
tested concentration range, so catalytic efficiencies were
determined at 0.8 mM for NAD.sup.+ and 80 mM for L-arabinitol
(Tables 24, 25). For cofactors, anLAD and tlLAD mutants showed
significantly higher preference for NADP.sup.+ over NAD.sup.+
(Table 25). The K.sub.m values of the anLAD and tlLAD mutants were
0.46.+-.0.09 and 0.10.+-.0.01 mM, and the k.sub.cat values were
55.7.+-.6.4 and 90.5.+-.9.2 min.sup.-1, respectively (Table 25) The
catalytic efficiencies of anLAD and tlLAD mutants were 130.+-.32
and 934.+-.72 mM.sup.-1min.sup.-1, and the ratios of the catalytic
efficiencies with NADP.sup.+ to NAD.sup.+ were 100 and 161,
respectively. For the tlLAD mutant, the ratio of catalytic
efficiency for NADP.sup.+ to NAD.sup.+ was increased by
2.5.times.10.sup.4 fold (Tables 21, 25). The pcLAD mutant showed no
activity with NAD.sup.+.
Table 24 shows kinetic parameters of LAD mutants for L-arabinitol
at saturated cofactor concentrations.
TABLE-US-00028 Specific activity K.sub.m k.sub.cat
k.sub.cat/K.sub.m (U/mg protein) (mM) (min.sup.-1) (mM.sup.-1
min.sup.-1) anLAD NAD.sup.+ --.sup.a -- -- .sup. 0.010 .+-.
0.002.sup.b mutant NADP.sup.+ -- -- -- 0.45 .+-. 0.20 tlLAD
NAD.sup.+ -- -- -- 0.050 .+-. 0.007 mutant NADP.sup.+ 3.9 .+-. 0.2
46 .+-. 4 170 .+-. 9 3.7 .+-. 0.2 pcLAD NAD.sup.+ -- -- -- --
mutant NADP.sup.+ -- -- -- 0.02 .+-. 0.02 .sup.aDash indicates not
determined due to high K.sub.m for indicated cofactor .sup.bError
indicates standard deviation from the mean, n = 3
Table 25 shows kinetic parameters of LAD mutants for NAD.sup.+ and
NADP.sup.+ at saturated L-arabinitol concentration.
TABLE-US-00029 K.sub.m k.sub.cat k.sub.cat/K.sub.m (mM)
(min.sup.-1) (mM.sup.-1 min.sup.-1) anLAD mutant NAD.sup.+ --.sup.a
-- .sup. 1.3 .+-. 0.3.sup.b NADP.sup.+ 0.46 .+-. 0.09 55.7 .+-. 6.4
130 .+-. 32 tlLAD mutant NAD.sup.+ -- -- 5.8 .+-. 0.8 NADP.sup.+
0.097 .+-. 0.011 90.5 .+-. 9.2 934 .+-. 72 pcLAD mutant NAD.sup.+
-- -- -- NADP.sup.+ -- -- 3.6 .+-. 1.0 .sup.aDash indicates not
determined due to high K.sub.m for indicated cofactor .sup.bError
indicates standard deviation from the mean, n = 3
Engineering of N. crassa XDH (ncXDH) with Altered Cofactor
Specificity
[0481] Cloning and Characterization of Putative ncXDH
[0482] A putative N. crassa xylitol dehydrogenase (ncXDH) sequence
was found using a protein BLAST search on the National Center for
Biotechnology Information website (webpage ncbi.nlm.nih.gov) using
the P. stipitis xylitol dehydrogenase (psXDH) enzyme as a query
sequence. The two enzymes were aligned fully using a ClustalW
algorithm and found to share 44% identity and 60% similarity (FIG.
56). The whole-genome sequence of Neurospora crassa has been
published (Galagan et al., 2003) and it was utilized to design
primers for cloning of the putative xylitol dehydrogenase (XDH)
gene.
[0483] RT-PCR performed on total RNA isolated from D-xylose-induced
N. crassa 10333 showed the expected size of gene product
(.about.1.1 kb). The RT-PCR product was cloned into the pET-28a
vector using NdeI and SacI restriction sites and was transformed
into E. coli BL21 (DE3). This construct (pET-28a ncXDH) expressed
ncXDH as an N-terminal His6-tagged fusion with a thrombin cleavage
site. Cell lysates of IPTG-induced cultures of these cells were
prepared, analyzed by SDS-PAGE, and assayed for XDH activities. The
XDH was then purified by immobilized metal ion affinity
chromatography (IMAC) using Talon.RTM. Co2+Superflow resin
(Clontech, Mountain View, Calif.) according to manufacturer's
protocol. The purified protein was desalted by ultrafiltration with
several washes of 50 mM
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer
(pH 7.25)+15% glycerol and stored frozen at -80.degree. C. Protein
concentrations were determined by the Bradford method (Bradford
1976).
[0484] ncXDH is a strictly NADtpreferring enzyme. ncXDH also
displays high stability (half-life of -200 min at 50.degree. C.)
and expression. Previous work by Watanabe et al. (2005b) was aimed
at reversing the cofactor specificity of psXDH.
[0485] Development of ncXDH with Altered Cofactor Specificity
[0486] Through sequence alignment, residues D204, 1205, and V206 of
ncXDH were targeted for site-directed mutagenesis to alanine,
arginine, and serine, respectively, to create ncXDH-ARS. Table 26
shows that ncXDH-ARS has completely reversed cofactor specificity,
now preferring NADP.sup.+. The affinity for substrate xylitol did
not suffer very much from the affinity-change for the
co-factor.
Table 26 shows kinetic parameters for N. crassa and P. stipitis XDH
and XDH-ARS with nicotinamide cofactors NAD.sup.+ and NADP.sup.+ at
saturated xylitol concentrations.
TABLE-US-00030 NAD.sup.+ NADP.sup.+ k.sub.cat/K.sub.m
k.sub.cat/K.sub.m k.sub.cat K.sub.m (mM.sup.-1 k.sub.cat K.sub.m
(mM.sup.-1 Enzyme (min.sup.-1) (mM) min.sup.-1) (min.sup.-1) (mM)
min.sup.-1) Source ncXDH-wt 2160 0.127 17000 --a ~5.6 ~68 This work
ncXDH- --a ~3.5 ~165 2080 0.325 6400 This work ARS psXDH 1050 0.381
2760 110 170 0.65 Watanabe et al. (2005b) psXDH- 240 1.3 181 2500
0.897 2790 Watanabe et ARS al. (2005b) aNot determined, cofactor
saturation not reached. All assays were performed at 25.degree. C.
in 50 mM Tris, pH 8.0.
[0487] Kinetic Analysis of ncXDH Mutant
[0488] The mutant ncXDH had a dramatic reversal of cofactor
specificity. The K.sub.m of the mutant ncXDH for NADP.sup.+ was
only about 2.5-fold higher than the K.sub.m of wild-type ncXDH for
NAD.sup.+ whereas the k, values were similar (Table 27).
Table 27 shows kinetic parameters of ncXDH mutants for substrate
xylitol.
TABLE-US-00031 k.sub.cat K.sub.m k.sub.cat/K.sub.m Enzyme
(min.sup.-1) (mM) (mM.sup.-1 min.sup.-1) ncXDH-wt 2170 .+-. 135 6.6
.+-. 2.0 330 ncXDH-ARS 2090 .+-. 35 4.3 .+-. 0.3 490 a Not
determined, cofactor saturation not reached. All assays were
performed at 25.degree. C. in 50 mM Tris, pH 8.0. All enzymes were
purified and characterized with N-His.sub.6-tag
[0489] As shown in FIG. 57, XDH activity exhibits a higher
tolerance to more acidic conditions with activity extending down to
pH 4.0, whereas LAD activity is abolished at pH 5.0 in the in vitro
activity assay.
Example 14
Expression of Xylose Isomerase from Bacteroides stercoris in S.
cerevisiae
[0490] Bacterial xylose isomerase (XI) is involved in converting
xylose into xylulose. Recently, three successful cases of
expressing active XI from two species of anaerobic fungi (Piromyces
sp. and Orpinomyces sp.) and from the anaerobic bacteria
(Clostridium phytofermentans) have been reported. A fungal XYLA
gene from Piromyces sp. E2 was functionally expressed in S.
cerevisiae and a maximum 1.1 U/mg-protein of XI activity was
obtained at 30.degree. C. (Kuyper et al., 2003). The second fungal
XYLA gene from Orpinomyces, which has 94% identity with that from
Piromyces sp., was also functionally expressed in S. cerevisiae
(Madhavan et al., 2009). Recently, the first prokaryotic xylA gene
from Clostridium phytofermentans was functionally expressed in S.
cerevisiae (Brat et al., 2009).
[0491] The isomerase gene xylA from the anaerobic bacteria
Bacteroides stercoris (BtXI) shares high sequence identity with the
isomerase gene from Piromyces sp. (82%). BtXI was cloned into the
pRS424TEF vector and transformed into the S. cerevisiae L2612
strain. The gene was also integrated into the S. cerevisiae D452-2
strain by using the pRS403TEF vector. Ethanol production was
observed in both strains expressing BtXI (5 g/L in L2612 and 7.8
g/L in D452-2) (FIG. 58-59). However, rates of production were
relatively low compared to that of engineered strains expressing
the XYL genes.
[0492] The low ethanol production could be attributed to the
inhibitory effect of any accumulated xylitol (formed from xylose by
endogenous yeast aldose reductase). To decrease xylitol
accumulation, XDH and XK were expressed in BTX1-expressing yeast
strain (DBtXI). The resulting strain had slightly improved ethanol
yield and decreased xylitol production (FIG. 60). Co-expression of
these two XYL genes in DBtXI resulted in ethanol production even
under aerobic conditions.
Example 15
Over-Expression of Enzymes in Pentose Phosphate Pathway (PPP)
[0493] The PPP enzymes glucose-6-phosphate dehydrogenase (ZWF1),
6-phosphogluconate dehydrogenase (GDN1), transaldolase (TALI), and
transketolase (TKT1) from P. stipitis were cloned into an
integration vector (pRS406) under the control of a strong promoter
(P.sub.GP). The plasmid was linearized by the enzyme StuI and
integrated into the chromosome of S. cerevisiae.
[0494] However, to get the beneficial effects of over-expressing
the PPP enzymes, there also had to be over-expression of XYL3 (XK)
(FIG. 61). Expression of XYL3 and the PPP enzymes also improved
ethanol production in YP-xylulose media.
Example 16
Expression of Aldose-I-Epimerase
[0495] Hydrolysis of cellobiose by .beta.-glucosidase releases
.beta.-D-glucose. However, yeast hexokinases prefer (or exclusively
use) .alpha.-D-glucose, and the rate of mutaroation of
.beta.-D-glucose to .alpha.-D-glucose could effectively slow down
metabolic rate. One way of enhancing the conversion was to
over-express the predicted aldose-1-epimerase NCU09705. This
hypothesis was tested by over-expressing NCU09705 homologs: galM in
E. coli; GAL10, YHR210C, and YNR071C in S. cerevisiae; and GAL 10
in P. stipitis. The strains were then tested for cellobiose
consumption and ethanol production (FIGS. 62A-B). The results
indicated that over-expression of the homologs in S. cerevisiae
caused a slight increase in cellobiose consumption and ethanol
production.
Example 17
Co-Fermentation of Xylose and Cellobiose
[0496] In this example a new strategy was used to overcome glucose
repression in which a dimer of glucose, cellobiose, was
co-fermented with xylose (a pentose). Cellobiose is an intermediate
product from enzymatic hydrolysis of cellulose, which is further
converted to glucose by .beta.-glucosidases in the cocktail of
cellulases including exocellulases, endocellulases, and
.beta.-glucosidases, whereas pentose sugars are the products of
dilute acid hydrolysis of hemicellulose. Wild type S. cerevisiae
cannot assimilate cellobiose because it lacks both a cellobiose
transporter and a .beta.-glucosidase capable of hydrolyzing
cellobiose into glucose. Hence, the newly discovered cellodextrin
transporter genes described in Example 9 and a .beta.-glucosidase
gene from N. crassa were co-expressed in S. cerevisiae and a
mixture of xylose and cellobiose was used as carbon source (FIGS.
63A-C). Similar approaches have employed either secretion, or cell
surface display, of .beta.-glucosidases to allow cellobiose
fermentation by S. cerevisiae (van Rooyen et al., 2005; Skory et
al., 1996; Kotaka et al., 2008; Katahira et al., 2006). In those
cases, cellobiose was hydrolyzed into glucose extracellularly
before being transported by the endogenous hexose transport system
of S. cerevisiae. In contrast, in this strategy, cellobiose was
hydrolyzed intracellularly following transport.
[0497] In the conventional methods for mixed sugar fermentation in
S. cerevisiae, a mixture of glucose and pentose sugars derived from
lignocellulose is used. However, in this new strategy, a mixture of
cellobiose and pentose sugars was used. The cellobiose was
transported inside yeast cells via the heterologous cellodextrin
transporters while pentose sugars were transported inside yeast
cells by endogenous hexose transporters, thus removing the direct
competition between glucose and pentose sugars for the same
transporters, a phenomenon that is partly responsible for glucose
repression. Once inside yeast cells, cellobiose was converted to
glucose by .beta.-glucosidase and immediately consumed by yeast
cells, which resulted in low intracellular glucose concentration,
thereby further alleviating glucose repression.
[0498] The engineered xylose-utilizing yeast strain L2612 was used
as a host to co-express cellodextrin transporter and
.beta.-glucosidase genes. In this strain, the D-xylose utilization
pathway consisting of xylose reductase, xylitol dehydrogenase, and
xylulokinase from Pichia stipitis was integrated into the
chromosome. The cellodextrin transporters from Neurospora crassa
including NCU008011, NCU08114, and, NCU00809, and two
.beta.-glucosidase genes, one from Neurospora crassa and the other
from Aspergillus aculeatus, were evaluated.
[0499] S. cerevisiae L2612 (MAT.alpha., leu2-3, leu2-112, ura3-52,
trp1-298, can1, cyn1, gal+) was cultivated in synthetic dropout
media to maintain plasmids (0.17% of Difco yeast nitrogen base
without amino acids and ammonium sulfate, 0.5% of ammonium sulfate,
0.05% of amino acid dropout mix). YPA medium (1% yeast extract, 2%
peptone, 0.01% adenine hemisulfate) with 2% of sugar was used to
grow yeast strains.
[0500] To integrate the D-xylose utilization pathway consisting of
D-xylose reductase, xylitol dehydrogenase, and xylulokinase from
Pichia stipitis, the corresponding genes were PCR-amplified and
cloned into the pRS416 plasmid using the DNA assembler method (Shao
et al., 2009). BamHI and HindIII were used to remove the DNA
fragment encoding the D-xylose utilization pathway and then ligated
into the pRS406 plasmid digested by the same two restriction
enzymes. The resulting plasmid was then linearized by ApaI and
integrated into the URA3 locus on the chromosome of L2612.
[0501] The pRS425 plasmid (New England Biolabs, Ipswich, Mass.) was
used to co-express a cellodextrin transporter gene and a
.beta.-glucosidase gene. As shown in FIG. 64, the pRS425 plasmid
was digested by BamHI and ApaI. The PYK1 promoter and the ADH1
terminator were added to N-terminus and C-terminus of the
cellodextrin transporter, respectively, while the TEF1 promoter and
the PGK1 terminator were added to the N-terminus and C-terminus of
the .beta.-glucosidase, respectively. These DNA fragments were
assembled into the linearized pRS425 shuttle vector using the DNA
assembler method (Shao et al., 2009). Three cellodextrin
transporter genes NCU00801 (XM.sub.--958708), NCU08114
(XM.sub.--958780), and NCU00809 (XM.sub.--959259) from Neurospora
crassa and two .beta.-glucosidase genes NCU00130 (XM.sub.--951090)
from Neurospora crassa and BGL1 (D64088) from Aspergillus aculeatus
were used. There were six combinations in total, each with one
cellodextrin transporter gene and one .beta.-glucosidase gene.
[0502] Yeast plasmids were then transferred into E. coli
DH5.alpha., which were used for recombinant DNA manipulation. The
transformants were plated on Luria broth plates containing 00 mg/L
ampicillin. Single colonies of E. coli transformants were then
inoculated into the liquid Luria broth media (Fisher Scientific,
Pittsburgh, Pa.) and grown at 37.degree. C. and 250 rpm. Plasmids
were isolated from E. coli using the QIAprep Spin Miniprep Kit
(QIAGEN). These plasmids were transformed into the L2612 strain
individually to yield the following strains: SL01 (contained the
plasmid harboring the NCU00801 cellodextrin transporter gene and
the NCU00130.beta.-glucosidase gene from Neurospora crassa), SL02
(contained the plasmid harboring the NCU00809 cellodextrin
transporter gene and the NCU00130.beta.-glucosidase gene from
Neurospora crassa), SL03 (contained the plasmid harboring the
NCU08114 cellodextrin transporter gene and the NCU00130
.beta.-glucosidase gene from Neurospora crassa), SL04 (contained
the plasmid harboring the NCU00801 cellodextrin transporter gene
and the BGL1 gene from Aspergillus aculeatus), SL05 (contained the
plasmid harboring the NCU00809 cellodextrin transporter gene and
the BGL1 gene from Aspergillus aculeatus), and SL06 (contained the
plasmid harboring the NCU08114 cellodextrin transporter gene and
the BGL1 gene from Aspergillus aculeatus). The empty pRS425 plasmid
was transformed into the L2612 strain to yield the SL00 strain,
which was used as a negative control. Yeast transformation was
carried out using the standard lithium acetate method (Gietz et
al., 1995). The resulting transformation mixtures were plated on
SC-Ura-Leu medium supplemented with 2% D-glucose.
[0503] To confirm the proper construction of plasmids using the DNA
assembler method, plasmids were isolated from yeast cells using the
Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research, Orange,
Calif.) and then transferred into E. coli DH5.alpha. cells. The
resulting cells were spread on LB plates containing 100 mg/L
ampicillin. Single E. coli colonies were inoculated into the LB
liquid media. Plasmids were isolated from E. coli using the QIAprep
Spin Miniprep Kit (QIAGEN, Valencia, Calif.) and checked by
diagnostic PCR or restriction digestion using ClaI and HindIII. All
restriction enzymes were obtained from New England Biolabs
(Ipswich, Mass.). All chemicals were purchased from Sigma Aldrich
or Fisher Scientific.
[0504] For each yeast strain, single colony was first grown up in 2
mL SC-Ura-Leu medium plus 2% glucose, and then inoculated into 50
mL of the same medium in a 250 mL shake flask to obtain enough
cells for mixed sugar fermentation studies. After one day of
growth, cells were spun down and inoculated into 50 mL of YPA
medium supplemented with 4% cellobiose and 5% D-xylose, or 4%
cellobiose, 5% xylose, and 0.5% glucose, or 4% cellobiose, 5%
xylose, and 1% glucose in a 250 mL unbaffled shake-flask. Starting
from an initial OD.sub.600.about.1, cell culture was grown at
30.degree. C. at 100 rpm for fermentation under oxygen limited
condition. OD.sub.600 reading and cell culture sample were taken at
various time points. Sugar concentrations were analyzed using HPLC,
while ethanol formation was analyzed using the Ethanol Kit
(R-biopharm, Darmstadt, Germany). For each data point, triplicate
samples were taken. The mixed sugar fermentation results for the
strains ranging from SL00 to SL06 are shown in FIGS. 65A-G. The
best strain SL01 was selected for further characterization.
[0505] A total of six different strains, ranging from SL01 to SL06,
were constructed by introducing a pRS425 plasmid harboring one of
the cellodextrin transporter genes and one of the
.beta.-glucosidase genes into the L2612 strain. In each plasmid,
the cellodextrin transporter gene and the .beta.-glucosidase gene
were added with a yeast promoter and terminator, respectively, and
assembled into the pRS425 multi-copy plasmid by the DNA 10
assembler method (Shao et al., 2009) (FIG. 64). The empty pRS425
plasmid was introduced into the L2612 strain to yield the SL00
strain, which was used as a negative control. All strains were
cultivated with a mixture of 40 g/L cellobiose and 50 g/L D-xylose
in shake-flasks, and their sugar consumption rates, cell growth
rates, and ethanol titers were determined (FIG. 65). Amongst all
strains, the SL01 strain containing the .beta.-glucosidase from
Neurospora crassa and the cellodextrin transporter NCU00801 showed
the highest sugar consumption rate and ethanol productivity. Thus,
this strain was selected for further characterization.
[0506] Both SL01 and SL00 were cultivated using a mixture of 40 g/L
cellobiose and 50 g/L D-xylose in both shake-flasks and bioreactors
(FIGS. 66A-D). In the shake-flask cultivation (FIG. 66a-b), 83%
cellobiose was consumed in 96 hours by SL01, with 41.2% higher
average D-xylose consumption rate compared to SL100 (from 0.33
g/L/h to 0.46 g/L/h). Consistent with the enhanced sugar
consumption rate, 1.32-fold increased average biomass growth rate
was observed (from 0.031 g dry cell weight/L/h to 0.072 g dry cell
weight/L/h). The ethanol productivity was increased by more than
2.1-fold, from 0.07 g/L/h to 0.23 g/L/h. The highest ethanol yield
of 0.31 g per g sugar was reached in 48 hours, and the average
ethanol yield was 0.28 g per g sugar, representing a 23% increase
compared to the SL00 strain. In the SL01 cultivation, a faster
D-xylose consumption rate was observed, without the lag phase that
is the hallmark of glucose repression in co-fermentation of glucose
and D-xylose. Moreover, enhanced biomass growth and ethanol
production were also observed.
[0507] The Multifors system (Infors-HT, Bottmingen, Switzerland)
was used for mixed sugar fermentation in bioreactors. Each vessel
had a total capacity volume of 750 mL. For each vessel, there was
one individual set of pO.sub.2 sensor, air sparger, exit gas
cooler, temperature sensor, inoculation port, spare port, dip tube,
antifoam sensor, pH sensor, drive shaft, heater block, rotameter,
and peristaltic pumps system. The whole bioreactor system was
equipped with a cooling system, ThermoFlex900 (Thermo Scientific,
Waltham, Mass.).
[0508] Single colonies of yeast strains were first grown up in 2 mL
SC-Ura-Leu medium plus 2% glucose, and then inoculated into 50 mL
of the same medium in a 250 mL shake flask to obtain enough cells
for mixed sugar fermentation studies. After one day of growth, 10
mL saturated culture were inoculated in 400 mL YPA medium
supplemented with 4% cellobiose and 5% D-xylose, or 4% cellobiose,
5% xylose, and 0.5% glucose, or 4% cellobiose, 5% xylose, and 1%
glucose. The temperature was maintained at 30.degree. C. and the pH
was maintained at 5.5, adjusted by addition of either 2 N
H.sub.2SO.sub.4 or 4 N NaOH. In the first 48 hours, the air flow
rate was maintained at 0.5 L/min, with the impeller speed at 250
rpm. Afterwards, the air flow rate was adjusted to 0.2 L/min to
achieve high ethanol production under oxygen limited condition.
Triplicate samples were taken at various time points and the
OD.sub.600, sugar concentration, and ethanol concentration were
determined as described above.
[0509] In the bioreactor cultivation (FIG. 66c-d), almost all
cellobiose and 66% D-xylose were consumed in 48 hours, representing
44% increased D-xylose consumption rate (from 0.47 g/L/h to 0.68
g/L/h) and 1.1-fold increased biomass growth rate (from 0.08 g dry
cell weight/L/h to 0.17 g dry cell weight/L/h). The ethanol
productivity was increased by more than 4.3-fold (from 0.09 g/L/h
to 0.50 g/L/h), and the ethanol yield was 0.39 g per g sugar.
Compared to shake-flask cultivations, sugar consumption rates in
the first 24 hours were lower, which was due to the low cell
density used in the beginning of batch cultivation.
[0510] Unexpectedly, a small amount of glucose was detected even
though there was no glucose added in fermentation (FIG. 66a-b). The
maximum glucose concentration was reached in approximately 24 hours
in both shake-flasks (12.1 g/L) and bioreactors (17.5 g/L) and then
dropped to a very low level. However, no obvious glucose repression
was observed even in the presence of such glucose. Because no
glucose was detected in the SL00 strain, the extracellular glucose
may result from the slow conversion of .beta.-glucose to its epimer
.alpha.-glucose, the main form of glucose used in glycolysis.
Typically, .beta.-glucose can be efficiently converted to
.alpha.-glucose either enzymatically or chemically because of its
relatively low concentration in glucose (Bouffard et al., 1994).
However, in the engineered SL01 strain, catalyzed by
.beta.-glucosidase, an excess amount of .beta.-glucose is produced
from cellobiose intracellularly and a small fraction may be
secreted outside cells, similar to what was observed with
.beta.-galactose (Bouffard et al., 1994).
[0511] Because a small amount of glucose (less than 10% of total
sugars) is typically present in lignocellulosic hydrolysates in
industrial settings, the fermentation performance of the engineered
SL01 strain was also investigated using a mixture of cellobiose,
D-xylose, and glucose. Two concentrations of glucose, 5 g/L or 10
g/L, were combined with 40 g/L cellobiose and 50 g/L D-xylose as
mixed carbon source in bioreactors. With 5 g/L glucose (FIG.
67a-b), 81.7% cellobiose was consumed by SL01, with 67.8% D-xylose
consumed at 48 hours in batch cultivation. The D-xylose consumption
rate was increased by 1.19-fold, from 0.32 g/L/h to 0.69 g/L/h. The
ethanol productivity was increased by 3.3-fold (from 0.11 g/L/h to
0.46 g/L/h) while the ethanol yield was increased from 0.26 g per g
sugar to 0.33 g per g sugar. With 10 g/L glucose (FIG. 67c-d),
83.8% cellobiose was consumed by SL01, with 74.7% D-xylose consumed
at 48 hour in batch cultivation. The D-xylose consumption rate was
increased by 68%, from 0.45 g/L/h to 0.76 g/L/h. The ethanol
productivity was increased by 2.1-fold (from 0.16 g/L/h to 0.50
g/L/h) and the ethanol yield was increased from 0.30 g per g sugar
to 0.33 g per g sugar. As expected, the engineered SL01 strain
showed both a higher efficiency of sugar consumption and a higher
rate of ethanol production than the SL00 wild type strain. More
importantly, there was no significant glucose repression in the
co-fermentation of three sugars even with glucose up to 10% of
total sugars (FIG. 67c-d) suggesting that this approach may be
viable for industrial applications.
[0512] A similar study was carried out in the S. cerevisiae strain
D452-2, where the three N. crassa cellodextrin transporters
NCU00801, NCU08114, and NCU00809 were introduced together with the
.beta.-glucosidase NCU00130. The transformants were selected on YSC
medium containing 20 g/liter cellobiose expressing an intracellular
.beta.-glucosidase (NCU00130). Strains and plasmids used in this
work are described in Table 17 (Ex. 12). The primers used are
listed in Table 28.
[0513] Table 28 shows the synthetic oligonucleotides used in the
study.
TABLE-US-00032 Name Sequences NCU00801-F
ATGGATCCAAAAATGTCGTCTCACGGCTCC SEQ ID NO: 92 NCU00801-R
ATGAATTCCTACAAATCTTCTTCAGAAATCAATTTTTG TTCAGCAACGATAGCTTCGGAC SEQ
ID NO: 93 NCU08114-F ATACTAGTAAAAATGGGCATCTTCAACAAGAAGC SEQ ID NO:
94 NCU08114-R GCATATCGATCTACAAATCTTCTTCAGAAATCAATTTT
TGTTCAGCAACAGACTTGCCCTCATG SEQ ID NO: 95 NCU00130-F
GCATACTAGTAAAAATGTCTCTTCCTAAGGATTTCCTCT SEQ ID NO: 96 NCU00130-R
ATACTGCAGTTAATGATGATGATGATGATGGTCCTTCTT GATCAAAGAGTCA AAG SEQ ID
NO: 97
[0514] Yeast were grown in YP medium containing 20 g/L of glucose
or 20 g/L of cellobiose to prepare inoculums for xylose or
cellobiose fermentation experiments, respectively. Cells at
mid-exponential phase from YP media containing 20 g/L of glucose or
cellobiose were harvested and inoculated after washing twice with
sterilized water. All of the flask fermentation experiments were
performed using 50 mL of YP medium containing 40 g/L or 80 g/L of
xylose in 250 mL flask at 30.degree. C. with initial OD.sub.600 of
1.0 under oxygen limited conditions. Bioreactor fermentations were
performed in 400 mL of YP medium containing appropriate amounts of
sugars using Sixfors Bioreactors (Appropriate Technical Resources,
Inc) at 30.degree. C. with an agitation speed of 200 rpm under
oxygen limited 250 conditions. Initial cell densities were adjusted
to OD.sub.600=1.0.
[0515] Cell growth was monitored by optical density (OD) at 600 nm
using UV-visible Spectrophotometer (Biomate 5, Thermo, NY).
Glucose, xylose, xylitol, glycerol, acetate, and ethanol
concentrations were determined by high performance liquid
chromatography 264 (HPLC, Agilent Technologies 1200 Series)
equipped with a refractive index detector using 265 a Rezex
ROA-Organic Acid H+(8%) column (Phenomenex Inc., Torrance, Calif.).
The column was eluted with 0.005 N of H.sub.2SO.sub.4 at a flow
rate of 0.6 mL/min at 50.degree. C.
[0516] All three transformants were able to grow and produce
ethanol when cellobiose was the sole carbon source (FIGS. 68A-C),
but the three transformants exhibited different cellobiose
fermentation rates (NCU00801>NCU08114>NCU00809). The fastest
cellulose-fermenting transformant (D801-130), expressing both
NCU00801 and NCU00130, consumed 40 g/L of cellobiose within 4
hours, producing 16.8 g/L of ethanol. The volumetric productivity
of cellobiose fermentation (P.sub.Ethanol/Cellobiose=0.7 g/L/h) was
lower than that of glucose fermentation (P.sub.Ethanol/Glucose=1.2
g/L/h), and ethanol yield from cellobiose
(Y.sub.Ethanol/Cellobiose=0.42 g/g) was about the same as ethanol
yield from glucose (Y.sub.Ethanol/Glucose=0.43 g/g) under the same
culture conditions. However, the observed cellobiose consumption
rate and ethanol yield by D801-130 were an improvement over S.
cerevisiae strains engineered to ferment cellobiose through surface
display of .beta.-glucosidase (Kotaka et al., 2008; Nakamura et
al., 2008). These results suggest that simultaneous expression of
NCU00801 and NCU00130 in S. cerevisiae can result in efficient
cellobiose fermentation.
[0517] After developing the efficient xylose fermenting strain
DA24-16 (described in Example 13), genes coding cellodextrin
transporter and .beta.-glucosidase (NCU00801 and NCU00130) enzyme
were introduced into the strain enabling it to consume cellobiose
and xylose simultaneously. It was hypothesized that glucose
repression of xylose utilization may be alleviated in this strain,
due to the intracellular hydrolysis of cellobiose. The NCU00801
gene was integrated into the genome of DA24-16, and NCU00130 was
expressed from a multi-copy plasmid. The resulting transformant,
DA24-16-BT3, was selected on an agar plate containing cellobiose as
the sole carbon source.
[0518] The DA24-16-BT3 strain grown in media containing various
amounts of cellobiose and xylose co-consumed cellobiose and xylose,
and produced ethanol with yields of 0.38-0.39 g/g in all conditions
tested (FIGS. 69A-C). The potential synergistic effects of
co-fermentation were tested by culturing DA2416-BT3 under three
different conditions: 40 g/L of cellobiose, 40 g/L of xylose, and
40 g/L of both sugars (total 80 g/L of sugars). Surprisingly,
DA24-16BT3 was able to co-consume 80 g/L of a cellobiose/xylose
mixture within the same period that was required to consume 40 g/L
of cellobiose or 40 g/L xylose separately (FIGS. 70A-C). Moreover,
DA24-16BT3 produced ethanol with a higher yield (0.39 g/g) from a
mixture of cellobiose and xylose as compared to ethanol yields
(0.31-0.33 g/g) from single sugar fermentations (cellobiose or
xylose). Ethanol productivity also drastically increased from 0.27
g/L/h to 0.65 g/L/h during co-fermentation. These results
demonstrated that co-fermentation of cellobiose and xylose can
enhance overall ethanol yield and productivity. Fermentation
experiments were also done to compare this engineered S. cerevisiae
strain (DA24-16BT3) to P. stipitis, which is capable of
co-fermenting cellobiose and xylose efficiently.
[0519] A simulated hydrolysate (10 g/L of glucose, 80 g/L of
cellobiose, 40 g/L of xylose) based on the composition of
energycane was used. The composition of different lignocellulosic
plants varies in a broad range. For instance, the US Department of
Energy biomass database lists the composition of more than 150
biomass samples (webpage
eere.energy.gov/biomass/m/feedstock_databases.html). The
cellulose-to-hemicellulose ratios of these samples are between 1.4
and 19, and the average is 2.3. Energy crops typically have higher
hemicellulose content than woody biomass. The average cellulose to
hemicellulose ratios of sugarcane bagasse, corn stover, sorghum are
2.0, 1.85 and 2.14, respectively. We therefore used a glucan/xylan
ratio of 2 in our simulated sugar experiment design. The engineered
yeast will likely be used in conjunction with traditional cellulase
cocktails that are deficient in .beta.-glucosidase activities for
the biofuels production. The biomass hydrolysis process may result
in small amounts of glucose in the lignocellulosic hydrolysates as
6-30% glucan-to-glucose conversions with incomplete cellulase
cocktails were reported (Medve et al., 1998). Considering all the
above factors, a sugar combination of 10 g/L glucose, 80 g/L
cellobiose, and 40 g/L xylose was chosen in the simulated sugar
experiments.
[0520] The DA24-16BT3 consumed glucose first before co-consuming
cellobiose and xylose rapidly. A total of 130 g/L of sugars was
consumed within 60 hours even though small inoculums were used
(OD.sub.600=1). In contrast, P. stipitis could not finish
fermenting the sugar mixture within the same period under identical
culture conditions (FIGS. 71A-B). DA24-16BT3 produced 48 g/L of
ethanol within 60 hours (Y.sub.Ethano/Sugars=0.37 g/g and
P.sub.Ethanol/Sugars=0.79 g/L/h).
[0521] A transient accumulation of cellodextrins in the medium
during cellobiose consumption was observed (FIG. 72-73). The
accumulated cellotriose and cellotetraose were again consumed after
depletion of cellobiose. It is likely that the accumulated
cellodextrins were generated by the trans-glycosylation activity
(Christakopoulos et al., 1994) of .beta.-glucosidase (NCU00130),
and secreted by the cellodextrin transporter (NCU00801), which
might facilitate the transport of cellodextrins in both directions
(intracellular.revreaction.extracellular). This transient
cellodextrin accumulation would probably not reduce product yields
since the accumulated cellodextrins would eventually be consumed by
the engineered yeast. However, it might decrease productivity
because the transport rates of cellotriose and cellotetraose might
be slower than that of cellobiose.
[0522] Small amounts of glucose were constantly detected in the
medium during co-fermentation. Since even low amounts of glucose
accumulation can repress xylose fermentation, glucose levels have
to be kept at a minimum. It can be hypothesized that the relative
expression levels of the cellodextrin transporter and
.beta.-glucosidase are likely to affect glucose accumulation. In
support of this, it was observed that more glucose was accumulated
in the medium when NCU00801 was introduced on a multi-copy plasmid
than when NCU00801 was integrated into the yeast genome. The strain
(DA24-16-BT). containing both NCU00801 and NCU00130 on multi-copy
plasmids, had relatively slower xylose utilization rates than those
observed in DA24-16-BT3, a potential reason being glucose
repression (FIGS. 74A-B). Further adjustments of the cellodextrin
transporter and .beta.-glucosidase expression levels, or the
identification of .beta.-glucosidases with reduced
trans-glycosylation activities, may be able to reduce the
accumulation of glucose and cellodextrin during
co-fermentation.
[0523] Co-fermentation of xylose and cellobiose could also be
achieved by mixed cultivation of two different yeast strains: the
xylose-fermenting DA24-16 strain and the cellobiose-fermenting
DA452BT (FIG. 75). As explained above, the yeast strain DA24-16
expressed the xylose-utilizing enzymes wild type xylose reductase
(XYL1), mutant xylose reductase R276H (mXYL1), xylitol
dehydrogenase (XYL2), and xylulokinase (XKS1) (Ex. 12; Table 17).
D452BT was formed by engineering D452 to express the cellodextrin
transporter NCU00801 and the .beta.-glucosidase NCU00130. In the
mixed culture, the DA24-16 strain took up xylose (xylose molecule
shown as a green pentagon in FIG. 75a) and metabolized it using the
enzymes XYL1 (wild type and mutant), XYL2, and XYL3, whereas the
other strain D452BT was able to take up cellobiose (cellobiose
molecule shown as two red hexagons in FIG. 75a) using the
transporter NCU00801 and convert the cellobiose into glucose using
the enzyme NCU00130. Hence, the mixed culture was able to
co-ferment both xylose and cellobiose to produce ethanol (FIG.
75b).
[0524] This study demonstrated a novel strategy to allow
co-fermentation of hexose and pentose sugars by S. cerevisiae. By
combining an efficient xylose utilization pathway with a
cellodextrin transport system, the problem caused by glucose
repression was over-come. As a result, the engineered yeast
co-fermented two non-metabolizable sugars in cellulosic
hydrolysates synergistically into ethanol. The new co-fermentation
method described herein advances lignocellulosic technologies on
both the saccharification and fermentation fronts. Most traditional
fungal cellulase cocktails are deficient in .beta.-glucosidase and
end the cellulose hydrolysis with cellobiose that is not fermented
efficiently by yeast. As a result, extra .beta.-glucosidase enzyme
must be added to convert cellobiose into glucose. The
cellobiose/xylose co-fermentation yeast makes it possible to use
these cellulase cocktails with limited.beta.-glucosidase
activities, lowering enzyme usage and cost associated with the
cellulose saccharification process. Further, the synergy between
cellobiose and xylose co-fermentation significantly increases
ethanol productivity, thus improving fermentation economics. The
presence of a small amount of glucose from the pre-treatment and
hydrolysis of lignocellulosic materials does not affect the
capacity of the engineered yeast to convert hexose and pentose
sugar mixtures into ethanol.
[0525] This study involved measuring the capacity of an engineered
S. cerevisiae strain to ferment various mixtures of sugars meant to
mimic hydrolysates from plant biomass. The ability of this strain
to co-ferment cellodextrins and xylose is particularly useful
during the simultaneous saccharification and co-fermentation (SSCF)
of pre-treated plant biomass. During SSCF, hemicellulose would
first be hydrolyzed by acid pre-treatment, resulting in formation
of xylose and still-crystalline cellulose. Then, fungal cellulases
and the yeast strain described herein would be added, allowing the
cellulases to co-convert xylose and cellobiose into ethanol.
Because of the limited extracellular glucose production in this
scheme, there will be reduced repression of xylose utilization and
co-fermentation will proceed rapidly and synergistically.
[0526] Although the S. cerevisiae strain used in this study was a
laboratory strain, the fermentation performance of the engineered
strain was very impressive when compared to published results. The
key fermentation parameters (yield and productivity) may be further
improved by the use of industrial yeast strains as a platform.
Applications of this co-fermentation strategy would not be limited
to ethanol production. Since it is a foundational technology, the
strategy presented here can be combined with any other product
diversification technologies to produce commodity chemicals and
advanced biofuels.
Example 18
Transcriptome Analysis of N. crassa Grown on Xylan
[0527] Lignocellulosic biomass is composed of cellulose,
hemicellulose, and lignin. Examples 1-3 describe the discovery of
genes critical for growth on cellulose through transcriptome and
secretome analysis of N. crassa. In this example the expression
profile of the N. crassa genome was examined during growth on xylan
to determine which genes are important for utilization of
hemicellulose.
[0528] Ten day old conidia of WT or .DELTA.xlnR strains were
inoculated at 10.sup.6 conidia/mL on 100 mL 1.times. Vogel's salts
minimal medium (2% sucrose), grown for 16 hours at 25.degree. C.
with constant light, and washed with 1.times. Vogel's only medium.
Conidia were then transferred into 100 mL 1.times. Vogel's salts
with 2% sucrose or 2% Beechwood xylan as the sole carbon source in
the medium and allowed to grow for 4 hours. Mycelia were harvested
by filtration and immediately flash frozen in liquid nitrogen.
Total RNA was isolated using TRIzol (Invitrogen) according to the
manufacturer's instructions and treated with DNase (Turbo DNA-free
kit; Ambion) (Kasuga, Townsend et al., 2005).
[0529] For cDNA synthesis and labeling, the Pronto kit (Catalog No.
40076; Corning) was used according to the manufacturer's
specifications except that the total RNA used was 10 .mu.g per
sample.
[0530] Microarray hybridization and data analysis were performed as
previously described (Tian, Kasuga et al., 2007). A GenePix 4000B
scanner (Axon Instruments) was used to acquire images, and GenePix
Pro6 software was used to quantify hybridization signals and
collect the raw data. Normalized expression values were analyzed by
using the BAGEL (Bayesian analysis of gene expression levels)
software program (Townsend and Hartl 2002; Townsend 2004). 354
genes were found to be induced greater than 2-fold in N. crassa
grown on xylan. The list is shown in FIG. 76.
Example 19
Secretome Analysis of N. crassa Grown on Xylan
[0531] The secretome of N. crassa during growth on xylan was
analyzed using a shotgun proteomics approach. Supernatants from
xylan cultures were digested with trypsin and analyzed by liquid
chromatography nano-electrospray ionization tandem mass
spectrometry.
[0532] Mass spectrometry samples were prepared as follows. N.
crassa wild type strain was grown on 2% xylan media for 4 or 7
days. Culture supernatants were isolated by centrifugation,
filtered through 0.22 .mu.m filters, and concentrated 10 times with
10 kDa MWCO PES spin concentrators. 3.36 mg of urea, 5 .mu.L of 1M
Tris pH 8.5, and 5 .mu.L of 100 mM DTT were then added to 100 .mu.L
of concentrated culture supernatant, and the mixture was heated at
60.degree. C. for 1 hour. After heating, 700 .mu.L of 25 mM
ammonium bicarbonate and 140 .mu.L of methanol were added to the
solution followed by treatment with 50 .mu.L of 100 .mu.g/mL
trypsin in 50 mM sodium acetate pH 5.0. The trypsin was left to
react overnight at 37.degree. C. with inverting for about 8-9 hours
at basal pH. After digestion the volume was reduced to dryness by
speedvac and washed with 300 .mu.l MilliQ water three times. The
final volume was 100.sub.111. TFA was added at 0.1-0.3% v/v.
Residual salts in the sample were removed by using OMIX
microextraction pipette tips according to the manufacturer's
instructions. The acetonitrile was removed by evaporation. The
sample solution was an aqueous solution with 0.1%-1% TFA, and the
final volume was 10 microliters or greater.
Example 20
Analysis of Xylan-Induced Genes Predicted to Encode Secreted
Proteins
[0533] The transcriptome and secretome analysis results indicated a
total of 71 genes, of which 55 were predicted to be secreted. The
list of these genes is in Table 29. Deletion strains were available
for 46 out of 69 genes. Out of these 46, six of the strains were
heterokaryons, thus the remaining 40 deletion strains were analyzed
for total secreted protein, amount of xylose present, and
azo-endo-xylanase activity. Results are shown in FIG. 77.
Table 29 shows xylan-induced N. crassa genes
TABLE-US-00033 Gene Name Signal P Data Annotation NCU00642 Y
Transcription probable beta-galactosidase NCU00695 Y Transcription
putative protein NCU00798 MS hypothetical protein NCU00937 Y
Transcription conserved hypothetical protein NCU01517 Y
Transcription glucan 1,4-alpha-glucosidase NCU02136 MS probable
transaldolase NCU02252 MS probable phosphoglyceromutase NCU02343 Y
Transcription related to alpha-L-arabinofuran- osidase A precursor
NCU02455 Y Transcription FK506-binding protein 2 precursor
(Peptidyl-prolyl cis-trans isomerase) NCU02583 Y Transcription
probable Alpha-glucosidase precursor (Maltase) NCU03013 Y
Transcription related to cytosolic Cu/Zn superoxide dismutase
NCU03222 Y Transcription putative protein NCU03636 Y Transcription
NCU03639 Y Transcription probable triacylglycerol lipase precursor
NCU04202 MS nucleoside-diphosphate kinase NCU04265 Y Transcription
related to beta-fructofuranosidase NCU04388 Y Transcription
probable phosphatidylglycerol/phosphatidyl- inositol transfer
protein NCU04395 MS beta-1,6-glucanase Neg1 NEG-1 NCU04415 Y
Transcription related to brefeldin A resistance protein NCU04431 Y
MS related to endo-1,3-beta-glucanase NCU04475 Y Transcription
probable lipase B precursor NCU04482 MS hypothetical protein
NCU04623 Y Transcription related to beta-galactosidase NCU04674 Y
Transcription related to alpha-glucosidase b NCU04675 Y
Transcription putative protein NCU04930 Y Transcription related to
triacylglycerol lipase NCU05137 Y Transcription conserved
hypothetical protein NCU05143 Y Transcription related to Rds1
protein NCU05159 Y Transcription probable acetylxylan esterase
precursor NCU05275 MS probable ubiquitin fusion protein
(ubiquitin/ribosomal protein) NCU05315 Y Transcription hypothetical
protein NCU05395 Y Transcription conserved hypothetical protein
NCU05686 Y MS probable cell wall protein UTR2 NCU05751 Y
Transcription related to acetylxylan esterase NCU05924 Y
Transcription probable endo-beta-1,4-D-xylanase NCU05965 Y
Transcription related to putative arabinase NCU05974 MS related to
cell wall protein (putative glycosidase) NCU06364 Y Transcription
hypothetical protein NCU06380 Y Transcription related to
catecholamines up protein NCU06650 Y Transcription conserved
hypothetical protein NCU06781 MS probable beta (1-3) glucanosyl-
transferase NCU06961 Y Transcription probable exopolygalacturonase
NCU07067 MS related to class I alpha- NCU07143 Y Transcription
mannosidase 1B NCU07190 Y Transcription related to cellulose
1,4-beta- cellobiosidase II precursor NCU07200 Y MS related to
metalloprotease MEP1 NCU07225 Y Transcription probable
endo-1,4-beta-xylanase B precursor NCU07281 MS probable
glucose-6-phosphate isomerase NCU07787 Y MS probable SnodProt1
precursor NCU08131 Y Transcription probable alpha-amylase precursor
NCU08171 Y MS conserved hypothetical protein NCU08189 Y
Transcription related to endo-1,4-beta-xylanase NCU08384 MS
probable D-xylose reductase NCU08418 MS related to
tripeptidyl-peptidase I NCU08457 Y Transcription hydrophobin Ccg-2
CCG-2 NCU08516 Y Transcription related to aldose 1-epimerase
NCU08750 Y Transcription related to isoamyl alcohol oxidase
NCU08752 Y Transcription related to esterase NCU08755 Y
Transcription hypothetical protein NCU08909 Y MS probable beta
(1-3) glucanosyl- transferase gel3p NCU08936 MS related to
sporulation-specific gene SPS2 NCU09024 Y MS related to choline
dehydrogenase NCU09133 Y Transcription putative protein NCU09170 Y
MS probable alpha-N-arabinofuranos- idase NCU09175 Y Transcription
related to glucan 1,3-beta- glucosidase precursor NCU09267 MS
related to glyoxal oxidase precursor NCU09491 MS feruloyl esterase
B precursor (subclass of the carboxylic acid esterases) NCU09923 Y
Transcription related to xylan 1,4-beta-xylosidase NCU09924 Y
Transcription conserved hypothetical protein NCU10040 Y
Transcription NCU10045 Y Transcription
[0534] Samples were prepared as follows. 10 day old conidia were
grown in 100 mL 2% xylan Vogel's media at 10.sup.6 conidia/mL. Two
replicates were prepared for each strain. Cultures were grown at
25.degree. C. with constant light and 220 rpm. Samples were
harvested on day 4. Supernatants were isolated by centrifugation
and used in assays.
[0535] Bradford protein concentrations were measured to determine
the total amount of secreted protein. Stocks were prepared with BSA
standards: 0 .mu.g/mL, 50 .mu.g/mL, 100 .mu.g/mL, 250 .mu.g/mL, and
500 .mu.g/mL. Bradford solution was diluted 1:4. A multichannel
pipette was used to pipette 200 .mu.L of Bradford solution into a
96-well plate. 10 .mu.L of sample and 10 .mu.L of each standard
were added. Samples were incubated at room temperature for 10
minutes. The absorbance was read at 595 nm, and the protein
concentration was determined.
[0536] The assay used to measure xylose was modified from Bailey et
al., 1992 (J Biotech 23: 257-270). Xylose standards were prepared
in H.sub.2O. For concentrated 0.8 M xylose (1.2 g in 10 mL), the
standards included 0 mM, 8 mM (1:100 dilution; 990 .mu.l+10 .mu.l),
20 mM (1:100 dilution; 975 .mu.l+25 .mu.l), 40 mM (1:100 dilution;
950 .mu.l+50 .mu.l), 80 mM (1:100 dilution; 900 .mu.l+100 .mu.l),
and 160 mM (1:100 dilution; 800 .mu.l+200 .mu.l). A multichannel
pipette was used to add 900 .mu.L of substrate solution to a deep
well 96-well plate. The substrate was allowed to incubate at
50.degree. C. for 10 minutes. One hundred .mu.L of culture
supernatant and the standards were added and allowed to incubate at
50.degree. C. for 5 minutes. Samples were centrifuged for 10
minutes at 3,400 rpm. A multichannel pipette was used to pipette 75
.mu.L DNS solution into a 96-well PCR plate. Five .mu.L of solution
was removed from the reaction and added to the PCR plate containing
DNS solution. The plate was heated at 99.degree. C. in the PCR
machine for 5 min. After the samples cooled, they were transferred
to clear flat-bottomed plates, and the absorbance was read at 540
nm. Substrate solution (500 mL) contained beechwood xylan (5 g; 10
mg/mL), 3M NaOAc, pH 5.0 (8.33 mL; 50 mM), water (491 mL), and was
autoclaved for 20 minutes. DNS solution (100 mL) contained
3,5-dinitrosalicylic acid (707 mg), NaOH (1.32 g), Rochelle salts
(Na K tartrate) (20.4 g), Sodium meta-bisulfate (553 mg), phenol
(507 .mu.L), and water (94.4 mL).
[0537] Azo-endo-xylanase activity was measured with a kit from
Megazyme. This assay indirectly measures the amount of
endo-xylanase activity in a sample by spectrophotometrically
measuring the amount of dye liberated from a xylan chain complexed
with the dye. The more enzymes that are present, the more dye will
be released. All supernatant samples were diluted 1:10 by adding 50
.mu.L of supernatant to 450 .mu.L of Na Acetate buffer (50 mM, pH
4.5) in separate 15 mL Falcon tubes. Next, Falcon tubes were
pre-warmed about 10 minutes. Substrate solution was added for all
samples (500 .mu.L/sample) to the tubes. Samples and substrate
solutions were added into a 40.degree. C. water bath for 10 minutes
to pre-equilibrate them. Five hundred .mu.L substrate solution was
added to each 1:10 diluted sample, vortexed for 10 seconds, and
incubated at 40.degree. C. for 10 minutes. The reaction was
terminated by adding 2.5 mL of precipitant solution (95% ethanol)
to each sample and vortexing for 10 seconds. Tubes were allowed to
stand at room temperature for 10 minutes. Tubes were vortexed for
10 seconds and then centrifuged at room temperature for 10 minutes
at 1,000 g. One mL of supernatant solution from each tube was
placed directly into a cuvette, and the absorbance was measured at
590 nm. The blank used for this procedure was the supernatant from
500 .mu.L substrate solution added to 2.5 mL of precipitant
solution.
[0538] In conclusion, it is anticipated that the modulation of
genes identified here that affect the degradation of hemicellulose
in N. crassa will facilitate engineering strains that have enhanced
capacity for plant cell wall breakdown and growth on plant cell
wall components such as hemicellulose. Genes of interest include
NCU01517, which encodes a predicted glucamylase; NCU02343, which
encodes a predicted arabinofuranosidase; NCU05137, which encodes a
conserved hypothetical protein; NCU05159, which encodes a predicted
acetylxylan esterase precursor; NCU09133, which encodes a conserved
hypothetical protein; and NCU10040, which encodes a hypothetical
protein.
[0539] The growth of a cell on hemicellulose will be increased by
providing a host cell that contains a recombinant polynucleotide
that encodes a polypeptide encoded by NCU01517, NCU09133, or
NCU10040. The host cell will be cultured in a medium that contains
hemicellulose such that the recombinant polynucleotide is
expressed. The host cell will grow at a faster rate in this medium
than a cell that does not contain the recombinant
polynucleotide.
Example 21
Further Analysis of the 4NCU05137 Strain
[0540] As described in Examples 1-3 and 18-20, NCU05137 is a
predicted secreted protein that was overexpressed during growth of
N. crassa on any of Miscanthus, Avicel, or xylan. A deletion strain
of N. crassa lacking NCU05137 grown on Avicel showed increased
endoglucanase, .beta.-glucosidase, and Avicelase activity. An
NCU05137 deletion strain grown on xylan showed increased
azo-endo-xylanase activity. As described in this example, the
complementation of .DELTA.NCU05137 was performed in order to verify
that the phenotypes observed in the .DELTA.NCU05137 strain were due
to the loss of the NCU05137 gene.
[0541] A plasmid containing NCU05137 with a C-terminal GFP tag
under the control of the ccgl promoter was generated. N. crassa
conidia were transformed with the NCU05137-GFP construct.
Experiments were performed according to standard Neurospora
procedures (webpage
fgsc.net/Neurospora/NeurosporaProtocolGuide.htm).
[0542] The total secreted protein and carboxymethyl cellulase (CMC)
activity of wild-type, .DELTA.NCU05137, and
.DELTA.NCU05137-NCU05137-GFP strains was measured. Total secreted
protein was measured by taking 100 .mu.L of supernatant from a
culture of each strain, adding it to 900 .mu.L Bradford Dye, and
measuring absorbance at 595 nm. CMC activity was measured with
20.times. diluted supernatant from each strain culture and an
azo-CMC kit (Megazyme SCMCL). .DELTA.NCU05137 knockout strains
displayed increased levels of secreted protein and CMC activity.
Introduction of the GFP-tagged NCU05137 into .DELTA.NCU05137
strains reduced these levels back to wild-type levels (FIG.
78).
[0543] In addition, the localization of NCU05137-GFP in
complemented strains was observed. NCU05137-GFP localized to the
cell wall of conidia and to the hypha tip (FIG. 79-80). These data
indicate that the GFP-tagged NCU05137 protein is fully functional
and can be used for purification and experiments addressing the
biochemical activity of this protein.
[0544] Thus, the normal function of NCU05137 may be to inhibit
signaling processes associated with induction of cellulase and
hemicellulase gene expression. Reduction of expression of NCU05137
or a homolog of NCU05137 in a cell is likely to increase cellulase
and hemicellulase activity in that cell and, consequently, growth
of the cell on cellulose or hemicellulose. The growth of a cell on
cellulose or hemicellulose will be increased by providing a host
cell that contains an endogenous polynucleotide that encodes a
polypeptide encoded by NCU05137. The expression of the endogenous
polynucleotide will be inhibited, and the cell will be cultured in
a medium containing cellulose and/or hemicellulose. The host cell
will grow at a faster rate in the medium than a cell in which
expression of the endogenous polynucleotide is not inhibited.
Example 22
Further Analysis of NCU07705
[0545] Expression of NCU07705 was found to be upregulated during
growth of N. crassa on cellulose. BLAST analysis of the polypeptide
encoded by NCU07705 revealed that the polypeptide has high
similarity to many C6 zinc finger domain containing transcription
factors (FIG. 1). To further investigate the role of NCU07705 in
the utilization of cellulose, the phenotype of a deletion strain
lacking NCU07705 was evaluated.
[0546] The .DELTA.NCU07705 strain was unable to grow on 2%
cellulose (Avicel), PASC, or CMC as a sole carbon source (Table 30)
but grew with similar kinetics to wild-type strain on sucrose,
xylan, and xylose. In order to determine whether NCU07705 plays a
role in regulating expression of cellulases, the expression of
cellulase and hemicellulase genes was examined during growth of
.DELTA.NCU07705 on cellulose. Ten-day-old conidia from wild-type
(FGSC 2489) and .DELTA.NCU07705 strains were inoculated into
Vogel's liquid MM (2% sucrose) (Vogel 1956) and grown for 16 hours.
Mycelia were centrifuged, washed with 1.times. Vogel's salts, and
then transferred into either Vogel's media with 2% sucrose or 2%
Avicel and grown in constant light for 4 hours. They were harvested
by filtration and immediately frozen in liquid nitrogen. Total RNA
was isolated using TRIzol (Invitrogen, Carlsbad, Calif.) according
to the manufacturer's instructions and treated with DNase (Turbo
DNA-free kit, Ambion/Applied Biosystems, Foster City, Calif.)
(Kasuga et al., 2005). ChipShot.TM. Indirect Labeling/Clean-Up
System (Catalog No. Z4000, Promega, Madison, Wis.) and CyDye
Post-Labeling Reactive Dye Pack (Catalog No. RPN5661, GE
Healthcare, Piscataway, N.J.) were used to synthesize and label
cDNA according to the manufacturer's instructions except the amount
of RNA used was 10 .mu.g. The Pronto! Hybridization Kit (Catalog
No. 40076, Corning, Lowell, Mass.) was used for microarray
hybridization according to the manufacturer's specifications.
[0547] Data analyses were performed as previously described (Tian
et al., 2007). A GenePix 4000B scanner (Axon Instruments, Union
City, Calif.) was used to acquire images, and GenePix Pro6 software
was used to quantify hybridization signals and collect the raw
data. Normalized expression values were analyzed by using BAGEL
(Bayesian Analysis of Gene Expression Levels) (Townsend and Hartl,
2002). None of the predicted cellulase genes were induced in the
.DELTA.NCU07705 strain, whereas induction of predicted
hemicellulase genes was unaffected (see Table 30 below). Thus,
NCU07705 has been named cdr-1, cellulose degradation regulator
1.
[0548] Therefore, the growth of a cell on cellulose will be
increased by providing a host cell that contains a recombinant
polynucleotide that encodes a polypeptide encoded by NCU07705. The
host cell will be cultured in a medium that contains cellulose such
that the recombinant polynucleotide is expressed. The host cell
will grow at a faster rate in this medium than a cell that does not
contain the recombinant polynucleotide.
[0549] Table 30 shows expression profile of genes in N. crassa
.DELTA.NCU07705 strain
TABLE-US-00034 7705- WT- Gene/ GH up in switch.sup.2 switch.sup.1
locus name Family Class Avi.sup.3 No 15 NCU00762 5 endo- 31.5 No No
NCU03996 6 CBHII like No 168 NCU07190 6 CBHII like 119 No 26
NCU09680 6 CBHII 251.3 No 18 NCU04854 7 CBHI like 10.8 No 3.8
NCU05057 7 CBHI like 7.4 No No NCU05104 7 CBHI like No 93 NCU07340
7 CBHI 382.2 No 2 NCU05121 45 endo- 17.2 No 5.8 NCU00836 61 endo-
31 No 3.7 NCU01050 61 endo- 382.1 No No NCU01867 61 endo- No 49
NCU02240 61 endo- 84 No No NCU02344 61 endo- 4.1 No 6.1 NCU02916 61
endo- 17.7 No No NCU03000 61 endo- No 17 NCU03328 61 endo- 23.8 No
No NCU05969 61 endo- 12.7 No No NCU07520 61 endo- No No NCU07760 61
endo- No 103 NCU07898 61 endo- 230 No No NCU07974 61 endo- No 25
NCU08760 61 endo- 44.7 .sup.1Expression levels of predicted
cellulase genes from an N. crassa (NCU07705) culture grown in
Vogel's/sucrose for 16 hours, filtered, and resuspended in
Vogel's/Avicel for 4 hours prior to RAN extraction.
.sup.2Expression levels of predicted cellulase gene from an N.
crassa (wild type FGSC 2489) culture grown in Vogel's/sucrose for
16 hours, filtered, and resuspended in Vogel's/sucrose for 4 hours
prior to RNA extraction. .sup.3Expression levels derived from
microarray analyses of wild type (FGSC 2489) cells grown for 30
hours in Avicel (Tian et al., 2009).
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Sequence CWU 1
1
97118PRTArtificial SequenceSequence motif 1Xaa Tyr Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15 Xaa
Asp217PRTArtificial SequenceSequence motif 2Xaa Xaa Xaa Gly Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15 Xaa33PRTArtificial
SequenceSequence motif 3Gly Arg Xaa1 49PRTArtificial
SequenceSequence motif 4Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn1 5
58PRTArtificial SequenceSequence motif 5Trp Arg Xaa Pro Xaa Xaa Xaa
Gln1 5 624PRTArtificial SequenceSequence motif 6Pro Glu Ser Pro Arg
Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala1 5 10 15 Xaa Xaa Xaa
Leu Xaa Xaa Tyr His 20 79PRTArtificial SequenceSequence motif 7Phe
Xaa Gln Xaa Ser Gly Asn Xaa Xaa1 5 818PRTArtificial
SequenceSequence motif 8Leu Xaa Xaa Xaa Xaa Xaa Xaa Glu Xaa Leu Xaa
Xaa Xaa Xaa Arg Xaa1 5 10 15 Lys Gly95PRTArtificial
SequenceTransmembrane helix 9Pro Glu Ser Pro Arg1 5
104PRTArtificial SequenceTransmembrane helix 10Pro Glu Thr Lys1
1118PRTArtificial SequenceSequence motif 11Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Gly Arg Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15 Xaa
Xaa1226PRTArtificial SequenceSequence motif 12Xaa Xaa Gly Xaa Xaa
Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 138PRTArtificial
SequenceSynthesized Construct 13Xaa Xaa Val Xaa Xaa Xaa Xaa Xaa1 5
149PRTArtificial SequenceSequence motif 14Xaa Xaa Xaa Xaa Glu Asn
Gly Xaa Xaa1 5 1518PRTArtificial SequenceSequence motif 15Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Asp Xaa1 5 10 15
Xaa Xaa1619DNAArtificial SequenceSynthesized Construct 16atctgggaag
cgaacaaag 191718DNAArtificial SequenceSynthesized Construct
17tagcggtcgt cggaatag 181818DNAArtificial SequenceSynthesized
Construct 18cccatcacca ctactacc 181918DNAArtificial
SequenceSynthesized Construct 19ccagccctga acaccaag
182018DNAArtificial SequenceSynthesized Construct 20tgatcttacc
gactacct 182118DNAArtificial SequenceSynthesized Construct
21cagagcttct ccttgatg 1822533PRTSporotrichum thermophile 22Met Gly
Leu Ser Thr Lys Ile Leu Gln Lys Ile Val Arg Asn Glu Ala1 5 10 15
Met Ala Ser Asp Pro Pro Glu Ile Tyr Gly Trp Arg Val Tyr Leu Leu 20
25 30 Ala Cys Ser Ala Cys Phe Gly Ala Met Ser Phe Gly Trp Asp Ser
Ser 35 40 45 Val Ile Gly Gly Val Ile Val Leu Pro Pro Phe Ile Arg
Asp Phe Asn 50 55 60 Leu Gly Asp Pro Lys Ser Gln Ala Ser Ala Asn
Leu Ser Ala Asn Ile65 70 75 80 Val Ser Thr Leu Gln Ala Gly Cys Phe
Leu Gly Ala Leu Val Ala Ser 85 90 95 Pro Met Thr Asp Arg Phe Gly
Arg Lys Trp Cys Leu Ile Gly Val Ser 100 105 110 Leu Ile Ile Ile Ile
Gly Ile Ile Met Gln Ala Ala Ala Ser Gly Asn 115 120 125 Leu Gly Pro
Met Tyr Ala Gly Arg Phe Ile Ala Gly Ala Gly Val Gly 130 135 140 Ala
Ala Ser Thr Ile Asn Pro Ile Tyr Val Ser Glu Asn Ala Pro Arg145 150
155 160 Ala Ile Arg Gly Leu Leu Thr Gly Leu Tyr Gln Leu Phe Ile Val
Thr 165 170 175 Gly Gly Met Ile Ala Phe Trp Ile Asn Tyr Ser Val Ser
Ile His Phe 180 185 190 Pro Glu Thr Lys Ile Met Tyr Val Phe Pro Leu
Ala Ile Gln Ala Leu 195 200 205 Pro Ala Ala Leu Leu Cys Leu Cys Met
Leu Leu Cys Gln Glu Ser Pro 210 215 220 Arg Trp Leu Ala Arg Arg Asp
Arg Trp Glu Asp Thr Lys Arg Val Leu225 230 235 240 Ser Arg Ile Arg
Asn Leu Pro Pro Asp His Pro Tyr Ile Gln Asp Glu 245 250 255 Phe Gln
Glu Ile Val Ala Gln Leu Glu His Glu Arg Arg Leu Ile Gly 260 265 270
Asp Ala Ser Phe Trp Asn Leu Gln Arg Glu Met Trp Thr Ile Ala Gly 275
280 285 Asn Arg Arg Arg Val Leu Ile Ser Ile Ile Leu Met Ile Cys Gln
Gln 290 295 300 Met Thr Gly Thr Asn Ala Ile Asn Thr Tyr Ala Pro Thr
Ile Phe Lys305 310 315 320 Asn Leu Gly Leu Thr Gly Thr Ser Thr Ser
Leu Phe Ser Thr Gly Val 325 330 335 Tyr Gly Ile Val Lys Val Thr Ser
Cys Ile Ile Phe Leu Leu Phe Met 340 345 350 Ala Asp Ser Leu Gly Arg
Arg Arg Ser Leu Leu Trp Thr Ser Ile Ala 355 360 365 Gln Gly Leu Ala
Met Phe Tyr Ile Gly Leu Tyr Val Arg Ile Ala Pro 370 375 380 Pro Lys
Glu Gly Glu Ser Val Pro Pro Ala Gly Tyr Phe Ala Leu Val385 390 395
400 Cys Ile Phe Leu Phe Ala Ala Phe Phe Gln Phe Gly Trp Gly Pro Ala
405 410 415 Cys Trp Ile Tyr Ala Ser Glu Ile Pro Ala Ala Arg Leu Arg
Ser Leu 420 425 430 Asn Val Ala Tyr Ala Ala Ala Thr Gln Trp Leu Phe
Asn Phe Val Val 435 440 445 Ala Arg Thr Val Pro Val Met Ile Val Thr
Met Gly Glu Gly Gly Tyr 450 455 460 Gly Thr Tyr Leu Leu Phe Gly Ser
Phe Cys Phe Ser Met Phe Val Phe465 470 475 480 Val Trp Phe Phe Val
Pro Glu Thr Lys Gly Val Ser Leu Glu Ala Met 485 490 495 Asp Lys Leu
Phe Gly Val Thr Asp Glu Ser Ser Lys Ser Leu Thr Val 500 505 510 Asp
Glu Asp Ala Lys Glu Lys Glu Lys Asp Gly Pro His Ala Arg Gln 515 520
525 Thr Glu Val Val Ala 530 23512PRTSporotrichum thermophile 23Met
Lys Lys Phe Leu Gly Leu Arg Gly Gln Ala Leu Asn Leu Ala Val1 5 10
15 Gly Thr Ile Ala Gly Cys Asp Phe Leu Leu Phe Gly Tyr Asp Gln Gly
20 25 30 Val Met Gly Gly Ile Leu Thr Leu Lys Val Phe Leu Asp Ala
Phe Pro 35 40 45 Met Ile Asn Pro Glu Ala Ala Gly Leu Ser His Asp
Glu Ser Ser Thr 50 55 60 Arg Ser Thr Tyr Gln Gly Ile Ala Val Ala
Ser Tyr Asn Leu Gly Cys65 70 75 80 Phe Leu Gly Ala Ile Ile Thr Ile
Phe Ile Gly Asn Pro Leu Gly Arg 85 90 95 Lys Arg Val Ile Met Leu
Gly Thr Ser Val Met Val Ile Gly Ala Ile 100 105 110 Leu Gln Ala Ser
Ser Thr Thr Leu Pro Gln Phe Ile Val Gly Arg Ile 115 120 125 Ile Thr
Gly Leu Gly Asn Gly Gly Asn Thr Ser Thr Val Pro Thr Trp 130 135 140
Gln Ser Glu Thr Ser Lys Ala His Lys Arg Gly Lys Met Ile Phe Phe145
150 155 160 Cys Ala Ile Ile Leu Ala Phe Ile Pro Phe Leu Pro Glu Ser
Pro Arg 165 170 175 Trp Leu Ile Leu Lys Gly Arg Glu Asp Glu Ala Arg
Glu Val Ile Ala 180 185 190 Ala Leu Glu Asp Thr Asp Thr Ser Asp Arg
Ile Val Glu Asn Glu Phe 195 200 205 Leu Ala Ile Lys Glu Thr Val Leu
Glu Met Ser Lys Gly Thr Phe Arg 210 215 220 Asp Leu Phe Thr Met Asp
Lys Asn Arg Asn Leu His Arg Thr Leu Leu225 230 235 240 Ala Tyr Phe
Asn Gln Val Phe Gln Gln Ile Ser Gly Ile Asn Leu Ile 245 250 255 Thr
Tyr Tyr Ala Ala Val Ile Tyr Lys Gly Leu Gly Met Ser Asp Phe 260 265
270 Leu Ser Arg Leu Leu Ala Ala Leu Asn Gly Thr Glu Tyr Phe Leu Ala
275 280 285 Ser Trp Pro Ala Val Phe Leu Val Glu Arg Val Gly Arg Arg
Asn Leu 290 295 300 Met Leu Phe Gly Ala Val Gly Gln Ala Ala Thr Met
Ala Ile Leu Ala305 310 315 320 Gly Val Asn Ser Arg Gln Glu Thr Gly
Phe Gln Ile Ala Gly Ile Val 325 330 335 Phe Leu Phe Val Phe Asn Thr
Phe Phe Ala Val Gly Trp Leu Gly Met 340 345 350 Thr Trp Leu Tyr Pro
Ala Glu Ile Val Pro Leu Arg Ile Arg Ala Pro 355 360 365 Ala Asn Ala
Leu Ser Thr Ser Ala Asn Trp Ile Phe Asn Phe Leu Val 370 375 380 Val
Met Ile Thr Pro Val Ala Phe Asn Asn Ile Gly Tyr Gln Thr Tyr385 390
395 400 Ile Ile Phe Ala Val Ile Asn Ala Phe Met Val Pro Cys Val Tyr
Phe 405 410 415 Phe Tyr Pro Glu Thr Ala Tyr Arg Ser Leu Glu Glu Met
Asp Asn Ile 420 425 430 Phe His Lys Val Ala Asp Gly Trp Lys Gly Val
Phe Thr Val Val His 435 440 445 Gln Ala Lys Val Glu Pro Arg Trp Tyr
Gly Lys Asn Gly Glu Leu Leu 450 455 460 Val Asp Tyr Gln Gln Thr Glu
Glu His Arg Arg His Leu Gln Gln Gln465 470 475 480 Glu Gly Ala Val
Thr Ala Ser Glu Lys Arg Ser Val Glu Gly Ala Gly 485 490 495 Ser Gly
Ser Gly Ser Gly Asp Val Lys Gln Asp Glu Tyr Lys Asp Cys 500 505 510
24519PRTSporotrichum thermophile 24Met Glu Ser Thr His Glu Pro Ala
Asp Pro Ile Ala Lys Gly Val Leu1 5 10 15 Ala Thr Ala Lys Gln Ser
Trp His Asp Leu Phe Ile Phe Lys Gln Arg 20 25 30 Val Val Val Thr
Asn Glu Leu Gly Glu Thr Ser Thr Glu Trp Ala Arg 35 40 45 Pro Val
Pro Leu Arg Asn Pro Ile Ser Leu Leu Ala Gln Leu Ser Ala 50 55 60
Arg Asn Trp Leu Phe Phe Ile Val Gly Phe Leu Ala Trp Val Ala Asp65
70 75 80 Ala Tyr Asp Phe His Ala Leu Ser Ile Gln Gln Val Lys Leu
Ala Glu 85 90 95 Phe Tyr Asn Thr Thr Lys Thr Asn Ile Ser Thr Ala
Ile Thr Leu Thr 100 105 110 Leu Leu Leu Arg Ser Val Gly Ala Ala Phe
Phe Gly Leu Ala Gly Asp 115 120 125 Lys Trp Gly Arg Lys Trp Pro Met
Val Ala Asn Met Ile Val Leu Gly 130 135 140 Val Leu Gln Ile Gly Thr
Ile Tyr Ser Val Thr Phe Ser Asp Phe Leu145 150 155 160 Ala Val Arg
Ala Leu Phe Gly Leu Phe Met Gly Gly Val Tyr Gly Asn 165 170 175 Ala
Ile Ala Met Ala Leu Glu Asn Ser Pro Pro Asp Ala Arg Gly Leu 180 185
190 Met Ser Gly Ile Leu Gln Gln Gly Tyr Ser Leu Gly Tyr Val Ile Ala
195 200 205 Ala Cys Ala Asn Leu Gly Val Gly Gly Gly Asp Asn Ser Trp
Lys Thr 210 215 220 Val Phe Trp Ile Gly Ala Gly Leu Ser Ile Gly Val
Gly Leu Val Arg225 230 235 240 Cys Phe Phe Pro Glu Ser Gln Gln Phe
Leu Glu Ala Arg Ala Ala Gly 245 250 255 Lys Gly Gln Ala Ser Ala Ser
Ala Phe Trp Lys Glu Thr Lys Met Met 260 265 270 Leu Ala Gln Glu Trp
Lys Met Cys Val Tyr Cys Ile Ile Leu Met Thr 275 280 285 Trp Phe Asn
Tyr Tyr Ser His Thr Ser Gln Asp Ser Tyr Thr Thr Phe 290 295 300 Met
Leu Thr Gln Lys Glu Leu Asp Asn Asp Gly Ala Ser Arg Ala Ser305 310
315 320 Ile Leu Met Lys Val Gly Ala Cys Val Gly Gly Thr Ile Ile Gly
Tyr 325 330 335 Ile Ser Gln Trp Phe Gly Arg Arg Arg Thr Ile Ile Val
Ala Ala Leu 340 345 350 Ile Ser Gly Leu Ile Ile Pro Ala Trp Ile Leu
Pro Glu Gly Glu Arg 355 360 365 Ser Leu Ser Val Thr Gly Phe Phe Met
Gln Phe Phe Val Gln Gly Ala 370 375 380 Trp Gly Val Ile Pro Ile His
Leu Asn Glu Leu Ser Pro Pro Ala Phe385 390 395 400 Arg Ser Ser Phe
Pro Gly Leu Thr Tyr Gln Leu Gly Asn Met Ile Ser 405 410 415 Ser Pro
Ser Ala Gln Ile Val Asn Ala Ile Ala Glu Ser His Ser Val 420 425 430
Thr Ser Lys Ser Gly Lys Ser Val Asn Ala Tyr Gly Pro Thr Met Gly 435
440 445 Ile Ala Thr Ala Ile Ile Ala Thr Gly Ile Ala Val Thr Thr Ala
Leu 450 455 460 Gly Pro Glu Lys Arg Gly Arg Glu Phe Glu Lys Thr Leu
Pro Ala Gly465 470 475 480 Met Asn Ile Ile Gln Gly Gly Lys Ala Val
Asp Asp Leu Glu Lys Gly 485 490 495 Asp Ser Arg Asp Glu Lys Pro Val
Val Gly Glu Val Glu Gly Gly Asn 500 505 510 Asp Gly Ser Gly Glu Lys
Lys 515 25488PRTSporotrichum thermophile 25Met Ala Asp Glu Lys Arg
Met Gly Ser Ser Asp Ser Asp Lys Ala Ala1 5 10 15 Val Gln His Ser
Glu Thr Leu Pro Gly Val Ser Ser Thr Ala Ala Glu 20 25 30 Arg Gly
Phe Ala Ala Thr Asp Gln Asn Gly Gln Pro Ile Val Gln Phe 35 40 45
Asp Leu Lys Ala Glu Ala Arg Leu Arg Arg Lys Leu Asp Leu Phe Ile 50
55 60 Val Pro Thr Val Ser Leu Leu Tyr Leu Phe Cys Phe Ile Asp Arg
Ala65 70 75 80 Asn Ile Gly Asn Ala Arg Ile Ala Gly Leu Glu Lys Asp
Leu Asn Leu 85 90 95 Thr Gly Tyr Asp Tyr Asn Ala Leu Leu Ser Val
Phe Tyr Ile Ser Tyr 100 105 110 Ile Val Phe Glu Ile Pro Ser Asn Ile
Ala Cys Lys Trp Ile Gly Pro 115 120 125 Gly Trp Phe Ile Pro Ala Ile
Ser Leu Gly Phe Gly Val Val Ser Leu 130 135 140 Ala Thr Ala Phe Val
Asp Asn Phe Ala Gln Ala Ala Gly Val Arg Phe145 150 155 160 Leu Leu
Gly Val Phe Glu Ala Gly Met Met Pro Gly Ile Ala Tyr Tyr 165 170 175
Leu Ser Arg Trp Tyr Arg Arg Ala Glu Leu Thr Phe Arg Leu Ser Leu 180
185 190 Tyr Ile Val Met Ala Pro Met Ala Gly Ala Phe Gly Gly Leu Leu
Ala 195 200 205 Ser Gly Ile Leu Ser Leu Asp His Val Gly Gly Val Thr
Gly Trp Arg 210 215 220 Met Ile Phe Val Val Glu Gly Ile Ile Thr Ile
Gly Leu Ser Val Ile225 230 235 240 Ser Phe Ile Thr Leu Thr Asp Arg
Pro Glu Thr Ala Arg Trp Leu Thr 245 250 255 Gln Glu Glu Lys Asp Leu
Ala Ile Ala Arg Val Lys Ser Glu Arg Val 260 265 270 Ala Thr Thr Glu
Val Leu Asp Arg Met Asp Thr Lys Lys Leu Ile Gln 275 280 285 Gly Ile
Leu Ser Pro Val Thr Leu Ala Thr Ser Phe Met Phe Leu Leu 290 295 300
Asn Asn Ile Thr Gln Leu Phe Thr Val Pro Pro Tyr Val Val Gly Gly305
310 315 320 Phe Phe Thr Leu Ala Leu Pro Leu Leu Ser Trp Tyr Leu Asp
Arg Arg 325 330 335 Gln Ile Ile Ile Leu Leu Ser Thr Pro Leu Val Ile
Val Gly Tyr Ser 340 345 350 Met Phe Leu Gly Thr Thr Asn Pro Ser Ala
Arg Tyr Gly Ala Thr Phe 355
360 365 Leu Leu Ser Ser Ser Leu Phe Ala Val Gly Ala Leu Ser Asn Ser
Gln 370 375 380 Val Ser Ala Asn Val Val Ser Asp Thr Ala Arg Ser Ser
Ala Ile Gly385 390 395 400 Leu Asn Val Met Met Gly Asn Val Gly Gly
Leu Ile Ala Thr Trp Ser 405 410 415 Tyr Leu Pro Trp Asp Gly Pro Asn
Tyr Lys Ile Gly Asn Gly Leu Asn 420 425 430 Leu Ala Ala Cys Cys Thr
Val Leu Ile Leu Ser Ala Val Thr Leu Leu 435 440 445 Trp Met Lys Trp
Asp Asn Arg Arg Arg Glu Gly Arg Asn Ala Glu Glu 450 455 460 Glu Leu
Ala Gly Met Ser Arg Gln Glu Ile Gln Asp Leu Asp Trp Lys465 470 475
480 His Pro Ala Phe Arg Trp Arg Pro 485 26546PRTSporotrichum
thermophile 26Met Pro Lys Ala Arg Ser Arg Val Pro Val Lys Val Asn
Val Gly Thr1 5 10 15 Ser Ala Asp Pro Ile Val Thr Arg Leu Val Gln
Glu Asp Lys Ile Pro 20 25 30 Trp Tyr Lys Lys Pro Asn Leu Arg Arg
Met Tyr Ile Phe Leu Phe Leu 35 40 45 Cys Cys Met Gly Val Glu Met
Thr Ser Gly Phe Asp Ser Gln Leu Ile 50 55 60 Asn Thr Leu Gln Tyr
Ala Glu Thr Phe His Lys Tyr Leu Gly Asn Gly65 70 75 80 Arg Lys Asp
Glu Asp Gly Asn Tyr Ala Ile Glu Pro Gly Leu Leu Gly 85 90 95 Phe
Val Asn Ser Cys Tyr Gln Leu Gly Ser Ile Phe Ala Val Pro Ile 100 105
110 Ala Pro Trp Phe Ala Gln Arg Phe Gly Arg Arg Trp Ser Ile Met Leu
115 120 125 Gly Ser Leu Ile Met Val Gly Gly Ala Ile Ile Gln Gly Phe
Ala Gln 130 135 140 His Val Ala Met Tyr Ile Ile Ala Arg Met Ile Leu
Gly Met Gly Ile145 150 155 160 Leu Phe Cys Ile Ile Ser Gly Ala Ala
Leu Ile Gly Glu Leu Gly His 165 170 175 Pro Lys Glu Arg Ala Val Leu
Thr Ser Leu Phe Asn Ser Ser Tyr Phe 180 185 190 Ile Gly Gln Ile Leu
Ala Ser Ala Ile Thr Ile Gly Thr Thr Glu Met 195 200 205 Lys Thr Asn
Trp Ala Trp Arg Leu Pro Ser Leu Leu Gln Ile Cys Pro 210 215 220 Ser
Leu Leu Gln Ile Val Thr Val Phe Phe Leu Pro Glu Ser Pro Arg225 230
235 240 Phe Leu Ile Ser Lys Asp Arg Asp Asp Asp Ala Lys Glu Val Leu
Ile 245 250 255 Lys Tyr His Ala Glu Gly Asp Ala Ser Ser Leu Leu Val
Gln Ala Glu 260 265 270 Ile Val Gln Ile Arg Glu Thr Ile Arg Thr Glu
Met Glu Val Ser Asn 275 280 285 Gln Ser Trp Met Glu Leu Val Ser Thr
Tyr Gly Met Arg Arg Arg Leu 290 295 300 Val Ile Thr Leu Phe Ile Gly
Leu Phe Thr Gln Leu Ser Gly Asn Thr305 310 315 320 Leu Leu Ser Tyr
Tyr Ser Gly Lys Leu Phe Glu Met Met Gly Tyr Thr 325 330 335 Glu Ala
Ser Val Lys Thr Arg Ile Asn Val Ala Asn Ala Cys Trp Ser 340 345 350
Leu Leu Asn Ala Thr Thr Ile Ala Phe Leu Val Pro Tyr Phe Lys Arg 355
360 365 Arg His Met Phe Met Thr Ser Ala Leu Ser Met Cys Ala Val Phe
Ile 370 375 380 Ala Ile Thr Val Ser Leu Glu Arg Thr Gln Ala Ala Gln
Asp Ala Gly385 390 395 400 Phe Lys Asn Thr Ala Ala Gly Ile Ser Gly
Leu Phe Trp Tyr Phe Ala 405 410 415 Phe Ala Pro Cys Tyr Asn Met Gly
Asn Asn Ala Leu Thr Tyr Thr Tyr 420 425 430 Leu Val Glu Leu Trp Pro
Tyr Ser His Arg Ser Arg Gly Ile Gly Val 435 440 445 Gln Gln Ile Phe
Gly Lys Leu Gly Gly Phe Phe Ser Thr Asn Val Asn 450 455 460 Ser Ile
Ala Leu Asp Ala Ile Arg Trp Lys Tyr Met Ala Ile Tyr Cys465 470 475
480 Gly Trp Ile Phe Phe Glu Phe Leu Ile Val Phe Phe Leu Tyr Pro Glu
485 490 495 Thr Ser Gly Arg Thr Leu Glu Glu Leu Ala Phe Leu Phe Glu
Asp Ala 500 505 510 Ser Leu Asn Glu Lys Ala Ala Ala Ala Val Glu Lys
Gln Ile His Tyr 515 520 525 Gly Asp Glu Lys Val Val His Glu Glu Gly
Gln Pro Ala Ala Lys Ser 530 535 540 Val Val545 27481PRTSporotrichum
thermophile 27Met Leu Ser Ser Gly Phe Trp Lys Arg Arg Ser Leu Arg
Val Pro Asp1 5 10 15 Asn Gln Arg Thr Lys Ala Ala Glu Leu Thr Leu
Arg Glu Ser Leu Tyr 20 25 30 Pro Leu Ser Leu Val Thr Ile Leu Phe
Phe Leu Trp Gly Phe Ser Tyr 35 40 45 Gly Leu Leu Asp Thr Leu Asn
Lys His Phe Gln Asn Thr Leu Gly Ile 50 55 60 Thr Lys Thr Arg Ser
Ser Gly Leu Gln Ala Ala Tyr Phe Gly Ala Tyr65 70 75 80 Pro Leu Ala
Ser Leu Gly His Ala Ala Trp Ile Leu Arg His Tyr Gly 85 90 95 Tyr
Arg Ala Val Phe Ile Trp Gly Leu Phe Leu Tyr Gly Leu Gly Ala 100 105
110 Leu Leu Ala Ile Pro Ser Ile Met His His Ser Phe Ala Gly Phe Cys
115 120 125 Val Cys Ile Phe Ile Ile Gly Asn Gly Leu Gly Ser Leu Glu
Thr Ala 130 135 140 Ala Asn Pro Tyr Ile Thr Val Cys Gly Pro Pro Lys
Phe Ser Glu Ile145 150 155 160 Arg Ile Asn Val Ala Gln Ala Phe Asn
Gly Ile Gly Thr Val Val Ala 165 170 175 Pro Val Leu Gly Ser Tyr Val
Phe Phe Thr Phe Asp Asp Gln Thr Ala 180 185 190 Leu Arg Asn Val Gln
Trp Val Tyr Leu Ala Ile Ala Cys Phe Val Phe 195 200 205 Leu Leu Ala
Gly Val Phe Phe Leu Ser Val Ile Pro Glu Ile Thr Asp 210 215 220 Ala
Asp Met Ala Phe Gln Ala Ala Glu Thr His Ala Gly Ala Asp Asp225 230
235 240 Arg Pro Phe His Thr Gln Tyr Arg Leu Phe His Ala Ala Phe Ala
Gln 245 250 255 Phe Cys Tyr Thr Gly Ala Gln Val Ala Ile Ala Gly Tyr
Phe Ile Asn 260 265 270 Tyr Ala Thr Glu Thr Arg Pro Asn Thr Asp Ser
Ser Leu Gly Ser Lys 275 280 285 Phe Leu Ala Gly Ser Gln Ala Gly Phe
Ala Val Gly Arg Phe Gly Gly 290 295 300 Ala Ala Met Met Gln Phe Ile
Lys Pro Arg Lys Val Phe Ala Leu Phe305 310 315 320 Met Thr Met Cys
Ile Val Phe Ser Ala Pro Ala Ile Thr Gln Arg Gly 325 330 335 Asn Ala
Gly Leu Ser Met Leu Tyr Leu Val Met Phe Phe Glu Ser Ile 340 345 350
Cys Phe Pro Thr Ile Ile Ala Leu Gly Met Arg Gly Leu Gly Arg His 355
360 365 Thr Lys Arg Gly Ser Gly Trp Ile Val Ala Gly Val Leu Gly Gly
Ala 370 375 380 Cys Val Pro Pro Leu Met Gly Ala Ala Ala Asp Ala Arg
Gly Thr Gly385 390 395 400 Phe Ser Met Leu Val Pro Leu Cys Phe Phe
Val Ala Ala Trp Thr Tyr 405 410 415 Ala Leu Ala Val Asn Phe Ala Pro
Pro Tyr Arg Ser Val Val Asp Ala 420 425 430 Phe Ser Thr Thr Asp Val
Gly Leu Arg Glu Lys Gln Arg Glu Asp Val 435 440 445 Gly Ala Glu Lys
Gly Gly Glu Ala Gly Gly Lys Gly Gly Val Thr Gly 450 455 460 Pro Glu
Asp Ala Ser Glu Asp Lys Pro Asp Val Val Asn Ser Glu Lys465 470 475
480 Val28419PRTSporotrichum thermophile 28Met Leu Ser Ser Leu Arg
Ile Ala Ser Arg Arg Ala Ala Val Ala Arg1 5 10 15 Asn Phe Ser Ala
Val Arg Ala Ala Ser Thr Trp Ala Asn Val Pro Gln 20 25 30 Gly Pro
Pro Val Cys Ile Thr Glu Ala Phe Lys Ala Asp Pro Phe Glu 35 40 45
Lys Lys Ile Asn Leu Gly Val Gly Ala Tyr Arg Asp Asp Lys Gly Lys 50
55 60 Pro Tyr Val Leu Pro Ser Val Arg Lys Ala Glu Glu Lys Val Ile
Ala65 70 75 80 Ser Arg Leu Asn Lys Glu Tyr Ala Gly Ile Thr Gly Val
Pro Glu Phe 85 90 95 Thr Lys Ala Ala Ala Val Leu Ala Tyr Gly Lys
Asp Ser Ser Ala Leu 100 105 110 Asp Arg Leu Ala Ile Thr Gln Ser Ile
Ser Gly Thr Gly Ala Leu Arg 115 120 125 Ile Gly Ala Ala Phe Leu Ser
Arg Phe Tyr Pro Gly Ala Lys Thr Ile 130 135 140 Tyr Ile Pro Thr Pro
Ser Trp Ala Asn His Ala Ala Val Phe Lys Asp145 150 155 160 Ser Gly
Leu Gln Val Glu Lys Tyr Ala Tyr Tyr Asn Lys Asp Thr Ile 165 170 175
Arg Leu Asp Phe Glu Gly Met Ile Ala Asp Ile Asn Lys Ala Pro Asn 180
185 190 Gly Ser Ile Phe Leu Phe His Ala Cys Ala His Asn Pro Thr Gly
Val 195 200 205 Asp Pro Thr Gln Glu Gln Trp Lys Glu Ile Glu Ala Ala
Val Lys Ala 210 215 220 Lys Gly His Phe Ala Phe Phe Asp Met Ala Tyr
Gln Gly Phe Ala Ser225 230 235 240 Gly Asp Ile His Arg Asp Ala Phe
Ala Val Arg Tyr Phe Val Glu Lys 245 250 255 Gly His Asn Ile Cys Leu
Ala Gln Ser Phe Ala Lys Asn Met Gly Leu 260 265 270 Tyr Gly Glu Arg
Thr Gly Ala Phe Ser Ile Val Cys Ala Asp Ala Glu 275 280 285 Glu Arg
Lys Arg Val Asp Ser Gln Ile Lys Ile Leu Val Arg Pro Met 290 295 300
Tyr Ser Asn Pro Pro Ile His Gly Ala Arg Ile Ala Ala Glu Ile Leu305
310 315 320 Asn Thr Pro Glu Leu Tyr Asp Gln Trp Leu Val Glu Val Lys
Glu Met 325 330 335 Ala Asn Arg Ile Ile Thr Met Arg Ala Leu Leu Lys
Glu Asn Leu Glu 340 345 350 Lys Leu Gly Ser Lys His Asp Trp Ser His
Ile Thr Ser Gln Ile Gly 355 360 365 Met Phe Ala Tyr Thr Gly Leu Thr
Pro Glu Gln Met Glu Lys Leu Ala 370 375 380 Lys Glu His Ser Val Tyr
Ala Thr Arg Asp Gly Arg Ile Ser Val Ala385 390 395 400 Gly Ile Thr
Thr Asp Asn Val Gly Arg Leu Ala Glu Ala Ile Phe Lys 405 410 415 Val
Lys Gly 29522PRTSporotrichum thermophile 29Met Gly Ile Phe Ala Phe
Asn Lys Gln Lys Pro Asn Ala Glu Ala Thr1 5 10 15 Ala Val Ala Gln
Glu Glu Ala Pro Gln Phe Glu Arg Val Asp Trp Lys 20 25 30 Arg Asp
Pro Gly Leu Arg Lys Leu Tyr Phe Tyr Ala Phe Val Leu Cys 35 40 45
Ile Ala Ser Ala Thr Thr Gly Tyr Asp Gly Met Phe Phe Asn Ser Val 50
55 60 Gln Asn Phe Glu Thr Trp Glu Asn Tyr Phe Asn His Pro Thr Gly
Ser65 70 75 80 Lys Leu Gly Val Leu Gly Ala Leu Tyr Gln Ile Gly Ser
Leu Ala Ser 85 90 95 Ile Pro Leu Val Pro Ile Ile Ala Asp Arg Val
Gly Arg Lys Ile Pro 100 105 110 Ile Ala Ile Gly Cys Val Ile Met Ile
Val Gly Ala Val Leu Gln Ala 115 120 125 Ala Cys Arg Asn Leu Gly Thr
Phe Met Gly Gly Arg Phe Leu Leu Gly 130 135 140 Phe Gly Asn Ser Leu
Ala Gln Leu Cys Ser Pro Met Leu Leu Thr Glu145 150 155 160 Leu Ala
His Pro Gln His Arg Gly Arg Leu Thr Thr Val Tyr Asn Cys 165 170 175
Leu Trp Asn Val Gly Ala Leu Val Val Ala Trp Val Ser Phe Gly Thr 180
185 190 Asp Tyr Leu Lys Ser Asp Trp Ser Trp Arg Ile Pro Ala Leu Ile
Gln 195 200 205 Ala Phe Pro Ser Val Ile Gln Leu Leu Phe Ile Phe Trp
Val Pro Glu 210 215 220 Ser Pro Arg Tyr Leu Met Ala Lys Asp Lys His
Glu Arg Ala Leu Ala225 230 235 240 Ile Leu Ala Lys Tyr His Ala Asn
Gly Asp Ala Asn His Pro Thr Val 245 250 255 Gln Phe Glu Tyr Arg Glu
Ile Lys Glu Thr Leu Arg Leu Glu Phe Glu 260 265 270 Ala Ser Lys Ser
Ser Ser Tyr Leu Asp Phe Val Arg Thr Arg Gly Asn 275 280 285 Arg Tyr
Arg Leu Ala Val Leu Ile Ser Leu Gly Ile Phe Ser Gln Trp 290 295 300
Ser Gly Asn Ala Ile Ile Ser Asn Tyr Ser Ser Lys Leu Tyr Asp Thr305
310 315 320 Ala Gly Val Thr Gly Ser Thr Gln Lys Leu Gly Leu Ser Ala
Gly Gln 325 330 335 Thr Gly Leu Ser Leu Ile Ile Ser Val Thr Met Ala
Leu Leu Val Asp 340 345 350 Lys Phe Gly Arg Arg Pro Met Phe Leu Thr
Ser Thr Ala Gly Met Phe 355 360 365 Cys Thr Phe Ile Phe Trp Thr Leu
Thr Ser Gly Leu Tyr Glu Glu His 370 375 380 Asn Ala Asp Gly Ala Arg
Tyr Ala Met Ile Leu Phe Ile Trp Ile His385 390 395 400 Gly Ile Phe
Tyr Ser Ile Ser Trp Ser Gly Leu Leu Val Gly Tyr Ala 405 410 415 Ile
Glu Val Leu Pro Tyr Lys Leu Arg Ala Lys Gly Leu Met Ile Met 420 425
430 Asn Leu Thr Val Gln Ala Ala Leu Thr Leu Asn Thr Tyr Ala Asn Pro
435 440 445 Val Ala Phe Asp Ala Phe Glu Gly His Ser Trp Lys Leu Tyr
Ile Ile 450 455 460 Tyr Thr Ile Trp Ile Phe Leu Glu Leu Cys Phe Val
Trp Lys Met Tyr465 470 475 480 Ile Glu Thr Lys Gly Pro Thr Leu Glu
Glu Leu Ala Lys Ile Ile Asp 485 490 495 Gly Asp Glu Ala Ala Val Ala
His Val Asp Ile Lys Gln Val Glu Lys 500 505 510 Glu Thr His Ile Asn
Glu Glu Lys Ser Val 515 520 30554PRTSporotrichum thermophile 30Met
Ser Ser Ser Glu Lys Glu Ala Thr Gly Pro Val Ala Ala His Val1 5 10
15 Gly Asn Leu Ala Thr Thr Gln Asp Val Glu Lys Ile Glu Ala Pro Val
20 25 30 Thr Trp Lys Ala Tyr Leu Ile Cys Ala Phe Ala Ser Phe Gly
Gly Ile 35 40 45 Phe Phe Gly Tyr Asp Ser Gly Tyr Ile Asn Gly Val
Leu Ala Ser Lys 50 55 60 Leu Phe Ile Asn Ala Val Glu Gly Ala Gly
Lys Asp Ala Ile Ser Glu65 70 75 80 Ser His Ser Ser Leu Ile Val Ser
Ile Leu Ser Cys Gly Thr Phe Phe 85 90 95 Gly Ala Leu Ile Ala Gly
Asp Leu Ala Asp Phe Ile Gly Arg Lys Tyr 100 105 110 Thr Val Ile Leu
Gly Cys Leu Ile Tyr Ile Ile Gly Cys Val Ile Gln 115 120 125 Ile Ile
Thr Gly Leu Gly Asn Ala Leu Gly Ala Ile Val Ala Gly Arg 130 135 140
Leu Ile Ala Gly Ile Gly Val Gly Phe Glu Ser Ala Ile Val Ile Leu145
150 155 160 Tyr Met Ser Glu Ile Cys Pro Arg Lys Val Arg Gly Ala Leu
Val Ala 165 170 175 Gly Tyr Gln Phe Cys Ile Thr Ile Gly Leu Met Leu
Ala Ser Cys Val 180 185 190 Val Tyr Gly Thr Gln Asn Arg Gln Asp
Thr Gly Gln Tyr Arg Ile Pro 195 200 205 Ile Gly Ile Gln Phe Ile Trp
Ala Leu Ile Leu Gly Gly Gly Leu Leu 210 215 220 Cys Leu Pro Asp Ser
Pro Arg Tyr Phe Val Lys Arg Gly Arg Leu Ala225 230 235 240 Asp Ala
Thr Ser Ala Leu Ser Arg Leu Arg Gly Gln Pro Glu Asp Ser 245 250 255
Glu Tyr Ile Gln Val Glu Leu Ala Glu Ile Val Ala Asn Glu Glu Tyr 260
265 270 Glu Arg Gln Leu Ile Pro Ser Thr Thr Trp Phe Gly Ser Trp Ala
Asn 275 280 285 Cys Phe Lys Gly Ser Leu Phe Lys Ala Asn Ser Asn Leu
Arg Lys Thr 290 295 300 Ile Leu Gly Thr Ser Leu Gln Met Met Gln Gln
Trp Thr Gly Val Asn305 310 315 320 Phe Ile Phe Tyr Tyr Ser Thr Pro
Phe Leu Lys Ser Thr Gly Ala Ile 325 330 335 Asp Asp Pro Phe Leu Met
Ser Met Val Phe Thr Ile Ile Asn Val Phe 340 345 350 Ser Thr Pro Ile
Ser Phe Tyr Thr Val Glu Arg Phe Gly Arg Arg Thr 355 360 365 Ile Leu
Phe Trp Gly Ala Leu Gly Met Leu Ile Cys Gln Phe Leu Val 370 375 380
Ala Ile Val Gly Val Thr Val Gly Phe Asn His Thr His Pro Ala Pro385
390 395 400 Thr Ala Asp Asp Pro Glu Ala Thr Leu Ala Asn Asn Ile Ser
Ala Val 405 410 415 Asn Ala Gln Ile Ala Phe Ile Ala Ile Phe Ile Phe
Phe Phe Ala Ser 420 425 430 Thr Trp Gly Pro Gly Ala Trp Ile Val Ile
Gly Glu Ile Phe Pro Leu 435 440 445 Pro Ile Arg Ser Arg Gly Val Gly
Leu Ser Thr Ala Ser Asn Trp Leu 450 455 460 Trp Asn Thr Ile Ile Ala
Val Ile Thr Pro Tyr Met Val Gly Glu Asp465 470 475 480 Arg Gly Asn
Met Lys Ser Ser Val Phe Phe Val Trp Gly Gly Leu Cys 485 490 495 Thr
Cys Ala Phe Val Tyr Thr Tyr Phe Leu Val Pro Glu Thr Lys Gly 500 505
510 Leu Ser Leu Glu Gln Val Asp Lys Met Met Glu Glu Thr Thr Pro Arg
515 520 525 Thr Ser Ala Lys Trp Lys Pro Thr Thr Thr Phe Ala Ala Ser
His Pro 530 535 540 Thr Asp Leu Lys Gln Gly Glu Ala Ala Val545 550
31537PRTSporotrichum thermophile 31Met Gly Thr Ser Arg Asp Glu Lys
Glu Thr Val Val Ala Asp His Ala1 5 10 15 Asp Asp Asp Ala Leu Arg
Glu Ala Asp Leu Ala Val Gln Val Ala His 20 25 30 Asp Ala Asp Gly
Thr Val Tyr Ser Pro Trp Ser Leu Arg Met Ile Arg 35 40 45 Leu Tyr
Leu Val Leu Ser Leu Ser Tyr Leu Cys Gly Cys Leu Asn Gly 50 55 60
Tyr Asp Gly Ser Leu Met Gly Gly Leu Asn Gly Met Thr Ser Tyr Gln65
70 75 80 Arg Tyr Phe His Met Ser Thr Ala Gly Ser Thr Thr Gly Leu
Ile Phe 85 90 95 Ala Met Tyr Asn Ile Gly Ser Val Ala Ala Val Phe
Phe Thr Gly Pro 100 105 110 Val Asn Asp Tyr Phe Gly Arg Arg Trp Gly
Met Phe Val Gly Ala Leu 115 120 125 Leu Val Ile Val Gly Thr Cys Val
Gln Ala Pro Cys Thr Thr Arg Gly 130 135 140 Gln Phe Leu Ala Gly Arg
Phe Val Leu Gly Phe Gly Val Ser Phe Cys145 150 155 160 Cys Val Ser
Ala Pro Cys Tyr Val Ser Glu Met Ala His Pro Lys Trp 165 170 175 Arg
Gly Thr Leu Thr Gly Leu Tyr Asn Cys Thr Trp Tyr Ile Gly Ser 180 185
190 Ile Val Ala Ser Trp Val Val Tyr Gly Cys Ser Tyr Ile Asp Thr Leu
195 200 205 Asp Ala Trp Arg Ile Pro Ile Trp Cys Gln Met Val Thr Ser
Gly Leu 210 215 220 Val Cys Leu Gly Val Phe Trp Leu Pro Glu Ser Pro
Arg Trp Leu Met225 230 235 240 Ala Gln Asp Arg His Asp Asp Ala Ala
Arg Val Leu Ala Thr Tyr His 245 250 255 Gly Glu Gly Arg Ala Asp His
Pro Leu Val Lys Leu Gln Met Gln Glu 260 265 270 Met Met Asn Gln Ile
Ser Thr Glu Ala Ser Asp Lys Lys Trp Tyr Asp 275 280 285 Tyr His Glu
Leu Trp Asn Thr His Ser Ala Arg Arg Arg Leu Ile Cys 290 295 300 Val
Ile Gly Met Ala Val Phe Gly Gln Ile Ser Gly Asn Ser Leu Ser305 310
315 320 Ser Tyr Tyr Leu Val Asn Met Leu Lys Ser Ala Gly Ile Thr Glu
Glu 325 330 335 Arg Arg Val Leu Ala Leu Asn Gly Val Asn Pro Ala Leu
Ser Phe Leu 340 345 350 Gly Ala Ile Leu Gly Ala Arg Met Thr Asp Val
Val Gly Arg Arg Pro 355 360 365 Leu Leu Leu Tyr Thr Ile Val Phe Ala
Ser Val Cys Phe Ala Val Ile 370 375 380 Thr Gly Thr Ser Lys Met Ala
Thr Asp Asp Pro Thr Arg Thr Ala Ala385 390 395 400 Ala Asn Ala Thr
Ile Ala Phe Ile Phe Ile Phe Gly Ile Val Phe Ser 405 410 415 Phe Gly
Trp Thr Pro Leu Gln Ser Met Tyr Ile Ala Glu Thr Leu Pro 420 425 430
Thr Ala Thr Arg Ala Lys Gly Thr Ala Val Gly Asn Phe Ser Ser Ser 435
440 445 Val Ala Ser Thr Ile Leu Gln Tyr Ala Ser Gly Pro Ala Phe Glu
Gly 450 455 460 Ile Gly Tyr Tyr Phe Tyr Leu Val Phe Val Phe Trp Asp
Leu Ile Glu465 470 475 480 Gly Ala Ile Met Tyr Phe Tyr Phe Pro Glu
Thr Lys Asp Arg Thr Leu 485 490 495 Glu Glu Leu Glu Glu Val Phe Ser
Ala Pro Asn Pro Val Lys Lys Ser 500 505 510 Leu Glu Lys Arg Ser Ala
Gln Thr Val Leu Asn Thr Val Gly Ala Ala 515 520 525 Gln Asn Glu Lys
Leu Ala Arg Asp Val 530 535 32566PRTSporotrichum thermophile 32Met
Ala Val Phe Ala Met Gly Trp Gln Lys Pro Asp Asn Val Ala Gly1 5 10
15 Ser Ser Ala Pro Ala Ile Met Val Gly Leu Phe Val Ala Thr Gly Gly
20 25 30 Leu Leu Phe Gly Tyr Asp Thr Gly Ala Ile Asn Gly Ile Leu
Ala Met 35 40 45 Asp Thr Phe Lys Glu Asp Phe Thr Thr Gly Tyr Thr
Asp Lys Gln Gly 50 55 60 Lys Pro Gly Leu Tyr Ala Ser Glu Val Ser
Leu Ile Val Ala Met Leu65 70 75 80 Ser Ala Gly Thr Ala Thr Gly Ala
Leu Leu Ser Ala Pro Met Gly Asp 85 90 95 Arg Trp Gly Arg Arg Leu
Ser Leu Ile Val Ala Ile Gly Val Phe Cys 100 105 110 Val Gly Ala Ile
Ile Gln Val Cys Ala Thr Asn Val Ala Met Leu Val 115 120 125 Val Gly
Arg Thr Leu Ala Gly Ile Gly Val Gly Val Val Ser Val Leu 130 135 140
Val Pro Leu Tyr Gln Ser Glu Met Ala Pro Lys Trp Ile Arg Gly Thr145
150 155 160 Leu Val Cys Ala Tyr Gln Leu Ser Ile Thr Ala Gly Leu Leu
Ala Ala 165 170 175 Ala Thr Val Asn Ile Leu Thr Tyr Lys Leu Lys Ser
Ala Ala Ala Tyr 180 185 190 Arg Ile Pro Ile Gly Leu Gln Leu Thr Trp
Ala Leu Val Leu Ala Leu 195 200 205 Gly Leu Val Ile Leu Pro Glu Thr
Pro Arg Tyr Leu Val Lys Arg Gly 210 215 220 Leu Lys Glu Ala Ala Ala
Leu Ser Leu Ser Arg Leu Arg Arg Leu Asp225 230 235 240 Ile Thr His
Pro Ala Leu Ile Glu Glu Leu Ala Glu Ile Glu Ala Asn 245 250 255 His
Glu Tyr Glu Met Ala Leu Gly Pro Asp Thr Tyr Lys Asp Ile Ile 260 265
270 Phe Gly Glu Pro His Leu Gly Arg Arg Thr Leu Thr Gly Cys Gly Leu
275 280 285 Gln Met Leu Gln Gln Leu Thr Gly Val Asn Phe Ile Met Tyr
Tyr Gly 290 295 300 Thr Thr Phe Phe Tyr Gly Ala Gly Ile Gly Asn Ala
Phe Thr Val Ser305 310 315 320 Leu Ile Met Gln Val Ile Asn Leu Val
Ser Thr Phe Pro Gly Leu Phe 325 330 335 Val Val Glu Ser Trp Gly Arg
Arg Lys Leu Leu Ile Val Gly Ser Val 340 345 350 Gly Met Ala Ile Cys
Gln Leu Leu Ile Ala Ser Phe Ala Thr Ala Ser 355 360 365 Gly Asn Asp
Asn Lys Pro Thr Gln Asn Gln Ile Leu Ile Ile Phe Val 370 375 380 Ala
Ile Tyr Ile Phe Phe Phe Ala Ala Ser Trp Gly Pro Val Val Trp385 390
395 400 Val Val Thr Ser Glu Ile Tyr Pro Leu Lys Val Arg Ala Lys Ser
Met 405 410 415 Ser Ile Ser Thr Ala Ser Asn Trp Val Leu Asn Phe Gly
Ile Ala Tyr 420 425 430 Gly Thr Pro Tyr Leu Val Asp Thr Ser Asp Gly
Ser Pro Asp Leu Gly 435 440 445 Ser Arg Val Phe Phe Val Trp Gly Ala
Phe Cys Ile Leu Ser Ile Ala 450 455 460 Phe Val Trp Tyr Met Val Tyr
Glu Thr Ser Lys Ile Ser Leu Glu Gln465 470 475 480 Ile Asp Glu Met
Tyr Glu Arg Val Ala His Ala Trp Asn Ser Arg Ser 485 490 495 Phe Glu
Pro Ser Trp Ser Phe Gln Gln Met Arg Asp Phe Gly Phe Ser 500 505 510
Asp Ser Gly Ile Pro Pro Ala Glu Pro Gln Leu Glu Leu Gln Gln Ser 515
520 525 Asn Ala Ser Thr Ser Gln Ser Asp Thr Gly Gly Ser Ser Ala Thr
His 530 535 540 Ala Thr Ala Ala Asn Pro Gln Asp Ala Lys Met Val Ser
Gln Leu Ala545 550 555 560 Asn Ile Asp Leu Ser Tyr 565
331404DNAPichia stipitis 33atgaagtatt ttcaaatctg gaaatcaggc
aaacaagtaa gctacgctgt tacattcact 60tgtgaattgg catttattct ttttggtatt
gaacagggta ttattggtaa tcttattaac 120aaccaggact tcctaaacac
ttttggaaac cccaccggta gttatttagg tattatcgtt 180tctatctata
ccttagggtg tttttttggt tgtgttatga acttcttcat tggtgatcga
240atgggcagaa gaagcaaaat tgcttcctca atgacagtta tcacaattgg
tgttgctctt 300caatgtagtt ccttttcagt tgaacaattg atgattggaa
gatttatcac tgggcttgga 360actggttggg aaacttctac ttgtccaatg
tatcaggcag aactttcacc tccaaaagtt 420agaggacgtt tggtgtgctc
agaagcattg tttgttggag ttggtttaat ctatgcatat 480tggtttgatt
atgctctttc tttcacttct ggtcctattg catggagact tcctcttgcc
540tctcagattg tgttcgcctt tgttgttttc tgtttcactt tcacaatacc
cgaatcccct 600agatacatgt tttacaaagg agagaaagaa gaagccaaaa
gaattttatc ttatgtcttt 660ggaaagccag gagatcatcc tgacattctt
aaggaatgga atgatattaa tgatgctgtt 720attttggaaa cttcagaagg
agctttctcg tgggcaaaac ttttcaagcc cgataaggca 780agaactggat
acagagtctt cttggcatac atgagcatgt ttgcgcaaca gttgagtggt
840gttaatgtag ttaattacta tattacattt gttttgatta acagtgttgg
catcgaagac 900aacttggccc taattcttgg tggtgttgcc gtcatctgtt
tcactgttgg ttcattagtt 960cctactttct ttgctgatag gatgggaaga
agattgcctt cagcagttgg agcttttggc 1020tgtggtgttt gtatgatgct
aatttcaatc ttattaagtt ttcaagacaa tccaaagttg 1080aagaagagca
gtggagctgg tgctgtggct ttctttttcg ttttccaact tgtcttcggc
1140tccactggta attgtattcc atggctgatg atttcagagc ttatccccct
tcatgcacgt 1200gctaaaggat cttcattagc tacatcaagt aactggcttt
ggaatttctt tgttgttgag 1260atcactccaa ctatcattga aaagttgaag
tggaaagcat atttgatctt tatgtgctgc 1320aacttctcct tcgtaccaat
gttttacttt ttctttcccg agacaaagaa ccttacttta 1380gaagccattg
acgatttgtt ctca 1404341653DNAPichia stipitis 34atgtcctcac
aagatttacc ctcgggtgct caaaccccaa tcgatggttc ttccatcctc 60gaagataaag
ttgagcaaag ttcgtcctca aatagccaaa gtgatttagc ttccattcca
120gcaacaggta tcaaagccta tctcttggtt tgtttcttct gcatgttggt
tgcctttggt 180ggcttcgtat tcggtttcga taccggtaca atttccggtt
tccttaatat gtctgatttc 240ctttccagat ttggtcaaga tggttctgaa
ggaaaatatt tgtctgatat cagagtcggt 300ttgattgttt ccatttttaa
cattggttgt gcaattggtg gtattttcct ttctaagata 360ggagatgttt
acggtagaag aattggtatc atttcagcta tggttgtcta cgtcgtcggt
420attatcatcc agatctcgtc ccaagacaag tggtaccaac ttacaattgg
acgtggagtt 480acaggattag ctgttggtac tgtttcagtg ttgtctccaa
tgttcattag tgaaagtgct 540ccaaagcatt tgagaggtac tttggtatac
tgttaccaat tatgtatcac cttaggtatt 600ttcattggtt actgtgtcac
ttatggaacc aaagatttaa atgattcaag acaatggaga 660gttcctttgg
gcttatgctt cctttgggct attttcttag ttgtcggtat gttggctatg
720ccagaatccc caagattctt aattgaaaag aagagaatcg aagaagccaa
gaagtccctt 780gcaagatcca acaagttatc tccggaagat ccaggtgtct
acactgaact tcaattgatt 840caggctggta ttgacagaga agctgctgca
ggttctgctt cgtggatgga attgatcact 900ggtaagccag ctattttcag
aagagttatc atgggaatta tcttgcagtc tttgcaacaa 960ttaactggtg
tcaactattt cttctattac ggaactacaa tcttccaagc tgttggtttg
1020caagattcct tccagacttc catcatctta ggtacagtca actttctttc
tacatttgtt 1080ggtatttggg ccattgaaag atttggaaga agacaatgtt
tgttagtcgg ttctgctggt 1140atgttcgttt gtttcatcat ttactccgtt
attggtacaa ctcatttgtt cattgatgga 1200gtagtagata acgacaacac
ccgtcaactg tctggtaatg ctatgatctt tatcacttgt 1260ttgttcatct
tcttctttgc ctgtacatgg gctggaggtg tttttaccat catttccgaa
1320tcatatccat tgagaatcag atccaaggca atgtctattg ctactgctgc
taactggatg 1380tggggcttct tgatttcctt ctgcactcca ttcattgtta
atgccatcaa cttcaagttc 1440ggctttgtgt ttactggttg tttactcttt
tcgttcttct atgtctactt ctttgtcagc 1500gaaaccaaag gtttgtcgtt
ggaagaagtt gatgagttgt acgctgaagg tattgcacca 1560tggaagtctg
gtgcatgggt tcctccttct gcccaacaac aaatgcaaaa ctccacttat
1620ggtgccgaag caaaagagca agagcaagtt tag 1653352025DNANeurospora
crassa 35atggcgtcga acccaacgaa caccgcggcc cctacgggtg gccttaccga
gaagaagcat 60gaccgccgtt caacatcgtc cgaatccgtc tcgggaaccg ggtttgcgga
acatgcagac 120cgcaccggca cttttaacca gaacgctcga ctagaggctt
caaaaaagat agcgaatcct 180ttggccggtc taagccctca gcgtctcgag
gccatgggag aagaatatgc aatgatggcc 240ggtctcacca gcgaggagga
catcagggcc tttcgactcg gagccagaat cgccggcgat 300gagagcaact
acgacctcat cccggagctt actgaacggg agaaagaggt gttggtgcgc
360gaaacaactc acaagtggtc taacccaccc atgctttact gggttgttgt
catttgctct 420ctatgcgccg ccgtccaagg aatggacgag acggtcgtca
acggcgccca gctcttctac 480aaggacaagt ttggcattgg tactgatagc
cagagagaca cttggcttct gggtctcgtc 540aactcagcgc cctacctttg
ctgtgccttt atcggctgct ggctcactga accgatgaac 600agaatctttg
gcagacgagg caccatcttt gtttcttgca tcatctcagc cgtagcttgc
660ttccaccagg cctttaccaa cacgtggtgg cacatgttca tcgcccgttt
ctacctcggc 720cttggcatcg gtcccaagtc agccaccacc cccatcttcg
ccgccgaatg ctcccctccc 780aagctccgcg gtgcgctggt catgcaatgg
cagatgtgga ccgccttcgg tatcatggtc 840ggctacattg ccgatctcgc
tttctacttc gttcccgatc acggcatcgg cttgggtctg 900aactggcgtc
tgatgatggg ctccgccatg attcccgccg tcatcgtcgt ctgcctcgcc
960ttcctctgcc cggagtcgcc ccgttggtac ctcagcaagg gccgacacca
agacgccttc 1020ggggcgctct gccgcctgcg tttcgaaaag gtccaagccg
cccgcgacct cttctacacc 1080cacaccctcc tagaagccga gaagcaagcc
atgtcgggcg tcaagaaggg taaccgcttt 1140aaggagctct tcaccgtgcg
tcgtaaccgc aacgcggtca ttgcctcgtc gggactcatg 1200ttcatgcagc
agttctgcgg cgtcaacatc atcgcctact actcctcggc ggtcttccga
1260gacgccggct tcagcgacgt ctcagcactg gccgcctcgc tcgggtttgg
cgtcgtcaac 1320tggctgtttg ccatcccggc catgtacacc atcgacactt
tcggccggcg caacctgctg 1380ctgaccacct tcccgctcat gtccctcttc
ctcttcttca ccggcttcag cttttggatc 1440cccgaggact ccaaagccca
catcggctgc atcgcgctag gcatttactt gttcggcatg 1500gtctactccc
ccggtgaagg gccggtgccg tttacttact cggccgaggc ctacccgctg
1560tacatccggc caatcggcat gtccctcgcc acggcgacta cctggttctt
caatttcatt 1620ctttccatca cctggcctag gatggtcacg gccttcaagc
cgcagggcgc gtttggctgg 1680tatgcagggt ggaatatcat tgggtttctc
tttaccctgt tcttggtccc cgagaccaag 1740ggcaagacgc tggaggagct
cgatcacgtg tttgacgtgc cgttgaagaa gttggtcaga 1800tacggggcgg
atcagagctt gtggtttttc cacaggggaa agaatggaaa tggaatgagg
1860ccgacggcgc ctagtgcgga gatgtatcat ggggatgcgg agcggatgaa
cgaggtggtt 1920agcgggcagc agcttgggga gggtgagagg gagaagaggt
ggaacaagga acaagagagg 1980gaagggggga ttatgggacg aggggatgct
gctgggaagg tgtag 2025361458DNANeurospora crassa 36atgtccgcca
tcgtcgtgac cgaccaatac ctcacctact tcaacaaccc ccatgatatc 60atccaaggag
ccatcggctc tgcccttgct gctggctccg tcgtcggttc cgccatcgcc
120ggtcctcttt ccgacaagat cggtcgtcgt gactccatct ttttcgcctg
cttcttctgg
180ctcattggta cctccgtcca ggttgcctgc aagaactatg gccagctcat
cgccggccgt 240gtgctcaacg gctttaccgt cggcatcact tcctcccagg
ttcccgtgta ccttgccgag 300atcgccaagg cagagaagcg tggttccttg
gtcatcatcc agcaactcgc catcgagttt 360ggtatcttga tcatgtactt
tatcggctac ggctgtgcgt cgatcgaggg ccctgcttcg 420ttccggaccg
cttggggcat tcagtttatc ccttgctttt tcctcatggt cggtcttccc
480ttcttgccta ggtcgcccag atggctggcc aaggtcggta gggaccagga
ggccattgct 540gtcctggcta acatccaggc tgatggcaac gttgatgacc
cgagagtcgt tgctgagtgg 600gaggagattg tcaccgttat gaacgccgag
cgtgaggccg gtaagggatg gaggaagttt 660gtcaagaacg gcatgtggaa
gcgaaccatg gctggcatga ctgtacaggc ttggcagcaa 720ctcgccggcg
ccaacgtaat cgtctactac ctaacctaca tcgcccaaat ggccggactc
780acaggcaacg tcgccatggt gacctcgggc atccaatacg ccgttttcat
catcttcacc 840ggcgtcatgt ggctcttcat cgacaagacc ggtcgtcgca
cccttttagt ttacggcgcc 900ttgggaatgg ccttctgcca ctttgtcgtc
ggcggcgtca tgggcgcgca ccacgacaac 960gttccggacg gcgtcggcgg
caacgccaac attgtcatta gcgtgcacaa gggcgcgccc 1020gccaacacgg
tcatcctgtt ctcgtacctg ctcattgtcg tctacgcctt gacgctcgct
1080cccgtctgct ggatctacgc cgccgaggtc tggtcgttgg gcactcgcgc
tacgggcatg 1140tccatggctg ccatgtccaa ctgggtgttc aactttgcgc
tgggcatgtt cacgccgccg 1200gcgtttgtca atattacgtg gaagctgttt
atcattttcg gggtgctttg cgtcacggcg 1260gcggtctggt tctggttgtt
ttacccggag acgtgtggta agacgctgga ggagattgag 1320atcctgtttg
gtgatcaggg tcctaagccg tggaagacaa agaagggcga gtcgagactt
1380acggcggaga ttgaggctgt caaggcgagg aagacggtgg agcacgagat
tgaggtgcat 1440gagcatgaga aggtttag 1458371407DNAPichia stipitis
37atgaagtatt ttcaaatctg gaaatcaggc aaacaagtaa gctacgctgt tacattcact
60tgtgaattgg catttattct ttttggtatt gaacagggta ttattggtaa tcttattaac
120aaccaggact tcctaaacac ttttggaaac cccaccggta gttatttagg
tattatcgtt 180tctatctata ccttagggtg tttttttggt tgtgttatga
acttcttcat tggtgatcga 240atgggcagaa gaagcaaaat tgcttcctca
atgacagtta tcacaattgg tgttgctctt 300caatgtagtt ccttttcagt
tgaacaattg atgattggaa gatttatcac tgggcttgga 360actggttggg
aaacttctac ttgtccaatg tatcaggcag aactttcacc tccaaaagtt
420agaggacgtt tggtgtgctc agaagcattg tttgttggag ttggtttaat
ctatgcatat 480tggtttgatt atgctctttc tttcacttct ggtcctattg
catggagact tcctcttgcc 540tctcagattg tgttcgcctt tgttgttttc
tgtttcactt tcacaatacc cgaatcccct 600agatacatgt tttacaaagg
agagaaagaa gaagccaaaa gaattttatc ttatgtcttt 660ggaaagccag
gagatcatcc tgacattctt aaggaatgga atgatattaa tgatgctgtt
720attttggaaa cttcagaagg agctttctcg tgggcaaaac ttttcaagcc
cgataaggca 780agaactggat acagagtctt cttggcatac atgagcatgt
ttgcgcaaca gttgagtggt 840gttaatgtag ttaattacta tattacattt
gttttgatta acagtgttgg catcgaagac 900aacttggccc taattcttgg
tggtgttgcc gtcatctgtt tcactgttgg ttcattagtt 960cctactttct
ttgctgatag gatgggaaga agattgcctt cagcagttgg agcttttggc
1020tgtggtgttt gtatgatgct aatttcaatc ttattaagtt ttcaagacaa
tccaaagttg 1080aagaagagca gtggagctgg tgctgtggct ttctttttcg
ttttccaact tgtcttcggc 1140tccactggta attgtattcc atggctgatg
atttcagagc ttatccccct tcatgcacgt 1200gctaaaggat cttcattagc
tacatcaagt aactggcttt ggaatttctt tgttgttgag 1260atcactccaa
ctatcattga aaagttgaag tggaaagcat atttgatctt tatgtgctgc
1320aacttctcct tcgtaccaat gttttacttt ttctttcccg agacaaagaa
ccttacttta 1380gaagccattg acgatttgtt ctcataa 1407381653DNAPichia
stipitis 38atgtcctcac aagatttacc ctcgggtgct caaaccccaa tcgatggttc
ttccatcctc 60gaagataaag ttgagcaaag ttcgtcctca aatagccaaa gtgatttagc
ttccattcca 120gcaacaggta tcaaagccta tctcttggtt tgtttcttct
gcatgttggt tgcctttggt 180ggattcgtat tcggtttcga taccggtaca
atttccggtt tccttaatat gtctgatttc 240ctttccagat ttggtcaaga
tggttctgaa ggaaaatatt tgtctgatat cagagtcggt 300ttgattgttt
ccatttttaa cattggttgt gcaattggtg gtattttcct ttctaagata
360ggagatgttt acggtagaag aattggtatc atttcagcta tggttgtcta
cgtcgtcggt 420attatcatcc agatctcgtc ccaagacaag tggtaccaac
ttacaattgg acgtggagtt 480acaggattag ctgttggtac tgtttcagtg
ttgtctccaa tgttcattag tgaaagtgct 540ccaaagcatt tgagaggtac
tttggtatac tgttaccaat tatgtatcac cttaggtatt 600ttcattggtt
actgtgtcac ttatggaacc aaagatttaa atgattcaag acaatggaga
660gttcctttgg gtttatgttt cctttgggct attttcttag ttgtcggtat
gttggctatg 720cctgaatccc caagattctt aattgaaaag aagagaatcg
aagaagccaa gaagtccctt 780gcaagatcca acaagttatc tccagaagat
ccaggtgtct acactgaagt tcaattgatt 840caggctggta ttgacagaga
agctgctgca ggttctgctt catggatgga attgatcact 900ggtaagccag
ctattttcag aagagttatc atgggaatta tcttacagtc tttgcaacaa
960ttaactggtg tcaactattt cttctattac ggaactacaa tcttccaagc
tgttggtttg 1020caagattcct tccagacttc catcatctta ggtacagtca
actttctttc tacatttgtt 1080ggtatttggg ccattgaaag atttggaaga
agacaatgtt tgttagtcgg ttctgctggt 1140atgttcgttt gtttcatcat
ttactccatt attggtacaa ctcatttgtt cattgatgga 1200gtagtagata
acgacaacac ccgtcaactg tctggtaatg ctatgatctt tatcacttgt
1260ttgttcatct tcttctttgc ctgtacatgg gctggaggtg tttttaccat
catttccgaa 1320tcatatccat tgagaatcag atccaaggca atgtctattg
ctactgctgc taactggatg 1380tggggcttct tgatttcctt ctgcactcca
ttcattgtta atgccatcaa cttcaagttc 1440ggctttgtgt ttactggttg
tttactcttt tcgttcttct atgtctactt ctttgtcagc 1500gaaaccaaag
gtttgtcgtt ggaagaagtt gatgagttgt acgctgaagg tattgcacca
1560tggaagtctg gtgcatgggt tcctccttct gcccaacaac aaatgcaaaa
ctccacttat 1620ggtgccgaag caaaagagca agagcaagtt tag
1653391641DNAPichia stipitis 39atggaattct ccagtgttga aaaaagtgct
gaaactgctt cctatacgtc gcaggtcagc 60gcaagcggct ctgcaaagac caacagctac
cttggcctca gaggccacaa acttaatttt 120gctgtctctt gttttgctgg
tgttggtttc ttacttttcg gttacgatca aggtgtcatg 180ggttcattgt
tgaccttgcc atccttcgaa aacactttcc cggccatgaa ggctagcaac
240aacgctacct tacaaggcgc cgttattgca ctttatgaaa tcggttgtat
gtcttcttct 300ttagcaacca tttaccttgg tgacagattg ggtagattga
agatcatgtt tattggctgt 360gtaattgtct gtattggtgc tgctttgcaa
gcttctgctt tcactattgc tcacttgact 420gttgctagaa ttatcactgg
tttaggtaca ggtttcatca cttctactgt tccagtttac 480caatcggagt
gctctccagc caagaaaaga ggacagttga tcatgatgga aggttctctt
540atcgcccttg gcattgccat ctcatactgg attgactttg gattttactt
tttgagaaac 600gatggtttgc actcctcggc ttcttggaga gcacctatcg
cgcttcaatg tgtcttcgct 660gtcttgttga tttccacagt cttcttcttc
ccagaatctc caagatggtt gctcaacaaa 720ggtaggaccg aagaagctag
agaagttttt tctgctcttt acgacttgcc agccgactct 780gaaaagattt
ctattcaaat tgaagaaatt caagctgcta tagatttaga aagacaagcc
840ggagaaggtt tcgtacttaa ggaattgttc actcagggcc cagccagaaa
cttgcagcgt 900gtggccttgt catgttggtc tcaaataatg caacaaatca
ctggtattaa cattattacg 960tactatgctg gtactatttt tgaatcatac
attggtatga gtccatttat gtcaagaatc 1020ttggctgcct tgaacggtac
tgaatatttc cttgtctctc ttattgcttt ctacaccgtc 1080gaaagattag
gtagaagatt ccttttgttc tggggtgcca tcgccatggc tcttgtcatg
1140gctggtttaa ctgttaccgt taaacttgcc ggtgaaggca acacccatgc
tggtgtcggt 1200gctgctgttc ttttgtttgc attcaactca ttcttcggcg
tctcctggtt aggtggatcc 1260tggttgttac cacctgaatt gttgtctttg
aaattgagag ctcctggtgc tgctttgtcg 1320accgcttcta actgggcttt
taacttcatg gttgtcatga tcactcctgt cggtttccaa 1380agtattggtt
cctacaccta ccttatcttt gctgccatca atttgttgat ggctccggtc
1440atctacttct tgtatcccga aaccaagggt agatcgttgg aagaaatgga
tatcattttc 1500aaccaatgtc ctgtttggga gccatggaag gttgtccaaa
ttgccagaga cctccctatt 1560atgcactcag aagttcttga ccacgaaaag
aatgtcatta ttaaaaaatc tagaatagag 1620catgtcgaaa acatcagcta a
1641401701DNAPichia stipitis 40atgcacggtg gtggtgacgg taacgatatc
acagaaatta ttgcagccag acgtctccag 60atcgctggta agtctggtgt ggctggttta
gtcgcaaact caagatcttt cttcatcgca 120gtctttgcat ctcttggtgg
attggtctac ggttacaatc aaggtatgtt cggtcaaatt 180tccggtatgt
actcattctc caaagctatt ggtgttgaaa agattcaaga caatcctact
240ttgcaaggtt tgttgacttc tattcttgaa cttggtgcct gggttggtgt
cttgatgaac 300ggttacattg ctgatagatt gggtcgtaag aagtcagttg
ttgtcggtgt tttcttcttc 360ttcatcggtg tcattgtaca agctgttgct
cgtggtggta actacgacta catcttaggt 420ggtagatttg tcgtcggtat
tggtgtgggt attctttcta tggttgtgcc attgtacaat 480gctgaaattt
ctccaccaga aattcgtggt tctttggttg ctttgcaaca attggctatt
540actttcggta ttatgatttc ttactggatt acctacggta ccaactacat
tggtggtact 600ggctctggtc aaagtaaagc ttcttggttg gttcctattt
gtatccaatt ggttccagct 660ttgctcttgg gtgttggtat cttcttcatg
cctgagtctc caagatggtt gatgaacgaa 720gacagagaag acgaatgttt
gtccgttctt tccaacttgc gttccttgag taaggaagat 780actcttgttc
aaatggaatt ccttgaaatg aaggcacaaa agttgttcga aagagaactt
840tctgcaaagt acttccctca cctccaagac ggttctgcca agagcaactt
cttgattggt 900ttcaaccaat acaagtccat gattactcac tacccaacct
tcaagcgtgt tgcagttgcc 960tgtttaatta tgaccttcca acaatggact
ggtgttaact tcatcttgta ctatgctcca 1020ttcatcttca gttctttagg
tttgtctgga aacaccattt ctcttttagc ttctggtgtt 1080gtcggtatcg
tcatgttcct tgctaccatt ccagctgttc tttgggtcga cagacttggt
1140agaaagccag ttttgatttc cggtgccatt atcatgggta tttgtcactt
tgttgtggct 1200gcaatcttag gtcagttcgg tggtaacttt gtcaaccact
ccggtgctgg ttgggttgct 1260gttgtcttcg tttggatttt cgctatcggt
ttcggttact cttggggtcc atgtgcttgg 1320gtccttgttg ccgaagtctt
cccattgggt ttgcgtgcta agggtgtttc tatcggtgcc 1380tcttctaact
ggttgaacaa cttcgctgtc gccatgtcta ccccagattt tgttgctaag
1440gctaagttcg gtgcttacat tttcttaggt ttgatgtgta ttttcggtgc
cgcatacgtt 1500caattcttct gtccagaaac taagggtcgt accttggaag
aaattgatga acttttcggt 1560gacacctctg gtacttccaa gatggaaaag
gaaatccatg agcaaaagct taaggaagtt 1620ggtttgcttc aattgctcgg
tgaagaaaat gcttctgaat ccgaaaacag caaggctgat 1680gtctaccacg
ttgaaaaata a 1701411656DNAPichia stipitis 41atgagagaag ttggtattct
tgatgttgcc catggcaacg ttgtaactat aatgatgaaa 60gatccagtag tatttttggt
gattttattt gcatcccttg gaggtttgct ttttggttat 120gatcaagggg
ttattagtgg cattgtcaca atggaatctt ttggtgcaaa attccccaga
180atttttatgg atgccgatta caagggttgg tttgtgtcta cttttttgct
atgcgcatgg 240tttggctcta ttattaatac tccaattgtt gataggtttg
gaagacgtga ttctatcaca 300atctcttgtg ttatttttgt cattggttct
gcgttccaat gtgctggcat taatacaagt 360atgttatttg gtgggcgtgc
tgttgctggt cttgcagtcg gtcaattaac catggtagtt 420ccaatgtaca
tgtcggaatt ggctcctcca tcggtgagag gtgggttggt tgtaattcag
480caactttcga ttacaattgg tatcatgatt tcctattggt tggattatgg
cactcatttt 540attggaggta ctagatgtgc tcctagtcac ccataccaag
gtgaaacttt taaccctaat 600gtggatgttc ctccaggtgg ctgctatggt
caaagtgatg ccagttggag aattcctttt 660ggtgttcaga ttgctccagc
agtgttgttg ggtattggaa tgatattttt cccaagatct 720cccagatggt
tactctctaa aggtcgcgac gaagaagctt ggagctcttt gaaatatctc
780agaagaaaga gtcatgagga tcaagtcgaa agagagtttg ctgaaattaa
ggcagaggtc 840gtttatgaag acaagtacaa ggaaaagaga ttccctggta
agactggagt tgctttaaca 900cttactggat actgggatat tcttactact
aaatctcact tcaagagagt ttttattgga 960tcagctgtca tgttcttcca
acaattcatt ggctgcaatg caataattta ttacgcacct 1020acaattttca
cacaattggg aatgaactct acaactactt ccttgcttgg tactggtctt
1080tatggtattg ttaattgtct ttccaccctt ccagcagtgt tcttgatcga
tagatgtgga 1140agaaagactt tgttaatggc aggtgctatt ggaactttta
tttccttggt tattgtcggc 1200gcaatcgttg gcaagtatgg cgatcgttta
tctgaattca agacagcagg gagaactgca 1260attgctttca ttttcattta
tgatgtgaat ttctcgtaca gttgggctcc aattggatgg 1320gttttaccct
cagagatttt cccaatcggc atcagatcca atgccatctc cataactacc
1380tcatctactt ggatgaataa ttttattatt ggcttggtca ctccacatat
gttagaaaca 1440atgaagtggg gcacttacat tttttttgca gcgtttgcta
ttattgcgtt ctttttcact 1500tggcttatca tcccggaaac caagggagtt
ccattggaag aaatggatgc cgtgtttggc 1560gatactgcag cattgcagga
aaagaatttg gttaccatta cgtcagtttc tgaatctgac 1620gccaaggatc
gcaactcgat tgaaatgtca gaataa 1656421590DNAPichia stipitis
42atggcatatc ttgattggtt aacagctaga accaacactt tcgggttgag gggcaagaag
60ttgagagcct tcatcactgt agtggctgtc actggtttct cattattcgg atatgatcaa
120gggttgatgt ccggaattat tactgctgat caattcaact ctgagtttcc
cgccactaga 180aataacagta ctatccaagg tgccgtcacc tcctgttacg
agcttggttg tttctttggt 240gctgtgtttg ccttgttaag aggtgaaaga
attggaagaa gacctcttgt gctttgtggc 300tcgcttatta tcatcttggg
aacagttatt tctgtaaccg ccttccatcc acactggtca 360ttaggtcagt
ttgttattgg tagagttatc actggtattg gtaatggtat gaatactgcc
420accattccag tttggcaatc ggaaatgtca agagctgaaa acagaggaag
attggtcaac 480ttggaaggtt ccgttgtcgc tgtgggtaca tgtattgcct
actggttgga tttcggtttg 540tcttatgtcg acaattcagt ttcctggaga
tttccagttg ctttccaaat agtgtttgct 600tccgttttat ttgtgggaat
gttgcaattg cccgactctc caagatggtt ggttgctaac 660cacagaagag
cagaggctct tcaagtgttg tctgctttga aagacttgcc cgaagacgac
720gaagaaattc ttaatgaagc tgaagttatt caggaaagtg tagacaagtt
tgctggacat 780gcttccgtca aggaagtgtt tactggtggt aagacccagc
actggcaaag aatggttatt 840ggatccagca cccaattctt tcagcagttc
actggttgta acgctgccat ttactattcc 900actgtcttgt ttcaagacac
tattggttta gaaagaagaa tggcattgat tatcggtggt 960gttttcgcaa
ctgtctacgc cattttcaca attccttcct tcttcttggt cgatactctt
1020ggacgtagaa acttgttctt gattggtgct atgggacaag gtattgcatt
cactatcacc 1080tttgcctgtt tgattgacga tactgaaaac aacgccaagg
gtgccgcagt tggtttattc 1140ttgtttattt gtttcttcgc cttcaccatc
ttgccattgc catgggtata cccaccagaa 1200atcaatcctt tgagaactag
aactatagct tctgcaattt ccacttgtac caactggatc 1260tgtaactttg
ctgttgttat gttcacccct gtctttgtca ctaacaccag atggggagcc
1320tatcttttct ttgctgtgat gaacttcctt ttcgttccta ttattttctt
cttctaccca 1380gaaacagctg gaagatcgtt ggaagaaatc gatatcatct
ttgcgaaggc attcgttgac 1440aaaagacagc catggagagt tgctgcaacc
atgccaaagt tgtccaacca cgaaattgaa 1500gacgaagcca acagattggg
cttgtttgac gatggtacat tcgacaagga agcatttgaa 1560accaaagaaa
acgcatccag cagctcttaa 1590431689DNANeurospora crassa 43atggcgcctc
caaagttcct gggcctctca ggccgaccgc tctctctagc tgtctcgact 60gtagccacca
cgggcttcct tctcttcggc tatgaccaag gtgtcatgag cggcatcatt
120accgcccccg ctttcaacaa cttcttcaca ccaaccaaag acaactcgac
catgcagggt 180ctcatcactg ccatctacga aattggatgc ttgattggtg
ccatgttcgt cctctggacc 240ggcgatttgt tgggtagacg caggaacatc
atggtgggcg ccttcattat ggctctcggt 300gtcattattc aggttacctg
tcaggctgga tccaaccctt ttgctcagct gttcgtcggc 360agagtcgtca
tgggtattgg caacggcatg aacacttcga ccattcccac ttatcaagcc
420gaatgctcaa agacatcgaa ccgcggtctt ttgatctgca ttgaaggcgg
tgtcattgcc 480tttggtactt tgattgctta ttggatcgac tatggtgcat
cttacggtcc cgatgacctc 540gtttggcgct tccccatcgc tttccagctt
ctcttcgcca tcttcatctg cgtccccatg 600ttttaccttc ccgagtcgcc
cagatggctc ctcagccatg gccggaccca agaagctgac 660aaggtcattg
ctgccctccg tggctacgag atcgatggtc ccgagaccat ccaagagcgc
720aacctcattg ttgactccct gcgtgcctct ggaggtttcg gccaaaagag
cactcccttc 780aaggccctct tcactggcgg caagacccag catttccgtc
gtctcttgct cggttccagc 840tcccagttca tgcagcaagt tggtggttgc
aacgccgtca tctactactt ccccattctg 900ttccaggatt ctattggcga
gtcccacaac atgtccatgt tgctgggcgg tatcaacatg 960atcgtctact
ccatcttcgc taccgtttcc tggttcgcca ttgagcgtgt cggtcgtcgt
1020cgtctgttct tgatcggcac cgttggccag atgctctcca tggtcatcgt
cttcgcctgc 1080ttgatccccg acgaccctat gaaggcccgc ggtgccgcgg
tcggtctctt cacttacatt 1140gcctttttcg gtgccacttg gcttcccctc
ccctggctct accccgccga ggttaacccc 1200atccgcacac gtggaaaggc
taacgccgtc tccacctgct ccaactggat gttcaacttc 1260ctcatcgtca
tggtcacccc catcatggtc gacaagattg gctggggaac ttacctcttc
1320ttcgcggtca tgaacggctg cttccttccc atcatttact tcttctaccc
cgagactgcg 1380aaccgctcgc tcgaggagat cgacatcatc ttcgccaagg
gcttcgtcga gaacatgtcg 1440tacgtcactg ccgccaagga gctgcctcac
ctcactgccg aggagatcga gtcctatgcc 1500aacaagtatg gcctcgtcga
ccgcgattcc aacggcgagg gcggcaaccg ccatgacgag 1560gagaagacgc
gcgaccgccc cgaccagagt gacagcgact cccccgctca cgtcgagatt
1620gatgttgtcg acgagcacgg tgtcgagtcc ggcttcggtg atggtattaa
caccaaggaa 1680acacgttaa 1689441626DNANeurospora crassa
44atggaattcg agcacgatca ctccgcctcc gacattgaga aggaggccgt cactgtggcc
60cggccacagg gcgatgtcac ccgcgttgag gctcccgtta ccctcaaggc gtacatgatg
120tgcgtctttg ccgctttcgg cggtatcttc tttggctacg attcaggtta
catctctggt 180gtcatgggca tgaagtactt tatcgaaacc atcaacggac
ccggcgccac cttcctgcca 240tccaaggaaa agtcgctcat cacctccatt
ctctctgccg gaaccttctt tggcgccctc 300atgggcggtg atctcgctga
ctgggttggc cgtcgtccta ccatcatctt cggctgcctc 360gtcttcatcg
tcggtgttgt tctccagact gcctcccaga gcttgggtct cattgtggcc
420ggccgtctcg tcgctggttt cggtgtcggt ttcgtctcgg ccattatcat
cctgtacatg 480tctgagatcg cgccccgcaa ggtccgcggt gctatggtgt
cgggctacca gttctgcatc 540tgcctgggtc tgctcctggc ctcgtgcgtt
gactacggca cccagaaccg caccgacagc 600ggctcttaca gaatcccgat
tggtctccag atggcctggg ccctcattct tgctactggt 660atctttttcc
ttcctgaatc ccctcgcttc ttcgtcaaga agggcaagct cgacaaggcc
720gccggcgtgc tctcccgcct gcgcgaccag ccgctcgatt ccgactacgt
cagggacgaa 780cttgccgaga tcgttgccaa ccacgaattc gaaatgaccg
tcgtccccta cggcaactac 840ttccagcagt gggccaactg cttccgcggc
tccatctggc agggtggttc ttacctccgc 900cgcaccattc tcggcacttc
gatgcagatg atgcagcagt ggacgggaat caactttatc 960ttttactttg
gaaccacctt cttccagcag ctcggcacca ttgacaaccc cttcctgatg
1020tctctggtca ctactcttgt caacgtctgc tccaccccca tctccttcta
caccatggag 1080aagctcggcc gtcgtaccct cctcatctgg ggcgctctcg
gcatgctgat ctgcgagttt 1140atcgtcgcca tcgttggtac ctgcaggccg
gatgatacca tggccatcaa ggccatgctc 1200gccttcatct gcatctacat
cttcttcttt gctaccacct ggggccctgc ttcctgggtc 1260gtcatcggcg
aggttttccc tcttcccatt cgtgccaagg gtgttgccct ttccaccgcc
1320tccaactggc tctgtaactg catcatcgcc gtcatcactc cctacatggt
cgacgaggac 1380aagggcaacc tgggccccaa ggtgttctac atctggggtg
gcctctgcac ctgctgcttc 1440atctacgcct acctgcttgt gcccgagacc
aagggcctca cgctcgagca ggtcgaccaa 1500atgctttccg agtctacccc
ccgcacctcg accaagtgga agcctcacac cacttatgct 1560gctgagatgg
gcatgaccga gaagactgtt gctggccacg ctgagaaccg cagcgatagc 1620gagtaa
1626451614DNANeurospora crassa 45atgggtcttt cgataggaaa taggatcctc
cggaaaattg tcaaaaatga ggccatggca 60gaagatcccc cagagatcta tggctggcgt
gtctatctcc tagcgtgctc tgcctgcttc 120ggcgccatgt ctttcggctg
ggattcctcc gtcatcggcg gcgtcatcga actcgaaccc 180tttaaacacg
actttggctt catcggcaac
gataaagcca aggccaacct gggcgccaat 240atcgtctcta ccctccaagc
cggctgcttc ctcggtgcgc tgatcgcctc acctataacc 300gatcgcttcg
gccgcaagtg gtgtctcatc gctgtctccc tggtcgtcat catcggtatc
360atcatgcaag ccgccgcctc aggcaacctc gcacccatgt acattggccg
tttcgtcgcc 420ggcgtgggcg tcggcgccgc cagctgcatc aaccccgtct
ttgtgtctga gaacgctccc 480cgctcgatcc gcggtctgtt gacgggcctc
taccaactct tcattgtcac cggcggcatg 540atcgcatttt ggatcaacta
ctccgtctct ctgcacttca agggcaaatc catgtacatc 600ttcccgctcg
ccatccaagg tcttcccgcc ggccttttgt gcgtctgcat gctcctctgc
660cacgaaagcc cgcgctggct ggcccgtcgt gaccgatggg aagaatgcaa
gtctgtgctg 720gcgcgcatcc gcaacctccc cccagaccac ccgtacatcg
tcgacgagtt ccgcgagatc 780caggaccagc tcgaacagga gcgtcgtctc
cagggcgacg ccacttactg ggacttgacc 840cgcgatatgt ggaccgtcgc
cggcaaccgc aagcgcgccc tgattagtat tttcttgatg 900atctgccagc
aaatgacggg caccaacgcc atcaacacgt acgcgcctac catcttcaag
960aacttgggta tcaccggcac gtcgactagc ttgtttagta ccggcatcta
tggtattgtc 1020aaggtcgtta gctgcgtcat tttcttgctg ttcttggccg
actcgctggg tcgtagacgt 1080tcgctgctgt ggacgtcgat tgcgcagggt
cttgctatgt tttatattgg cctttatgtc 1140cgcatctcgc cgccgattga
tggccagccg gtgccgcctg cgggttatgt agcgttggtg 1200tgcatctttc
tgtttgccgc tttcttccaa tttggctggg gtcctgcctg ctggatctac
1260gcctcggaaa tccccgccgc ccgcctgcgc tccctcaacg tgtcctacgc
cgccgcgacg 1320cagtggctgt tcaatttcgt cgtggcccgc gccgtgccta
ctatgctggt cacggtcggc 1380ccccacggtt acggcaccta cctcatcttt
ggcagcttct gcctcagcat gtttgtcttt 1440gtctggttct tcgtgcccga
gacaaagggt atctcgcttg agcacatgga tgagctgttt 1500ggcgttactg
atgggcctgc cgctgagaag tcgtcggtgc atggtggaga tgatgtcggg
1560tcggagatgg ggaaggggga tcagaagtcg aagcatgtgg aggtttatgt ttaa
1614461587DNAPichia stipitis 46atgtcttcgt tattgactaa cgaatacttc
aaagactact accacaaccc gactcctgtt 60gaagtgggta ctatgattgc tatcttagag
atcggcgcac ttttttcctc cttcatagct 120ggaagagtag gtgacatcgt
tggcagaaga agaaccatta gatacgggtc tttcattttt 180gtagtaggcg
gtcttgtaca agctacttcg gtcaatattg tcaatctctc actaggaaga
240ttgattgccg gtattgccat tggctttttg acaaccatca tcccatgcta
ccagtctgaa 300atcagccccc cagacgatag aggtttctat gcctgtttgg
agttcaccgg aaatatcatt 360ggatatgcta gtagtatttg ggtagactac
gggttttcat ttttagacaa tgatttcagc 420tggaggagcc cattgtatgt
tcaggttgtt attggctcca tgttatttat tggttcattc 480cttattgtag
aaacccctag atggctcttg gatcacaacc atgatatcga aggcatgatt
540gtcatttctg acttgtatgc agatggtgat gtggaagacg atgatgctat
tgctgagtac 600agaaacataa aggaaagtgt cttgatagcc agagttgaag
gcggagagag atcgtaccag 660tatttgttca ccaaatatac caagagactt
tctgtggcat gcttttcgca aatgtttgcc 720cagatgaatg gtataaacat
ggtatcttac tatgctccta tgatcttcga atctgctggc 780tgggttggta
gacaagctat cttgatgact ggtatcaact ccattatcta catctttagt
840accattcctc catggtactt agttgattct tggggcagaa aacctttgct
tttatctgga 900tctgtgctca tgggtgttcc gctcttaacc attgcttgtt
cgttattctt aaacaacaca 960tacacacccg gggttgtggt tggcagtgta
atcgtattca atgctgcttt tggatacagt 1020tggggtccaa ttccttggct
catgagcgaa gtgttcccta actcagttag atcaaaaggt 1080gctgccatgt
ctactgcaac caactggctc tttaacttta ttgttggaga gatgacacct
1140attttgttgg atacaattac ctggagaact tacttgatcc cggcaacttc
gtgtgtatta 1200tcgttttttg ctgttggatt tttatttcca gagaccaagg
gtttagcatt ggaggatatg 1260ggctccgtat tcgatgataa ttcgtcaata
ttttcatatc actcaacttc ttccactggg 1320tatggtgcga ccgagtctaa
cagtaatgcc aggagagcaa gtgtcatctc ttcagaaaac 1380taccaggata
gtttgcatca gacagcggct tcattggcta gaaatccttc aagcatgagg
1440cctgattacg atggcataat cacaggagct gctacccttt cgccagtacc
accattaaaa 1500ccaataaaca tttccagcaa tattccgcag gaaattgaac
caccaacctt tgatgaaatc 1560tttaagtaca agttgaatga gatggaa
1587471257DNAPichia stipitis 47atgacttttg cagttaactt gtatgtgttt
gcagttggta gagtgctttc tggggtgggt 60gtaggagttc tatcgactat ggtgccgtcc
tatcaatgcg aaattagtcc cagcgaagaa 120agaggcaagt tggtgtgtgg
agagttcacg ggaaatatca ctggttatgc tctcagtgta 180tgggccgatt
acttctgcta ctttattcaa gatataggtg atgcaaggga gaagcctcat
240agcttctttg cccacttgtc ctggcgattg cctctattca tccaggtggt
gatagcggct 300gttctctttg ttgggggatt ttttattgtc gagtcacctc
gttggttatt agatgtagac 360caggaccaac aaggattcca tgtattagcg
ttgctctatg attcacatct agatgataac 420aaaccacgtg aagagttctt
tatgatcaaa aactccatct tgttagaaag agaaactaca 480cctaagagcg
aacgaacttg gaaacatatg ttcaagaact acatgacccg agtgcttata
540gcttgttcag cacttggctt tgcacagttc aacggcataa atatcatttc
gtactatgcc 600cccatggtat ttgaagaagc aggcttcaac aactccaagg
ctttacttat gacaggcatc 660aactctatag tatattggtt cagtacgatt
cctccgtggt ttctcgtgga tcattggggt 720agaaagccaa ttttgatatc
cgggggttta tctatgggaa tatgtattgg tttgattgcg 780gtggtaattc
tactagacaa gtcgttcaca ccgtctatgg ttgcggtatt ggtgataatc
840tacaatgcat cttttggcta cagttggggt cctatcggat tcttgatccc
gccggaggtg 900atgccattgg cagttagatc gaaaggtgtt tctatttcta
cggctacaaa ctggtttgcc 960aattttgttg tgggtcagat gacgccaatt
ctacagcaga gattgggctg gggaacttat 1020ctattcccgg ctggtagttg
tatcatctcg gtgatagtgg tgattttctt ctatccagag 1080acaaagggtg
cagagctaga ggatatggac tctgtgttcg agagctttta caactacaag
1140tctccgttca agatttcacg aaagagacac cagaatgatg gccaggcgta
ccaaagggta 1200gagaacgata tccgccacaa cgatgtagaa atggacgatt
tggacgattt ggactaa 1257481757DNANeurospora crassa 48atggaattcg
gtggcggagg cggctccggc gcagctggtt tttatgatgc ggctcttcag 60aggcgtgagg
cagtgatggg gaagagtggc cctgcagcac ttgtcaagaa cttccgggtc
120ttttctattg catgcttcgc atgtatcggt ggtgtgctct atgggtacaa
ccaagggatg 180ttttcgggtg tcctcgccat gccagccttt cagaaacaca
tgggcgaata cgatccgata 240gacgagaacg cgagtcagac aaagaagggc
tggctaaccg caattcttga gctcggtgct 300tggcttggta cgcttctgtc
tgggttcatg gcagaggttc tctcgagaaa gtacggtgtg 360ctagtggcgt
gcttggtttt catgctgggt gtggtcatcc aagccacgtc tatctctgga
420ggacatgaga ccattcttgc cggacggttt atcacgggta tgggtgtcgg
atccttagcc 480atgatcattc ccatttacaa ctcggaagtt gcaccacctg
aggtccgtgg agctcttgtt 540gctctccagc agttggctat ctgcttcggt
atcatggtca gcttctggat tgactacgga 600accaactata tcggcggcac
caagctcgag acccaatccg acgccgcctg gcttgtaccc 660gtctgcctgc
aactcgcccc tgctctcatt ctgtttttcg gcatgatgtt catgcccttc
720tccccacgct ggctcatcca ccatggccgc gaggcggaag ctcgaaagat
cctctccacc 780cttcgcggtc taccccaaga ccacgagctt gtcgagctcg
agttcctcga aataaaggct 840cagtctctct tcgaaaaacg cagcattgcc
gagttgtttc ccgaattgcg cgagcagact 900gcctggaata cctttaagct
ccagtttgtc gccatagaga agcttttccg gacaaaggca 960atgttccgac
gcgttgtcgt ggcaaccgta accatgttct tccagcagtg gtccggcatc
1020aatgcgattc tctactacgc cccgcaaatc ttcaagcagc ttggactgag
cggtaacaca 1080acctcactcc tggctacggg tgtagtaggc atcgtcatgt
tcatcgcaac ggttcctgcc 1140gtgctgtgga tcgaccgtgt tggtcgcaag
cccgtgctta ctatcggtgc cctcgggatg 1200gctacctgcc atatcatcat
cgctgtcatt gttgccaaga acgtggacca atgggagact 1260cataaggctg
ctggatgggc tgctgtagcc atggtctggc tattcgtcat tcactttgga
1320tattcatggg gtccatgtgc ctggatcatt gttgctgaga tctggccgtt
gagtacgagg 1380ccatatggtg tctctctagg agcttcgagc aactggatga
acaactttat cgtcggtcag 1440gtcacgccgg atatgttaaa ggcgatcccg
tacggaacgt atatcatctt cgggttgttg 1500actatatggg tgccgccttt
atttggttct ttgtgccgga aacgaagaga ttaaccttgg 1560aagagatgga
catgatcttc ggatccgaag gcactgcaca agccgacaat gagcgcatgg
1620aggagatcaa tgctgagatt ggtcttaccc gattcctgca aggtggtagt
ggtgcaaacc 1680aaggtgctgc tgatggaagc gatactggtt atgatgcgga
gaagggcaag agcgaacact 1740attctcagca tgtctaa
1757491584DNANeurospora crassa 49atgggcttgt cactcaagaa gcctgaaggt
gtgccgggca agtcatggcc cgccattgtc 60attggcttgt ttgtcgcctt tggtggtgta
ctctttgggt atgacactgg cactattggc 120ggtatccttg ctatgcccta
ttggcaagat ttgttttcga caggttacag aaacccagag 180catcacttgg
acgttaccgc gtcgcagtct gccactatcg tctccattct gtctgctgga
240accttctttg gcgctcttgg tgccgctccc cttgccgact gggctggacg
acgcttgggc 300cttattctgt cgtcgtttgt gtttatcttc ggtgtcatcc
tgcagaccgc agccgtcagc 360attcctcttt ttctggctgg ccgattcttt
gctggattgg gagttggtct catatcggca 420accatccccc tctatcaatc
cgagactgcc ccgaaatgga ttcgtggtgt catcgtcggg 480tcctatcagc
tagccattac catcggtctt cttcttgcct ccattgtcaa caatgccacg
540cataacatgc agaacaccgg ctgctatcgc atccccatag ctgtccaatt
tgcatgggcg 600atcatcctga tcgttggcat gatcattctt cccgaaactc
cacgctttca tatcaagaga 660gacaatctcc cagccgccac taggtctcta
gctatcctcc gccgtctgga gcagaaccat 720ccagcgatca tcgaagagct
ttccgagatc caagccaatc atgaatttga gaagagcctc 780gggaaggcga
cctacttgga ctgcctcaag ggcaatttac tcaagcggct ccttactggc
840tgttttctcc agagcctgca gcagttgact ggcatcaact ttatcttcta
ctacggcaca 900cagttcttca aaaactccgg attctcagac tcgtttctga
tatccttgat cactaatctt 960gtcaatgtcg tgtcgaccct tcccggactc
tacgccatcg acaaatgggg ccggaggcct 1020gttttactct ggggagctgt
tgggatgtgt gtctgccagt tcatcgttgc tattcttggg 1080acaacaacga
caagtcaaga tgcaagcgga atgatcattg tgcataatct cgccgcacag
1140aaagcagcta ttgcattcat ctgcttctac atctttttct tcgctgcatc
ttggggtcca 1200gttgcctggg tcgttacagg cgagatcttc ccccttaaag
tccgcgccaa gtcgctctcc 1260ataactacag cgtcgaattg gctgctcaac
tgggccattg cttacagcac accttacctt 1320gtcaactacg gccctggcaa
tgcgaacctg cagtccaaga tcttcttcgt ctggggcgga 1380tgctgcttca
tctgcatcgc attcgtttac ttcatgatct atgagacaaa aggtctcaca
1440ctggagcagg ttgacgagct atatgaagag gtctcggatg ccaggaagag
tattggttgg 1500gtgccgacca tcactttccg ggagatccgg gaggaaaaga
aagtaaggga tccagttgtt 1560gatatcactg aagaggcagc ttga
1584501968DNANeurospora crassa 50atgggccaca atccagacct ggacagtagc
ggcactgccg gagaacccaa aggtgtcacc 60ggctcgcaca ttgaacaaac ctcgtccaac
ctcgaagcca acatcaacct cgaagccaag 120ctcaagaacc cgctcgacgg
cctttcccgc gtcgagctcc tgtcacgcgt cgagaccttt 180tgcgccgaaa
agaacctaac cgagcacctc cctcttttcc gtaagggagc actcatcgcc
240cagtccccgg acagctatgc gtccatctcg ggcccggaag ccttggacga
tgaggagaag 300gcagtacttt tgaaggaggt cgaacacaag tggcggctgc
cggcaagact gttcctgacg 360attgctactt gctcgatcgg tgctgctgtc
caaggttggg atcagacggg cacgaatggc 420gcgaatatct tctttcccaa
ctattacggt atcggaggcg acactgcgag ggagaagttg 480cttgtcggat
tgatcaatgc tgggccctat attgggagcg cattcatcgg ttgctggctt
540tctgatccca tcaacaactg gattggtcgt cgtggtgtta tctttgtctc
tgctcacttc 600tgtatctggc ccgtcatcgg ttctgctttc tgtcacacat
ggccccagca actggcctgc 660cgtctgctga tgggtatcgg tatgggtgtg
aaggcatcaa cggtgccgat ctatgccgcg 720gaaaactcgc ctgcttctat
tcgaggtgcg ctggtcatgt catggcagat gtggacagcc 780ttcggcatct
tcttgggcac tgcctttaac cttgccgtct tccacgccag ctccaacgtt
840aactggcgcc tcatgctcgg tgcccccttc attcccgccg tacccctgct
tctgctcatc 900tatctttgcc ccgagtcccc gcgctggtac atgaagaagg
gccgctaccc agaagcctgg 960aaatccatgg tcaagctgcg caaccacccc
atccaagttg cccgcgacat gttctacatc 1020cactcgcaat tggaagtcga
gcaccagctc ctcgccggct ccaactatgc caagcgcttc 1080gtcgagctct
tcaccgtccc tcgtgttcgc cgcgccaccc tcgccgcttt caccgtcatg
1140attgcccagc agatgtgcgg aatcaacatc atcgcctttt acagcaccac
catcttcaag 1200gattccggct ccaccgaatt ccaagccctg ctttcctcct
tcggcttcgg tctagtcaat 1260tggctctttg ccttccccgc cttctggact
atcgacactt ttggccggcg ctctctgctt 1320ctttttacct tcccgcaaat
gatgtggacc ctgctagcag ccggcctctt caccttgctc 1380gacatgggtc
ccgcccggac cgggctcgtc gccttattcg tcttcctctt cgccgcgttc
1440tactcacccg gtgaaggtcc tgtccccttc acctactcgg ccgaagtctt
ccccctctct 1500cacagagaag taggcatggg cttcgccgtc gccacctgcc
tcttctgggc atctgttttg 1560ggtattacct tccccttctt gcttgactct
ctcggcaccg tcggcgcctt tggtctgtac 1620gcgggcttca acctagtggc
gtttattgcc atcttcttgg tcgtgccgga gacgaagcag 1680aagacgctcg
aggagttgga ttatgtcttt gctgtgaaga cgagcaagtt catgtcgtat
1740cagtgcacca aggcgctgcc gtggttcatc aagaggtggg tgttttggca
gaggaatgca 1800aagctggagc cactgtatga gtttgatcgg atcaaggagg
cggagaagga gaggagagca 1860gaggaggaga gaagggcaaa ggagacggga
acgatcacct ctactgctac aggagctgag 1920ttggatgaga agaagggact
gagtcatgtt aatgctccta attcttag 1968511725DNASaccharomyces
cerevisiae 51atggcagttg aggagaacaa tatgcctgtt gtttcacagc aaccccaagc
tggtgaagac 60gtgatctctt cactcagtaa agattcccat ttaagcgcac aatctcaaaa
gtattctaat 120gatgaattga aagccggtga gtcagggtct gaaggctccc
aaagtgttcc tatagagata 180cccaagaagc ccatgtctga atatgttacc
gtttccttgc tttgtttgtg tgttgccttc 240ggcggcttca tgtttggctg
ggataccggt actatttctg ggtttgttgt ccaaacagac 300tttttgagaa
ggtttggtat gaaacataag gatggtaccc actatttgtc aaacgtcaga
360acaggtttaa tcgtcgccat tttcaatatt ggctgtgcct ttggtggtat
tatactttcc 420aaaggtggag atatgtatgg ccgtaaaaag ggtctttcga
ttgtcgtctc ggtttatata 480gttggtatta tcattcaaat tgcctctatc
aacaagtggt accaatattt cattggtaga 540atcatatctg gtttgggtgt
cggcggcatc gccgtcttat gtcctatgtt gatctctgaa 600attgctccaa
agcacttgag aggcacacta gtttcttgtt atcagctgat gattactgca
660ggtatctttt tgggctactg tactaattac ggtacaaaga gctattcgaa
ctcagttcaa 720tggagagttc cattagggct atgtttcgct tggtcattat
ttatgattgg cgctttgacg 780ttagttcctg aatccccacg ttatttatgt
gaggtgaata aggtagaaga cgccaagcgt 840tccattgcta agtctaacaa
ggtgtcacca gaggatcctg ccgtccaggc agagttagat 900ctgatcatgg
ccggtataga agctgaaaaa ctggctggca atgcgtcctg gggggaatta
960ttttccacca agaccaaagt atttcaacgt ttgttgatgg gtgtgtttgt
tcaaatgttc 1020caacaattaa ccggtaacaa ttattttttc tactacggta
ccgttatttt caagtcagtt 1080ggcctggatg attcctttga aacatccatt
gtcattggtg tagtcaactt tgcctccact 1140ttctttagtt tgtggactgt
cgaaaacttg ggacatcgta aatgtttact tttgggcgct 1200gccactatga
tggcttgtat ggtcatctac gcctctgttg gtgttactag attatatcct
1260cacggtaaaa gccagccatc ttctaaaggt gccggtaact gtatgattgt
ctttacctgt 1320ttttatattt tctgttatgc cacaacctgg gcgccagttg
cctgggtcat cacagcagaa 1380tcattcccac tgagagtcaa gtcgaaatgt
atggcgttgg cctctgcttc caattgggta 1440tgggggttct tgattgcatt
tttcacccca ttcatcacat ctgccattaa cttctactac 1500ggttatgtct
tcatgggctg tttggttgcc atgttttttt atgtcttttt ctttgttcca
1560gaaactaaag gcctatcgtt agaagaaatt caagaattat gggaagaagg
tgttttacct 1620tggaaatctg aaggctggat tccttcatcc agaagaggta
ataattacga tttagaggat 1680ttacaacatg acgacaaacc gtggtacaag
gccatgctag aataa 1725521908DNAPichia stipitis 52atgagtgctg
acgaaaaagt cgctgctgcc ggccaggacg gcttgtttga acacaacagt 60tccacttcga
gcatcgagga caagaagccc tccaagagct ccgatgtcga ttccgtgaac
120tcgcaattag tagacaactc ggtagagggc aacatcttgt cccagtacac
cgaaagtcag 180gtgatgcaga tgggtagaag ctatgccacc aagcacggct
tggacccaga attgttcgcc 240aaggcagctg ctgttgccag aactcctctt
ggtttcaact ccatgccctt cttgacagag 300gaagagaagg ttggtttgaa
tgccgaagcc actaataagt ggcacattcc acccagattg 360atcggggtta
ttgccttggg ttctatggcc gctgctgtgc agggtatgga cgaatcggtc
420attaacggtg ccaacttgtt ctaccccaag gctttcggag tcgacaccat
gcacaattcg 480gacttgattg aaggtttgat caatggtgct ccttaccttt
gctgtggtat tctttcctgt 540tggttgtctg acgcttgtaa ccgtcgtctt
ggtagaaaat ggaccatttt ctggtgttgt 600gtcatttctg ccatcacctg
tgtctggcaa ggtcttgtca acaactggta ccatttgttc 660attgctcgtt
tcttccttgg atttggtgtt ggtatcaagt ccgccactgt tcctgcctac
720tctgccgaat gtactcctaa acacatcaga ggttcgttag tcatgttgtg
gcaattcttc 780acagctgttg gtattatgtt tggttatgtt gcttccttgg
ctttctacaa tgtcggagat 840agaggaatcc attacgggtt gaactggaga
ttgatgcttg gttcggccgc tattcctgct 900gtcatcatct tgttccaaat
tcctttcgct cctgaatctc cacgttggtt aatgggtaag 960gacagacacc
ttgaagcctt tgagtccttg aagcaattga gatacgaaga acttgctgct
1020gctcgtgact gtttctacca gtacgtcttg ttagctgaag aaggttctta
caagatccca 1080accctcacca gatttaagga aatgttcacc aagagaagaa
acagaaacgg tgccatcggt 1140gcatttattg tcatgttcat gcaacagttc
tgtggtatca acgtcattgc ttactactct 1200tcgtctatct ttgtccaatc
tggtttctct caaacttctg ctttgatcgc ttcttggggt 1260ttcggtatgc
ttaacttcac ctttgccatt cctgccttct tcacaatcga tcgtttcggt
1320agaagatcct tattgttggt taccttcccc ttgatggcta ttttcttatt
gattgccggt 1380ttcggtttct tgataaacga agaaacaaac tccaagggaa
gattgggaat gatcatcatc 1440ggtatctata tgttcaccat ctgttactct
tccggtgaag gtccagttcc tttcacctac 1500tctgccgaag ccttcccatt
gtacatcaga gacttgggta tgtcttttgc tactgccacc 1560tgttggactt
tcaacttcat cttggccttc acctggaaca gattggtcaa tgcattcaca
1620tctactggtg ccttcggctt ctacgctgct tggaacatca ttggtttctt
cttggtctta 1680tggttcttgc cagaaaccaa gggcttgacc ttggaagaat
tggacgaagt cttcgccgtt 1740tccgccgtcc aacacgccaa gtaccaaacc
aagagtttga tcaacttcat ccaaagatac 1800gttttacgtt ccaaggtggc
tccattgcct ccattgtacg accaccagag attggctgtc 1860accaacccag
aatggaacga caagccagaa gtctcttatg tcgagtag 19085340DNAArtificial
SequenceSynthesized Construct 53atatatgagc tcgtgagtaa ggaaagagtg
aggaactatc 405450DNAArtificial SequenceSynthesized Construct
54atatatacta gttgttttat atttgttgta aaaagtagat aattacttcc
505530DNAArtificial SequenceSynthesized Construct 55atggatccaa
aaatgtcgtc tcacggctcc 305660DNAArtificial SequenceSynthesized
Construct 56atgaattcct acaaatcttc ttcagaaatc aatttttgtt cagcaacgat
agcttcggac 605734DNAArtificial SequenceSynthesized Construct
57atactagtaa aaatgggcat cttcaacaag aagc 345864DNAArtificial
SequenceSynthesized Construct 58gcatatcgat ctacaaatct tcttcagaaa
tcaatttttg ttcagcaaca gacttgccct 60catg 645942DNAArtificial
SequenceSynthesized Construct 59tattaaatcg atggtagtgg tagtgtgagc
aagggcgagg ag 426036DNAArtificial SequenceSynthesized Construct
60tattaagtcg acctacttgt acagctcgtc catgcc 366128DNAArtificial
SequenceSynthesized Construct 61gcatggatcc atgtcgtctc acggctcc
286231DNAArtificial SequenceSynthesized Construct 62tataatgaat
tcagcaacga tagcttcgga c 316334DNAArtificial SequenceSynthesized
Construct 63tattaaacta gtatgggcat cttcaacaag aagc
346433DNAArtificial SequenceSynthesized Construct 64ttataagaat
tcagcaacag acttgccctc atg 336539DNAArtificial SequenceSynthesized
Construct 65gcatactagt aaaaatgtct cttcctaagg atttcctct
396655DNAArtificial SequenceSynthesized Construct 66atactgcagt
taatgatgat gatgatgatg gtccttcttg atcaaagagt caaag
55676PRTArtificial SequenceSynthesized Construct 67Pro Glu Ser Pro
Arg Phe1 5 684PRTArtificial SequenceSynthesized Construct 68Pro Glu
Ser Pro1 694PRTArtificial SequenceSynthesized Construct 69Pro Glu
Ser Pro1 7010PRTArtificial SequenceGS-linker 70Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser1 5 10 7129DNAArtificial SequenceSynthesized
Construct 71gacatcgatg acatatgcgc taccgcaac 297224DNAArtificial
SequenceSynthesized Construct 72gtgcacgtcg gacccgcaga ttcc
247324DNAArtificial SequenceSynthesized Construct 73ggaatctgcg
ggtccgacgt gcac 247428DNAArtificial SequenceSynthesized Construct
74cagaagattt aaggatcctg aacgtaga 287526DNAArtificial
SequenceSynthesized Construct 75gacatcagtg acatatgtcg ccttcc
267626DNAArtificial SequenceSynthesized Construct 76cctggattga
ggatcctgaa cgtata 267728DNAArtificial SequenceSynthesized Construct
77gacatcgatg acatatggct tccgcaac 287829DNAArtificial
SequenceSynthesized Construct 78ccagaagtat tgagaattct gaacgtaga
297927DNAArtificial SequenceSynthesized Construct 79gacatcgatg
acatatggcg actctgc 278029DNAArtificial SequenceSynthesized
Construct 80ggatacagaa tgaggatcct gaacgtaga 298120DNAArtificial
SequenceSynthesized Construct 81taatacgact cactataggg
208219DNAArtificial SequenceSynthesized Construct 82gctagttatt
gctcagcgg 198336DNAArtificial SequenceSynthesized Construct
83cctatcgtca ttacctcacg tgacgagggg cggctg 368436DNAArtificial
SequenceSynthesized Construct 84cagccgcccc tcgtcacgtg aggtaatgac
gatagg 368529DNAArtificial SequenceSynthesized Construct
85ccttcgaaac ggctacaaac cccaagacg 298634DNAArtificial
SequenceSynthesized Construct 86gcttgtcatc acatcacgtt cagagagccg
tctg 348734DNAArtificial SequenceSynthesized Construct 87cagacggctc
tctgaacgtg atgtgatgac aagc 348830DNAArtificial SequenceSynthesized
Construct 88gcatttgaga cgtcaacaga tcccaagagc 308936DNAArtificial
SequenceSynthesized Construct 89cctattgtca tcacttcacg tgacgagggc
cgcttg 369036DNAArtificial SequenceSynthesized Construct
90caagcggccc tcgtcacgtg aagtgatgac aatagg 369133DNAArtificial
SequenceSynthesized Construct 91cctttgagac tgccacaaac cctaagaccg
gtg 339230DNAArtificial SequenceSynthesized Construct 92atggatccaa
aaatgtcgtc tcacggctcc 309360DNAArtificial SequenceSynthesized
Construct 93atgaattcct acaaatcttc ttcagaaatc aatttttgtt cagcaacgat
agcttcggac 609434DNAArtificial SequenceSynthesized Construct
94atactagtaa aaatgggcat cttcaacaag aagc 349564DNAArtificial
SequenceSynthesized Construct 95gcatatcgat ctacaaatct tcttcagaaa
tcaatttttg ttcagcaaca gacttgccct 60catg 649639DNAArtificial
SequenceSynthesized Construct 96gcatactagt aaaaatgtct cttcctaagg
atttcctct 399755DNAArtificial SequenceSynthesized Construct
97atactgcagt taatgatgat gatgatgatg gtccttcttg atcaaagagt caaag
55
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