U.S. patent application number 14/923147 was filed with the patent office on 2016-02-11 for pentose fermentation by a recombinant microorganism.
The applicant listed for this patent is SHELL OIL COMPANY. Invention is credited to Ryan C. Fong, Ezhilkani Subbian, Xiyun Zhang.
Application Number | 20160040139 14/923147 |
Document ID | / |
Family ID | 46927737 |
Filed Date | 2016-02-11 |
United States Patent
Application |
20160040139 |
Kind Code |
A1 |
Zhang; Xiyun ; et
al. |
February 11, 2016 |
PENTOSE FERMENTATION BY A RECOMBINANT MICROORGANISM
Abstract
The present invention provides methods and compositions suitable
for use in the conversion of xylose to xylitol and xylulose,
including nucleic acid constructs, recombinant fungal host cells,
and related materials.
Inventors: |
Zhang; Xiyun; (Fremont,
CA) ; Fong; Ryan C.; (Redwood City, CA) ;
Subbian; Ezhilkani; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHELL OIL COMPANY |
Houston |
TX |
US |
|
|
Family ID: |
46927737 |
Appl. No.: |
14/923147 |
Filed: |
October 26, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14154488 |
Jan 14, 2014 |
9169468 |
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14923147 |
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13429710 |
Mar 26, 2012 |
8663962 |
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14154488 |
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61469505 |
Mar 30, 2011 |
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61496152 |
Jun 13, 2011 |
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Current U.S.
Class: |
435/254.21 ;
435/254.11; 435/254.2; 435/254.22; 435/254.23; 435/254.3;
435/254.4; 435/254.5; 435/254.6; 435/254.7; 435/254.8;
435/254.9 |
Current CPC
Class: |
C12N 9/0006 20130101;
C12Y 101/01307 20130101 |
International
Class: |
C12N 9/04 20060101
C12N009/04 |
Claims
1. A recombinant fungal host cell comprising at least one
polynucleotide sequence encoding at least one xylose reductase
and/or xylitol dehydrogenase variant, wherein said variant is has
xylose reductase activity and comprises a substitution at one or
more positions selected from 2, 3, 7, 11, 14, 17, 19, 20, 23, 24,
33, 36, 46, 47, 49, 56, 62, 63, 68, 86, 89, 97, 102, 107, 108, 114,
116, 123, 132, 134, 143, 152, 155, 157, 162, 168, 184, 206, 219,
224, 225, 226, 227, 228, 246, 231, 232, 233, 236, 240, 242, 245,
246, 249, 252, 255, 261, 264, 266, 267, 270, 271, 272, 275, 276,
279, 281, 282, 283, 285, 297, 299, 301, 302, 303, and/or 318,
wherein the positions are numbered by correspondence with the amino
acid sequence set forth in SEQ ID NO:2.
2. The recombinant fungal host cell of claim 1, wherein said
variant has xylose reductase activity and further comprises a
substitution at one or more positions selected from P2T, S3H, S3R,
S3W, S3X, N7L, D11K, A14V, F17W, C19L, W20X, D23E, V24G, R33L,
R33V, K36Q, E46C, E46K, D47G, D47N, A49G, K52R, A56E, A56Y, I62V,
D63X, K68G, K68M, K68R, D86N, E89N, E89V, S97R, S97T, D102T, F107Y,
L108Y, T114S, K116Q, K123C, K132A, K132N, D134E, D134H, D134V,
I143L, K152A, K152E, K152H, K152Q, K155A, K155D, K155I, K155R,
K155Y, G157R, I162L, P168S, S184A, R206S, R206V, Q219H, Q219L,
Q219T, L224A, L224S, L224V, N225D, N225E, N225K, N225S, N225Y,
Q226D, Q226E, Q226S, Q226V, G227D, R228T, N231G, N231H, N231L,
N231S, T232A, T232C, T232S, T232V, S233C, S233F, S233G, S233I,
S233K, S233V, S233X, F236L, T240V, K242L, A245S, A246L, A246S,
G249D, P252C, V255I, S261A, S261C, S261N, S261T, G264D; A266V,
A266C, I267V, I267X, K270R, S271G, N272P, P275A, R276M, R276F,
R276W, W276M, E279Q, K281L, K281V, D282C, D282G, D282R, V283H,
S285E, S285T, A297H, A297S, L299X, I301C, I301Y, N302D, N302G,
N302S, L303I, L303V, and V318C, wherein the positions are numbered
by correspondence with the amino acid sequence set forth in SEQ ID
NO:2.
3. The recombinant fungal host cell of claim 1, wherein said
variant has xylose reductase activity and comprises at least one of
the following substitutions and/or substitution sets
P2T/S3H/A49G/P168S/S233K/R276W;
P2T/S3X/A49G/S233X/1267X/R276W/L299X;
P2T/C19L/E46C/A49G/F107Y/K132N/S233K/1267V/R276W;
P2T/C19L/E46C/A49G/K132N/S233K/1267V/R276W;
P2T/W20X/A49G/D63X/R276W; P2T/V24G/A49G; P2T/V24G/A49G/R276W;
P2T/K36Q/A49G/G227D/R276W;
P2T/E46C/A49G/F107Y/K132N/S233K/I267V/R276W;
P2T/E46C/A49G/K132N/V164I/S233K/I267V/R276W; P2T/A49G;
P2T/A49G/K52R/K152E/S233K/I267V/R276W;
P2T/A49G/K52R/R206S/I267V/R276W; P2T/A49G/K52R/R276W;
P2T/A49G/K68G/S261N/R276W/V318C; P2T/A49G/K68G/S261N/V318C;
P2T/A49G/D86N/K132N/R276W; P2T/A49G/K132N;
P2T/A49G/K132N/P168S/S233K/I267V/R276W;
P2T/A49G/K132N/G227D/P252C/R276W; P2T/A49G/K132N/G227D/R276W;
P2T/A49G/K132N/S233K/I267V/R276W; P2T/A49G/K132N/S233K/R276W;
P2T/A49G/K132N/I267V/R276W; P2T/A49G/K132N/R276W; PT2/A49G/K152E;
P2T/A49G/K152E/P168S/S233V/I267V/R276W; P2T/A49G/K152E/I267V/R276W;
P2T/A49G/K152E/R276W; P2T/A49G/P168S/R206S/I267V/R276W;
P2T/A49G/P168S/S233K/R276W; P2T/A49G/P168S/R276W;
P2T/A49G/R206S/S233K/I267V/R276W; P2T/A49G/R206S;
P2T/A49G/R206S/R276W; P2T/A49G/G227D/P252C/G264D/R276W;
P2T/A49G/G227D/P252C/R276W; P2T/A49G/G227D/I267V/R276W;
P2T/A49G/G227D/R276W; P2T/A49G/S233K/R276W;
P2T/A49G/S233K/I267V/R276W; P2T/A49G/S233V/A246L/R276W/N302S;
P2T/A49G/S233V/A246L/N302S; P2T/A49G/S233V/I267V/R276W;
P2T/A49G/S233V/R276W; P2T/A49G/P252C/R276W; P2T/A49G/S261N;
P2T/A49G/S261N/R276W; P2T/A49G/I267V/R276W; P2T/A49G/R276W;
P2T/A46G/R276W/N302S; P2T/A49G/R276W/V318C; P2T/A49G/N302S;
P2T/A49G/V319C; E46K/D47N; A49G/K132A; A49G/K132A/K152E;
A49G/K132A/K152E/R276W; A49G/K132A/R206S; A49G/K132A/R276W;
A49G/K132A/R206S/R276W; A49G/K132N/K152E/R276W;
A49G/K132N/K152E/R276W/N302S; A49G/K132N/K152E/N302S;
A49G/K132N/R206S/S233V/W276M/N302S;
A49G/K132N/R206S/S233V/R276M/N302S; A49G/K152E; A49G/K152E/R276W;
A49G/K152E; and A49G/R276W, wherein the positions are numbered by
correspondence with the amino acid sequence set forth in SEQ ID
NO:2.
4. The recombinant fungal host cell of claim 1, wherein, said
variant comprises the polypeptide sequence set forth in SEQ ID
NOS:55, 57, or 59.
5. A recombinant fungal host cell comprising at least one
polynucleotide sequence encoding at least one xylose reductase,
wherein said polynucleotide sequence comprises SEQ ID NOS:1, 3, 4,
40, 42, 44, and/or 46; and at least one polynucleotide encoding at
least one xylitol dehydrogenase, wherein said polynucleotide
sequence comprises SEQ ID NOS:5, 7, 8, and/or 48.
6. The recombinant fungal host cell of claim 5, further comprising
at least one polynucleotide encoding at least one xylulokinase,
wherein said polynucleotide comprises SEQ ID NO: 11 and/or 12.
Description
[0001] The present application is a Divisional of U.S. patent
application Ser. No. 14/154,488, filed on Jan. 14, 2014, which is a
Divisional of U.S. patent application Ser. No. 13/429,710, filed on
Mar. 26, 2012, which claims priority to U.S. Pat. App. No.
61/469,505, filed on Mar. 30, 2011, and U.S. Pat. App. No.
61/496,152, filed on Jun. 13, 2011, each of which is incorporated
by reference in their entirety herein.
FIELD OF THE INVENTION
[0002] The present invention provides methods and compositions
suitable for use in the conversion of xylose to xylitol and
xylulose, including nucleic acid constructs, recombinant fungal
host cells, and related materials.
BACKGROUND
[0003] Ethanol and ethanol fuel blends are widely used in Brazil
and in the United States as a transportation fuel. Combustion of
these fuels is believed to produce fewer of the harmful exhaust
emissions (e.g., hydrocarbons, nitrogen oxide, and volatile organic
compounds [VOCs]) that are generated by the combustion of
petroleum. Bioethanol is a particularly favored form of ethanol
because the plant biomass from which it is produced utilizes
sunlight, an energy source that is renewable. In the United States,
ethanol is used in gasoline blends that are from 5% to 85% ethanol.
Blends of up to 10% ethanol (E10) are approved for use in all
gasoline vehicles in the U.S. and blends of up to 85% ethanol (E85)
can be utilized in specially engineered flexible-fuel vehicles
(FFV). The Brazilian government has mandated the use of
ethanol-gasoline blends as a vehicle fuel, and the mandatory blend
has been 25% ethanol (E25) since 2007.
[0004] Bioethanol is currently produced by the fermentation of
hexose sugars that are obtained from carbon feedstocks. Currently,
only the sugar from sugar cane and starch from feedstock such as
corn can be economically converted. There is, however, much
interest in using lignocellulosic feedstocks where the cellulose
part of a plant is broken down to sugars and subsequently converted
to ethanol. Lignocellulosic biomass is made up of cellulose,
hemicelluloses, and lignin. Cellulose and hemicellulose can be
hydrolyzed in a saccharification process to sugars that can be
subsequently converted to ethanol via fermentation. The major
fermentable sugars from lignocelluloses are glucose and xylose. For
economical ethanol yields, a strain that can effectively convert
all the major sugars present in cellulosic feedstock would be
highly desirable.
SUMMARY OF THE INVENTION
[0005] The present invention provides methods and compositions
suitable for use in the conversion of xylose to xylitol and
xylulose, including nucleic acid constructs, recombinant fungal
host cells, and related materials.
[0006] In some embodiments, the present invention provides
recombinant nucleic acid constructs comprising at least one
polynucleotide sequence that encodes at least one xylose reductase,
wherein the polynucleotide is selected from: (a) a polynucleotide
that encodes a polypeptide comprising an amino acid sequence having
at least about 70% identity to SEQ ID NO:2, wherein the amino acid
sequence comprises at least one substitution set forth herein; and
(b) a polynucleotide that hybridizes under stringent hybridization
conditions to the complement of a polynucleotide that encodes a
polypeptide having the amino acid sequence of SEQ ID NO:2, and
wherein the amino acid sequence comprises at least one substitution
set forth herein. In some embodiments, the polynucleotide sequence
encodes a polypeptide comprising an amino acid sequence at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least
about 97%, at least about 98%, or at least about 99% identical to
SEQ ID NO:2, wherein the amino acid sequence comprises at least one
substitution set forth herein. In some alternative embodiments, the
polynucleotide sequence encodes a polypeptide comprising a portion
of the amino acid sequence set forth in SEQ ID NO:2, wherein the
amino acid sequence comprises at least one substitution set forth
herein. In some further embodiments, the polynucleotide sequence
encodes a polypeptide having an amino acid sequence that comprises
at least one substitution at position 2, 3, 7, 11, 14, 17, 23, 24,
33, 36, 46, 47, 49, 56, 62, 68, 89, 97, 102, 108, 114, 116, 123,
132, 134, 143, 152, 155, 157, 162, 168, 184, 206, 219, 224, 225,
226, 228, 246, 231, 232, 233, 236, 240, 242, 245, 246, 249, 252,
255, 261, 266, 267, 275, 276, 279, 281, 282, 283, 285, 297, 301,
302, 303, and/or 318, wherein the positions are numbered by
correspondence with the amino acid sequence of P. stipitis xylose
reductase set forth in SEQ ID NO:2. In some additional embodiments,
the polynucleotide sequence encodes a polypeptide having an amino
acid sequence that comprises at least one substitution selected
from P2, S3, N7, D11, A14, F17, D23, V24, R33, K36, E46, D47, A49,
A56, 162, K68, E89, S97, D102, L108, T114, K116, K123, K132, D134,
I143, K152, K155, G157, I162, P168, S184, R206, Q219, L224, N225,
Q226, R228, A246, N231, T232, S233, F236, T240, K242, A245, A246,
G249, P252, V255, S261, A266, I267, P275, R276, E279, K281, D282,
V283, S285, A297, 1301, N302, L303, and/or V318, wherein the
positions are numbered by correspondence with the amino acid
sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2.
In yet some additional embodiments, the polynucleotide sequence
encodes a polypeptide having an amino acid sequence that comprises
at least one substitution selected from P2T, S3H, S3R, S3W, N7L,
D11K, A14V, F17W, D23E, V24G, R33L, R22V, K36Q, E46K, D47G, D47N,
A49G, A56E, A56Y, I62V, K68G, K68M, K68R, E89N, E89V, S97R, S97T,
D102T, L108Y, T114S, K116Q, K123C, K132A, K132N, D134E, D134H,
D134V, I143L, K152A, K152E, K152H, K152Q, K155A, K155D, K155I,
K155R, K155Y, G157R, I162L, P168S, S184A, R206S, R206V, Q219H,
Q219L, Q219T, L224A, L224S, L224V, N225D, N225E, N225K, N225S,
N225Y, Q226D, Q226E, Q226S, Q226V, R228T, N231G, N231H, N231L,
N231S, T232A, T232C, T232S, T232V, S233C, S233F, S233G, S233I,
S233K, S233V, F236L, T240V, K242L, A245S, A246L, A246S, G249D,
P252C, V255I, S261A, S261C, S261N, S261T, A266V, A266C, I267V,
P275A, R276M, R276W, E279Q, K281L, K281V, D282C, D282G, D282R,
V283H, S285E, S285T, A297H, A297S, I301C, I301Y, N302D, N302G,
N302S, L303I, L303V, and/or V318C, wherein the positions are
numbered by correspondence with the amino acid sequence of P.
stipitis xylose reductase set forth in SEQ ID NO:2. In some
embodiments, the polynucleotide sequence is at least about 80%,
about 85%, about 90%, or at least 95% identical to SEQ ID NO:1, and
wherein the polynucleotide sequence comprises at least one mutation
set forth herein. In some additional embodiments, the
polynucleotide sequence comprises at least one mutation selected
from t82a, t99a, g156a, c201t, a280c, c306t, t354g, t358c, a378t,
c408t, a426t, c438t, a478c, t511c, a585g, t670c, t688c, t703c,
t747c, t751a, t766c, t811a, c816t, a826c, c849g, c855t, and/or
c906t, wherein the nucleotide position is determined by alignment
with SEQ ID NO:1. In some additional embodiments, the present
invention provides nucleic acid constructs comprising SEQ ID NO:3
and/or SEQ ID NO:4.
[0007] The present invention also provides isolated xylose
reductase variants, wherein the variants have xylose reductase
activity and comprise a substitution at one or more positions
selected from 2, 3, 7, 11, 14, 17, 23, 24, 33, 36, 46, 47, 49, 56,
62, 68, 89, 97, 102, 108, 114, 116, 123, 132, 134, 143, 152, 155,
157, 162, 168, 184, 206, 219, 224, 225, 226, 228, 246, 231, 232,
233, 236, 240, 242, 245, 246, 249, 252, 255, 261, 266, 267, 275,
276, 279, 281, 282, 283, 285, 297, 301, 302, 303, and/or 318,
wherein the positions are numbered by correspondence with the amino
acid sequence of P. stipitis xylose reductase set forth in SEQ ID
NO:2. In some embodiments, the variant has xylose reductase
activity and comprises a substitution at one or more positions
selected from P2, S3, N7, D11, A14, F17, D23, V24, R33, K36, E46,
D47, A49, A56, 162, K68, E89, S97, D102, L108, T114, K116, K123,
K132, D134, I143, K152, K155, G157, I162, P168, S184, R206, Q219,
L224, N225, Q226, R228, A246, N231, T232, S233, F236, T240, K242,
A245, A246, G249, P252, V255, S261, A266, I267, P275, R276, E279,
K281, D282, V283, S285, A297, I301, N302, L303, and/or V318,
wherein the positions are numbered by correspondence with the amino
acid sequence of P. stipitis xylose reductase set forth in SEQ ID
NO:2. In some additional embodiments, the variant has xylose
reductase activity and comprises a substitution at one or more
positions selected from P2T, S3H, S3R, S3W, N7L, D11K, A14V, F17W,
D23E, V24G, R33L, R22V, K36Q, E46K, D47G, D47N, A49G, A56E, A56Y,
I62V, K68G, K68M, K68R, E89N, E89V, S97R, 597T, D102T, L108Y,
T114S, K116Q, K123C, K132A, K132N, D134E, D134H, D134V, I143L,
K152A, K152E, K152H, K152Q, K155A, K155D, K155I, K155R, K155Y,
G157R, I162L, P168S, S184A, R206S, R206V, Q219H, Q219L, Q219T,
L224A, L224S, L224V, N225D, N225E, N225K, N225S, N225Y, Q226D,
Q226E, Q226S, Q226V, R228T, N231G, N231H, N231L, N231S, T232A,
T232C, T232S, T232V, S233C, S233F, S233G, S233I, S233K, S233V,
F236L, T240V, K242L, A245S, A246L, A246S, G249D, P252C, V255I,
S261A, S261C, S261N, S261T, A266V, A266C, I267V, P275A, R276M,
R276W, E279Q, K281L, K281V, D282C, D282G, D282R, V283H, S285E,
S285T, A297H, A297S, I301C, I301Y, N302D, N302G, N302S, L303I,
L303V, and/or V318C, wherein the positions are numbered by
correspondence with the amino acid sequence of P. stipitis xylose
reductase set forth in SEQ ID NO:2. In some further embodiments,
the isolated xylose reductase variants comprise the sequence set
forth in SEQ ID NO:41, 43, 45, and/or 47. The present invention
also provides isolated nucleic acid sequences comprising SEQ ID
NO:3, 4, 40, 42, 44, and/or 46.
[0008] The present invention also provides recombinant nucleic acid
constructs comprising at least one polynucleotide sequence that
encodes a xylitol dehydrogenase, wherein the polynucleotide is
selected from: (a) a polynucleotide that encodes a polypeptide
comprising an amino acid sequence having at least 70% identity to
SEQ ID NO:6, wherein the amino acid sequence comprises at least one
substitution set forth herein; and (b) a polynucleotide that
hybridizes under stringent hybridization conditions to the
complement of a polynucleotide that encodes a polypeptide having
the amino acid sequence of SEQ ID NO:6, wherein the amino acid
sequence comprises at least one substitution set forth herein. The
present invention further provides nucleic acid constructs, wherein
the polynucleotide sequence encodes a polypeptide comprising an
amino acid sequence at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 91%, at least
about 92%, at least about 93%, at least about 94%, at least about
95%, at least about 96%, at least about 97%, at least about 98%, or
at least about 99% identical to SEQ ID NO:6, wherein the amino acid
sequence comprises at least one substitution set forth herein. In
some embodiments, the polynucleotide sequence encodes a polypeptide
comprising at least a portion of the amino acid sequence set forth
in SEQ ID NO:6, wherein the amino acid sequence comprises at least
one substitution set forth herein. In some further embodiments, the
polynucleotide sequence encodes a polypeptide having an amino acid
sequence that comprises at least one substitution at position 5,
13, 19, 49, 81, 149, 187, 189, 202, 205, 206, 210, 215, 218, 226,
227, 228, 229, 230, 231, 235, 239, 241, 251, 252, 256, 260, 286,
287, 296, 307, 327, 350, and/or 352, wherein the positions are
numbered by correspondence with the amino acid sequence of P.
stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some
additional embodiments, the polynucleotide sequence encodes a
polypeptide having an amino acid sequence that comprises at least
one substitution selected from P5, D13, T19, A49, S81, H149, V187,
L189, G202, V205, V206, I208, F209, D210, N211, M215, D218, F226,
N227, S228, K229, T230, G231, E235, A239, G241, C251, T252, P256,
L260, T286, V287, F296, K307, D327, A350, and/or K352, wherein the
positions are numbered by correspondence with the amino acid
sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID
NO:6. In some further embodiments, the polynucleotide sequence
encodes a polypeptide having an amino acid sequence that comprises
at least one substitution selected from P5E, PSR, D13G, T19L, T19Q,
A49Q, S81G, H149R, V187M, L189C, G202D, V205C, V205H, V206A, I208R,
F209S, D210W, N211K, N211R, N211S, M215A, M215C, D218R, F226V,
N227T, S228P, K229R, T230V, G231A, G231H, E235K, E235Q, A239C,
G241W, C251G, C251T, T252S, P256A, P256Q, L260Q, T286V, V287L,
F296R, K307R, D327A, A350D, and/or K352E, wherein the positions are
numbered by correspondence with the amino acid sequence of P.
stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some
still further embodiments, the polynucleotide sequence is at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least
about 97%, at least about 98%, or at least about 99% identical to
SEQ ID NO:5, and wherein the polynucleotide sequence comprises at
least one mutation set forth herein. In some embodiments, the
polynucleotide sequence comprises mutations at at least one
position selected from t24g, c630t, a732g, a768t, a780g, wherein
nucleotide position is determined by alignment with SEQ ID NO:5. In
some additional embodiments, the nucleic acid construct comprises
SEQ ID NO:7 and/or SEQ ID NO:8. In some further embodiments, the
present invention provides an isolated nucleic acid sequence
comprising SEQ ID NO:7, 8, and/or 48.
[0009] The present invention also provides isolated xylitol
dehydrogenase variants comprising the sequence set forth in SEQ ID
NO:49. In some additional embodiments, the present invention
provides isolated xylitol dehydrogenase variants, wherein the
variants have xylitol dehydrogenase activity and comprise a
substitution at one or more positions selected from 5, 13, 19, 49,
81, 149, 187, 189, 202, 205, 206, 210, 215, 218, 226, 227, 228,
229, 230, 231, 235, 239, 241, 251, 252, 256, 260, 286, 287, 296,
307, 327, 350, and/or 352, wherein the positions are numbered by
correspondence with the amino acid sequence of P. stipitis xylitol
dehydrogenase set forth in SEQ ID NO:6. In some embodiments, the
xylitol dehydrogenase variant have xylitol dehydrogenase activity
and comprise a substitution at one or more positions selected from
P5, D13, T19, A49, S81, H149, V187, L189, G202, V205, V206, I208,
F209, D210, N211, M215, D218, F226, N227, S228, K229, T230, G231,
E235, A239, G241, C251, T252, P256, L260, T286, V287, F296, K307,
D327, A350, and/or K352, wherein the positions are numbered by
correspondence with the amino acid sequence of P. stipitis xylitol
dehydrogenase set forth in SEQ ID NO:6. In some further
embodiments, the isolated xylitol dehydrogenase variant has xylitol
dehydrogenase activity and comprises a substitution at one or more
positions selected from PSE, PSR, D13G, T19L, T19Q, A49Q, S81G,
H149R, V187M, L189C, G202D, V205C, V205H, V206A, I208R, F209S,
D210W, N211K, N211R, N211S, M215A, M215C, D218R, F226V, N227T,
S228P, K229R, T230V, G231A, G231H, E235K, E235Q, A239C, G241W,
C251G, C251T, T252S, P256A, P256Q, L260Q, T286V, V287L, F296R,
K307R, D327A, A350D, and/or K352E, wherein the positions are
numbered by correspondence with the amino acid sequence of P.
stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. The
present invention also provides recombinant nucleic acid constructs
comprising SEQ ID NO:11 and/or SEQ ID NO:12.
[0010] The present invention also provides recombinant nucleic acid
constructs comprising polynucleotide sequences encoding at least
one xylose reductase and at least one xylitol dehydrogenase,
wherein the polynucleotide sequences are selected from SEQ ID
NOS:1, 3, 4, 40, 42, 44, and 46; and SEQ ID NOS:5, 7, 8, 48.
[0011] The present invention further provides recombinant nucleic
acid constructs further comprising at least one polynucleotide
sequence encoding at least one xylulokinase, wherein the
polynucleotide sequence comprises SEQ ID NO:11 and/or SEQ ID NO:12.
In some embodiments, the recombinant nucleic acid constructs
comprise at least one sequence selected from SEQ ID NOS:1, 3, 4, 5,
7, 8, 11, 12, 40, 42, 44, 46, and 48. In some further embodiments,
the constructs comprise SEQ ID NO:46 and/or 48. In some additional
embodiments, the nucleic acid constructs further comprise at least
one genetic element that facilitates stable integration into a
fungal host genome. In some additional embodiments, the nucleic
acid constructs further comprise a genetic element that facilitates
stable integration into a fungal host genome. In some embodiments,
the genetic element facilitates integration into a fungal host
genome by homologous recombination. In yet some additional
embodiments, the nucleic acid constructs comprise a fungal origin
of replication. In some embodiments, the fungal origin of
replication is a yeast origin of replication. In some further
embodiments, the polynucleotide sequence is operatively linked to a
promoter sequence that is functional in a fungal cell. In some
embodiments, the promoter sequence is a fungal promoter sequence.
In some additional embodiments, the fungal promoter sequence is a
yeast promoter sequence. In still some further embodiments, the
polynucleotide sequence is operatively linked to a transcription
termination sequence that is functional in a fungal cell. In some
embodiments, the polynucleotide sequence contains codons optimized
for expression in a yeast cell. In some further embodiments, the
polynucleotide sequence comprises SEQ ID NO:3, 4, 7, 8, 11, and/or
12.
[0012] The present invention also provides recombinant fungal host
cells comprising at least one polynucleotide sequence encoding at
least one xylose reductase, wherein the polynucleotide sequences
comprise at least one mutation relative to SEQ ID NO:1, as set
forth herein. In some embodiments, the recombinant fungal host cell
comprises at least one polynucleotide sequence selected from SEQ ID
NOS:3, 4, 40, 42, 44, and 46. In some embodiments, the recombinant
fungal host cells comprise at least one polynucleotide sequence
encoding at least one xylitol dehydrogenase, wherein the
polynucleotide sequence comprises at least one mutation relative to
SEQ ID NO:5, as set forth herein. In some embodiments, at least one
polynucleotide sequence is selected from SEQ ID NOS:7, 8, and
48.
[0013] The present invention also provides recombinant fungal host
cells comprising at least one polynucleotide sequence encoding at
least one xylulokinase, wherein the polynucleotide sequences
comprise at least one mutation relative to SEQ ID NO:9. In some
embodiments, at least one polynucleotide sequence is selected from
SEQ ID NOS:11 and 12.
[0014] The present invention also provides recombinant fungal host
cells comprising at least one polynucleotide sequence encoding at
least one xylose reductase, wherein the polynucleotide sequence
comprises SEQ ID NO:1, 3, 4, 40, 42, 44, and/or 46; and at least
one polynucleotide encoding at least one xylitol dehydrogenase,
wherein the polynucleotide sequence comprises SEQ ID NO:5, 7, 8,
and/or 48. In some embodiments, the recombinant fungal host cells
further comprise at least one polynucleotide encoding at least one
xylulokinase, wherein the polynucleotide comprises SEQ ID NO:11
and/or 12. In some embodiments, at least one polynucleotide is
integrated into the host cell genome. In some additional
embodiments, the host cell is a yeast cell. In some embodiments,
the host cell has had one or more native genes deleted from its
genome. In some further embodiments, the deletion results in one or
more phenotypes selected from increased transport of xylose into
the host cell, increased xylulose kinase activity, increased flux
through the pentose phosphate pathway, decreased sensitivity to
catabolite repression, increased tolerance to ethanol, increased
tolerance to acetate, increased tolerance to increased osmolarity,
increased tolerance to low pH, and reduced production of by
products, wherein comparison is made with respect to the
corresponding host cell without the deletion(s). In some additional
embodiments, the host cell is altered to overexpress one or more
polynucleotides. In some further embodiments, overexpression
results in one or more phenotypes selected from increased transport
of xylose into the host cell, increased xylulose kinase activity,
increased flux through the pentose phosphate pathway, decreased
sensitivity to catabolite repression, increased tolerance to
ethanol, increased tolerance to acetate, increased tolerance to
increased osmolarity, increased tolerance to low pH, and reduced
product of by products, wherein comparison is made to the
corresponding unaltered host cell. In still some further
embodiments, the host cell comprises at least one xylose reductase
variant and at least one xylitol dehydrogenase variant. In some
additional embodiments, the recombinant fungal host cells further
comprise at least one xylulokinase. In some embodiments, the
xylulokinase is encoded by SEQ ID NO:11 and/or 12. In some
additional embodiments, the recombinant fungal host cell is
transformed using nucleic acid constructs comprising from about two
to about fifty copies of at least one xylose reductase and/or
xylitol dehydrogenase and/or xylulokinase. It is not intended that
the present invention be limited to any particular copy number of
xylose reductase and/or xylitol dehydrogenase genes and/or
xylulokinase, as any suitable number finds use in the present
invention. In some embodiments, the nucleic acid constructs
comprise any combination of wild-type and/or variant xylose
reductase and/or xylitol dehydrogenase and/or xylulokinase.
Furthermore, any suitable method for introducing multiple copies of
either xylose reductase and/or xylitol dehydrogenase and/or
xylulokinase find use in the present invention.
[0015] The present invention also provides processes for producing
a fermentation product, comprising: (a) providing at least one
recombinant fungal host cell provided herein; (b) providing a
fermentation medium comprising; and (c) contacting the fermentation
medium with the recombinant fungal host cell under conditions
suitable for generating the fermentation product. In some
embodiments, the process further comprises (d) recovering the
fermentation product. In some additional embodiments, the
fermenting step is carried out under conditions selected from
microaerobic or aerobic conditions. In some embodiments, the
fermenting step is carried out under anaerobic conditions. In some
further embodiments, the fermentation product is selected from an
alcohol, a fatty acid, lactic acid, acetic acid, 3-hydroxypropionic
acid, acrylic acid, succinic acid, citric acid, malic acid, fumaric
acid, succinic acid, an amino acid, 1,3-propanediol, ethylene,
glycerol, and a .beta.-lactam. In some additional embodiments, the
fermentation product is an alcohol selected from ethanol and
butanol. In some embodiments, the fermentation product is ethanol.
In some embodiments, the fermentation medium comprises product from
a saccharification process. In some further embodiments, the
fermentation medium comprises hemicellulosic feedstock.
DESCRIPTION OF THE FIGURES
[0016] FIG. 1 depicts xylose conversion pathways. In yeast and
filamentous fungi, D-xylose is initially reduced to xylitol by
NAD(P)H-dependent xylose reductase ("XR"). Xylitol is subsequently
oxidized to D-xylulose by NAD+-dependent xylitol dehydrogenase
("XDH" or "XD"). Xylulokinase ("XK") subsequently phosphorylates
D-xylulose to produce D-xylulose 5-phosphate, which is then further
metabolized through the pentose phosphate pathway ("PPP"). In
bacteria, D-xylose is directly converted to D-xylulose by a xylose
isomerase ("XI").
[0017] FIGS. 2A-C depict the metabolic pathways for converting
D-xylulose-5-P to ethanol.
[0018] FIG. 2A depicts the pentose phosphate pathway (PPP). The
substrates and products are shown. The enzymes are represented by
numbers as follows: 6. Ribulose-5-phosphate 3-epimerase; 7.
Transketolase (TKL1); 8. Transaldolase (TALI); 9.
Ribose-5-phosphate ketoisomerase (RKI1); 10. 6-phosphogluconate
dehydrogenase (GND1); 11. 6-phosphogluconalactonase (SOL3); and 12.
Glucose-6-phosphate-1-dehydrogenase (ZWF).
[0019] FIG. 2B depicts the pathway of glycolysis. The substrates
and products are shown. The enzymes are represented by numbers as
follows: 13. Hexokinase; 14. Phosphoglucose isomerase; 15.
Phosphofructokinase; 16. Aldolase; 17. Triose phosphate isomerase;
18. Glyceraldehyde 3-phosphate dehydrogenase; 19.
3-Phosphoglycerate kinase; 20. Phosphoglyceromutase; 21. Enolase;
and 22. Pyruvate kinase.
[0020] FIG. 2C depicts the metabolic pathway for converting
pyruvate to ethanol. The substrates and products are shown. The
enzymes are represented by numbers as follows: 23. Pyruvate
decarboxylase; 24. Aldehyde dehydrogenase; and 25. Alcohol
dehydrogenase.
[0021] FIG. 3 provides a map of plasmid PLS2802, comprising XR.2,
XD.2, and XK.2 genes.
DESCRIPTION OF THE INVENTION
[0022] The present invention provides methods and compositions
suitable for use in the conversion of xylose to xylitol and
xylulose, including nucleic acid constructs, recombinant fungal
host cells, and related materials.
[0023] All patents and publications, including all sequences
disclosed within such patents and publications, referred to herein
are expressly incorporated by reference. Unless otherwise
indicated, the practice of the present invention involves
conventional techniques commonly used in molecular biology,
fermentation, microbiology, and related fields, which are known to
those of skill in the art. Unless defined otherwise herein, all
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention belongs. Although any methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of the present invention, the preferred methods and
materials are described. Indeed, it is intended that the present
invention not be limited to the particular methodology, protocols,
and reagents described herein, as these may vary, depending upon
the context in which they are used. The headings provided herein
are not limitations of the various aspects or embodiments of the
present invention.
[0024] Nonetheless, in order to facilitate understanding of the
present invention, a number of terms are defined below. Numeric
ranges are inclusive of the numbers defining the range. Thus, every
numerical range disclosed herein is intended to encompass every
narrower numerical range that falls within such broader numerical
range, as if such narrower numerical ranges were all expressly
written herein. It is also intended that every maximum (or minimum)
numerical limitation disclosed herein includes every lower (or
higher) numerical limitation, as if such lower (or higher)
numerical limitations were expressly written herein.
[0025] As used herein, the term "comprising" and its cognates are
used in their inclusive sense (i.e., equivalent to the term
"including" and its corresponding cognates).
[0026] As used herein and in the appended claims, the singular "a",
"an" and "the" include the plural reference unless the context
clearly dictates otherwise. Thus, for example, reference to a "host
cell" includes a plurality of such host cells.
[0027] Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation; amino acid sequences are written
left to right in amino to carboxy orientation, respectively. The
headings provided herein are not limitations of the various aspects
or embodiments of the invention that can be had by reference to the
specification as a whole. Accordingly, the terms defined below are
more fully defined by reference to the specification as a
whole.
[0028] As used herein, the terms "isolated" and "purified" are used
to refer to a molecule (e.g., an isolated nucleic acid,
polypeptide, etc.) or other component that is removed from at least
one other component with which it is naturally associated.
[0029] As used herein, the term "reference enzyme" refers to an
enzyme to which a variant enzyme of the present invention is
compared in order to determine the presence of an improved property
in the variant enzyme being evaluated, including but not limited to
improved thermoactivity, improved thermostability, or improved
stability. In some embodiments, a reference enzyme is a wild-type
enzyme (e.g., wild-type xylose reductase or xylitol dehydrogenase).
In some embodiments, a reference enzyme is another variant enzyme
(e.g., another variant xylose reductase or xylitol dehydrogenase
enzyme of the present invention).
[0030] As used herein, the term "recombinant" refers to a
polynucleotide or polypeptide that does not naturally occur in a
host cell. In some embodiments, recombinant molecules contain two
or more naturally-occurring sequences that are linked together in a
way that does not occur naturally. A recombinant cell contains a
recombinant polynucleotide or polypeptide.
[0031] As used herein, the terms "enzyme variant" and "variant
enzyme" are used in reference to enzymes that are similar to a
reference enzyme, particularly in their function, but have
mutations in their amino acid sequence that make them different in
sequence from the wild-type or another reference enzyme. Enzyme
variants (e.g., "xylose reductase variants" and/or "xylitol
dehydrogenase variants") can be made by a wide variety of different
mutagenesis techniques well known to those skilled in the art. In
addition, mutagenesis kits are also available from many commercial
molecular biology suppliers. Methods are available to make specific
substitutions at defined amino acids (site-directed), specific or
random mutations in a localized region of the gene (regio-specific)
or random mutagenesis over the entire gene (e.g., saturation
mutagensis). Numerous suitable methods are known to those in the
art to generate enzyme variants, including but not limited to
site-directed mutagenesis of single-stranded DNA or double-stranded
DNA using PCR, cassette mutagenesis, gene synthesis, error-prone
PCR, shuffling, and chemical saturation mutagenesis, or any other
suitable method known in the art. After the variants are produced,
they can be screened for the desired property (e.g., high or
increased; or low or reduced activity, increased thermal and/or
alkaline stability, etc.).
[0032] As used herein, "combinatorial variant" refers to any
variant that has a combination of two or more mutations (e.g.,
substitutions). In some embodiments, the combination of mutations
results in changes in enzyme activity (e.g., improved
thermostability, improved thermoactivity, improved specific
activity, etc.).
[0033] As used herein, the term "overexpress" is intended to
encompass increasing the expression of a protein to a level greater
than the cell normally produces. It is intended that the term
encompass overexpression of endogenous, as well as heterologous
proteins.
[0034] For clarity, reference to a cell of a particular strain
refers to a parental cell of the strain as well as progeny and
genetically modified derivatives. Genetically modified derivatives
of a parental cell include progeny cells that contain a modified
genome or episomal plasmids that confer for example, antibiotic
resistance, improved fermentation, the ability to utilize xylose as
a carbon source, etc.
[0035] A nucleic acid construct, nucleic acid (e.g., a
polynucleotide), polypeptide, or host cell is referred to herein as
"recombinant" when it is non-naturally occurring, artificial or
engineered.
[0036] The terms "xylose reductase" and "xylose reductase
polypeptide" are used interchangeably herein to refer to an enzyme
that is capable of catalyzing xylose to xylitol. The ability to
catalyze xylose to xylitol is referred to herein as "xylose
reductase activity".
[0037] The terms "protein" and "polypeptide" are used
interchangeably herein to refer to a polymer of amino acid
residues. The term "xylose reductase polynucleotide" refers to a
polynucleotide that encodes a xylose reductase polypeptide.
[0038] In some embodiments, xylose reductase polynucleotides
employed in the practice of the present invention encode a
polypeptide comprising an amino acid sequence that is at least
about 71% identical, at least about 72% identical, at least about
73% identical, at least about 74% identical, at least about 75%
identical, at least about 76% identical, at least about 77%
identical, at least about 78% identical, at least about 79%
identical, at least about 80% identical, at least about 81%
identical, at least about 82% identical, at least about 83%
identical, at least about 84% identical, at least about 85%
identical, at least about 86% identical, at least about 87%
identical, at least about 88% identical, at least about 89%
identical, at least about 90% identical, at least about 91%
identical, at least about 92% identical, at least about 93%
identical, at least about 94% identical, at least about 95%
identical, at least about 96% identical, at least about 97%
identical, at least about 98% identical, or at least about 99%
identical to SEQ ID NO:2, wherein the sequence comprises at least
one substitution set forth herein. In some embodiments, the xylose
reductase polynucleotide encodes a polypeptide having an amino acid
sequence comprising SEQ ID NO:41, 43, 45, and/or 47.
[0039] In some embodiments, xylose reductase polynucleotides
employed in the practice of the present invention comprise
polynucleotide sequences that are at least about 70% identical, at
least about 71% identical, at least about 72% identical, at least
about 73% identical, at least about 74% identical, at least about
75% identical, at least about 76% identical, at least about 77%
identical, at least about 78% identical, at least about 79%
identical, at least about 80% identical, at least about 81%
identical, at least about 82% identical, at least about 83%
identical, at least about 84% identical, at least about 85%
identical, at least about 86% identical, at last about 87%
identical, at least about 88% identical, at least about 89%
identical, at least about 90% identical, at least about 91%
identical, at least about 92% identical, at least about 93%
identical, at least about 94% identical, at least about 95%
identical, at least about 96% identical, at least about 97%
identical, at least about 98% identical, or at least about 99%
identical to SEQ ID NO:1, 3 and/or 4.
[0040] The terms "xylitol dehydrogenase" and "xylitol dehydrogenase
polypeptide" are used interchangeably herein to refer to an enzyme
that is capable of catalyzing xylitol to xylulose. The ability to
catalyze xylitol to xylulose is referred to herein as "xylitol
dehydrogenase activity". Also, as used herein, the term "xylitol
dehydrogenase polynucleotide" refers to a polynucleotide that
encodes a xylitol dehydrogenase polypeptide.
[0041] In some embodiments, xylitol dehydrogenase polynucleotides
employed in the practice of the present invention encode
polypeptides comprising amino acid sequences that are at least
about 71% identical, at least about 72% identical, at least about
73% identical, at least about 74% identical, at least about 75%
identical, at least about 76% identical, at least about 77%
identical, at least about 78% identical, at least about 79%
identical, at least about 80% identical, at least about 81%
identical, at least about 82% identical, at least about 83%
identical, at least about 84% identical, at least about 85%
identical, at least about 86% identical, at least about 87%
identical, at least about 88% identical, at least about 89%
identical, at least about 90% identical, at least about 91%
identical, at least about 92% identical, at least about 93%
identical, at least about 94% identical, at least about 95%
identical, at least about 96% identical, at least about 97%
identical, at least about 98% identical, or at least about 99%
identical to SEQ ID NO:6, wherein SEQ ID NO:6 further comprises at
least one substitution as set forth herein. In some embodiments,
the xylitol dehydrogenase polynucleotide encodes a polypeptide
having an amino acid sequence comprising SEQ ID NO:6 and/or 49,
wherein SEQ ID NO:6 further comprises at least one substitution as
set forth herein
[0042] In some embodiments, xylulokinase polynucleotides employed
in the practice of the present invention comprise polynucleotides
sequence that are at least about 70% identical, at least about 71%
identical, at least about 72% identical, at least about 73%
identical, at least about 74% identical, at least about 75%
identical, at least about 76% identical, at least about 77%
identical, at least about 78% identical, at least about 79%
identical, at least about 80% identical, at least about 81%
identical, at least about 82% identical, at least about 83%
identical, at least about 84% identical, at least about 85%
identical, at least about 86% identical, at last about 87%
identical, at least about 88% identical, at least about 89%
identical, at least about 90% identical, at least about 91%
identical, at least about 92% identical, at least about 93%
identical, at least about 94% identical, at least about 95%
identical, at least about 96% identical, at least about 97%
identical, at least about 98% identical, or at least about 99%
identical to SEQ ID NO:9, 11 and/or 12, wherein SEQ ID NO:9 further
comprises at least one substitution.
[0043] The terms "percent identity," "% identity", "percent
identical," and "% identical," are used interchangeably herein to
refer to the percent amino acid or polynucleotide sequence identity
that is obtained by ClustalW analysis (version W 1.8 available from
European Bioinformatics Institute, Cambridge, UK), counting the
number of identical matches in the alignment and dividing such
number of identical matches by the length of the reference
sequence, and using the following ClustalW parameters to achieve
slow/accurate pairwise optimal alignments--DNA/Protein Gap Open
Penalty:15/10; DNA/Protein Gap Extension Penalty:6.66/0.1; Protein
weight matrix: Gonnet series; DNA weight matrix: Identity; Toggle
Slow/Fast pairwise alignments=SLOW or FULL Alignment; DNA/Protein
Number of K-tuple matches:2/1; DNA/Protein number of best
diagonals: 4/5; DNA/Protein Window size:4/5.
[0044] Two sequences are "aligned" when they are aligned for
similarity scoring using a defined amino acid substitution matrix
(e.g., BLOSUM62), gap existence penalty and gap extension penalty
so as to arrive at the highest score possible for that pair of
sequences. Amino acid substitution matrices and their use in
quantifying the similarity between two sequences are well known in
the art (See, e.g., Dayhoff et al., in Dayhoff [ed.], Atlas of
Protein Sequence and Structure," Vol. 5, Suppl. 3, Natl. Biomed.
Res. Round., Washington D.C. [1978]; pp. 345-352; and Henikoff et
al., Proc. Natl. Acad. Sci. USA, 89:10915-10919 [1992], both of
which are incorporated herein by reference). The BLOSUM62 matrix is
often used as a default scoring substitution matrix in sequence
alignment protocols such as Gapped BLAST 2.0. The gap existence
penalty is imposed for the introduction of a single amino acid gap
in one of the aligned sequences, and the gap extension penalty is
imposed for each additional empty amino acid position inserted into
an already opened gap. The alignment is defined by the amino acid
position of each sequence at which the alignment begins and ends,
and optionally by the insertion of a gap or multiple gaps in one or
both sequences so as to arrive at the highest possible score. While
optimal alignment and scoring can be accomplished manually, the
process is facilitated by the use of a computer-implemented
alignment algorithm (e.g., gapped BLAST 2.0; See, Altschul et al.,
Nucleic Acids Res., 25:3389-3402 [1997], which is incorporated
herein by reference), and made available to the public at the
National Center for Biotechnology Information Website). Optimal
alignments, including multiple alignments can be prepared using
readily available programs such as PSI-BLAST (See e.g., Altschul et
al., supra).
[0045] The present invention also provides a recombinant nucleic
acid construct comprising a xylose reductase polynucleotide
sequence that hybridizes under stringent hybridization conditions
to the complement of a polynucleotide which encodes a polypeptide
having the amino acid sequence of SEQ ID NO:2, wherein the
polypeptide is capable of catalyzing the xylose to xylitol, and the
polypeptide comprises at least one substitution set forth
herein.
[0046] In some embodiments, the polynucleotide that hybridizes to
the complement of a polynucleotide which encodes a polypeptide
having the amino acid sequence of SEQ ID NO:2, wherein the
polypeptide comprises at least one substitution as set forth
herein, does so under high or very high stringency conditions to
the complement of a reference sequence having the sequence of SEQ
ID NO:1 (e.g., over substantially the entire length of the
reference sequence). In some embodiments, the polynucleotide that
hybridizes to the complement of a polynucleotide which encodes a
polypeptide having the amino acid sequence of SEQ ID NO:2, wherein
the polypeptide comprises at least one substitution as set forth
herein, does so under high or very high stringency conditions to
the complement of a reference sequence having the sequence of SEQ
ID NO:3 (e.g., over substantially the entire length of the
reference sequence). In some embodiments, the polynucleotide that
hybridizes to the complement of a polynucleotide which encodes a
polypeptide having the amino acid sequence of SEQ ID NO:2, wherein
the amino acid sequence comprises at least one substitution as set
forth herein, does so under high or very high stringency conditions
to the complement of a reference sequence having the sequence of
SEQ ID NO:4 (e.g., over substantially the entire length of the
reference sequence).
[0047] The present invention also provides a recombinant nucleic
acid construct comprising a xylitol dehydrogenase polynucleotide
sequence that hybridizes under stringent hybridization conditions
to the complement of a polynucleotide which encodes a polypeptide
having the amino acid sequence of SEQ ID NO:6, wherein the
polypeptide comprises at least one substitution set forth herein,
and wherein the polypeptide is capable of catalyzing xylitol to
xylulose.
[0048] Nucleic acids "hybridize" when they associate, typically in
solution. There are numerous texts and other reference materials
that provide details regarding hybridization methods for nucleic
acids (See e.g., Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology-Hybridization with Nucleic Acid Probes," Part 1,
Chapter 2, Elsevier, New York, [1993], which is incorporated herein
by reference). For polynucleotides of at least 100 nucleotides in
length, low to very high stringency conditions are defined as
follows: prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon
sperm DNA, and either 25% formamide for low stringencies, 35%
formamide for medium and medium-high stringencies, or 50% formamide
for high and very high stringencies, following standard Southern
blotting procedures. For polynucleotides of at least 200
nucleotides in length, the carrier material is finally washed three
times each for 15 minutes using 2.times.SSC, 0.2% SDS at least at
50.degree. C. (low stringency), at least at 55.degree. C. (medium
stringency), at least at 60.degree. C. (medium-high stringency), at
least at 65.degree. C. (high stringency), and at least at
70.degree. C. (very high stringency).
[0049] The terms "corresponding to", "reference to" and "relative
to" when used in the context of the numbering of a given amino acid
or polynucleotide sequence refers to the numbering of the residues
of a specified reference sequence when the given amino acid or
polynucleotide sequence is compared to the reference sequence.
[0050] The "position" is denoted by a number that sequentially
identifies each amino acid in the reference sequence based on its
position relative to the N-terminus. Owing to deletions,
insertions, truncations, fusions, and the like that must be taken
into account when determining an optimal alignment, in general the
amino acid residue number in a test sequence determined by simply
counting from the N-terminal will not necessarily be the same as
the number of its corresponding position in the reference sequence.
For example, in a case where there is a deletion in an aligned test
sequence, there will be no amino acid that corresponds to a
position in the reference sequence at the site of deletion. Where
there is an insertion in an aligned reference sequence, that
insertion will not correspond to any amino acid position the
reference sequence. In the case of truncations or fusions there can
be stretches of amino acids in either the reference or aligned
sequence that do not correspond to any amino acid in the
corresponding sequence.
[0051] As used herein, the terms "transformed" and "transformation"
used in reference to a cell refer to a cell that has a non-native
nucleic acid sequence integrated into its genome or has an episomal
plasmid that is maintained through multiple generations.
[0052] As used herein, the term "by-product" refers to an organic
molecule that is an undesired product of a particular fermentation
process.
[0053] As used herein, the term "xylose pathway" refers to the
steps of conversion of xylose to xylulose phosphate which is then
metabolized through the pentose phosphate pathway. In some
embodiments, this involves the reduction of xylose to xylitol,
oxidation of xylitol to xylulose and subsequent conversion of
xylulose to xylulose phosphate. In some other embodiments the
xylose is directly converted to xylulose which is then
phosphorylated to xylulose phosphate.
[0054] As used herein the term "xylose pathway enzymes" refers to
the enzymes that catalyze the conversion of xylose to
xylulose-phosphate. In some embodiments, these enzymes comprise
xylose reductase, xylitol dehydrogenase and/or xylulose kinase. In
some other embodiments, the enzymes comprise xylose isomerase and
xylulose kinase.
DETAILED DESCRIPTION OF THE INVENTION
[0055] The present invention provides methods and compositions
suitable for use in the conversion of xylose to xylitol and
xylulose, including nucleic acid constructs, recombinant fungal
host cells, and related materials.
[0056] The initial metabolic pathways for xylose utilization in
fungi and bacteria differ. In most fungi, including
xylose-fermenting yeasts (e.g., Pichia stipitis, Pachysolen
tannophilus, and Candida shehatae), D-xylose is converted to
D-xylulose by two oxidoreductases involving cofactors NAD(P)H and
NAD(P)+. (See, Matsushika et al., Appl. Microbiol. Biotechnol.,
84:37-53 [2009]). In these organisms, D-xylose is initially reduced
to xylitol by NAD(P)H-dependent xylose reductase ("XR") (EC
1.1.1.21). Xylitol is subsequently oxidized to D-xylulose by
NAD+-dependent xylitol dehydrogenase ("XDH" or "XD") (EC 1.1.1.9).
Xylulokinase ("XK") (EC 2.7.1.17) subsequently phosphorylates
D-xylulose to produce D-xylulose 5-phosphate ("X5P"), which is then
further metabolized through the pentose phosphate pathway ("PPP").
FIG. 1 provides a schematic of the pathway.
[0057] However, most strains of S. cerevisiae cannot utilize xylose
even though the genes encoding XR, XDH, and XK are present in its
genome, as the expression levels of these enzymes are too low to
allow xylose utilization (See, Matsushika et al., supra). Some
strains have been shown to natively utilize xylose but at very low
rates and fermentation to ethanol has not been detected (See,
Wenger et al., PLoS Genet., 6(5):e1000942 [2010]). Even when the
endogenous genes are overexpressed in S. cerevisiae, only slow
growth on xylose has been observed (See, Matsushika et al.
supra).
[0058] In contrast, most bacteria (e.g., Escherichia coli and
Streptomyces species) can isomerize D-xylose directly to D-xylulose
by using a xylose isomerase ("XI") (EC 5.3.1.5) (See, Matsushika et
al., supra). In bacteria, as in fungi, the D-xylulose is
phosphorylated to D-xylulose 5-phosphate by XK, which is then
further metabolized through the pentose phosphate pathway.
[0059] Xylose utilization by these host cells results in useful
products that are produced metabolically by the host cell. In these
host cells, D-xylulose may be phosphorylated by a native or
recombinant xylulokinase to xylulose-5-P, as depicted in FIG. 1.
The xylulose-5-P may be further metabolized by enzymes in the
pentose phosphate pathway to products such as glucose-6-P,
fructose-6-P, glyceraldehydes-3-P, and the like. The pentose
phosphate pathway and relevant enzymes and products are depicted in
FIG. 2A. As used herein, the terms "enzyme from the pentose
phosphate pathway" and "pentose phosphate pathway enzyme" are used
interchangeably to refer to an enzyme from the group of enzymes
involved in the pentose phosphate pathway, (i.e., 6.
ribulose-5-phosphate ketoisomerase (RK11); 7. transketolase (TKL1);
8. transaldolase (TALI); 9. ribose-5-phosphate ketolisomerase
(RK11); 10. 6-phosphogluconate dehydrogenase (GND1); 11.
6-phosphogluconalactonase (SOL3); and/or 12.
glucose-6-phosphate-1-dehydrogenase (ZWF); the reference numbers
correspond to those in FIG. 2A).
[0060] Products of the pentose phosphate pathway may be further
metabolized through the process of glycolysis. The metabolic
process of glycolysis is depicted in FIG. 2B. As used herein, the
term "glycolytic enzyme" refers to an enzyme from the group of
enzymes involved in glycolysis (i.e.: 13. hexokinase; 14.
phosphoglucose isomerase; 15. phosphofructokinase; 16. aldolase;
17. triose phosphate isomerase; 18. glyceraldehyde phosphate
dehydrogenase; 19. phosphoglycerate kinase; 20.
phosphglyceromutase; 21. enoase; and/or 22. pyruvate kinase; the
reference numbers correspond to those in FIG. 2B).
[0061] Pyruvate from the glycolytic pathway (i.e., glycolysis) may
be further metabolized to ethanol as shown in FIG. 2C by
ethanologenic enzymes. As used herein, the term "ethanologenic
enzyme" refers to an enzyme involved in the conversion of pyruvate
to ethanol, (e.g., a pyruvate decarboxylase, an aldehyde
dehydrogenase, and/or an alcohol dehydrogenase). The term
"ethanologenic pathway" refers to the pathway depicted in FIG.
2C.
[0062] Recombinant host cells transformed with xylose reductase and
xylitol dehydrogenase genes are hence capable of converting xylose
to xylitol and then converting xylitol to xylulose, which can lead
to the production of desirable fermentation products (e.g., an
alcohol, such as ethanol, butanol, and the like, including, but not
limited to a fatty alcohol [e.g., a C8-C20 fatty alcohol], a fatty
acid [e.g., a C8-C20 fatty acid], lactic acid, 3-hydroxpropionic
acid, acrylic acid, acetic acid, succinic acid, citric acid, malic
acid, fumaric acid, an amino acid, 1,3-propanediol, ethylene,
glycerol, a .beta.-lactam, and the like). However, cells
transformed with wildtype xylose reductase and xylitol
dehydrogenase genes from Pichia stipitis convert xylose
inefficiently and with accumulation of xylitol (Matsushika et al.,
Appl. Environ Microbiol., 81:243-55 [2008]). The present
application provides improved xylose reductase and xylitol
dehydrogenase variants that significantly increase the efficiency
of xylose conversion.
Recombinant Nucleic Acid Constructs
[0063] The present invention provides recombinant nucleic acid
constructs comprising polynucleotide sequences that encode at least
one polypeptide comprising an amino acid sequence having at least
70% identity to SEQ ID NO:2, wherein the polypeptide is capable of
catalyzing xylose to xylitol, and wherein the polypeptide comprises
at least one substitution set forth herein. SEQ ID NO:2 corresponds
to the amino acid sequence encoding xylose reductase from the
yeast, Pichia stipitis. SEQ ID NO:1 corresponds to the native P.
stipitis polynucleotide sequence that encodes a P. stipitis xylose
reductase (SEQ ID NO:2), while SEQ ID NOS:3 and 4 correspond to
codon-optimized sequences encoding the xylose reductase. In some
embodiments, the nucleic acid construct comprises SEQ ID NO:40,
while in other embodiments, the nucleic acid construct comprises
SEQ ID NO:42, in still other embodiments, the nucleic acid
construct comprises SEQ ID NO:44, and in additional embodiments,
the nucleic acid construct comprises SEQ ID NO:46. In some
embodiments, the polypeptide comprises SEQ ID NO:41, while in other
embodiments, the polypeptide comprises SEQ ID NO:43, in still other
embodiments, the polypeptide comprises SEQ ID NO:45, and in further
embodiments, the polypeptide comprises SEQ ID NO:47.
[0064] The present invention also provides a recombinant nucleic
acid constructs comprising polynucleotide sequences that encode at
least one polypeptide comprising an amino acid sequence having at
least 70% identity to SEQ ID NO:6, wherein the polypeptide is
capable of catalyzing xylitol to xylulose, and wherein the
polypeptide comprises at least one substitution set forth herein.
SEQ ID NO:6 corresponds to the amino acid sequence encoding xylitol
dehydrogenase from the yeast, Pichia stipitis. SEQ ID NO:5
corresponds to the native P. stipitis polynucleotide sequence that
encodes a P. stipitis xylitol dehydrogenase (SEQ ID NO:6), while
SEQ ID NOS:7 and 8 correspond to codon-optimized sequences encoding
the xylitol dehydrogenase. In some embodiments, the nucleic acid
construct comprises SEQ ID NO:48, while in other embodiments, the
nucleic acid construct encodes a polypeptide comprising SEQ ID
NO:49.
[0065] The present invention also provides recombinant nucleic acid
constructs comprising polynucleotide sequences that encode
polypeptide sequences having xylose reductase and xylitol
dehydrogenase activity. In some embodiments, the nucleic acid
constructs comprise at least one of SEQ ID NO:40, 42, 44, and/or
46, and SEQ ID NO:48.
[0066] The present invention also provides nucleic acid constructs
comprising codon-optimized polynucleotides encoding xylulokinase.
In some embodiments, the present invention provides SEQ ID NO:11,
while in other embodiments, the present invention provides SEQ ID
NO:12.
[0067] The present invention also provides nucleic acid constructs
comprising polynucleotides encoding xylose reductase, xylitol
dehydrogenase and xylulokinase. In some embodiments, the nucleic
acid constructs comprise SEQ ID NO:1, 3, 4, 40, 42, 44, and/or 46,
as well as SEQ ID NO: 5, 7, 8, and/or 48, and SEQ ID NO:9, 11,
and/or 12.
[0068] In some embodiments, recombinant nucleic acid constructs of
the present invention further comprise a polynucleotide sequence
(genetic) element that facilitates integration into a fungal host
cell genome, by homologous or non-homologous recombination. In some
embodiments, the nucleic acid construct of the present invention
further comprises an origin of replication that is functional in a
fungal cell (e.g., a yeast origin of replication). Typically, the
fungal host cell is a yeast or filamentous fungal cell, more
typically, a yeast cell. In some embodiments, nucleic acid
constructs of the present invention comprise a transcriptional
regulatory element that is functional in a fungal cell. For
example, in some embodiments the recombinant nucleic acid construct
comprises a promoter sequence and/or transcription terminator
sequence that is functional in a fungal cell such that the xylose
reductase and/or xylitol dehydrogenase polynucleotide is
operatively linked to the promoter sequence and/or transcription
terminator sequences.
[0069] Xylose reductase and xylitol dehydrogenase polynucleotides
that are suitable for use in the practice of the present invention
include those encoding variants of SEQ ID NO:2 and/or 6,
respectively. These variants include those having amino acid
sequences with one or more conservative or non-conservative
substitutions relative to the amino acid sequence of SEQ ID NO:2
and/or SEQ ID NO:6. As used herein, the term "conservative
substitution" refers to the substitution of a residue for another
residue that does not generally alter the specific activity of the
encoded polypeptide. An exemplary conservative substitution is a
substitution that is within the same group of basic amino acids
(arginine, lysine and histidine), acidic amino acids (glutamic acid
and aspartic acid), polar amino acids (glutamine and asparagine),
hydrophobic amino acids (leucine, isoleucine and valine), aromatic
amino acids (phenylalanine, tryptophan and tyrosine), and small
amino acids (glycine, alanine, serine, threonine, proline, cysteine
and methionine). Amino acid substitutions that do not generally
alter the specific activity are well-known in the art. The most
commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu,
Thr/Ser, Ala/Gly, Ala/Thr. Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe,
Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly,
as well as these in reverse.
[0070] In some embodiments, polynucleotides encoding conservatively
substituted variations of the P. stipitis xylose reductase and/or
xylitol dehydrogenase employed in the practice of the present
invention include substitutions of a small percentage, typically
less than about 5%, more typically less than 2%, and often less
than about 1% of the amino acids of the polypeptide sequence, with
a conservatively selected amino acid of the same conservative
substitution group.
[0071] Other xylose reductase and/or xylitol dehydrogenase
polynucleotides suitable for use in the practice of the present
invention include those encoding variants of P. stipitis xylose
reductase and/or xylitol dehydrogenase generated by mutagenesis,
recombination, or other protein engineering method followed by
screening of the variants for xylose utilization using a method,
such as that described in the Examples. In some embodiments, the
resulting variants comprise one or more substitutions (conservative
or non-conservative), deletions, and/or insertions. The present
invention thus provides methods for making improved P. stipitis
xylose reductase and xylitol dehydrogenase polynucleotide variants,
wherein the method comprises introducing one or more modifications
into a polynucleotide encoding SEQ ID NO:1, 3 and/or 4; and/or 5, 7
and/or 8, respectively, to produce a modified polynucleotide,
wherein the modification is selected from at least one
substitution, at least one deletion, and/or at least one insertion;
transforming a host cell with the modified polynucleotide; and
screening the transformed host cell for an improvement in a desired
phenotype relative to the corresponding untransformed host cell. In
some embodiments, the improved variants are screened to assess
improvements in a desired phenotype relative to a transformed host
cell that has been transformed with wild-type sequences, while in
other embodiments, transformed host cells are compared with other
transformed host cells. Exemplary phenotypes include improved
utilization of a pentose sugar (e.g., xylose, arabinose, etc.),
stability, specific activity, lower Ki for xylitol, ethanol/acetate
tolerance and/or tolerance to low pH, decreased by-product
formation, and/or increased ethanol yield. Exemplary desirable
xylose utilization phenotypes include the ability to ferment xylose
to ethanol, the ability to ferment xylose to other metabolic
intermediates/products, the ability to undergo aerobic or anaerobic
growth on xylose, and the like.
[0072] Methods for generating variant libraries of polynucleotides
encoding modified polypeptides are well known in the art. For
example, mutagenesis and directed evolution methods can be readily
applied to polynucleotides encoding the xylose reductase
polypeptide of SEQ ID NO:2 and/or xylitol dehydrogenase of SEQ ID
NO:6, to generate variant libraries that can be expressed,
screened, and assayed using the methods described herein.
Mutagenesis and directed evolution methods are well known in the
art (See e.g., Ling et al., Anal. Biochem., 254(2):157-78 [1997];
Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev.
Genet., 19:423-462 [1985]; Botstein et al., Science, 229:1193-1201
[1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell,
38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull
et al., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians et al.,
Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature,
391:288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438
[1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509
[1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996];
Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci.
USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966;
WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of
which are incorporated herein by reference).
[0073] In some embodiments, the present invention provides P.
stipitis polypeptide variants that comprise at least one
modification that is a substitution, insertion, and/or deletion
relative to SEQ ID NO:2. Typically, the polypeptide variant has
from 1 to 2, 1 to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, up
to about 50, 75, 100, or 130 modifications.
[0074] In some embodiments, the xylose reductase variants of the
present invention comprise a substitution at position 276 of
wild-type P. stipitis xylose reductase. In some alternative
embodiments, the xylose reductase variants comprise at least one
substitution at positions 2, 49, 132, 233, 267, and/or 276, wherein
the positions are numbered by correspondence with the amino acid
sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2.
In some additional embodiments, the substitutions comprise
substitutions at R276, P2, A49, K132, S233, I267, and/or R276. In
some further embodiments, the substitutions comprise R276W, P2T,
A49G, K132N, S233K, I267V, and/or R276W, wherein the positions are
numbered by correspondence with the amino acid sequence of P.
stipitis xylose reductase set forth in SEQ ID NO:2. In some
embodiments, the variants comprise substitutions P2T, A49G, and
R276W, wherein the positions are numbered by correspondence with
the amino acid sequence of P. stipitis xylose reductase set forth
in SEQ ID NO:2. In some additional embodiments, the variants
comprise substitutions P2T, A49G, K132N, S233K, I267V, and R276W,
wherein the positions are numbered by correspondence with the amino
acid sequence of P. stipitis xylose reductase set forth in SEQ ID
NO:2. In some additional embodiments, the variants comprise at
least one substitution selected from position 2, 3, 7, 11, 14, 17,
23, 24, 33, 36, 46, 47, 49, 56, 62, 68, 89, 97, 102, 108, 114, 116,
123, 132, 134, 143, 152, 155, 157, 162, 168, 184, 206, 219, 224,
225, 226, 228, 246, 231, 232, 233, 236, 240, 242, 245, 246, 249,
252, 255, 261, 266, 267, 275, 276, 279, 281, 282, 283, 285, 297,
301, 302, 303, and 318, wherein the positions are numbered by
correspondence with the amino acid sequence of P. stipitis xylose
reductase set forth in SEQ ID NO:2.
[0075] In still some further embodiments, the variants comprise at
least one substitution selected from P2, S3, N7, D11, A14, F17,
D23, V24, R33, K36, E46, D47, A49, A56, 162, K68, E89, S97, D102,
L108, T114, K116, K123, K132, D134, I143, K152, K155, G157, I162,
P168, S184, R206, Q219, L224, N225, Q226, R228, A246, N231, T232,
S233, F236, T240, K242, A245, A246, G249, P252, V255, S261, A266,
I267, P275, R276, E279, K281, D282, V283, S285, A297, 1301, N302,
L303, and V318, wherein the positions are numbered by
correspondence with the amino acid sequence of P. stipitis xylose
reductase set forth in SEQ ID NO:2.
[0076] In some additional embodiments, the substitutions comprise
at least one substitution selected from P2T, S3H, S3R, S3W, N7L,
D11K, A14V, F17W, D23E, V24G, R33L, R22V, K36Q, E46K, D47G, D47N,
A49G, A56E, A56Y, I62V, K68G, K68M, K68R, E89N, E89V, S97R, S97T,
D102T, L108Y, T114S, K116Q, K123C, K132A, K132N, D134E, D134H,
D134V, I143L, K152A, K152E, K152H, K152Q, K155A, K155D, K155I,
K155R, K155Y, G157R, I162L, P168S, S184A, R206S, R206V, Q219H,
Q219L, Q219T, L224A, L224S, L224V, N225D, N225E, N225K, N225S,
N225Y, Q226D, Q226E, Q226S, Q226V, R228T, N231G, N231H, N231L,
N231S, T232A, T232C, T232S, T232V, S233C, S233F, S233G, S233I,
S233K, S233V, F236L, T240V, K242L, A245S, A246L, A246S, G249D,
P252C, V255I, S261A, S261C, S261N, S261T, A266V, A266C, I267V,
P275A, R276M, R276W, E279Q, K281L, K281V, D282C, D282G, D282R,
V283H, S285E, S285T, A297H, A297S, I301C, I301Y, N302D, N302G,
N302S, L303I, L303V, and V318C, wherein the positions are numbered
by correspondence with the amino acid sequence of P. stipitis
xylose reductase set forth in SEQ ID NO:2.
[0077] In some embodiments, the present invention provides
polynucleotide sequences encoding xylose reductase variants
comprising SEQ ID NO:40, as provided below. In some additional
embodiments, the present invention provides polypeptide sequences
comprising SEQ ID NO:41, as provided below.
TABLE-US-00001 (SEQ ID NO: 40)
ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTT
CGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTACCGTG
CTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGCCAAC
GAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGT
CAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACC
ACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAA
GTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTT
CGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAAGGGTG
ACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTT
GAAAAGTTGGTCAAGGCCGGTAAGATCAGGTCTATCGGTGTTTCTAACTT
CCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCAT
CTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATC
GAGTTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGG
TCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTC
CATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGTAAG
TCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCAT
CATTCCAAAGTCCAACACTGTCCCATGGTTGTTGGAAAACAAGGATGTCA
ACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGAC
ATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTT CGTCTAA (SEQ ID
NO: 41) MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDYAN
EKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQ
VDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKAL
EKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLI
EFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGK
SPAQVLLRWSSQRGIAIIPKSNTVPWLLENKDVNSFDLDEQDFADIAKLD
INLRFNDPWDWDKIPIFV
[0078] In some embodiments, the present invention provides
polynucleotide sequences encoding xylose reductase variants
comprising SEQ ID NO:42, as provided below. In some additional
embodiments, the present invention provides polypeptide sequences
comprising SEQ ID NO:43, as provided below.
TABLE-US-00002 (SEQ ID NO: 42)
ATGACCTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTT
CGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTACCGTG
CTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGGCAAC
GAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGT
CAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACC
ACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAA
GTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTT
CGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAAGGGTG
ACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTT
GAAAAGTTGGTCAAGGCCGGTAAGATCAGGTCTATCGGTGTTTCTAACTT
CCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCAT
CTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATC
GAGTTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGG
TCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTC
CATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGTAAG
TCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCAT
CATTCCAAAGTCCAACACTGTCCCATGGTTGTTGGAAAACAAGGATGTCA
ACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGAC
ATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTT CGTCTAA (SEQ ID
NO: 43) MTSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDYGN
EKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQ
VDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKAL
EKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLI
EFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGK
SPAQVLLRWSSQRGIAIIPKSNTVPWLLENKDVNSFDLDEQDFADIAKLD
INLRFNDPWDWDKIPIFV
[0079] In some embodiments, the present invention provides
polynucleotide sequences encoding xylose reductase variants
comprising SEQ ID NO:44, as provided below. In some additional
embodiments, the present invention provides polypeptide sequences
comprising SEQ ID NO:45, as provided below.
TABLE-US-00003 (SEQ ID NO: 44)
ATGACCTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTT
CGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTACCGAG
CTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGGCAAC
GAAAAATTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGT
CAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACC
ACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAA
GTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTT
CGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAACGGTG
ACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTT
GAAAAGTTGGTCAAGGCCGGTAAGATCAGGTCTATCGGTGTTTCTAACTT
CCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCAT
CTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATC
GAGTTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGG
TCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTAAGC
CATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGCAAG
AGCCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCGT
TATTCCAAAGTCCAACACTGTCCCATGGTTGTTGGAAAACAAGGATGTCA
ACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGAC
ATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTT CGTCTAA (SEQ ID
NO: 45) MTSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDYGN
EKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQ
VDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGNGDNFDYEDVPILETWKAL
EKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLI
EFAQSRGIAVTAYSSFGPQSFVELNQGRALNTKPLFENETIKAIAAKHGK
SPAQVLLRWSSQRGIAVIPKSNTVPWLLENKDVNSFDLDEQDFADIAKLD
INLRFNDPWDWDKIPIFV
[0080] In some embodiments, the present invention provides
polynucleotide sequences encoding xylose reductase variants
comprising SEQ ID NO:46, as provided below. In some additional
embodiments, the present invention provides polypeptide sequences
comprising SEQ ID NO:47, as provided below.
TABLE-US-00004 (SEQ ID NO: 46)
ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTT
CGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTACCGTG
CTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGCCAAC
GAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGT
CAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACC
ACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAA
GTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTT
CGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAAGGGTG
ACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTT
GAAAAGTTGGTCAAGGCCGGTAAGATCAGGTCTATCGGTGTTTCTAACTT
CCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCAT
CTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATC
GAGTTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGG
TCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTC
CATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGTAAG
TCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCAT
CATTCCAAAGAGCAATACTGTCCCATTCTTGTTGGAAAACAAGGATGTCA
ACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGAC
ATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTT CGTCTAA (SEQ ID
NO: 47) MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDYAN
EKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQ
VDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKAL
EKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLI
EFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGK
SPAQVLLRWSSQRGIAIIPKSNTVPFLLENKDVNSFDLDEQDFADIAKLD
INLRFNDPWDWDKIPIFV
[0081] In some embodiments, the present invention also provides
nucleic acid substitutions in sequences encoding xylose reductase
variants (e.g., SEQ ID NOS:1, 3 and/or 4). In some embodiments, the
nucleic acids comprise substitutions at positions 42, 82, 99, 156,
201, 280, 306, 354, 358, 378, 408, 438, 478, 511, 585, 670, 688,
703, 747, 751, 766, 855, 849, and/or 906, wherein the positions are
numbered by correspondence with the nucleic acid sequence of P.
stipitis xylose reductase set forth in SEQ ID NO:1, 3 and/or 4. In
some embodiments, the substitutions comprise t82a, t99a, g156a,
c201t, a280c, c306t, t354g, t358c, a378t, c408t, a426t, c438t,
a478c, t511c, a585g, t670c, t688c, t703c, t747c, t751a, t766c,
c849g, c855t, and/or c906t, wherein the positions are numbered by
correspondence with the nucleic acid sequence of P. stipitis xylose
reductase set forth in SEQ ID NO:1, 3 and/or 4.
[0082] In some embodiments, the present invention further provides
xylitol dehydrogenase variants comprising at least one substitution
at positions 5, 13, 19, 49, 81, 149, 187, 189, 202, 205, 206, 208,
209, 211, 215, 218, 226, 227, 228, 229, 231, 235, 239, 241, 251,
252, 256, 260, 287, 296, 307, 327, 350, and/or 352, wherein the
positions are numbered by correspondence with the amino acid
sequence of P. stipitis xylose reductase set forth in SEQ ID
NO:6.
[0083] In some embodiments, the xylitol dehydrogenase variants
comprise substitution at position 208, wherein the positions are
numbered by correspondence with the amino acid sequence of P.
stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some
embodiments, the substitution at position 208 is I208X. In some
further embodiments, the substitution at position 208 is I208R. In
some embodiments, the xylitol dehydrogenase variants comprise
substitutions at position 211, wherein the positions are numbered
by correspondence with the amino acid sequence of P. stipitis
xylitol dehydrogenase set forth in SEQ ID NO:6. In some
embodiments, the substitution at position 211 is N211X. In some
further embodiments, the substitution is N211K, while in other
embodiments, the substitution is N211 S, and in still other
embodiments, the substitution is N211K.
[0084] In some embodiments, the xylitol dehydrogenase variants
comprise substitutions at positions 208 and 211, wherein the
positions are numbered by correspondence with the amino acid
sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID
NO:6. In some embodiments, the substitution at position 208 is
I208X and the substitution at position 211 is N211X. In some
further embodiments, the substitutions comprise I208R and N211K,
while in some other embodiments, the substitutions comprise 1208R
and N211S.
[0085] In some embodiments, the xylitol dehydrogenase variants
comprise substitutions at positions 209 and 211, wherein the
positions are numbered by correspondence with the amino acid
sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID
NO:6. In some embodiments, the substitutions are F209X and
N211X.
[0086] In some embodiments, the xylitol dehydrogenase variants
comprise substitutions at positions 208, 209 and 211, wherein the
positions are numbered by correspondence with the amino acid
sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID
NO:6. In some embodiments, the substitutions are I208X, F209X, and
N211X. In some further embodiments, the substitutions are I208R,
F209S, and N211 S.
[0087] In some embodiments, the present invention also provides
nucleic acid substitutions in sequences encoding xylitol
dehydrogenase variants (e.g., SEQ ID NOS:5, 7, and/or 8). In some
embodiments, the nucleic acids comprise substitutions at positions
t24g, c630t, a732g, a768t, and/or a780g, wherein the positions are
numbered by correspondence with the nucleic acid sequence of P.
stipitis xylose reductase set forth in SEQ ID NO:5, 7 and/or 8.
[0088] In some embodiments, the present invention provides
polypeptides that comprise XR and XD substitutions, including, but
not limited to combinations of substitutions at positions 271, 270,
272, and/or 276 in XR (wherein the positions are numbered by
correspondence with SEQ ID NO:2) and substitutions at positions
208, 209, and/or 211 in XD (wherein the positions are numbered by
correspondence with SEQ ID NO:6). In some embodiments, the
substitutions comprise substitutions S271X, K270, N272, and/or R276
in XR (wherein the positions are numbered by correspondence with
SEQ ID NO:2) and substitutions I208X, F209X, and/or N211X in XD
(wherein the positions are numbered by correspondence with SEQ ID
NO:6). In some embodiments, the substitutions comprise
substitutions S271G, K270R, N272P, and/or R276F in XR (wherein the
positions are numbered by correspondence with SEQ ID NO:2) and
substitutions I208R, F209S, and/or N211K in XD (wherein the
positions are numbered by correspondence with SEQ ID NO:6).
[0089] In some embodiments, the substitutions comprise R276F in XR
(wherein the position is numbered by correspondence with SEQ ID
NO:2), and I208R in XD (wherein the position is numbered by
correspondence with SEQ ID NO:6). In some embodiments, the
substitutions comprises R276F in XR (wherein the position is
numbered by correspondence with SEQ ID NO:2), and F209S and N211K
in XD (wherein the positions are numbered by correspondence with
SEQ ID NO:6). In some embodiments, the substitutions comprise S271
G in XR (wherein the position is numbered by correspondence with
SEQ ID NO:2), and I208R and N211K in XD (wherein the positions are
numbered by correspondence with SEQ ID NO:6). In some embodiments,
the substitutions comprise S271G in XR (wherein the position is
numbered by correspondence with SEQ ID NO:2), and I208R and N211S
in XD (wherein the positions are numbered by correspondence with
SEQ ID NO:6). In some embodiments, the substitutions comprise R276F
in XR (wherein the position is numbered by correspondence with SEQ
ID NO:2), and I208R, F209S and N211K in XD (wherein the positions
are numbered by correspondence with SEQ ID NO:6). In some
embodiments, the substitutions comprise S271 G in XR (wherein the
position is numbered by correspondence with SEQ ID NO:2), and I208R
in XD (wherein the positions are numbered by correspondence with
SEQ ID NO:6). In some embodiments, the substitutions comprise K270R
in XR (wherein the position is numbered by correspondence with SEQ
ID NO:2), and I208R and N211R in XD (wherein the positions are
numbered by correspondence with SEQ ID NO:6). In some embodiments,
the substitutions comprise K270R in XR (wherein the position is
numbered by correspondence with SEQ ID NO:2), and F209S and N211K
in XD (wherein the positions are numbered by correspondence with
SEQ ID NO:6). In some embodiments, the substitutions comprise N272P
and R276F in XR (wherein the position is numbered by correspondence
with SEQ ID NO:2), and F209S and N211K in XD (wherein the positions
are numbered by correspondence with SEQ ID NO:6). In some
embodiments, the substitutions comprise K270R, N272P and R276F in
XR (wherein the position is numbered by correspondence with SEQ ID
NO:2), and N211K in XD (wherein the positions are numbered by
correspondence with SEQ ID NO:6).
[0090] In some additional embodiments, the present invention
provides XR and XD variants having amino acid and nucleic acid
substitutions, wherein amino acid substitutions in SEQ ID NO:2
comprises R276F, amino acid substitutions in SEQ ID NO:6 comprise
I208R, and the nucleic acid substitutions comprise t811a and c816t
in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the
present invention provides XR and XD variants having amino acid and
nucleic acid substitutions, wherein amino acid substitutions in SEQ
ID NO:2 comprise R276F, amino acid substitutions in SEQ ID NO:6
comprise F209S and N211K, and the nucleic acid substitutions
comprise t811a and c816t in SEQ ID NO:1, 3 and/or 4. In some
additional embodiments, the present invention provides XR and XD
variants having amino acid and nucleic acid substitutions, wherein
amino acid substitutions in SEQ ID NO:2 comprise S271G, amino acid
substitutions in SEQ ID NO:6 comprise I208R and N211K, and the
nucleic acid substitutions comprise c816t and a826c in SEQ ID NO:1,
3 and/or 4. In some additional embodiments, the present invention
provides XR and XD variants having amino acid and nucleic acid
substitutions, wherein amino acid substitutions in SEQ ID NO:2
comprise S271G, amino acid substitutions in SEQ ID NO:6 comprise
I208R and N211S, and the nucleic acid substitutions comprise c816t
and a826c in SEQ ID NO:1, 3 and/or 4. In some additional
embodiments, the present invention provides XR and XD variants
having amino acid and nucleic acid substitutions, wherein amino
acid substitutions in SEQ ID NO:2 comprise R276F, amino acid
substitutions in SEQ ID NO:6 comprise I208R, F209S and N211K, and
the nucleic acid substitutions comprise t811a and c816t in SEQ ID
NO:1, 3 and/or 4. In some additional embodiments, the present
invention provides XR and XD variants having amino acid and nucleic
acid substitutions, wherein amino acid substitutions in SEQ ID NO:2
comprise S271G, amino acid substitutions in SEQ ID NO:6 comprise
I208R, and the nucleic acid substitutions comprise c816t and a826c
in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the
present invention provides XR and XD variants having amino acid and
nucleic acid substitutions, wherein amino acid substitutions in SEQ
ID NO:2 comprise K270R, amino acid substitutions in SEQ ID NO:6
comprise I208R and N211R, and the nucleic acid substitutions
comprise t811a, c816t, and a826c, in SEQ ID NO:1, 3 and/or 4. In
some additional embodiments, the present invention provides XR and
XD variants having amino acid and nucleic acid substitutions,
wherein amino acid substitutions in SEQ ID NO:2 comprise K270R,
amino acid substitutions in SEQ ID NO:6 comprise F209S and N211K,
and the nucleic acid substitutions comprise t811a, c816t, and
a826c, in SEQ ID NO:1, 3 and/or 4. In some additional embodiments,
the present invention provides XR and XD variants having amino acid
and nucleic acid substitutions, wherein amino acid substitutions in
SEQ ID NO:2 comprise K272P and R276F, amino acid substitutions in
SEQ ID NO:6 comprise F209S and N211K, and the nucleic acid
substitutions comprise t811a in SEQ ID NO:1, 3 and/or 4. In some
additional embodiments, the present invention provides XR and XD
variants having amino acid and nucleic acid substitutions, wherein
amino acid substitutions in SEQ ID NO:2 comprise K270R, N272P, and
R276F, amino acid substitutions in SEQ ID NO:6 comprise N211K, and
the nucleic acid substitutions comprise t811a in SEQ ID NO:1, 3
and/or 4. In some additional embodiments, the present invention
provides XR and XD variants having amino acid and nucleic acid
substitutions, wherein amino acid substitutions in SEQ ID NO:6
comprise I208R, and the nucleic acid substitutions comprise t811a,
c816t, and a826c, in SEQ ID NO:1, 3 and/or 4.
[0091] In some embodiments, the present invention provides
polynucleotide sequences encoding xylitol dehydrogenase variants
comprising SEQ ID NO:48, as provided below. In some additional
embodiments, the present invention provides polypeptide sequences
comprising SEQ ID NO:49, as provided below.
TABLE-US-00005 (SEQ ID NO: 48)
ATGACCGCTAATCCCTCTCTTGTTTTGAATAAGATTGACGACATTTCTTT
TGAAACTTACGATGCTCCCGAAATTAGCGAACCCACAGACGTTTTAGTTC
AAGTTAAAAAAACTGGTATCTGCGGTTCTGACATCCACTTCTACGCTCAT
GGAAGGATCGGCAACTTCGTCTTAACAAAGCCAATGGTTCTGGGTCATGA
AAGCGCGGGTACTGTTGTTCAAGTCGGTAAAGGTGTTACTTCACTGAAGG
TTGGTGATAACGTCGCAATCGAGCCCGGTATTCCATCTAGGTTCAGTGAT
GAGTACAAATCTGGTCACTACAACCTGTGTCCACACATGGCATTTGCTGC
TACTCCCAATTCTAAAGAGGGTGAACCAAACCCACCAGGAACTCTATGTA
AGTACTTCAAATCTCCAGAAGACTTCCTGGTTAAGTTACCCGATCATGTT
TCTTTGGAGTTGGGTGCTTTGGTCGAGCCACTATCTGTTGGGGTCCATGC
TAGTAAATTAGGCTCCGTTGCATTTGGCGATTACGTTGCTGTTTTTGGTG
CTGGTCCAGTAGGATTACTGGCTGCCGCTGTCGCTAAGACATTTGGTGCC
AAGGGTGTGATTGTCGTTGATATATCTGACAAGAAGCTGAAGATGGCCAA
AGACATAGGTGCCGCTACACATACCTTCAACTCCAAGACGGGAGGTAGTG
AAGAATTGATCAAAGCCTTCGGTGGTAATGTACCAAATGTTGTCTTGGAA
TGTACTGGGGCTGAACCATGTATTAAGCTAGGTGTTGATGCCATCGCACC
AGGTGGTAGATTCGTGCAAGTTGGTAATGCTGCTGGTCCCGTGTCCTTTC
CCATAACAGTGTTCGCTATGAAAGAACTTACTTTGTTTGGTTCATTTCGT
TATGGTTTCAACGACTATAAGACAGCCGTGGGTATCTTTGATACTAACTA
CCAGAACGGTAGAGAGAATGCTCCCATTGACTTTGAACAGCTTATCACGC
ACAGATACAAATTCAAAGACGCCATTGAAGCCTACGACCTAGTAAGAGCA
GGTAAAGGGGCTGTCAAGTGTTTGATTGATGGTCCAGAATAA (SEQ ID NO: 49)
MTANPSLVLNKIDDISFETYDAPEISEPTDVLVQVKKTGICGSDIHFYAH
GRIGNFVLTKPMVLGHESAGTVVQVGKGVTSLKVGDNVAIEPGIPSRFSD
EYKSGHYNLCPHMAFAATPNSKEGEPNPPGTLCKYFKSPEDFLVKLPDHV
SLELGALVEPLSVGVHASKLGSVAFGDYVAVFGAGPVGLLAAAVAKTFGA
KGVIVVDISDKKLKMAKDIGAATHTFNSKTGGSEELIKAFGGNVPNVVLE
CTGAEPCIKLGVDAIAPGGRFVQVGNAAGPVSFPITVFAMKELTLFGSFR
YGFNDYKTAVGIFDTNYQNGRENAPIDFEQLITHRYKFKDAIEAYDLVRA
GKGAVKCLIDGPE
[0092] In some embodiments, the present invention provides
polynucleotide sequences encoding xylose reductase variants
comprising SEQ ID NO:46 and xylitol dehydrogenase variants
comprising SEQ ID NO:48. In some additional embodiments, the
present invention provides polypeptide sequences comprising SEQ ID
NO:47 and SEQ ID NO:49.
[0093] Also suitable for use in the practice of the present
invention are polynucleotides encoding a truncated variant of P.
stipitis xylose reductase and/or a truncated variant xylitol
dehydrogenase capable of catalyzing xylose to xylitol and/or
xylitol to xylulose. In some embodiments, these truncation variants
are truncated at the carboxy (C)-terminus and/or the amino
(N)-terminus. Typically, the truncation is from about 1 to about 50
amino acid residues in length.
[0094] Those having ordinary skill in the art will understand that
due to the degeneracy of the genetic code, a multitude of
nucleotide sequences that encode the xylose isomerase polypeptides
described herein exist. Table 1 provides the standard triplet
genetic code for each amino acid. For example, the codons AGA, AGG,
CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at
every position in the nucleic acids referred to herein, where an
arginine is specified by a codon, the codon can be altered to any
of the corresponding codons described above without altering the
encoded polypeptide. It is understood that U in an RNA sequence
corresponds to T in a DNA sequence. The invention contemplates and
provides each and every possible variation of nucleic acid sequence
encoding a polypeptide of the invention that could be made by
selecting combinations based on possible codon choices.
TABLE-US-00006 TABLE 1 Genetic Code Amino Acids Codon Alanine Ala A
GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly
G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC
AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
[0095] In some embodiments, DNA sequences are designed for high
codon usage bias (i.e., codons that are used at higher frequency in
the protein coding regions than other codons that code for the same
amino acid). In some embodiments, the preferred codons are
determined in relation to codon usage in a single gene, a set of
genes of common function or origin, highly expressed genes, the
codon frequency in the aggregate protein coding regions of the
whole organism, codon frequency in the aggregate protein coding
regions of related organisms, or combinations thereof. Codons whose
frequency increases with the level of gene expression are typically
optimal codons for expression. In particular, a DNA sequence can be
optimized for expression in a particular host organism. References
providing preference information for a wide range of organisms are
readily available (See e.g., Henaut and Danchin in Neidhardt et al.
[eds.], Escherichia coli and Salmonella, ASM Press, Washington
D.C., [1987], p. 2047-2066, which is incorporated herein by
reference).
[0096] A variety of methods are known for determining the codon
frequency (e.g., codon usage, relative synonymous codon usage) and
codon preference in specific organisms, including multivariate
analysis, for example, using cluster analysis or correspondence
analysis, and the effective number of codons used in a gene (See,
GCG CodonPreference, Genetics Computer Group Wisconsin Package;
Peden, Codon W, University of Nottingham; McInerney, Bioinform.,
14:372-73 [1998]; Stenico et al., Nucl. Acids Res. 222437-46
[1994]; Wright, Gene 87:23-29 [1990]; Wada et al., Nucl. Acids
Res., 20:2111-2118 [1992]; Nakamura et al., Nucl. Acids Res.,
28:292 [2000]; and Henaut and Danchin, supra; all of which are
incorporated herein by reference). The data source for obtaining
codon usage may rely on any available nucleotide sequence capable
of coding for a protein. These data sets include nucleic acid
sequences actually known to express proteins (e.g., complete
protein coding sequences-CDS), expressed sequence tags (ESTs), or
predicted coding regions of genomic sequences (See e.g., Mount,
Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., [2001];
Uberbacher, Methods Enzymol., 266:259-281 [1996]; and Tiwari et
al., Comput. Appl. Biosci. 13:263-270 [1997]; all of which are
incorporated herein by reference).
[0097] In some embodiments, the xylose reductase and xylitol
dehydrogenase polynucleotides contain codons optimized for
expression in a fungal cell, particularly a yeast cell. In some
embodiments, codon-optimized xylose reductase polynucleotide
sequence is provided as SEQ ID NOS:3 and 4. In addition,
codon-optimized xylitol dehydrogenase polynucleotides are provided
herein (SEQ ID NOS:7 and 8), as well as codon-optimized
xylulokinase polynucleotides (SEQ ID NOS:11 and 12). In some
embodiments, the codon-optimized sequences provide various
advantages, as compared to the corresponding wild-type S.
cerevisiae sequence(s).
[0098] Certain silent mutations have been identified in P. stipitis
xylose reductase and xylitol dehydrogenase polynucleotide variants
that appear to confer the property of greater xylose utilization in
transformed Saccharomyces cerevisiae. These variants are described
in the Examples. The silent mutations include t24g, t82a, t99a,
g156a, c201t, a280c, c306t, t354g, t358c, a378t, c408t, a426t,
c438t, a478c, t511c, a585g, c630t, t670c, t688c, t703c, a732g,
t747c, t751a, t766c, a768t, a780g, t811a, c816t, a826c, c849g,
c855t, and c906t (where the nucleotide position is determined by
alignment with SEQ ID NO:1). However, it is not intended that the
present invention be limited to these particular substitutions as
alternative substitutions find use in the present invention.
[0099] In some embodiments, the xylose reductase and/or xylitol
dehydrogenase polynucleotides are employed in recombinant nucleic
acid constructs that comprise a vector (e.g., a plasmid, a cosmid,
a phage, a virus, a yeast artificial chromosome (YAC), and the
like), into which a xylose reductase and/or xylitol dehydrogenase
polynucleotide sequence has been inserted. The xylose reductase and
xylitol dehydrogenase polynucleotides provided herein find use when
incorporated into any one of a variety of vectors. Suitable vectors
include, but are not limited to chromosomal, nonchromosomal and
synthetic DNA sequences, yeast plasmids, vectors derived from
combinations of plasmids and phage DNA, and many others. Any
suitable vector that transduces genetic material into a cell, and,
if replication is desired, which is replicable and viable in the
relevant host find use in the present invention.
[0100] Nucleic acid constructs of the present invention find use in
transforming a host cell to permit the host to express the xylose
reductase and/or xylitol dehydrogenase polypeptide. Methods for
recombinant expression of proteins in fungi are well known in the
art, and a number of vectors are available or can be constructed
using routine methods (See e.g., Zhu et al., Plasmid 6:128-33
[2009], incorporated herein by reference; and the many standard
reference works in this field).
[0101] In some embodiments, recombinant nucleic acid constructs of
the present invention further comprise a transcriptional regulatory
element that is functional in a fungal cell. In some embodiments,
the nucleic acid construct comprises the xylose reductase and/or
xylitol dehydrogenase polynucleotide operatively linked to a
transcriptional regulatory sequence (e.g., a promoter,
transcription termination sequence, and the like), that is
functional in a fungal cell. Examples of promoters that are
functional in a fungal host cell include, but are not limited to
promoters from yeast and filamentous fungi. Promoters that are
suitable for use in the practice of the present invention include
endogenous or heterologous promoters and include both constitutive
and inducible promoters that are natural or modified. Particularly
useful promoters are those that are insensitive to catabolite
(glucose) repression and/or do not require xylose for induction.
Such promoters are well known in the art. In some embodiments, a
promoter sequence is operably linked to the 5' region of the xylose
isomerase or xylitol dehydrogenase coding sequence using routine
methods that are well known in the art.
[0102] Promoters that are suitable for use in the practice of the
present invention include, but are not limited to yeast promoters
from glycolytic genes (e.g., yeast phosphofructokinase (PFK),
triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate
dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK),
phosphoglycerate kinase (PGK) promoters, and the like; See e.g., WO
93/03159, which is incorporated herein by reference); promoters of
glucose transporters; ribosomal protein encoding gene promoters;
alcohol dehydrogenase promoters (e.g., ADH1, ADH4, and the like),
and the enolase promoter (ENO).
[0103] Exemplary promoters that are useful for directing the
transcription of the nucleic acid constructs of the present
invention in yeast host cells include, but are not limited to those
from the genes for Saccharomyces cerevisiae enolase (eno-1),
Saccharomyces cerevisiae galactokinase (gal1), Saccharomyces
cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate
dehydrogenase (ADH1/ADH2/GAP), and Saccharomyces cerevisiae
3-phosphoglycerate kinase, Saccharomyces cerevisiae transcription
elongation factor (TEF), Saccharomyces cerevisiae fructose
1,6-bisphosphate aldolase (FBA1), and Saccharomyces cerevisiae
3-phosphate glycerate kinase (PGK1). Other useful promoters for
yeast host cells are well known in the art (See e.g., Romanos et
al., Yeast 8:423-488 [1992], which is incorporated herein by
reference).
[0104] Suitable filamentous fungal promoters that are useful in the
practice of the present invention include, but are not limited to
promoters obtained from the genes for Aspergillus oryzeae TAKA
amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger
neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, and Fusarium oxysporum trypsin-like protease (See
e.g., WO 96/00787, which is incorporated herein by reference), as
well as the NA2-tpi promoter (a hybrid of the promoters from the
genes for Aspergillus niger neutral alpha-amylase and Aspergillus
oryzae triose phosphate isomerase), promoters such as cbh1, cbh2,
egl1, egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (See, Nunberg et
al., Mol. Cell Biol., 4:2306-2315 [1984]; Boel et al., EMBO J.
3:1581-85 [1984]; and EP 0 137 280A, all of which are incorporated
herein by reference), and mutant, truncated, and hybrid promoters
thereof. Promoters associated with chitinase production in fungi
also find use in sme embodiments (See e.g., Blaiseau and Lafay,
Gene 120:243-248 [1992] [filamentous fungus Aphanocladium album];
and Limon et al., Curr. Genet., 28:478-83 [1995] [Trichoderma
harzianum]; both of which are incorporated herein by
reference).
[0105] Any other suitable promoter sequence that drives expression
in a fungal host cell, particularly a yeast host cell finds use in
the present invention. Suitable promoter sequences can be
identified using well known methods. In one approach, a putative
promoter sequence is linked 5' to a sequence encoding a reporter
protein, the construct is transfected into the host cell and the
level of expression of the reporter is measured. Expression of the
reporter can be determined by measuring, for example, mRNA levels
of the reporter sequence, an enzymatic activity of the reporter
protein, or the amount of reporter protein produced. For example,
promoter activity may be determined by using the green fluorescent
protein as coding sequence (See, Henriksen et al., Microbiol.,
145:729-34 [1999], which is incorporated herein by reference) or a
lacZ reporter gene (See, Punt et al., Gene, 197:189-93 [1997],
which is incorporated herein by reference). In some embodiments,
functional promoters are derived from naturally occurring promoter
sequences by directed evolution methods (See e.g., Wright et al.,
Hum. Gene Ther., 16:881-892 [2005], which is incorporated herein by
reference).
[0106] Exemplary transcription termination sequences (terminators)
that are functional in a fungal host cell, include transcription
termination sequences from yeast and filamentous fungi, that are
well known in the art. In some embodiments, the transcription
termination sequence is from a yeast. Exemplary yeast transcription
termination sequences include, but are not limited to CYC1, ADH1t,
ADH2t, etc. In some embodiments, the nucleic acid constructs of the
present invention contain a ribosome binding site for translation
initiation. In some embodiments, the construct includes appropriate
sequences for amplifying expression (e.g., an enhancer). Such
elements are well known in the art and any suitable enhancers
and/or transcription termination sequences, and/or ribosome binding
sites find use in the present invention.
[0107] In some additional embodiments, nucleic acid constructs of
the present invention contain one or more selectable marker genes
to provide a phenotypic trait for selection of transformed host
cells. Suitable marker genes include, but are not limited to those
coding for antimicrobial resistance such as, ampicillin (ampR),
kanamycin, chloramphenicol, tetracycline, streptomycin or
spectinomycin (e.g., the aada gene); including but not limited to
the streptomycin phosphotransferase (spt) gene coding for
streptomycin resistance, the neomycin phosphotransferase (nptII)
gene encoding kanamycin or geneticin resistance, the nourseothricin
actetyltransferase (nat1) gene coding for nourseothricin
resistance, the hygromycin phosphotransferase (hpt) gene coding for
hygromycin resistance, genes encoding dihydrofolate reductase,
phleomycin, or neomycin resistance for eukaryotic cell culture, and
tetracycline or ampicillin resistance in E. coli, as well as other
marker genes that are well known in the art.
[0108] Nucleic acid constructs of the present invention typically
comprise a fungal origin of replication, such as, for example, a
filamentous fungal or yeast origin of replication. Typically, the
recombinant nucleic acid constructs of the present invention
comprise a yeast origin of replication. Examples include, but are
not limited to constructs containing autonomous replicating
sequences, constructs containing 2 micron DNA including the
autonomous replicating sequence and rep genes, constructs
containing centromeres like the CEN6, CEN4, CEN11, CDN3 and
autonomous replicating sequences, and other like sequences that are
well known in the art. Exemplary nucleic acid constructs include
constructs suitable for transforming yeast. These include, but are
not limited to episomal constructs based on the yeast 2.mu. or CEN
origin based plasmids like pYES2/CT, pYES3/CT, pESC/His, pESC/Ura,
pESC/Trp, pES/Leu, p427TEF, pRS405, pRS406, pRS413, and other
yeast-based constructs that are known in the art.
[0109] In some embodiments, the nucleic acid constructs of the
present invention comprise elements to facilitate integration of
the xylose reductase and/or xylitol dehydrogenase polynucleotide
into a fungal host chromosome (i.e., the genome), by either
homologous or non-homologous recombination and either site-directed
or random mutagenesis. In some embodiments, the nucleic acid
constructs comprise elements that facilitate homologous
integration. In some embodiments, the xylose reductase and/or
xylitol dehydrogenase polynucleotide is integrated at one or more
site and is present in one or more copies. In some embodiments, the
nucleic acid construct comprises the xylose reductase and/or
xylitol dehydrogenase polynucleotide(s) and no promoter that is
operatively linked to the xylose reductase and/or xylitol
dehydrogenase polynucleotide. This type of construct typically
comprises genetic elements to facilitate integration into the
fungal host chromosome at a location that is downstream of a native
promoter (i.e., in the host chromosome). In some embodiments, a
second nucleic acid construct is employed which comprises a
promoter and genetic elements to facilitate integration into the
fungal host chromosome in a location upstream of the targeted
integration site of the xylose reductase and/or xylitol
dehydrogenase polynucleotide. In some embodiments, the nucleic acid
construct comprises the xylose reductase and/or xylitol
dehydrogenase polynucleotide operatively linked to a promoter or
promoter and terminator sequences such that all are integrated into
the host chromosome (genome).
[0110] Genetic elements that facilitate integration by homologous
recombination are those having sequence homology to targeted
integration sites in the fungal host chromosome (genome). Suitable
sites that find use as targets for integration include, but are not
limited to the TY1 loci, the RDN loci, the ura3 locus, the GPD
locus, aldose reductase (GRE3) locus, etc. Those having ordinary
skill in the art appreciate that additional sites for integration
can be readily identified using methods known in the art, including
but not limited to microarray analysis, metabolic flux analysis,
comparative genome hybridization analysis, etc.
[0111] Genetic elements or techniques which facilitate integration
by non-homologous recombination include, but are not limited to
restriction enzyme-mediated integration (REMI) (See e.g.,
Manivasakam et al., Mol. Cell Biol., 18(3):1736-1745 [1998], which
is incorporated herein by reference), transposon-mediated
integration, and other elements and methods that are well known in
the art.
[0112] In some embodiments, the nucleic acid constructs of the
present invention comprise at least one further recombinant
polynucleotide that is capable of conferring a desired phenotype to
a fungal host cell, particularly in the context of xylose
fermentation. In some embodiments, the recombinant polynucleotide
that is capable of conferring an improved phenotype to the fungal
host cell is a non-coding polynucleotide such as a regulatory
polynucleotide, a coding polynucleotide, or combination
thereof.
[0113] Exemplary further desired phenotypes include, but are not
limited to increased transport of xylose into the host cell,
increased xylulose kinase activity, increased flux through the
pentose phosphate pathway, decreased sensitivity to catabolite
repression, increased tolerance to ethanol, increased tolerance to
increased osmolarity, increased tolerance to organic acids, reduced
production of by-products, and other similar properties related to
increasing flux through the pentose phosphate and glycolysis
pathways to produce a desired metabolic product/intermediate at
higher levels as compared to the corresponding wild-type host cell.
Typically, the desired metabolic product is an alcohol (e.g.,
ethanol).
[0114] In some embodiments, nucleic acid constructs comprising at
least one further polynucleotide that is capable of conferring a
desired phenotype to a fungal host cell comprise a polynucleotide
encoding a protein known to impact the desired phenotype, wherein
the polynucleotide is either native or heterologous to the fungal
host cell. In some embodiments, this polynucleotide is operatively
linked to its native promoter, or to a heterologous promoter (i.e.,
a promoter that is not associated with the polynucleotide in the
corresponding native gene). In some embodiments, the at least one
further polynucleotide is overexpressed. In some embodiments, the
nucleic acid constructs comprise multiple copies of a least one
polynucleotide. Suitable polynucleotides include, but are not
limited to those that facilitate overexpression of proteins known
to have an impact on the desired phenotype.
[0115] Exemplary recombinant polynucleotides that are capable of
conferring a desired phenotype to a fungal host cell include
recombinant polynucleotides (either wild-type or mutated forms)
which encode a xylose or hexose transporter, a xylulose kinase
(XKS), an enzyme from the pentose phosphate pathway (See e.g., FIG.
2A), a glycolytic enzyme (i.e., from the glycolytic metabolic
pathway; See e.g., FIG. 2B), and an ethanologenic enzyme (See e.g.,
FIG. 2C), regulatory sequences that enhance expression of these
sequences, and combinations thereof. Additional recombinant
polynucleotides (either wild-type or mutated forms) that find use
in the present invention include those that encode additional
proteins involved in the pentose phosphate, glycolysis, and
ethanologenic pathways (See e.g., FIGS. 2A-C).
[0116] Exemplary transporters include, but are not limited to GXF1,
SUT1 and At6g59250 from Candida intermedia, Pichia stipitis and
Arabidopsis thaliana, respectively (See e.g., Runquist et al.,
Biotechnol. Biofuels., 3:5 [2010], which is incorporated herein by
reference), as well as HXT4, HXT5, HXT7, GAL2, AGT1, GXF2 (See
e.g., Matsushika et al., Appl. Microbiol. Biotechnol., 84:37-53
[2009], which is incorporated herein by reference). In some
embodiments, overexpression of native S. cerevisiae transporters is
desirable, particularly HXT5 and HXT7.
[0117] Particularly suitable recombinant polynucleotides include
those which encode: a xylulose kinase (XK); an enzyme from the
pentose phosphate pathway (e.g., a ribulose-5-phosphate 3-epimerase
(RPE1), a ribose-5-phosphate ketol-isomerase (RKI1), a
transketolase (TKL1), a transaldolase (TALI), etc.); a glycolytic
enzyme (e.g., a hexokinase (HXK1/HXK2), a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pyruvate kinase
(PVK2), etc.); and an ethanologenic enzyme (e.g., a pyruvate
decarboxylase, an alcohol dehydrogenase, etc.).
[0118] Exemplary regulatory polynucleotides include promoters,
enhancer, terminator, and other regulatory elements that function
to improve the expression of polynucleotides in a fungal host cell,
particularly, a yeast host cell. These include, but are not limited
to the regulatory elements described hereinabove.
[0119] The nucleic acid constructs described herein are useful for
transforming fungal host cells to confer to these cells the
property of xylose utilization.
Recombinant Fungal Host Cells
[0120] The present invention provides recombinant fungal host cells
comprising at least one xylose reductase and/or xylitol
dehydrogenase polynucleotide provided herein. More specifically,
the recombinant fungal host cell comprises a polynucleotide
sequence that encodes a polypeptide which is capable of catalyzing
xylose to xylitol, wherein the polynucleotide is selected from: (a)
a polynucleotide that encodes a polypeptide comprising an amino
acid sequence that is at least about 70% identical to SEQ ID NO:2,
wherein the polypeptide comprises at least one substitution set
forth herein; and (b) a polynucleotide that hybridizes under
stringent hybridization conditions to the complement of a
polynucleotide encoding a polypeptide having the amino acid
sequence of SEQ ID NO:2, wherein the polypeptide comprises at least
one substitution set forth herein. In some other embodiments, the
recombinant fungal host cell comprises a polynucleotide sequence
that encodes a polypeptide which is capable of catalyzing xylitol
to xylulose, wherein the polynucleotide is selected from: (a) a
polynucleotide that encodes a polypeptide comprising an amino acid
sequence that is at least about 70% identical to SEQ ID NO:6,
wherein the polypeptide comprises at least one substitution as set
forth herein; and (b) a polynucleotide that hybridizes under
stringent hybridization conditions to the complement of a
polynucleotide encoding a polypeptide having the amino acid
sequence of SEQ ID NO:6, wherein the polypeptide comprises at least
one substitution set forth herein. In some embodiments, the
recombinant fungal host cell comprises polynucleotide sequences
that encode a polypeptide which is capable of catalyzing xylose to
xylitol and a polypeptide capable of catalyzing xylitol to
xylulose-5-P, wherein the polynucleotides are selected from: (a)
polynucleotides that encode polypeptides comprising amino acid
sequences that are at least about 70% identical to SEQ ID NO:2
and/or 6, wherein each of the polypeptides comprise at least one
substitution set forth herein; and (b) polynucleotides that
hybridize under stringent hybridization conditions to the
complement of polynucleotides encoding polypeptides having the
amino acid sequences of SEQ ID NO:2 and/or 6, wherein each of the
polypeptides comprise at least one substitution set forth
herein.
[0121] In some embodiments, the recombinant fungal host cell
further comprises at least one xylulokinase, including but not
limited to the xylulokinase provided herein (SEQ ID NO:10), encoded
by SEQ ID NO:9, and by codon-optimized sequences SEQ ID NOS:11 and
12.
[0122] In some embodiments, the recombinant fungal host cell
comprises at least one xylose reductase, at least one xylitol
dehydrogenase, and at least one xylulokinase. In some embodiments,
the xylose reductase comprises SEQ ID NO:2, 41, 43, 45, and/or 47,
In some embodiments, the xylitol dehydrogenase comprises SEQ ID
NO:6 and/or 42. In some embodiments, the xylulokinase comprises SEQ
ID NO:10.
[0123] In some embodiments, the present invention provides a
recombinant fungal host cell comprising or transformed with a
nucleic acid construct of the present invention. In some
embodiments, the xylose reductase and/or xylitol dehydrogenase
and/or xylulokinase polynucleotide is integrated into the host cell
genome. Typically, the recombinant fungal host cell is a
filamentous fungal or yeast host cell. More typically, the
recombinant fungal host cell is a yeast host cell.
[0124] The present invention also provides methods for producing a
recombinant fungal host cell, wherein the method comprises: (a)
providing at least one nucleic acid construct of the present
invention, wherein the nucleic acid construct comprises at least
one xylose reductase and/or xylitol dehydrogenase and/or at least
one xylulokinase polynucleotide provided herein; and (b)
transforming a fungal host cell with the nucleic acid construct to
produce a recombinant fungal host cell. In some additional
embodiments, the recombinant fungal host cell is transformed using
nucleic acid constructs comprising from about two to about fifty
copies of at least one xylose reductase and/or xylitol
dehydrogenase and/or xylulokinase. It is not intended that the
present invention be limited to any particular copy number of
xylose reductase and/or xylitol dehydrogenase genes and/or
xylulokinase, as any suitable number of copies finds use in the
present invention. In some embodiments, the nucleic acid constructs
comprise any combination of wild-type and/or variant xylose
reductase and/or xylitol dehydrogenase and/or xylulokinase.
Furthermore, any suitable method for introducing multiple copies of
either xylose reductase and/or xylitol dehydrogenase and/or
xylulokinase find use in the present invention.
[0125] Introduction of the expression construct of the present
invention into the host cell can be accomplished using any suitable
method, including but not limited to calcium phosphate
transfection, DEAE-dextran mediated transfection, electroporation,
or any other suitable technique. Indeed, there are numerous methods
known in the art and described in various standard reference texts.
In some embodiments, the xylose reductase and/or xylitol
dehydrogenase and/or xylulokinase polynucleotide sequence is
integrated into the host cell genome.
[0126] Suitable fungal host cells include yeast and filamentous
fungal host cells. In some embodiments, the fungal host cell is a
yeast cell. Exemplary yeast host cells that are useful in the
practice of the present invention include, but are not limited to
Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia,
Kluyveromyces, and Yarrowia. In some embodiments of the invention,
the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae,
Saccharomyces carlsbergensis, Saccharomyces diastaticus,
Saccharomyces norbensis, Saccharomyces kluyveri,
Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica,
Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens,
Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia
quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica,
Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia
lipolytica. In some embodiments, the yeast host cell is
Saccharomyces species. In some additional embodiments, the yeast
host cell is Saccharomyces cerevisiae.
[0127] Any suitable yeast strain finds use in the present
invention, including but not limited to strains such as those
commercially available from companies such as Lallemand (e.g.,
Superstart.TM. THERMOSACCO, etc.); RED STAR and ETHANOL REDO
(Fermentis/Lesaffre); FALI (AB Maui); (Baker's Best Yeast, Baker's
Compressed Yeast, etc. (Fleishmann's Yeast); BIOFERM AFT, XP, CF,
and XR (North American Bioproducts Corp.); Turbo Yeast (Gert Strand
AB); FERMIOLO (DSM Specialties); BY4741 (e.g., ATCC 201388); Y108-1
(ATCC PTA.10567) and LNH-ST (See, US Pat. Publ. No. 2011/0159560);
NRRL YB-1952 (ARS Culture Collection), as well as any additional
suitable strains.
[0128] In some embodiments, the filamentous fungal host cell is a
species of Achlya, Acremonium, Aspergillus, Aureobasidium,
Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium,
Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus,
Coriolus, Diplodia, Endothia, Fusarium, Gibberella, Gliocladium,
Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium,
Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus,
Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus,
Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium,
Volvariella, or teleomorphs, or anamorphs, and synonyms or
taxonomic equivalents thereof. However, it is not intended that the
present invention be limited to any particular species of
filamentous fungal host cell. Exemplary filamentous fungal host
cells that find use in the present invention include, but are not
limited to a filamentous fungal host cell of the Trichoderma
species (e.g., T. longibrachiatum, T. viride [e.g., ATCC 32098 and
32086], T. reesei [NRRL 15709, ATTC 13631, 56764, 56765, 56466,
56767, and RL-P37 and derivatives thereof; See e.g., Sheir-Neiss et
al., Appl. Microbiol. Biotechnol., 20:46-53 [1984], incorporated
herein by reference), T. koningii, and T. harzianum), as well as
Hypocrea jecorina. The term "Trichoderma" refers to any fungal
strain that was previously classified as Trichoderma or is
currently classified as Trichoderma.
[0129] In some embodiments of the present invention, the
filamentous fungal host cell is an Aspergillus species (e.g., A.
awamori, A. fumigatus, A. japonicas, A. nidulans, A. niger. A.
aculeatus, A. foetidus, A. oryzae, A. sojae, or A. kawachi (See
e.g., Kelly and Hynes, EMBO J., 4:475479 [1985]; NRRL 3112, ATCC
11490, 22342, 44733, and 14331; Yelton et al., Proc. Natl. Acad.
Sci. USA, 81, 1480-1474 [1984]; Tilburn et al., Gene 26, 205-221
[1982]; and Johnston et al., EMBO J., 4:1307-1311 [1985], all of
which are incorporated herein by reference). In some embodiments of
the invention, the filamentous fungal host cell is a Fusarium
species (e.g., F. bactridioides, F. cerealis, F. crookwellense, F.
culmorum, F. graminaearum, F. graminum, F. oxysporum, F. rosium, or
F. venenatum). In some embodiments of the invention, the
filamentous fungal host cell is of a Neurospora species (e.g., N.
crassa; See e.g., Case, et al., Proc. Natl. Acad. Sci. USA,
76:5259-5263 [1979]; U.S. Pat. No. 4,486,553; and Kinsey and
Rambosek, Mol. Cell. Biol., 4:117-122 [1984], all of which are
incorporated herein by reference). In some embodiments of the
invention, the filamentous fungal host cell is of a Humicola
species (e.g., H. insolens. H. grisea, or H. lanuginose). In some
embodiments of the invention, the filamentous fungal host cell is a
Mucor species (e.g., M. miehei or M. circinelloides). In some
embodiments of the invention, the filamentous fungal host cell is a
Rhizopus species (e.g., R. oryzae or R. niveus). In some
embodiments of the invention, the filamentous fungal host cell is
of a Penicillum species (e.g., P. purpurogenum, P. chrysogenum, or
P. verruculosum). In some embodiments of the invention, the
filamentous fungal host cell is a Thielavia species (e.g., T.
terrestris). In some embodiments of the invention, the filamentous
fungal host cell is a Tolypocladium species (e.g., T. inflatum or
T. geodes). In some embodiments of the invention, the filamentous
fungal host cell is a Trametes species (e.g., T. villosa or T.
versicolor). In some embodiments of the invention, the filamentous
fungal host cell is a Chrysosporium species, (e.g., C. lucknowense,
C. keratinophilum, C. tropicum, C. merdarium, C. Mops, C.
pannicola, or C. zonatum).
[0130] Strains that find use in the present invention include those
that are readily accessible to the public from a number of culture
collection, including but not limited to the American Type Culture
Collection (ATCC), Deutsche Sammlung von Mikroorganismen and
Zellkutlturen GmbH (DSM), Centraalbureau Voor Schimmelcultures
(CBS), and Agricultural Research Service Patent Culture Collection,
Northern Regional Research Center (NRRL).
[0131] Recombinant fungal host cells of the present invention are
capable of fermenting xylose when provided with a xylose based
culture medium. Typically the recombinant fungal host cells
described herein are capable of fermenting xylose at a faster rate
compared to the corresponding wild-type fungal host cell. In some
embodiments, the recombinant fungal host cells provided herein are
capable of fermenting xylose at a faster rate compared to fungal
host cells transformed with a nucleic acid construct comprising
xylose reductase and/or xylitol dehydrogenase genes. In some other
embodiments, the recombinant fungal host cells provided herein are
capable of fermenting xylose at a faster rate compared to fungal
host cells transformed with a nucleic acid construct comprising of
xylose reductase, xylitol dehydrogenase and/or xylulose kinase
genes. In some embodiments, the recombinant fungal host cells are
capable of fermenting xylose at a rate of at least about 1 g/L/h
and sometimes at a rate of at least about 2g/L/h. Exemplary
xylose-based culture media include culture media which have been
formulated to contain xylose, as well as feedstock from cellulosic
saccharification processes and/or feedstock from a hemicellulose
pre-treatment process (i.e., a "hemicellulosic feedstock").
[0132] In some embodiments, the fungal host cell is a wild-type
fungal cell, while in other embodiments, it is a mutated or
otherwise altered or engineered form of a wild-type fungal cell.
Typically, the fungal host cell (either wild-type or otherwise
altered or engineered) comprises polynucleotides encoding a
xylulokinase and one or more enzymes in the pentose phosphate,
glycolytic, and/or ethanologenic pathways. In some embodiments, the
fungal host cell comprises polynucleotides encoding a xylulokinase
and all of the enzymes in the pentose phosphate, glycolytic, and
ethanologenic pathways. In some embodiments, the fungal host cell
comprises recombinant polynucleotides encoding enzymes that are
heterologous to the fungal host cell (i.e., not native to the
fungal host cell). In some additional embodiments, the fungal host
cell is engineered to comprise other metabolic pathways that
utilize products/intermediates from the pentose phosphate,
glycolytic, and/or enthanologenic pathways to produce other
desirable products. For example, in some embodiments, the fungal
host cell is engineered to comprise a metabolic pathway for the
biosynthesis of a fatty alcohol or fatty acid (See e.g., WO
2007/136762, which is incorporated herein by reference). In some
embodiments, the fatty alcohol or fatty acid is a C8-C20 fatty acid
or fatty alcohol. In some embodiments, the fungal host cell is
altered or engineered to overexpress any one or more of the
polynucleotides encoding the enzymes in one or more of these
metabolic pathways.
[0133] In some embodiments, the recombinant fungal host cell of the
present invention further comprises genetic modifications in
addition to the xylose reductase and/or xylitol dehydrogenase
polynucleotide. In some embodiments, in addition to having a xylose
reductase and/or xylitol dehydrogenase polynucleotide described
herein, the recombinant host cell comprises at least one different
recombinant polynucleotide that is capable of conferring a further
desired phenotype to the fungal host cell. In some embodiments, the
present invention provides a recombinant fungal host cell
comprising at least one P. stipitis xylose reductase and/or xylitol
dehydrogenase polynucleotide or variant thereof as described
herein, and at least one recombinant polynucleotide that encodes a
polypeptide which differs from the P. stipitis xylose reductase
and/or xylitol dehydrogenase and/or variant(s) thereof, wherein the
recombinant polynucleotide imparts a desired phenotype to the
fungal host cell. It is contemplated that in some embodiments, the
recombinant polynucleotide that is capable of conferring a desired
phenotype to the fungal host cell is introduced to the fungal host
cell on the same nucleic construct as the xylose reductase and/or
xylitol dehydrogenase polynucleotide, or on a separate nucleic acid
construct. Nucleic acid constructs of the present invention
comprising at least one xylose reductase and xylitol dehydrogenase
polynucleotides and at least one further recombinant polynucleotide
capable of conferring a desired phenotype to the fungal host cell
are also provided by the present invention.
[0134] In some embodiments, the recombinant polynucleotide that is
capable of conferring a desired phenotype to the fungal host cell
is a non-coding polynucleotide (e.g., a regulatory polynucleotide,
a coding polynucleotide, or a combination thereof). As described
above, exemplary further desired phenotypes include, but are not
limited to increased transport of xylose into the host cell,
increased xylulose kinase activity, increased flux through the
pentose phosphate pathway, decreased sensitivity to catabolite
repression, increased tolerance to ethanol, increased tolerance to
increased osmolarity, increased tolerance to organic acids, reduced
production of by-products, and other like properties related to
increasing flux through the pentose phosphate, glycolysis, and/or
ethanologenic pathways to produce the desired metabolic
product/intermediate at higher levels as compared to the
corresponding wild-type host cell. In some embodiments, the desired
metabolic product is an alcohol (e.g., ethanol).
[0135] In some embodiments, recombinant fungal host cells
comprising at least one further polynucleotide capable of
conferring a desired phenotype to the fungal host cell comprise at
least one polynucleotide encoding a protein known to impact the
desired phenotype, wherein the polynucleotide is either native or
heterologous to the fungal host cell. In some embodiments, the
polynucleotide(s) are operatively linked to its native promoter,
while in other embodiments, the polynucleotide is operatively
linked to a heterologous promoter (i.e., one not associated with
the polynucleotide in the corresponding native gene). In some
embodiments, the polynucleotide is overexpressed. In some
embodiments, the recombinant fungal host cell comprises multiple
copies of the polynucleotide. Suitable polynucleotides include, but
are not limited to those that facilitate overexpression of proteins
known to have an impact on the desired phenotype. Therefore, in
some embodiments, the fungal host cell is altered or engineered to
overexpress one or more polynucleotides.
[0136] In some embodiments, recombinant polynucleotides that are
capable of imparting a desired phenotype to a fungal host cell
include, but are not limited to recombinant polynucleotides which
encode a xylose or hexose transporter, a xylulose kinase (XKS), an
enzyme from the pentose phosphate pathway (See e.g., FIG. 2A), a
glycolytic enzyme (i.e., from the metabolic pathway of glycolysis;
See e.g., FIG. 2B), and an ethanologenic enzyme (See e.g., FIG.
2C), the regulatory sequences associated with these sequences, and
any combination thereof.
[0137] Exemplary transporters that find use in the present
invention include, but are not limited to GXF1, SUT1 and At6g59250
from Candida intermedia, Pichia stipitis, and Arabidopsis thaliana,
respectively (See e.g., Runquist et al., 84:37-53 [2010],
incorporated herein by reference), HXT4, HXT5, HXT7, GAL2, AGT1,
and GXF2, (See e.g., Matsushika et al., Appl. Microbiol.
Biotechnol., 84:37-53 [2009]). In some embodiments, overexpression
of native S. cerevisiae transporters is desirable, particularly
HXT5 and HXT7.
[0138] Particularly suitable recombinant polynucleotides include,
but are not limited to those that encode: a xylulose kinase (XK);
an enzyme from the pentose phosphate pathway (e.g., a
ribulose-5-phosphate 3-epimerase (RPE1), a ribose-5-phosphate
ketol-isomerase (RKI1), a transketolase (TKL1), a transaldolase
(TALI), etc.); a glycolytic enzyme (e.g., a hexokinase (HXK1/HXK2),
a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pyruvate
kinase (PVK2), etc.; and an ethanologenic enzyme (e.g., a pyruvate
decarboxylase, an alcohol dehydrogenase, etc.).
[0139] Exemplary regulatory polynucleotides include promoters,
enhancer, terminator, and other regulatory elements that function
to improve the expression of polynucleotides in a fungal host cell,
particularly, a yeast host cell, as described above.
[0140] In some embodiments, recombinant host cells of the present
invention comprise one or more native genes deleted from its
genome. In some embodiments, the deletion(s) cause removal or
diminishment of a biological activity that is otherwise exhibited
by the fungal host cell. In some embodiments, the cumulative effect
of the deletion(s) also leads to an improvement in a phenotype of
the fungal host cell. Any suitable method for deleting gene finds
use in the present invention. There are numerous methods well known
in the art.
[0141] For example, in some embodiments, recombinant host cells of
the present invention have certain native genes deleted from the
host genome in order to improve the utilization of pentose sugars
(e.g., xylose), increase transport of xylose into the host cell,
increase xylulose kinase activity, increase flux through the
pentose phosphate pathway, decrease sensitivity to catabolite
repression, increase tolerance to ethanol/acetate, increase
tolerance to increased osmolarity, increase tolerance to organic
acids (low pH), reduce production of by-products, and other like
properties related to increasing flux through the relevant pathways
to produce ethanol and other desired metabolic products at higher
levels, where comparison is made with respect to the corresponding
host cell without the deletion(s). Genes targeted for deletion
include, but are not limited to genes encoding enzymes in the
pentose phosphate pathway, a glycolytic enzyme, and/or an
ethanologenic enzyme.
[0142] In some embodiments, other genes are targeted for deletion,
including but not limited to those encoding aldose reductase (GRE3)
(See e.g., Matsushika et al., Appl. Microbiol. Biotechnol.,
84:37-53 [2009]), sorbitol dehydrogenases (SOR1/SOR2), a glutamate
dehydrogenase (GDH1), a 6-phosphogluconate dehydrogenase (GND), a
glucose-5-phosphate dehydrogenase (ZWF1), and any enzyme for which
its deletion is known in the art to improve the utilization of a
pentose sugar, decrease by-product formation, and/or increase the
ethanol yield of a fungal host cell. The genes encoding these
enzymes in many fungi are known in the art. Those having ordinary
skill in the art appreciate that additional genes encoding these
enzymes can be readily identified by microarray analysis (See e.g.,
Sedlak et al., Yeast 21:671-684 [2004]), metabolic flux analysis
(See e.g Sonderegger et al., Appl. Environ. Microbiol.,
70(4):2307-2317 [2004]), in silico modeling (See e.g Hjersted et
al., Biotechnol. Bioengineer. 97(5):1190-1204 [2007]),
chemogenomics (See e.g Teixeira et al., Appl. Environ. Microbiol.,
75(18):5761-5772 [2009]), and other well known methods.
[0143] In some embodiments, the host cells employed in the practice
of the present invention are mutagenized and/or evolved to exhibit
further desired phenotypes, for example, further improvement in the
utilization of pentose sugars (e.g., xylose, arabinose, etc.),
increased transport of xylose into the host cell, increased
xylulose kinase activity, increased flux through the pentose
phosphate pathway, decreased sensitivity to catabolite repression,
increased tolerance to ethanol/acetate, increased tolerance to
increased osmolarity, increased tolerance to organic acids (low
pH), reduced production of by-products, and other like properties
related to increasing flux through the pentose phosphate and
glycolysis pathways to produce a desired metabolic
product/intermediate at higher levels. In some embodiments, the
desired metabolic product is an alcohol (e.g., ethanol). In some
embodiments, the host cells are mutagenized and/or evolved using
known methods either prior to or after transformation with the
xylose reductase and/or xylitol dehydrogenase polynucleotide. These
methods include, but are not limited to classical mutagenesis,
whole genome shuffling, evolutionary engineering methods, which
employ screening and/or selection methods, or any combination of
such well known methods.
[0144] Classical mutagenesis methods include, but are not limited
to treatment of the host cell with a mutagen such as a chemical
mutagen or irradiation exposure (e.g., ultraviolet or
gamma-irradiation). Whole genome shuffling methods involving, for
example, recombination of genomic DNA between native genomic DNA
sequences and/or variants thereof, can be facilitated by sexual
mating, protoplast fusion methods and other methods well known in
the art (See e.g., WO 98/31837 and WO 2000/04190, incorporated
herein by reference). These methods are coupled with screening
and/or selection methods to identify altered fungal host cells that
exhibit the desired phenotype. For example, such methods find use
in altering or engineering a fungal host cell to overexpress one or
more desired polynucleotides.
[0145] Mutagenesis may be performed in accordance with any of the
techniques known in the art, including random and site-specific
mutagenesis. Directed evolution can be performed with any of the
techniques known in the art to screen for improved promoter
variants including shuffling. Mutagenesis and directed evolution
methods are well known in the art (See e.g., U.S. Pat. Nos.
5,605,793, 5,830,721, 6,132,970, 6,420,175, 6,277,638, 6,365,408,
6,602,986, 7,288,375, 6,287,861, 6,297,053, 6,576,467, 6,444,468,
5,811,238, 6,117,679, 6,165,793, 6,180,406, 6,291,242, 6,995,017,
6,395,547, 6,506,602, 6,519,065, 6,506,603, 6,413,774, 6,573,098,
6,323,030, 6,344,356, 6,372,497, 7,868,138, 5,834,252, 5,928,905,
6,489,146, 6,096,548, 6,387,702, 6,391,552, 6,358,742, 6,482,647,
6,335,160, 6,653,072, 6,355,484, 6,03,344, 6,319,713, 6,613,514,
6,455,253, 6,579,678, 6,586,182, 6,406,855, 6,946,296, 7,534,564,
7,776,598, 5,837,458, 6,391,640, 6,309,883, 7,105,297, 7,795,030,
6,326,204, 6,251,674, 6,716,631, 6,528,311, 6,287,862, 6,335,198,
6,352,859, 6,379,964, 7,148,054, 7,629,170, 7,620,500, 6,365,377,
6,358,740, 6,406,910, 6,413,745, 6,436,675, 6,961,664, 7,430,477,
7,873,499, 7,702,464, 7,783,428, 7,747,391, 7,747,393, 7,751,986,
6,376,246, 6,426,224, 6,423,542, 6,479,652, 6,319,714, 6,521,453,
6,368,861, 7,421,347, 7,058,515, 7,024,312, 7,620,502, 7,853,410,
7,957,912, 7,904,249, and all related US and non-US counterparts;
Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale et al.,
Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet.,
19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985];
Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell,
38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull
et al., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians et al.,
Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature,
391:288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438
[1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509
[1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996];
Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci.
USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966;
WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of
which are incorporated herein by reference).
[0146] Evolutionary engineering can be done by prolonged
cultivation and selection of strains under desired conditions
through chemostat, turbidostat or batch cultures. Evolutionary
engineering methods can be practiced under either aerobic or
anaerobic conditions. Selection strategies can be optimized by
varying culture conditions, for example, carbon source, nitrogen
source, aeration, pH and temperature. Methods for evolutionary
engineering are well known in the art (See e.g., Wisselink et al.,
Appl. Environ. Microbiol., 75(4):907-914 [2009]; Kuyper et al.,
FEMS Yeast Res., 5:399-409 [2005]; and Sauer, Adv. Biochem.
Engineer. Biotechnol., 73:129-169 [2001], all of which are
incorporated herein by reference).
[0147] Therefore, in some embodiments, the recombinant fungal host
cell comprising a xylose reductase and/or xylitol dehydrogenase
polynucleotide exhibits an improved phenotype relative to the
corresponding fungal host cell without the xylose reductase and/or
xylitol dehydrogenase polynucleotide.
[0148] In some embodiments, whole genome shuffling and evolutionary
engineering methods result in increased copy number of xylose
reductase and/or xylitol dehydrogenase and/or xylulokinase from two
to fifty copies. It is not intended that the present invention be
limited to any particular copy number of xylose reductase and/or
xylitol dehydrogenase genes and/or xylulokinase, as any suitable
number of copies finds use in the present invention. In some
embodiments, the nucleic acid constructs comprise any combination
of wild-type and/or variant xylose reductase and/or xylitol
dehydrogenase and/or xylulokinase. Furthermore, any suitable method
of increasing copies of either xylose reductase and/or xylitol
dehydrogenase and/or xylulokinase finds use in the present
invention.
[0149] In some embodiments, the improved phenotype comprises
further improvement in the utilization of pentose sugars (e.g.,
xylose, arabinose, etc.), increased transport of xylose into the
host cell, increased xylulose kinase activity, increased flux
through the pentose phosphate pathway, decreased sensitivity to
catabolite repression, increased tolerance to ethanol/acetate,
increased tolerance to increased osmolarity, increased tolerance to
organic acids (low pH), and reduced production of by products, or
other properties.
Fermentation
[0150] The present invention provides processes for producing
fermentation products, wherein the method comprises: (a) providing
the recombinant fungal cell of the present invention; (b) providing
a fermentation medium comprising xylose; (c) contacting the
fermentation medium with the recombinant fungal cell under
conditions suitable for generating the fermentation product; and
optionally (d) recovering the fermentation product. In some
embodiments, the fermentation product is an alcohol (e.g., ethanol,
butanol, etc.), a fatty alcohol (e.g., a C8-C20 fatty alcohol), a
fatty acid (e.g., a C8-C20 fatty acid), lactic acid,
3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propanediol, ethylene, glycerol, and/or a .beta.-lactam (e.g.,
cephalosporin). However, it is contemplated that other fermentation
products will be produced using the methods of the present
invention.
[0151] In some embodiments, the fermentation medium is feedstock
from a cellulosic saccharification process and/or feedstock from a
hemicellulose pre-treatment process. Such feedstocks include, but
are not limited to carbohydrates (e.g., lignocellulose, xylans,
cellulose, starch, etc.), other sugars (e.g., glucose, xylose,
arabinose, etc.), and other compositions. Compositions of
fermentation media suitable for the growth of yeast and filamentous
fungi are well known in the art and there are various reference
texts that provide recipes for these media.
[0152] Fermentation conditions suitable for generating desired
fermentation products are well known in the art and any suitable
method finds use in the present invention. In some embodiments, the
fermentation process is carried out under aerobic or microaerobic
(i.e., where the concentration of oxygen is less than that in air),
or anaerobic conditions. In some embodiments, fermentation is
conducted under anaerobic conditions (i.e., no detectable oxygen),
or less than about 5, about 2.5, or about 1 mmol/L/h oxygen. In the
absence of oxygen, the NADH produced in glycolysis cannot be
oxidized by oxidative phosphorylation. Under anaerobic conditions,
pyruvate or a derivative thereof may be utilized by the host cell
as an electron and hydrogen acceptor in order to generated NAD+. In
some embodiments of the present invention, when the fermentation
process is carried out under anaerobic conditions, pyruvate may be
reduced to a fermentation product such as ethanol, butanol, lactic
acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic
acid, citric acid, malic acid, fumaric acid, an amino acid,
1,3-propanediol, ethylene, glycerol, and/or a .beta.-lactam (e.g.,
a cephalosporin).
[0153] The fermentation process is typically run at a temperature
that is optimal for the recombinant fungal cell. For example, in
some embodiments, the fermentation process is performed at a
temperature in the range of from about 25.degree. C. to about
42.degree. C. Typically the process is carried out a temperature
that is less than about 38.degree. C., less than about 35.degree.
C., less than about 33.degree. C., or less than about 38.degree.
C., but at least about 20.degree. C., 22.degree. C., or 25.degree.
C.
[0154] In some embodiments, recombinant host cells of the present
invention are grown under batch or continuous fermentation
conditions. Classical batch fermentation is a closed system,
wherein the composition of the medium is set at the beginning of
the fermentation and is not subject to artificial alterations
during the fermentation. A variation of the batch system is a
fed-batch fermentation, which also finds use in the present
invention. In this variation, the substrate is added in increments
as the fermentation progresses. Fed-batch systems are useful when
catabolite repression is likely to inhibit the metabolism of the
cells and/or where it is desirable to have limited amounts of
substrate in the medium. Batch and fed-batch fermentations are
common and well known in the art. Continuous fermentation is an
open system where a defined fermentation generally maintains the
culture at a constant high density where cells are primarily in log
phase growth. Continuous fermentation systems strive to maintain
steady state growth conditions. Methods for modulating nutrients
and growth factors for continuous fermentation processes, as well
as techniques for modulating nutrients and growth factors for
continuous fermentation processes as well as techniques for
maximizing the rate of product formation are well known in the art
of industrial microbiology.
[0155] The foregoing and other aspects of the invention may be
better understood in connection with the following non-limiting
examples.
EXPERIMENTAL
[0156] The present invention is described in further detail in the
following Examples, which are not in any way intended to limit the
scope of the invention as claimed.
[0157] In the experimental disclosure below, the following
abbreviations apply: ppm (parts per million); M (molar); mM
(millimolar), uM and .mu.M (micromolar); nM (nanomolar); mol
(moles); gm and g (gram); mg (milligrams); ug and .mu.g
(micrograms); L and l (liter); ml and mL (milliliter); cm
(centimeters); mm (millimeters); um and .mu.m (micrometers); sec.
(seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units);
MW (molecular weight); rpm (rotations per minute); .degree. C.
(degrees Centigrade); DNA (deoxyribonucleic acid); RNA (ribonucleic
acid); HPLC (high pressure liquid chromatography); MES
(2-N-morpholino ethanesulfonic acid); FIOPC (fold improvements over
positive control); YPD (10g/L yeast extract, 20g/L peptone, and
20g/L dextrose); SOE-PCR (splicing by overlapping extension PCR);
ARS (ARS Culture Collection or NRRL Culture Collection, Peoria,
Ill.); Axygen (Axygen, Inc., Union City, Calif.); Lallemand
(Lallemand Ethanol Technology, Milwaukee, Wis.); Dual Biosystems
(Dual Biosystems AG, Schlieven, Switzerland); Megazyme (Megazyme
International Ireland, Ltd., Wicklow, Ireland); Dasgip (Dasgip
Biotools, LLC, Shrewsbury, Mass.); Difco (Difco Laboratories, BD
Diagnostic Systems, Detroit, Mich.); PCRdiagnostics
(PCRdiagnostics, by E. coli SRO, Slovak Republic); Agilent (Agilent
Technologies, Inc., Santa Clara, Calif.); and Bio-Rad (Bio-Rad
Laboratories, Hercules, Calif.).
[0158] The following sequences find use in the present invention.
SEQ ID NO:2 provides the polypeptide sequence of P. stipitis xylose
reductase ("XR.1"), while SEQ ID NO:1 provides the native
polynucleotide sequence, SEQ ID NO:3 provides one codon optimized
DNA sequence ("XR.2"), and SEQ ID NO:4 provides an alternative
codon optimized DNA sequence ("XR.3"). SEQ ID NO:6 provides the
polypeptide sequence of P. stipitis xylitol dehydrogenase ("XD.1"),
while SEQ ID NO:5 provides the native polynucleotide sequence, SEQ
ID NO:7 provides one codon optimized DNA sequence ("XD.2"), and SEQ
ID NO:8 provides an alternative codon optimized DNA sequence
("XD.3"). SEQ ID NO:10 provides the polypeptide sequence of S.
cerevisiae xylulokinase ("XK.1"), SEQ ID NO:9 provides the native
polynucleotide sequence, SEQ ID NO:11 provides one codon optimized
DNA sequence ("XK.2"), and SEQ ID NO:12 provides an alternative
codon optimized DNA sequence ("XK.3").
TABLE-US-00007 (SEQ ID NO: 2)
MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQIYRAIKTGYRLFDGAEDYAN
EKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQ
VDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKAL
EKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLI
EFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGK
SPAQVLLRWSSQRGIAIIPKSNTVPRLLENKDVNSFDLDEQDFADIAKLD
INLRFNDPWDWDKIPIFV (SEQ ID NO: 1)
ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTT
CGGCTGTTGGAAAGTCGACGTCGACACCTGTTCTGAACAGATCTACCGTG
CTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGCCAAC
GAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGT
CAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACC
ACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAA
GTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTT
CGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAAGGGTG
ACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTT
GAAAAGTTGGTCAAGGCCGGTAAGATCAGATCTATCGGTGTTTCTAACTT
CCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCAT
CTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATC
GAATTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGG
TCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTC
CATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGTAAG
TCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCAT
CATTCCAAAGTCCAACACTGTCCCAAGATTGTTGGAAAACAAGGACGTCA
ACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGAC
ATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTT CGTCTAATAA (SEQ
ID NO: 3) ATGCCCTCCATAAAGCTAAACTCTGGTTATGATATGCCTGCCGTTGGATT
CGGTTGTTGGAAAGTAGATGTTGATACCTGCTCAGAACAGATTTATAGGG
CTATTAAAACAGGTTACAGGTTGTTCGACGGTGCCGAGGACTACGCGAAT
GAGAAGTTAGTTGGAGCTGGTGTTAAGAAGGCCATCGACGAGGGCATTGT
TAAGAGGGAGGACCTGTTCTTAACGTCTAAACTGTGGAATAACTATCACC
ACCCAGACAATGTTGAAAAAGCCCTGAATAGAACTTTGAGTGATTTGCAG
GTGGATTACGTTGACTTGTTTCTAATCCATTTCCCCGTTACTTTCAAGTT
CGTCCCTTTAGAGGAGAAGTATCCACCAGGTTTCTATTGTGGTAAGGGCG
ACAACTTCGACTATGAAGATGTTCCTATTCTGGAGACCTGGAAGGCTCTG
GAGAAACTGGTCAAGGCAGGCAAAATAAGGTCCATAGGCGTCTCTAACTT
CCCAGGAGCACTACTGCTTGACTTGTTGAGGGGAGCTACCATCAAACCTT
CAGTCTTGCAAGTTGAACACCACCCCTATTTGCAACAACCCAGGCTGATA
GAGTTTGCTCAATCCAGAGGTATAGCTGTTACTGCATACTCTTCCTTTGG
ACCTCAATCCTTCGTCGAACTTAACCAAGGTAGAGCGTTGAATACTTCCC
CCTTGTTCGAGAATGAAACTATCAAGGCTATTGCGGCTAAGCACGGTAAG
TCACCAGCTCAAGTGTTACTTAGATGGTCTTCCCAAAGGGGTATCGCAAT
CATTCCAAAGTCTAACACTGTTCCCAGATTGTTGGAGAATAAAGATGTTA
ATTCTTTCGATTTGGACGAACAAGACTTTGCAGACATAGCCAAACTAGAT
ATTAATCTGAGATTCAATGATCCCTGGGATTGGGACAAGATACCAATCTT CGTTTAATAA (SEQ
ID NO: 4) ATGCCATCTATTAAGTTGAACTCTGGTTACGACATGCCAGCTGTCGGTTT
CGGTTGTAAGGTTGACGTTGACACCTGTTCTGAACAAATTTACAGAGCTA
TTAAGACCGGTTACAGATTGTTCGACGGTGCTGAAGACTACGCTAACGAA
AAGTTGGTTGGTGCTGGTGTCAAGAAGGCTATTGACGAAGGTATTGTCAA
GAGAGAAGACTTGTTCTTGACCTCTAAGTTGAACAACTACCACCACCCAG
ACAACGTCGAAAAGGCTTTGAACAGAACCTTGTCTGACTTGCAAGTTGAC
TACGTTGACTTGTTCTTGATTCACTTCCCAGTCACCTTCAAGTTCGTCCC
ATTGGAAGAAAAGTACCCACCAGGTTTCTACTGTGGTAAGGGTGACAACT
TCGACTACGAAGACGTTCCAATTTTGGAAACCAAGGCTTTGGAAAAGTTG
GTTAAGGCTGGTAAGATTAGGTCTATTGGTGTTTCTAACTTCCCAGGTGC
TTTGTTGTTGGACTTGTTGAGAGGTGCTACCATTAAGCCATCTGTTTTGC
AAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATTGAGTTCGCT
CAATCTAGGGGTATTGCTGTCACCGCTTACTCTTCTTTCGGTCCACAATC
TTTCGTCGAATTGAACCAAGGTAGAGCTTTGAACACCTCTCCATTGTTCG
AAAACGAAACCATTAAGGCTATTGCTGCTAAGCACGGTAAGTCTCCAGCT
CAAGTCTTGTTGAGGTCTTCTCAAAGAGGTATTGCTATTATTCCAAAGTC
TAACACCGTCCCAAGATTGTTGGAAAACAAGGACGTTAACTCTTTCGACT
TGGACGAACAAGACTTCGCTGACATTGCTAAGTTGGACATTAACTTGAGA
TTCAACGACCCAGACGACAAGATTCCAATTTTCGTTTAATAA (SEQ ID NO: 6)
MTANPSLVLNKIDDISFETYDAPEISEPTDVLVQVKKTGICGSDIHFYAH
GRIGNFVLTKPMVLGHESAGTVVQVGKGVTSLKVGDNVAIEPGIPSRFSD
EYKSGHYNLCPHMAFAATPNSKEGEPNPPGTLCKYFKSPEDFLVKLPDHV
SLELGALVEPLSVGVHASKLGSVAFGDYVAVFGAGPVGLLAAAVAKTFGA
KGVIVVDIFDNKLKMAKDIGAATHTFNSKTGGSEELIKAFGGNVPNVVLE
CTGAEPCIKLGVDAIAPGGRFVQVGNAAGPVSFPITVFAMKELTLFGSFR
YGFNDYKTAVGIFDTNYQNGRENAPIDFEQLITHRYKFKDAIEAYDLVRA GKGAVKCLIDGPE
(SEQ ID NO: 5) ATGACTGCTAACCCTTCCTTGGTGTTGAACAAGATCGACGACATTTCGTT
CGAAACTTACGATGCCCCAGAAATCTCTGAACCTACCGATGTCCTCGTCC
AGGTCAAGAAAACCGGTATCTGTGGTTCCGACATCCACTTCTACGCCCAT
GGTAGAATCGGTAACTTCGTTTTGACCAAGCCAATGGTCTTGGGTCACGA
ATCCGCCGGTACTGTTGTCCAGGTTGGTAAGGGTGTCACCTCTCTTAAGG
TTGGTGACAACGTCGCTATCGAACCAGGTATTCCATCCAGATTCTCCGAC
GAATACAAGAGCGGTCACTACAACTTGTGTCCTCACATGGCCTTCGCCGC
TACTCCTAACTCCAAGGAAGGCGAACCAAACCCACCAGGTACCTTATGTA
AGTACTTCAAGTCGCCAGAAGACTTCTTGGTCAAGTTGCCAGACCACGTC
AGCTTGGAACTCGGTGCTCTTGTTGAGCCATTGTCTGTTGGTGTCCACGC
CTCTAAGTTGGGTTCCGTTGCTTTCGGCGACTACGTTGCCGTCTTTGGTG
CTGGTCCTGTTGGTCTTTTGGCTGCTGCTGTCGCCAAGACCTTCGGTGCT
AAGGGTGTCATCGTCGTTGACATTTTCGACAACAAGTTGAAGATGGCCAA
GGACATTGGTGCTGCTACTCACACCTTCAACTCCAAGACCGGTGGTTCTG
AAGAATTGATCAAGGCTTTCGGTGGTAACGTGCCAAACGTCGTTTTGGAA
TGTACTGGTGCTGAACCTTGTATCAAGTTGGGTGTTGACGCCATTGCCCC
AGGTGGTCGTTTCGTTCAAGTCGGTAACGCTGCTGGTCCAGTCAGCTTCC
CAATCACCGTTTTCGCCATGAAGGAATTGACTTTGTTCGGTTCTTTCAGA
TACGGATTCAACGACTACAAGACTGCTGTTGGAATCTTTGACACTAACTA
CCAAAACGGTAGAGAAAATGCTCCAATTGACTTTGAACAATTGATCACCC
ACAGATACAAGTTCAAGGACGCTATTGAAGCCTACGACTTGGTCAGAGCC
GGTAAGGGTGCTGTCAAGTGTCTCATTGACGGCCCTGAGTAATAA (SEQ ID NO: 7)
ATGACCGCTAATCCCTCTCTTGTTTTGAATAAGATTGACGACATTTCTTT
TGAAACTTACGATGCTCCCGAAATTAGCGAACCCACAGACGTTTTAGTTC
AAGTTAAAAAAACTGGTATCTGCGGTTCTGACATCCACTTCTACGCTCAT
GGAAGGATCGGCAACTTCGTCTTAACAAAGCCAATGGTTCTGGGTCATGA
AAGCGCGGGTACTGTTGTTCAAGTCGGTAAAGGTGTTACTTCACTGAAGG
TTGGTGATAACGTCGCAATCGAGCCCGGTATTCCATCTAGGTTCAGTGAT
GAGTACAAATCTGGTCACTACAACCTGTGTCCACACATGGCATTTGCTGC
TACTCCCAATTCTAAAGAGGGTGAACCAAACCCACCAGGAACTCTATGTA
AGTACTTCAAATCTCCAGAAGACTTCCTGGTTAAGTTACCCGATCATGTT
TCTTTGGAGTTGGGTGCTTTGGTCGAGCCACTATCTGTTGGGGTCCATGC
TAGTAAATTAGGCTCCGTTGCATTTGGCGATTACGTTGCTGTTTTTGGTG
CTGGTCCAGTAGGATTACTGGCTGCCGCTGTCGCTAAGACATTTGGTGCC
AAGGGTGTGATTGTCGTTGATATATTTGACAACAAGCTGAAGATGGCCAA
AGACATAGGTGCCGCTACACATACCTTCAACTCCAAGACGGGAGGTAGTG
AAGAATTGATCAAAGCCTTCGGTGGTAATGTACCAAATGTTGTCTTGGAA
TGTACTGGGGCTGAACCATGTATTAAGCTAGGTGTTGATGCCATCGCACC
AGGTGGTAGATTCGTGCAAGTTGGTAATGCTGCTGGTCCCGTGTCCTTTC
CCATAACAGTGTTCGCTATGAAAGAACTTACTTTGTTTGGTTCATTTCGT
TATGGTTTCAACGACTATAAGACAGCCGTGGGTATCTTTGATACTAACTA
CCAGAACGGTAGAGAGAATGCTCCCATTGACTTTGAACAGCTTATCACGC
ACAGATACAAATTCAAAGACGCCATTGAAGCCTACGACCTAGTAAGAGCA
GGTAAAGGGGCTGTCAAGTGTTTGATTGATGGTCCAGAATAATAA (SEQ ID NO: 8)
ATGACCGCTAACCCATCTTTGGTCTTGAACAAGATTGACGACATTTCTTT
CGAAACCTACGACGCTCCAGAAATTTCTGAACCAACCGACGTTTTGGTTC
AAGTCAAGAAGACCGGTATTTGTGGTTCTGACATTCACTTCTACGCTCAC
GGTAGAATTGGTAACTTCGTCTTGACCAAGCCAATGGTCTTGGGTCACGA
ATCTGCTGGTACCGTTGTTCAAGTCGGTAAGGGTGTCACCTCTTTGAAGG
TCGGTGACAACGTCGCTATTGAACCAGGTATTCCAAGTAGATTCTCTGAC
GAATACAAGTCTGGTCACTACAACTTGTGTCCACACATGGCTTTCGCTGC
TACCCCAAACTCTAAGGAAGGTGAACCAAACCCACCAGGTACCTTGTGTA
AGTACTTCAAGTCTCCAGAAGACTTCTTGGTTAAGTTGCCAGACCACGTT
TCTTTGGAATTGGGTGCTTTGGTTGAACCATTGTCTGTTGGTGTTCACGC
TTCTAAGTTGGGTTCTGTTGCTTTCGGTGACTACGTTGCTGTTTTCGGTG
CTGGTCCAGTTGGTTTGTTGGCTGCTGCTGTTGCTAAGACCTTCGGTGCT
AAGGGTGTCATTGTCGTTGACATTTTCGACAACAAGTTGAAGATGGCTAA
GGACATTGGTGCTGCTACCCACACCTTCAACTCTAAGACCGGTGGTTCTG
AAGAATTGATTAAGGCTTTCGGTGGTAACGTCCCAAACGTCGTCTTGGAA
TGTACCGGTGCTGAACCATGTATTAAGTTGGGTGTTGACGCTATTGCTCC
AGGTGGTAGATTCGTCCAAGTTGGTAACGCTGCTGGTCCAGTTTCTTTCC
CAATTACCGTTTTCGCTATGAAGGAATTGACCTTGTTCGGTTCTTTCAGA
TACGGTTTCAACGACTACAAGACCGCTGTTGGTATTTTCGACACCAACTA
CCAAAACGGTAGAGAAAACGCTCCAATTGACTTCGAACAATTGATTACCC
ACAGATACAAGTTCAAGGACGCTATTGAAGCTTACGACTTGGTTAGAGCT
GGTAAGGGTGCTGTTAAGTGTTTGATTGACGGTCCAGAATAATAA (SEQ ID NO: 9)
ATGTTGTGTTCAGTAATTCAGAGACAGACAAGAGAGGTTTCCAACACAAT
GTCTTTAGACTCATACTATCTTGGGTTTGATCTTTCGACCCAACAACTGA
AATGTCTCGCCATTAACCAGGACCTAAAAATTGTCCATTCAGAAACAGTG
GAATTTGAAAAGGATCTTCCGCATTATCACACAAAGAAGGGTGTCTATAT
ACACGGCGACACTATCGAATGTCCCGTAGCCATGTGGTTAGAGGCTCTAG
ATCTGGTTCTCTCGAAATATCGCGAGGCTAAATTTCCATTGAACAAAGTT
ATGGCCGTCTCAGGGTCCTGCCAGCAGCACGGGTCTGTCTACTGGTCCTC
CCAAGCCGAATCTCTGTTAGAGCAATTGAATAAGAAACCGGAAAAAGATT
TATTGCACTACGTGAGCTCTGTAGCATTTGCAAGGCAAACCGCCCCCAAT
TGGCAAGACCACAGTACTGCAAAGCAATGTCAAGAGTTTGAAGAGTGCAT
AGGTGGGCCTGAAAAAATGGCTCAATTAACAGGGTCCAGAGCCCATTTTA
GATTTACTGGTCCTCAAATTCTGAAAATTGCACAATTAGAACCAGAAGCT
TACGAAAAAACAAAGACCATTTCTTTAGTGTCTAATTTTTTGACTTCTAT
CTTAGTGGGCCATCTTGTTGAATTAGAGGAGGCAGATGCCTGTGGTATGA
ACCTTTATGATATACGTGAAAGAAAATTCAGTGATGAGCTACTACATCTA
ATTGATAGTTCTTCTAAGGATAAAACTATCAGACAAAAATTAATGAGAGC
ACCCATGAAAAATTTGATAGCGGGTACCATCTGTAAATATTTTATTGAGA
AGTACGGTTTCAATACAAACTGCAAGGTCTCTCCCATGACTGGGGATAAT
TTAGCCACTATATGTTCTTTACCCCTGCGGAAGAATGACGTTCTCGTTTC
CCTAGGAACAAGTACTACAGTTCTTCTGGTCACCGATAAGTATCACCCCT
CTCCGAACTATCATCTTTTCATTCATCCAACTCTGCCAAACCATTATATG
GGTATGATTTGTTATTGTAATGGTTCTTTGGCAAGGGAGAGGATAAGAGA
CGAGTTAAACAAAGAACGGGAAAATAATTATGAGAAGACTAACGATTGGA
CTCTTTTTAATCAAGCTGTGCTAGATGACTCAGAAAGTAGTGAAAATGAA
TTAGGTGTATATTTTCCTCTGGGGGAGATCGTTCCTAGCGTAAAAGCCAT
AAACAAAAGGGTTATCTTCAATCCAAAAACGGGTATGATTGAAAGAGAGG
TGGCCAAGTTCAAAGACAAGAGGCACGATGCCAAAAATATTGTAGAATCA
CAGGCTTTAAGTTGCAGGGTAAGAATATCTCCCCTGCTTTCGGATTCAAA
CGCAAGCTCACAACAGAGACTGAACGAAGATACAATCGTGAAGTTTGATT
ACGATGAATCTCCGCTGCGGGACTACCTAAATAAAAGGCCAGAAAGGACT
TTTTTTGTAGGTGGGGCTTCTAAAAACGATGCTATTGTGAAGAAGTTTGC
TCAAGTCATTGGTGCTACAAAGGGTAATTTTAGGCTAGAAACACCAAACT
CATGTGCCCTTGGTGGTTGTTATAAGGCCATGTGGTCATTGTTATATGAC
TCTAATAAAATTGCAGTTCCTTTTGATAAATTTCTGAATGACAATTTTCC
ATGGCATGTAATGGAAAGCATATCCGATGTGGATAATGAAAATTGGGATC
GCTATAATTCCAAGATTGTCCCCTTAAGCGAACTGGAAAAGACTCTCATC TAA (SEQ ID NO:
10) MLCSVIQRQTREVSNTMSLDSYYLGFDLSTQQLKCLAINQDLKIVHSETV
EFEKDLPHYHTKKGVYIHGDTIECPVAMWLEALDLVLSKYREAKFPLNKV
MAVSGSCQQHGSVYWSSQAESLLEQLNKKPEKDLLHYVSSVAFARQTAPN
WQDHSTAKQCQEFEECIGGPEKMAQLTGSRAHFRFTGPQILKIAQLEPEA
YEKTKTISLVSNFLTSILVGHLVELEEADACGMNLYDIRERKFSDELLHL
IDSSSKDKTIRQKLMRAPMKNLIAGTICKYFIEKYGFNTNCKVSPMTGDN
LATICSLPLRKNDVLVSLGTSTTVLLVTDKYHPSPNYHLFIHPTLPNHYM
GMICYCNGSLARERIRDELNKERENNYEKTNDWTLFNQAVLDDSESSENE
LGVYFPLGEIVPSVKAINKRVIFNPKTGMIEREVAKFKDKRHDAKNIVES
QALSCRVRISPLLSDSNASSQQRLNEDTIVKFDYDESPLRDYLNKRPERT
FFVGGASKNDAIVKKFAQVIGATKGNFRLETPNSCALGGCYKAMWSLLYD
SNKIAVPFDKFLNDNFPWHVMESISDVDNENWDRYNSKIVPLSELEKTLI (SEQ ID NO: 11)
ATGCTGTGCTCCGTTATACAAAGGCAAACAAGAGAAGTATCCAACACTAT
GTCTTTAGATAGTTATTATCTAGGATTCGATTTAAGTACACAACAATTGA
AATGTCTTGCTATAAACCAGGATCTAAAGATCGTCCATTCCGAAACTGTC
GAGTTCGAGAAGGACTTACCACATTATCACACCAAGAAAGGCGTCTACAT
TCATGGTGACACCATCGAATGCCCAGTTGCTATGTGGTTAGAAGCCCTGG
ATCTTGTCCTGTCCAAATATAGGGAGGCAAAGTTCCCACTGAACAAGGTC
ATGGCTGTTTCCGGTTCTTGTCAGCAGCATGGCTCCGTCTACTGGTCATC
ACAGGCTGAATCTCTGTTAGAACAACTGAACAAGAAGCCAGAGAAGGACC
TGTTACACTACGTCTCCTCTGTTGCATTTGCCAGACAAACTGCTCCTAAT
TGGCAAGACCATTCCACTGCTAAACAATGTCAGGAGTTCGAAGAGTGTAT
TGGTGGACCAGAGAAAATGGCCCAGTTAACTGGTTCCCGTGCTCATTTCA
GGTTCACAGGCCCACAAATCCTGAAGATTGCTCAGTTAGAACCAGAGGCT
TATGAAAAGACTAAGACCATCTCTTTGGTCTCTAATTTCTTAACTTCCAT
TCTGGTTGGTCACTTGGTCGAACTGGAAGAAGCTGATGCGTGTGGTATGA
ACCTGTACGACATCCGTGAGAGGAAGTTCTCTGACGAACTGCTGCATCTT
ATCGACTCCTCCTCTAAGGACAAGACCATCAGGCAGAAACTGATGAGGGC
ACCAATGAAGAACCTGATTGCCGGTACTATTTGCAAGTACTTCATCGAAA
AGTATGGCTTCAACACCAACTGCAAAGTCTCCCCTATGACTGGCGATAAC
CTAGCCACCATTTGTAGCTTGCCCTTAAGAAAAAACGATGTTCTTGTGTC
TTTGGGTACTTCCACAACCGTCTTGTTGGTTACCGACAAATATCACCCTT
CACCAAACTACCACCTGTTCATCCACCCGACGTTGCCTAACCACTACATG
GGCATGATCTGCTACTGCAATGGCAGTTTAGCAAGGGAAAGGATAAGGGA
CGAGTTGAACAAGGAGAGGGAGAACAACTACGAGAAGACCAACGATTGGA
CCCTGTTCAACCAAGCTGTCCTGGATGATAGCGAATCCTCCGAGAATGAA
CTGGGCGTTTACTTTCCACTAGGCGAGATCGTTCCATCTGTCAAGGCCAT
CAACAAGAGAGTAATCTTCAACCCCAAGACTGGCATGATCGAAAGGGAAG
TCGCCAAGTTCAAGGACAAGAGACATGACGCCAAGAACATCGTTGAATCT
CAAGCCTTATCTTGCCGTGTTAGGATTTCTCCCCTACTAAGCGACTCCAA
TGCTTCTTCCCAGCAACGTTTGAACGAGGATACGATTGTTAAATTCGACT
ACGACGAGAGTCCATTGAGAGACTACTTGAACAAACGTCCTGAGAGGACA
TTCTTTGTTGGTGGCGCATCCAAGAACGATGCTATTGTTAAGAAGTTTGC
TCAGGTCATAGGAGCAACCAAAGGTAACTTTCGTTTAGAAACTCCAAACT
CATGCGCTTTAGGTGGTTGCTACAAGGCTATGTGGTCTTTGTTGTATGAT
AGCAATAAAATCGCTGTTCCTTTCGACAAGTTCCTAAACGATAACTTCCC
TTGGCACGTCATGGAATCCATCAGCGATGTAGACAACGAGAATTGGGATA
GATACAATTCTAAAATAGTTCCCTTGTCTGAGTTAGAGAAGACCTTGATT TAATAA (SEQ ID
NO: 12) ATGTTGTGTTCTGTCATTCAAAGACAAACCAGAGAAGTTTCTAACACCAT
GTCTTTGGACTCTTACTACTTGGGTTTCGACTTGTCTACCCAACAATTGA
AGTGTTTGGCTATTAACCAAGACTTGAAGATTGTCCACTCTGAAACCGTT
GAGTTCGAAAAGGACTTGCCACACTACCACACCAAGAAGGGTGTCTACAT
TCACGGTGACACCATTGAATGTCCAGTCGCTATGTTGGAAGCTTTGGACT
TGGTTTTGTCTAAGTACAGAGAAGCTAAGTTCCCATTGAACAAGGTCATG
GCTGTCTCTGGTTCTTGTCAACAACACGGTTCTGTCTACTCTTCTCAAGC
TGAATCTTTGTTGGAACAATTGAACAAGAAGCCAGAAAAGGACTTGTTGC
ACTACGTCTCTTCTGTCGCTTTCGCTAGACAAACCGCTCCAAACCAAGAC
CACTCTACCGCTAAGCAATGTCAAGAGTTCGAAGAATGTATTGGTGGTCC
AGAAAAGATGGCTCAATTGACCGGTAGTAGAGCACACTTCAGATTCACCG
GTCCACAAATTTTGAAGATTGCTCAATTGGAACCAGAAGCTTACGAAAAG
ACCAAGACCATTTCTTTGGTCTCTAACTTCTTGACCTCTATTTTGGTCGG
TCACTTGGTCGAATTGGAAGAAGCTGACGCTTGTGGTATGAACTTGTACG
ACATTAGAGAAAGAAAGTTCTCTGACGAATTGTTGCACTTGATTGACTCT
TCTTCTAAGGACAAGACCATTAGACAAAAGTTGATGAGGGCTCCAATGAA
GAACTTGATTGCTGGTACCATTTGTAAGTACTTCATTGAAAAGTACGGTT
TCAACACCAACTGTAAGGTCTCTCCAATGACCGGTGACAACTTGGCTACC
ATTTGTTCTTTGCCATTGAGAAAGAACGACGTTTTGGTTTCTTTGGGTAC
CTCTACCACCGTCTTGTTGGTTACCGACAAGTACCACCCATCTCCAAACT
ACCACTTGTTCATTCACCCAACCTTGCCAAACCACTACATGGGTATGATT
TGTTACTGTAACGGTTCTTTGGCTAGAGAAAGAATTAGAGACGAATTGAA
CAAGGAAAGAGAAAACAACTACGAAAAGACCAACGACACCTTGTTCAACC
AAGCTGTTTTGGACGACTCTGAATCTTCTGAAAACGAATTGGGTGTCTAC
TTCCCATTGGGTGAAATTGTTCCATCTGTCAAGGCTATTAACAAGAGAGT
CATTTTCAACCCAAAGACCGGTATGATTGAAAGAGAAGTCGCTAAGTTCA
AGGACAAGAGACACGACGCTAAGAACATTGTCGAATCTCAAGCTTTGTCT
TGTAGAGTTAGAATTTCTCCATTGTTGTCTGACTCTAACGCTTCTTCTCA
ACAAAGATTGAACGAAGACACCATTGTCAAGTTCGACTACGACGAATCTC
CATTGAGAGACTACTTGAACAAGAGACCAGAAAGAACCTTCTTCGTTGGT
GGTGCTTCTAAGAACGACGCTATTGTTAAGAAGTTCGCTCAAGTCATTGG
TGCTACCAAGGGTAACTTCAGATTGGAAACCCCAAACTCTTGTGCTTTGG
GTGGTTGTTACAAGGCTATGTCTTTGTTGTACGACTCTAACAAGATTGCT
GTTCCATTCGACAAGTTCTTGAACGACAACTTCCCACACGTCATGGAATC
TATTTCTGACGTTGACAACGAAAACGACAGATACAACTCTAAGATTGTTC
CATTGTCTGAATTGGAAAAGACCTTGATTTAATAA
Example 1
Construction of Plasmid PLS2802 with XR, XD and XK Genes
[0159] Xylose reductase from Pichis stipitis (XR.2; SEQ ID NO:3),
xylitol dehydrogenase from Pichia stipitis (XD.2; SEQ ID NO:7), and
xylulose kinase (XK.2; SEQ ID NO:11) from Saccharomyces cerevisiae
were synthesized with codon optimization for expression in yeast.
The genes were synthesized with the following 5' and 3' flanks to
introduce specific restriction enzyme sites:
TABLE-US-00008 XR - (SEQ ID NO: 13) 5' GGATCCCAAACAAA; and (SEQ ID
NO: 14) 3' CATATG to introduce 5'-BamH1 and 3'-Nde1; XD - (SEQ ID
NO: 15) 5' ACTAGTCAAACAAA; and (SEQ ID NO: 16) 3'-GACGTC to
introduce 5'SpeI and 3' AatII; and XK - (SEQ ID NO: 17) 5'
GCGGCCGCCAAACAAA; and (SEQ ID NO: 18) 3' CTCGAG to introduce 5'
NotI and 3' XhoI
[0160] The yeast vector p427TEF (Dualsystems Biotech AG) was used
for gene expression. The vector contains a kanamycin resistance
gene that allows for selection in yeast, an ampicillin resistance
gene that allows for selection in E. coli, and a 2 micron origin of
replication that allows for propagation of plasmids in high copy
numbers in yeast. For cloning the pathway genes, p427TEF was
digested with Sac' and XhoI restriction enzymes. The larger
fragment (6235 bp) was ligated with an oligomer of the following
sequence,
5'GAGCTCACGGATCCGTCATATGCTAGATCTCTGAATTCTTACTAGTTCGACGTCTACCTAG
GCAGTCGACACGCGGCCGCTTCTCGAG 3' (SEQ ID NO:19) to introduce a new
multiple cloning site (MCS) with desired restriction sites. Using
the new MCS, the TEF1 promoter of S. cerevisiae was re-introduced
in the vector using SacI/BamHI restriction sites to create PLS1567.
To clone all 3 genes into one vector, additional promoters (Adh1p,
GPDp) and terminators (Adh2t, Adh1t) were cloned into PLS1567 using
yeast recombinational cloning using methods commonly used in the
art. The promoters and terminators were each amplified using the
primer sets shown in Table 1-1.
TABLE-US-00009 TABLE 1-1 Primers Used to Amplify Yeast Promoters
and Terminators for PLS1448 Construction Cassette Primer 1 Primer 2
ADH2 GCA TAG CAA TCT AAT CTA TTG TAT GTA CCT GTC TGA ATT terminator
AGT TTT GGA TCC GTC ATA CAG AGA TCT AGC CCT GAG AAA TGG CGG ATC TCT
TAT GTC TTT CTA TAT GAG GGT (SEQ ID NO: 21) ACG (SEQ ID NO: 20)
ADH1 ACC CTC ATA TAG TTT CTC TCA TAA ATC ATA AGA AAT TCG promoter
AGG GCT AGA TCT CTG AAT CGA CGT CGA ACT AGT TGT ATA TCA GAC AGG TAC
ATA CAA TGA GAT AGT TGA TTG TAT GC (SEQ ID NO: 22) (SEQ ID NO: 23)
ADH1 GCA TAC AAT CAA CTA TCT TGA TAA ACT CGA AGT CGA CTG terminator
CAT ATA CAA CTA GTT CGA CCT AGG CAT GCC GGT AGA GGT CGT CGC GAA TTT
CTT ATG ATT GTG GT (SEQ ID NO: 25) TAT GA (SEQ ID NO: 24) GPD ACC
ACA CCT CTA CCG GCA GCG TGA CAT AAC TAA TTA CAT promoter TGC CTA
GGC AGT CGA CTT GAC TCG AGA AGC GGC CGC TTT CGA GTT TAT CA (SEQ ID
NO: 26) GTT TGT TTA TGT GTGT (SEQ ID NO: 27)
[0161] SOE-PCR was used to combine the ADH2 terminator with the
ADH1 promoter, and the ADH1 terminator with the GPD1 promoter. An
internal AatII site in the ADH2 terminator was removed by changing
its sequence from GACGTC (SEQ ID NO:28) to GATGTC (SEQ ID NO:29).
For yeast recombinational cloning, PLS1567 was digested with
BamHI/XhoI, and a 3:1 mass ratio of each insert to digested PLS1567
was used. The transformants were selected on culture plates
containing G418. The resulting plasmid (PLS1448) with the 3
promoters and terminators was confirmed by sequencing. The 2 micron
origin in PLS1448 was replaced with the CEN6/ARS4 origin of
replication to yield PLS1565 with lower copy number and greater
stability.
[0162] XR.2, XD.2 and XK.2 genes were then cloned into PLS1565
using yeast recombinational cloning. To amplify all the pieces, the
primer sets provided in Table 1-2 were used.
TABLE-US-00010 TABLE 1-2 Primers Used to Amplify Cassettes for
PLS2802 Construction Cassette Primer 1 Primer 2 PS.XR.2 CTT GCT CAT
TAG AAA GAA CTA TAA ATC GTA AAG ACA TAA AGC ATA GCA ATC TAA TCT GAG
ATC CGC CAT ATG TTA TTA AAG TTT TGG ATC CCA AAC AAC GAA GAT TGG TAT
CTT G (SEQ AAA ATG CCC TCC A (SEQ ID ID NO: 31) NO: 30) ADH2t- CAA
GAT ACC AAT CTT CGT GAG GGA TTA GCG GTC ATT TTG ADH1p TTA ATA ACA
TAT GGC GGA TTT GAC TAG TTG TAT ATG AGA TCT CTT ATG TCT TTA CGA TTT
TAG TTG ATT GTA TGC TTG GTA ATA G (SEQ ID NO: 32) (SEQ ID NO: 33)
PS.XD.2 TAC CAA GCA TAC AAT CAA TAA TAA AAA TCA TAA ATC ATA CTA TCT
CAT ATA CAA CTA GTC AGA AAT TCG CGA CGT CTT ATT AAA CAA AAT GAC CGC
TAA ATT CTG GAC CAT CAA TCA (SEQ TCC CTC (SEQ ID NO: 34) ID NO: 35)
ADH1t- TGA TTG ATG GTC CAG AAT TTT GTA TAA CGG AGC ACA GCA GPDp AAT
AAG ACG TCG CGA ATT TTT TGT TTG GCG GCC GCT TTG TCT TAT GAT TTA TGA
TTT TTA TTT GTT TAT GTG TGT TTA T (SEQ TTA (SEQ ID NO: 36) ID NO:
37) SC.XK.2 ATA AAC ACA CAT AAA CAA GGG GAG GGC GTG AAT GTA AGC ACA
AAG CGG CCG CCA AAC GTG ACA TAA CTA ATT ACA TGA AAA ATG CTG TGC TCC
GTT CTC GAG TTA TTA AAT CAA GGT ATA CAA A (SEQ ID NO: 38) CTT CTC T
(SEQ ID NO: 39)
[0163] For yeast recombinational cloning, PLS1565 was digested with
BamHI/XhoI and gel purified. Then, a 3:1 mass ratio of each
insert:digested PLS1565 was used. Transformants were selected using
G418 and confirmed by sequencing. The resulting plasmid (PLS2802)
with the 3 genes cloned is depicted in FIG. 3.
Example 2
Transformation of PLS2802 into Yeast and Fermentation
[0164] PLS2802 was used to transform S. cerevisiae
(THERMOSACC.RTM.; LYCC6825; Lallemand). Transformants were selected
on YPD agar plates with 200 ug/ml G418 at 30.degree. C. for 48 hrs.
Single colonies were used to inoculate 250 .mu.l of defined media
(60g/L glucose; 3g/L potassium phosphate, 5g/L ammonium sulphate,
0.5 g/L magnesium sulphate, 100 mM MES pH 5.5) in Axygen 96-well
plates. The cultures were grown at 30.degree. C. for 72 hrs with
85% relative humidity. Then, 40 .mu.l of the culture was used to
inoculate 360 .mu.l of 3:1 mixture of defined media (60g/L glucose;
3g/L potassium phosphate, 5g/L ammonium sulphate, 0.5 g/L magnesium
sulphate, 100 mM MES pH 5.5) to plant (wheat straw) biomass
hydrolysates containing xylose in 96-well Costar deep well plates
for propagating the cells. The cultures were grown at 30.degree. C.
for 40 hrs with 250 rpm shaking. At 40 hrs, the cells were spun
down at 22.degree. C. for 10 mins and evaluated for xylose
fermentation activity.
[0165] For fermentation, cells were re-suspended in 400 ul of plant
(wheat straw) biomass hydrolysates containing xylose and the plates
were sealed with mats. Plates were incubated at 30.degree. C., with
100 rpm shaking. At different timepoints, cells were harvested and
the residual sugars and ethanol in the supernatant were measured by
a standard HPLC based method known in the art (See e.g., DuPont et
al., Carb. Polym., 68:1-16 [2007], which is incorporated herein by
reference). In some experiments, the residual xylose in the
supernatant was measured using a spectrophotometric assay (e.g.,
Megazyme xylose assay; Cat no. K-XYLOSE) performed according to the
manufacture's protocol. Strains transformed with the empty vector
were used as a control. Strains transformed with the xylose pathway
consumed significantly higher amounts of xylose at each of the
timepoints tested compared to the vector control.
Example 3
Effect of Codon Optimization on XR, XD Activity
[0166] To evaluate the effect of codon optimization, alternate
sequences of XR and XD genes were tested. Each of the genes were
synthesized with either their native Pichia stiptis sequence (XR.1,
XD.1) or with codon optimization towards highly expressed yeast
sequences (XR.3; XD.3) (Brat et al Appl. Environ. Microbiol.,
75:2304-2311 [2009]). To evaluate the effect of the optimization,
XR and XD genes were cloned into vector PLS1448 with different
combinations (XR.1-XD.1-XK.2; XR.2-XD.1-XK.2; XR.3-XD.1-XK.2;
XR.1-XD.2-XK.2; XR.1-XD.3-XK.2). The variants were used to
transform S. cerevisiae (THERMOSACC.RTM.; LYCC6469; Lallemand).
Transformants were selected on YPD agar plates containing 200 ug/ml
G418 at 30.degree. C. for 48 hrs. Single colonies were used to
inoculate 250 .mu.l of YPD in Axygen 96-well plates. The cultures
were grown at 30.degree. C. for 72 hrs with 85% relative humidity.
Then, 40 .mu.l of the culture was used to inoculate 360 .mu.l of
YPD with 20g/L xylose in 96 well Costar deep well plates for
propagating the cells. The cultures were grown at 30.degree. C. for
40 hrs with 250 rpm shaking At 40 hrs, the cells were spun down at
22.degree. C. for 10 mins and evaluated for xylose fermentation
activity.
[0167] For fermentation, cells were re-suspended in 400 ul of YPD
with 20g/L xylose and the plates were sealed with mats. Plates were
incubated at 30.degree. C. at 100 rpm shaking. At 48 hrs, cells
were harvested and the residual xylose in the supernatant was
measured by a spectrophotometric assay as described above. The best
xylose consumption was seen with XR.1-XD.2-XK.2 (PLS6058) variant
(See, Table 3-1).
TABLE-US-00011 TABLE 3-1 Xylose Consumption by Yeast Strain
Transformed With Xylose Pathway Strain Xylose Consumed (g/L)
THERMOSACC .RTM. + PLS1448 2.5 (vector control) THERMOSACC .RTM. +
XR.2-XD.2-XK.2 12.5 THERMOSACC .RTM. + XR.1-XD.2-XK.2 15.8
THERMOSACC .RTM. + XR.3-XD.2-XK.2 2.5 THERMOSACC .RTM. +
XR.2-XD.1-XK.2 13.8 THERMOSACC .RTM. + XR.2-XD.3-XK.2 13.5
Example 4
Construction of XR and XD Variant Libraries
[0168] Pichia stipitis xylose reductase and xylitol dehydrogenase
were subjected to directed evolution algorithms to improve xylose
utilization activity using methods as known in the art (See e.g.,
WO 2010/144103). Purified PCR products encoding XR and/or XD
variants (i.e., comprising amino acid substitutions) and the vector
portion of plasmid PLS2802 were pooled and used to transform S.
cerevisiae (THERMOSACC.RTM.; LYCC6825; Lallemand) to produce a
library via homologous recombination (See e.g., Oldenburg et al.,
Nucl. Acids Res., 25(2):451-452 [1997], which is incorporated
herein by reference). The library was screened for xylose
fermentation activity as described in Example 2. The xylose
utilization was measured relative to the positive control
(XR.1-XD.2-XK.2). Strains that had activity significantly above the
positive control were retested in replicate and the variants
sequenced to identify mutations. The results are provided in the
following Tables.
TABLE-US-00012 TABLE 4-1 XR and XD Variants Exhibiting Improved
Activity Xylose utilization Activity XR XD compared Active Silent
Active Silent to WT Variant Mutations wrt Mutations wrt Mutations
wrt Mutations P. stipitis No: XR WT XR WT XD WT wrt XD WT XR/XD 1
(WT & 1.0 Positive Control) 2 R276F t811a/c812g/c816t I208R
.gtoreq.1.0 3 R276F t811a/c812g/c816t F209S/N211K .gtoreq.1.0 4
S271G c816t/a826c/a828t I208R/N211K .gtoreq.1.0 5 S271G
c816t/a826c/a828t I208R/N211S .gtoreq.1.0 6 R276F t811a/c812g/c816t
I208R/F209S/N211S .gtoreq.1.0 7 S271G c816t/a826c/a828t; I208R
.gtoreq.1.0 8 K270R t811a/c812g/c816t/ I208R/N211R .gtoreq.1.0
a826c/a828t 9 K270R t811a/c812g/c816t/ F209S/N211K .gtoreq.1.0
a826c/a828t 10 N272P/R276F t811a/c812g F209S/N211K .gtoreq.1.0 11
K270R/N272P/R276F t811a/c812g N211K .gtoreq.1.0 12
t811a/c812g/c816t/ I208R .gtoreq.1.0 a826c/a828t
TABLE-US-00013 TABLE 4-2 XR Variants with Improved Activity Xylose
Utilization XR-Active XR-Silent Activity Mutations wrt Mutations
Compared to WT Variant No: XR WT wrt XR WT P. stipitis XR/XD 1 (WT
and 1.00 Positive Control) 13 R276W .gtoreq.1.5 14 A266V
.gtoreq.1.4 15 T232V .gtoreq.1.4 16 I267V .gtoreq.1.4 17 V255I
.gtoreq.1.4 18 P2T/A266C .gtoreq.1.4 19 S233V .gtoreq.1.4 20 K152E
.gtoreq.1.3 21 L224S .gtoreq.1.3 22 A246L .gtoreq.1.3 23
a280c/a282t .gtoreq.1.3 24 K132N .gtoreq.1.3 25 R276M .gtoreq.1.3
26 K132A .gtoreq.1.3 27 N302S .gtoreq.1.3 28 R206S .gtoreq.1.3 29
A246S .gtoreq.1.3 30 A49G .gtoreq.1.3 31 S261N .gtoreq.1.2 32 K36Q
.gtoreq.1.2 33 T232A .gtoreq.1.2 34 K155R .gtoreq.1.2 35 c438t
.gtoreq.1.2 36 L224A .gtoreq.1.2 37 S261A .gtoreq.1.2 38 K152H
.gtoreq.1.2 39 D134E .gtoreq.1.2 40 c906t .gtoreq.1.2 41 I62V
.gtoreq.1.2 42 V283H .gtoreq.1.2 43 L108Y .gtoreq.1.2 44 c201t
.gtoreq.1.2 45 D11K .gtoreq.1.2 46 S233C .gtoreq.1.2 47 D134V
.gtoreq.1.2 48 S285E .gtoreq.1.2 49 A245S .gtoreq.1.2 50 S233I
.gtoreq.1.2 51 S3R .gtoreq.1.2 52 I162L .gtoreq.1.2 53 c306t
.gtoreq.1.2 54 G157R .gtoreq.1.2 55 T114S .gtoreq.1.2 56 L224V
.gtoreq.1.2 57 t511c/g513t .gtoreq.1.2 58 R33L .gtoreq.1.2 59 V24G
.gtoreq.1.2 60 S233K .gtoreq.1.2 61 t358c/a360g .gtoreq.1.2 62
t82a/c83g .gtoreq.1.2 63 K68G .gtoreq.1.2 64 N302D .gtoreq.1.2 65
D282G .gtoreq.1.1 66 R206V .gtoreq.1.1 67 a426t .gtoreq.1.1 68
G249D .gtoreq.1.1 69 t511c/g513t .gtoreq.1.1 70 V318C .gtoreq.1.1
71 L303V .gtoreq.1.1 72 P252C .gtoreq.1.1 73 E89V .gtoreq.1.1 74
a478c/g480t .gtoreq.1.1 75 t703c .gtoreq.1.1 76 a378t .gtoreq.1.1
77 S97T .gtoreq.1.1 78 K155Y c855t .gtoreq.1.1 79 N231L .gtoreq.1.1
80 N225K .gtoreq.1.1 81 N225S .gtoreq.1.1 82 S3H .gtoreq.1.1 83
A56E .gtoreq.1.1 84 c201t .gtoreq.1.1 85 K152A .gtoreq.1.1 86 P168S
.gtoreq.1.1 87 D134H .gtoreq.1.1 88 N225D .gtoreq.1.1 89 Q226D
.gtoreq.1.1 90 S233G .gtoreq.1.1 91 D282C .gtoreq.1.1 92 E89N
.gtoreq.1.1 93 t670c/g672t .gtoreq.1.1 94 K281L .gtoreq.1.1 95
N302G .gtoreq.1.1 96 A297H .gtoreq.1.1 97 Q226E .gtoreq.1.1 98
S233F .gtoreq.1.1 99 K123C .gtoreq.1.1 100 S261T .gtoreq.1.1 101
K152Q .gtoreq.1.1 102 R33V .gtoreq.1.1 103 A14V .gtoreq.1.1 104
S97R .gtoreq.1.1 105 a585g .gtoreq.1.1 106 K155D .gtoreq.1.1 107
Q226S .gtoreq.1.1 108 Q219T .gtoreq.1.1 109 I143L .gtoreq.1.1 110
D102T; .gtoreq.1.1 111 N231H; .gtoreq.1.1 112 T232S .gtoreq.1.0 113
N225E .gtoreq.1.0 114 S184A .gtoreq.1.0 115 S3W .gtoreq.1.0 116
D282R .gtoreq.1.0 117 Q219L .gtoreq.1.0 118 T232C .gtoreq.1.0 119
R228T .gtoreq.1.0 120 K281V .gtoreq.1.0 121 S285T .gtoreq.1.0 122
t688c .gtoreq.1.0 123 N231S .gtoreq.1.0 124 c849g .gtoreq.1.0 125
K155I .gtoreq.1.0 126 N231G .gtoreq.1.0 127 K242L .gtoreq.1.0 128
t354g .gtoreq.1.0 129 A297S .gtoreq.1.0 130 K68M .gtoreq.1.0 131
c408t .gtoreq.1.0 132 t766c .gtoreq.1.0 133 T240V .gtoreq.1.0 134
E46K/ .gtoreq.1.0 D47N 135 I301Y .gtoreq.1.0 136 I301C .gtoreq.1.0
137 Q226V .gtoreq.1.0 138 L303I .gtoreq.1.0 139 E279Q .gtoreq.1.0
140 P275A .gtoreq.1.0 141 D23E/K68R .gtoreq.1.0 142 F236L
.gtoreq.1.0 143 Q219H .gtoreq.1.0 144 N225Y .gtoreq.1.0 145 N7L
.gtoreq.1.0 146 A56Y .gtoreq.1.0 147 F17W .gtoreq.1.0 148 K155A
.gtoreq.1.0 149 K116Q .gtoreq.1.0 150 S261C .gtoreq.1.0
TABLE-US-00014 TABLE 4-3 Additional XR Variants with Improved
Activity Xylose Utilization Activity Variant XR-Active Mutations
wrt XR-Silent Mutations wrt (Compared to No. XR WT XR WT Positive
Control) 13 (Positive R276W 1.00 Control) 151 P2T/R206S/R276W
.gtoreq.1.4 152 P2T/A49G/R276W .gtoreq.1.4 153 P2T/A49G/R206S/R276W
.gtoreq.1.4 154 P2T/R276W .gtoreq.1.3 155 A49G/R276W .gtoreq.1.3
156 P2T/K132N/K152E/R276W .gtoreq.1.3 157 P2T/A49G/R276W a280c
.gtoreq.1.2 158 G227D/R276W t6g .gtoreq.1.2 159
A49G/K132A/R206S/R276W .gtoreq.1.2 160 P2T/P168S/R276W
a853t/g854c/c954g .gtoreq.1.2 161 A49G; K152E; R276W a280c
.gtoreq.1.2 162 R276W c54t .gtoreq.1.2 163 P2T/A49G/R276W/V318C
a853t/g854c .gtoreq.1.2 164 P2T/A49G/K132N/R276W .gtoreq.1.2 165
P2T/K132N/R276W a280c .gtoreq.1.2 166 P2T/K132N/R276W .gtoreq.1.2
167 P2T/G157R/R276W g324a/c402t/a853t/g854c/c954g .gtoreq.1.2 168
R33L/A56E/R276W t7a/c8g/g456a .gtoreq.1.1 169 A49G/R276W t753c
.gtoreq.1.1 170 S3H/R33L/A56E/R276W .gtoreq.1.1 171 A49G/R276W
a280c .gtoreq.1.1 172 R276W t82a/c83g/c201t .gtoreq.1.1 173
P2T/R276W c954g .gtoreq.1.1 174 S3H/T114S/R276W/N302S t699c/a756c
.gtoreq.1.1 175 P2T/R276W t552c .gtoreq.1.1 176
A49G/K132N/R206S/S233V/R276M/ .gtoreq.1.1 N302S 177
A49G/K132A/R276W a280c .gtoreq.1.1 178 R276W/V318C
t6g/t781a/c782g/a853t/g854c .gtoreq.1.1 179 P2T/A246L/R276W
t781a/c782g/c954g .gtoreq.1.1 180 P2T/A49G/K152E/R276W .gtoreq.1.1
181 K155R/R206S/R276W .gtoreq.1.1 182 R276W g204a .gtoreq.1.1 183
R276W a853t/g854c/c954g .gtoreq.1.1 184 R276W c201t/a280c
.gtoreq.1.1 185 T114S/R276W .gtoreq.1.1 186 R33L/R276W t7a/c8g
.gtoreq.1.1 187 R276W c954g .gtoreq.1.1 188 P2T/R276W/V318C
.gtoreq.1.1 189 A49G/K152E/R276W .gtoreq.1.1 190 D11K/V255I/R276M
g231a/c801t .gtoreq.1.1 191 K132N/R276W a280c .gtoreq.1.1 192
K52R/R276W .gtoreq.1.0 193 P2T/A49G/S233V/A246L/R276W/ .gtoreq.1.0
N302S 194 A49G/K132A/K152E/R276W a280c .gtoreq.1.0 195 S3H/R276W
t99g/c168a .gtoreq.1.0 196 P2T/V24G/A49G/R276W c954g .gtoreq.1.0
197 R276W c201t .gtoreq.1.0 198 R276W/V318C t6g .gtoreq.1.0 199
R33L/A56E/R276W t7a/c8g .gtoreq.1.0 200 K132N/R276W
g465a/t618c/t907c .gtoreq.1.0 201 K132N/K155R/R276W/V283H t907c
.gtoreq.1.0 202 K36Q/R276W .gtoreq.1.0 203 R276W a478c .gtoreq.1.0
204 R276W c855t .gtoreq.1.0 205 R276W t82a/c83g .gtoreq.1.0 206
P2T/A49G/S261N/R276W a853t/g854c/c954g .gtoreq.1.0 207
P2T/K132A/R276W a280c .gtoreq.1.0 208 K132N/R276W .gtoreq.1.0 209
R276W t7a/c8g .gtoreq.1.0 210 P168S/R276W .gtoreq.1.0 211
A56E/T114S/R276W t7a/c8g .gtoreq.1.0 212 P2T/A49G/K68G/S261N/R276W/
a853t/g854c .gtoreq.1.0 V318C 213 R276W c906t .gtoreq.1.0 214
A49G/K132N/K152E/R276W/ a280c .gtoreq.1.0 N302S 215 R276W
g108a/t907c .gtoreq.1.0 216 K132N/R276W t289a/c290g/t291c
.gtoreq.1.0 217 D11K/R276W g108a/g465a .gtoreq.1.0 218 R276W
t82a/c83g/c201t/a280c/t358c/ .gtoreq.1.0 a378t/t511c 219
P252C/R276W .gtoreq.1.0 220 R276W c21t .gtoreq.1.0 221
P2T/A49G/R276W/N302S .gtoreq.1.0 222 P2T/V24G/R276W/V318C
.gtoreq.1.0 223 D11K/K155Y/R206S/R276W g108a/t289a/c290g/t291c
.gtoreq.1.0 224 S3H/R33L/T114S/R276W c168a .gtoreq.1.0
TABLE-US-00015 TABLE 4-4 Additional XR Variants with Improved
Activity Xylose Utilization Activity (Compared Variant XR-Active
Mutations wrt XR-Silent Mutations wrt to Positive No: XR WT XR WT
Control) 272 P2T/A49G/ t99a/g156a/t747c/t751a/c752g, 1.00 (Positive
K132N/S233K/I267V/R276W t753c Control) 377
P2T/E46C/A49G/F107Y/K132N/ t99a/g156a/t747c/t751a/c752g/t753c
.gtoreq.1.2 S233K/I267V/R276W 378 P2T/C19L/E46C/A49G/F107Y/
t99a/g156a/t747c/t751a/c752g/t753c .gtoreq.1.1
K132N/S233K/I267V/R276W 379 P2T/A49G/K132N/S233K/I267V/
t99a/g156a/t552g/t747c/t751a/c752g/ .gtoreq.1.1 R276W
t753c/a756t/c816t 380 P2T/A49G/K132N/S233K/I267V/
t99a/g108a/g156a/t322c/t747c/t751a/ .gtoreq.1.0 R276W c752g/t753c
381 P2T/A49G/K132N/S233K/I267V/ t99a/g156a/t552g/t747c/t751a/c752g/
.gtoreq.1.0 R276W t753c 382 P2T/E46C/A49G/K132N/V164I/
t99a/g156a/t747c/t751a/c752g/t753c .gtoreq.1.0 S233K/I267V/R276W
383 P2T/C19L/E46C/A49G/K132N/ t99a/g156a/t747c/t751a/c752g/t753c
.gtoreq.1.0 S233K/ I267V/R276W 384 P2T/A49G/K132N/S233K/R276W
t99a/g108a/g156a/t747c/t751a/c752g/ .gtoreq.1.0 t753c/c801t 385
P2T/A49G/K132N/P168S/S233K/ t99a/g156a/t322c/t747c/t751a/c752g/
.gtoreq.1.0 I267V/R276W t753c 386 P2T/A49G/K132N/P168S/S233K/
t99a/g108a/g156a/t322c/g513a/t747c/ .gtoreq.1.0 I267V/R276W
t751a/c752g/t753c/c816t
[0169] In some additional experiments, the Pichia stipitis xylitol
dehydrogenase (XD.2) was subjected to directed evolution to improve
xylose utilization activity using PCR amplification known in the
art (See e.g., WO 2010/144103). Purified PCR products were pooled
and transformed into S. cerevisiae (THERMOSACC.RTM.; LYCC6825;
Lallemand) to produce the library via homologous recombination (See
e.g., Oldenburg et al., Nucl. Acids Res., 25(2):451-452 [1997],
which is incorporated herein by reference). The library was
screened for xylose fermentation activity as described in Example
2. Variants that exhibited activity significantly above the
positive control were retested and sequenced. Variants that
exhibited activity improved above background are provided in Table
4-5.
TABLE-US-00016 TABLE 4-5 XD Variants with Improved Activity Xylose
Utilization XD-Silent Activity XD-Active Mutations Mutations wrt
(Compared to Variant No: wrt XD WT XD WT Positive Control) 13
(Positive 1.00 Control) 225 E235K .gtoreq.1.2 226 V206A .gtoreq.1.2
227 A49Q .gtoreq.1.2 228 K352E .gtoreq.1.2 229 M215C .gtoreq.1.1
230 P256A .gtoreq.1.1 231 t24g .gtoreq.1.1 232 S81G .gtoreq.1.1 233
a768t .gtoreq.1.1 234 T19L .gtoreq.1.1 235 G202D .gtoreq.1.1 236
L189C .gtoreq.1.1 237 P5R .gtoreq.1.1 238 G231H .gtoreq.1.1 239
D210W .gtoreq.1.1 240 T230V .gtoreq.1.1 241 a780g .gtoreq.1.1 242
a732g .gtoreq.1.1 243 T286V .gtoreq.1.1 244 c630t .gtoreq.1.1 245
G231A .gtoreq.1.1 246 P5E .gtoreq.1.1 247 D218R .gtoreq.1.1 248
S228P .gtoreq.1.0 249 T19Q .gtoreq.1.0 250 V287L .gtoreq.1.0 251
V205C .gtoreq.1.0 252 V205H .gtoreq.1.0 253 V187M .gtoreq.1.0 254
H149R .gtoreq.1.0 255 F296R .gtoreq.1.0 256 A239C .gtoreq.1.0 257
L260Q .gtoreq.1.0 258 G241W/K307R .gtoreq.1.0 259 E235Q .gtoreq.1.0
260 D13G .gtoreq.1.0 261 A350D .gtoreq.1.0 262 T252S .gtoreq.1.0
263 C251T .gtoreq.1.0 264 N227T .gtoreq.1.0 265 M215A .gtoreq.1.0
266 C251G .gtoreq.1.0 267 K229R .gtoreq.1.0 268 F226V .gtoreq.1.0
269 P256Q .gtoreq.1.0 270 D327A .gtoreq.1.0
[0170] The following Table (Table 4-6) provides results for XR and
XD combinatorial variants with improved activity compared to a
positive control having the active mutations indicated in the
Table.
TABLE-US-00017 TABLE 4-6 XR & XD Combinatorial Variants with
Improved Activity Xylose Utilization XR XD Activity Silent Silent
Improvement Mutations Active Mutations (Relative to Variant Active
Mutations wrt XR Mutations wrt XD Positive No. wrt XR WT WT wrt XD
WT WT Control) 152 P2T/A49G/R276W 1.00 (Positive Control) 271
P2T/K132N/ F209S/ .gtoreq.1.2 K152E/R276W N211K 272 P2T/A49G/
t99a/g156a/ .gtoreq.1.2 K132N/S233K/ t747c/t751a/ I267V/R276W
c752g/t753c 273 P2T/A49G/K132N/I267V/ t99a/g204a/ .gtoreq.1.2 R276W
t618c/t697a/ c698g/t699c/ t747c/t751a/ c752g/t753c 274
P2T/A49G/P168S/R206S/ g456a/t895c .gtoreq.1.2 I267V/ R276W 275
P2T/A49G/K52R/K152E/ g231a/t322c .gtoreq.1.2 S233K/ I267V/R276W 276
P2T/A49G/ .gtoreq.1.2 K132N/I267V/ R276W 277 P2T/A49G/ G232L
.gtoreq.1.2 R276W 278 P2T/A49G/ t99a/g204a .gtoreq.1.2 K132N/R276W
279 P2T/A49G/ t99a/t747c/ .gtoreq.1.2 K132N/I267V/ t751a/c752g/
R276W t753c 280 P2T/A49G/ t99g/g108a/ .gtoreq.1.2 K132N/R276W
a853t/g854c/ c855g 281 P2T/A49G/S233K/I267V/ t99a/g513a/
.gtoreq.1.2 R276W t747c/t751a/ c752g/t753c 282
P2T/A49G/R206S/S233K/ t895c .gtoreq.1.2 I267V/ R276W 283 P2T/A49G/
g156a/g204a/ .gtoreq.1.2 K132N/R276W c801t 284 P2T/A49G/
t753g/a756t/ .gtoreq.1.2 K132N/R276W a853t/g854c/ c855g 285
P2T/A49G/S233K/I267V/ t895c .gtoreq.1.2 R276W 286
P2T/A49G/K152E/P168S/ t895c .gtoreq.1.1 S233V/I267V/R276W 287
P2T/A49G/S233K/I267V/ t618c/t747c/ .gtoreq.1.1 R276W t751a/c752g/
t753c 288 P2T/A49G/R276W T225X a693w .gtoreq.1.1 289 P2T/A49G/R276W
c336t .gtoreq.1.1 290 P2T/A49G/S233K/I267V/ t322c/t895c .gtoreq.1.1
R276W 291 P2T/A49G/R276W E235K .gtoreq.1.1 292
P2T/A49G/S233K/I267V/ t618c/t895c .gtoreq.1.1 R276W 293
P2T/A49G/R276W K214A .gtoreq.1.1 294 P2T/A49G/K132N/I267V/
t99a/g156a .gtoreq.1.1 R276W 295 P2T/A49G/R276W K229V .gtoreq.1.1
296 P2T/A49G/R276W .gtoreq.1.1 297 P2T/A49G/R206S/ t9c/t895c
.gtoreq.1.1 S233K/I267V/R276W 298 P2T/A49G/I267V/ t99a/t697a/
.gtoreq.1.1 R276W c698g/t699c/ t747c/t751a/ c752g/t753c 299
P2T/A49G/P168S/ t9c/t618c .gtoreq.1.1 S233K/R276W 300
P2T/A49G/S233K/ g834a/t895c .gtoreq.1.1 I267V/R276W 301
P2T/A49G/G227D/ t753g .gtoreq.1.1 P252C/G264D/R276W 302
P2T/A49G/K132N/ a853t/g854c/ .gtoreq.1.1 R276W c855g 303
P2T/S3H/A49G/P168S/ g456a/c801t/ .gtoreq.1.1 S233K/R276W t895c 304
P2T/A49G/S233K/ t99a/t618c/ .gtoreq.1.1 I267V/R276W t747c/t751a/
c752g/t753c 305 P2T/R206S/R276W A49Q .gtoreq.1.1 306
P2T/A49G/I267V/ g156a .gtoreq.1.1 R276W 307 P2T/A49G/I267V/ t895c/
.gtoreq.1.1 R276W 308 P2T/A49G/R206S/ a504g .gtoreq.1.1 S233V/R276W
309 P2T/A49G/I267V/ t99a .gtoreq.1.1 R276W 310 P2T/A49G/S233K/
c801t/t895c .gtoreq.1.1 R276W 311 P2T/A49G/R206S/ t697a/c698g/
.gtoreq.1.1 R276W c801t/t895c 312 P2T/A49G/P252C/ t753g/a853t/
.gtoreq.1.1 R276W g854c/c855g 313 P2T/K132N/K152E/ K352E
.gtoreq.1.1 R276W 314 P2T/A49G/I267V/ t747c/t751a/ .gtoreq.1.1
R276W c752g/t753c 315 P2T/A49G/I267V/ .gtoreq.1.1 R276W 316
P2T/A49G/R276W C251A .gtoreq.1.1 317 P2T/A49G/R276W G232R
.gtoreq.1.1 318 P2T/A49G/R206S/ c801t/t895c .gtoreq.1.1 S233V/R276W
319 P2T/A49G/K132N/ g204a/t753g/ .gtoreq.1.1 R276W a756t/a853t/
g854c/c855g 320 P2T/A49G/R206S/ t697a/c698g/ .gtoreq.1.1 R276W
t699c 321 P2T/A49G/R276W K229M .gtoreq.1.1 322 P2T/A49G/S233K/
t322c/t618c/ .gtoreq.1.1 I267V/R276W t895c 323 P2T/K132N/K152E/
A49Q/E235K .gtoreq.1.1 R276W 324 P2T/A49G/R276W a780t .gtoreq.1.1
325 P2T/A49G/I267V/ t99a/g156a/ .gtoreq.1.1 R276W g204a 326
P2T/A49G/R276W D327X .gtoreq.1.1 327 P2T/K132N/K152E/ A49Q
.gtoreq.1.1 R276W 328 P2T/A49G/P168S/ .gtoreq.1.1 R276W 329
P2T/A49G/R276W V206A .gtoreq.1.0 330 P2T/A49G/R276W V206A
.gtoreq.1.0 331 P2T/A49G/R276W K337R .gtoreq.1.0 332
P2T/A49G/K152E/ a504g .gtoreq.1.0 I267V/R276W 333 P2T/A49G/K132N/
a478c/g480t .gtoreq.1.0 R276W 334 P2T/K36Q/A49G/ t99g/t753g/
.gtoreq.1.0 G227D/R276W a756t/a853t/ g854c/c855g 335 P2T/A49G/R276W
V287L .gtoreq.1.0 336 P2T/A49G/D86N/ g204a/a478c/ .gtoreq.1.0
K132N/R276W g480t/t753g/ a756t/a853t/ g854c/c855g 337
P2T/A49G/R206S/ A49Q .gtoreq.1.0 R276W 338 P2T/A49G/R276W G241E
.gtoreq.1.0 339 P2T/A49G/G227D/ t753g/a756t/ .gtoreq.1.0 R276W
a853t/g854c/ c855g 340 P2T/S3X/A49G/S233X/ t84w .gtoreq.1.0
I267X/R276W/L299X 341 P2T/A49G/I267V/ a504g/c816t/ .gtoreq.1.0
R276W t895c 342 P2T/A49G/R276W H112Q .gtoreq.1.0 343 P2T/A49G/R276W
K238G .gtoreq.1.0 344 P2T/A49G/P168S/ t895c .gtoreq.1.0 R276W 345
P2T/A49G/G227D/ .gtoreq.1.0 I267V/R276W 346 P2T/A49G/K132N/
t753g/a853t/ .gtoreq.1.0 G227D/P252C/R276W g854c/c855g 347
P2T/A49G/R276W K352Q .gtoreq.1.0 348 P2T/A49G/I267V/ g204a/t697a/
.gtoreq.1.0 R276W c698g/t699c/ t747c/t751a/ c752g/t753c 349 R276F
t811a/c812g/ I208R/N211K .gtoreq.1.0 c816t 350 P2T/A49G/I267V/
g231a/t322c/ .gtoreq.1.0 R276W t895c 351 P2T/A49G/R276W K259S
.gtoreq.1.0 352 P2T/A49G/K132N/ a478c/g480t/ .gtoreq.1.0
G227D/R276W t753g/a756t/ a853t/g854c/ c855g 353 P2T/A49G/I267V/
g156a/t747c/ .gtoreq.1.0 R276W t751a/c752g/ t753c 354
P2T/A49G/S233V/ g156a/g231a/ .gtoreq.1.0 I267V/R276W t322c/
g456a/t895c 355 P2T/A49G/G227D/ t753g/a853t/ .gtoreq.1.0
P252C/R276W g854c/c855g 356 P2T/A49G/R276W K352G .gtoreq.1.0 357
P2T/A49G/R276W a853t/g854c/ .gtoreq.1.0 c855g 358 P2T/K132N/K152E/
E235K .gtoreq.1.0 R276W 359 P2T/A49G/K52R/ t9c/t84a/ .gtoreq.1.0
R206S/I267V/R276W g231a/t697a/ c698g/t895c 360 P2T/A49G/K132N/
.gtoreq.1.0 R276W 361 P2T/A49G/R276W K352E .gtoreq.1.0 362
P2T/A49G/K52R/ t99a .gtoreq.1.0 R276W 363 P2T/A49G/R276W H112A
.gtoreq.1.0 364 P2T/A49G/S233V/ t895c .gtoreq.1.0 R276W 365
P2T/R206S/R276W E235K .gtoreq.1.0 366 P2T/A49G/R276W t99a/g156a/
.gtoreq.1.0 g204a 367 P2T/A49G/R276W S26A .gtoreq.1.0 368
P2T/A49G/R276W D210A .gtoreq.1.0 369 P2T/A49G/R206S/ K352E
.gtoreq.1.0 R276W 370 P2T/A49G/R276W t618c/t697a/ .gtoreq.1.0
c698g/c801t/ t895c 371 P2T/A49G/S233V/ a504g/t895c .gtoreq.1.0
I267V/R276W 372 P2T/W20X/A49G/ c111n/c822t/ .gtoreq.1.0 D63X/R276W
a825c/a853t/ g854c/c855g 373 P2T/A49G/R276W t84a .gtoreq.1.0 374
P2T/A49G/R276W g204a/c801t .gtoreq.1.0 375 P2T/A49G/R276W
t552g/c801t/ .gtoreq.1.0 t895c 376 P2T/A49G/R276W c801t
.gtoreq.1.0
[0171] The sequences of some variants included in the above
table(s) are provided below. Variant 3 has active substitutions in
both the XR and XD (R276F and F209S/N211K, respectively), as well
as XR silent mutations (t811a/c812g/c816t), as shown in SEQ ID
NOS:50 and 51 (XR DNA and amino acid sequences, respectively), and
SEQ ID NOS:52 and 53 (XD DNA and amino acid sequences,
respectively).
TABLE-US-00018 (SEQ ID NO: 50)
ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTT
CGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTACCGTG
CTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGCCAAC
GAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGT
CAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACC
ACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAA
GTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTT
CGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAAGGGTG
ACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTT
GAAAAGTTGGTCAAGGCCGGTAAGATCAGGTCTATCGGTGTTTCTAACTT
CCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCAT
CTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATC
GAGTTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGG
TCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTC
CATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGTAAG
TCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCAT
CATTCCAAAGAGCAATACTGTCCCATTCTTGTTGGAAAACAAGGATGTCA
ACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGAC
ATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTT CGTCTAA (SEQ ID
NO: 51) MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDYAN
EKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQ
VDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKAL
EKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLI
EFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGK
SPAQVLLRWSSQRGIAIIPKSNTVPFLLENKDVNSFDLDEQDFADIAKLD
INLRFNDPWDWDKIPIFV (SEQ ID NO: 52)
ATGACCGCTAATCCCTCTCTTGTTTTGAATAAGATTGACGACATTTCTTT
TGAAACTTACGATGCTCCCGAAATTAGCGAACCCACAGACGTTTTAGTTC
AAGTTAAAAAAACTGGTATCTGCGGTTCTGACATCCACTTCTACGCTCAT
GGAAGGATCGGCAACTTCGTCTTAACAAAGCCAATGGTTCTGGGTCATGA
AAGCGCGGGTACTGTTGTTCAAGTCGGTAAAGGTGTTACTTCACTGAAGG
TTGGTGATAACGTCGCAATCGAGCCCGGTATTCCATCTAGGTTCAGTGAT
GAGTACAAATCTGGTCACTACAACCTGTGTCCACACATGGCATTTGCTGC
TACTCCCAATTCTAAAGAGGGTGAACCAAACCCACCAGGAACTCTATGTA
AGTACTTCAAATCTCCAGAAGACTTCCTGGTTAAGTTACCCGATCATGTT
TCTTTGGAGTTGGGTGCTTTGGTCGAGCCACTATCTGTTGGGGTCCATGC
TAGTAAATTAGGCTCCGTTGCATTTGGCGATTACGTTGCTGTTTTTGGTG
CTGGTCCAGTAGGATTACTGGCTGCCGCTGTCGCTAAGACATTTGGTGCC
AAGGGTGTGATTGTCGTTGATATATCTGACAAGAAGCTGAAGATGGCCAA
AGACATAGGTGCCGCTACACATACCTTCAACTCCAAGACGGGAGGTAGTG
AAGAATTGATCAAAGCCTTCGGTGGTAATGTACCAAATGTTGTCTTGGAA
TGTACTGGGGCTGAACCATGTATTAAGCTAGGTGTTGATGCCATCGCACC
AGGTGGTAGATTCGTGCAAGTTGGTAATGCTGCTGGTCCCGTGTCCTTTC
CCATAACAGTGTTCGCTATGAAAGAACTTACTTTGTTTGGTTCATTTCGT
TATGGTTTCAACGACTATAAGACAGCCGTGGGTATCTTTGATACTAACTA
CCAGAACGGTAGAGAGAATGCTCCCATTGACTTTGAACAGCTTATCACGC
ACAGATACAAATTCAAAGACGCCATTGAAGCCTACGACCTAGTAAGAGCA
GGTAAAGGGGCTGTCAAGTGTTTGATTGATGGTCCAGAATAA (SEQ ID NO: 53)
MTANPSLVLNKIDDISFETYDAPEISEPTDVLVQVKKTGICGSDIHFYAH
GRIGNFVLTKPMVLGHESAGTVVQVGKGVTSLKVGDNVAIEPGIPSRFSD
EYKSGHYNLCPHMAFAATPNSKEGEPNPPGTLCKYFKSPEDFLVKLPDHV
SLELGALVEPLSVGVHASKLGSVAFGDYVAVFGAGPVGLLAAAVAKTFGA
KGVIVVDISDKKLKMAKDIGAATHTFNSKTGGSEELIKAFGGNVPNVVLE
CTGAEPCIKLGVDAIAPGGRFVQVGNAAGPVSFPITVFAMKELTLFGSFR
YGFNDYKTAVGIFDTNYQNGRENAPIDFEQLITHRYKFKDAIEAYDLVRA
GKGAVKCLIDGPE
[0172] The DNA and amino acid sequences of variant 152
(P2T/A49G/R276W in XR) are provided below (SEQ ID NOS:54 and
55).
TABLE-US-00019 (SEQ ID NO: 54)
ATGACCTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTT
CGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTACCGTG
CTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGGCAAC
GAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGT
CAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACC
ACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAA
GTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTT
CGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAAGGGTG
ACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTT
GAAAAGTTGGTCAAGGCCGGTAAGATCAGGTCTATCGGTGTTTCTAACTT
CCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCAT
CTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATC
GAGTTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGG
TCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTC
CATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGTAAG
TCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCAT
CATTCCAAAGTCCAACACTGTCCCATGGTTGTTGGAAAACAAGGATGTCA
ACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGAC
ATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTT CGTCTAA (SEQ ID
NO: 55) MTSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDYGN
EKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQ
VDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKAL
EKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLI
EFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGK
SPAQVLLRWSSQRGIAIIPKSNTVPWLLENKDVNSFDLDEQDFADIAKLD
INLRFNDPWDWDKIPIFV
[0173] The DNA and amino acid sequences of variant 272
(P2T/A49G/K132N/S233K/1267V/R276W in XR) are provided below (SEQ ID
NOS:56 and 57).
TABLE-US-00020 (SEQ ID NO: 56)
ATGACCTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTT
CGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTACCGAG
CTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGGCAAC
GAAAAATTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGT
CAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACC
ACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAA
GTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTT
CGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAACGGTG
ACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTT
GAAAAGTTGGTCAAGGCCGGTAAGATCAGGTCTATCGGTGTTTCTAACTT
CCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCAT
CTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATC
GAGTTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGG
TCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTAAGC
CATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGCAAG
AGCCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCGT
TATTCCAAAGTCCAACACTGTCCCATGGTTGTTGGAAAACAAGGATGTCA
ACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGAC
ATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTT CGTCTAA (SEQ ID
NO: 57) MTSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDYGN
EKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQ
VDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGNGDNFDYEDVPILETWKAL
EKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLI
EFAQSRGIAVTAYSSFGPQSFVELNQGRALNTKPLFENETIKAIAAKHGK
SPAQVLLRWSSQRGIAVIPKSNTVPWLLENKDVNSFDLDEQDFADIAKLD
INLRFNDPWDWDKIPIFV
[0174] The DNA and amino acid sequences of variant 377
(P2T/E46C/A49G/F107Y/K132N/S233K/1267V/R276W in XR) are provided
below (SEQ ID NOS:58 and 59).
TABLE-US-00021 (SEQ ID NO: 58)
ATGACCTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTT
CGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTACCGAG
CTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGGCAAC
GAAAAATTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGT
CAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACC
ACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAA
GTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTT
CGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAACGGTG
ACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTT
GAAAAGTTGGTCAAGGCCGGTAAGATCAGGTCTATCGGTGTTTCTAACTT
CCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCAT
CTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATC
GAGTTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGG
TCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTAAGC
CATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGCAAG
AGCCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCGT
TATTCCAAAGTCCAACACTGTCCCATGGTTGTTGGAAAACAAGGATGTCA
ACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGAC
ATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTT CGTCTAA (SEQ ID
NO: 59) MTSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDYGN
EKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQ
VDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGNGDNFDYEDVPILETWKAL
EKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLI
EFAQSRGIAVTAYSSFGPQSFVELNQGRALNTKPLFENETIKAIAAKHGK
SPAQVLLRWSSQRGIAVIPKSNTVPWLLENKDVNSFDLDEQDFADIAKLD
INLRFNDPWDWDKIPIFV
[0175] While particular embodiments of the present invention have
been illustrated and described, it will be apparent to those
skilled in the art that various other changes and modifications can
be made without departing from the spirit and scope of the present
invention. Therefore, it is intended that the present invention
encompass all such changes and modifications with the scope of the
present invention.
[0176] The present invention has been described broadly and
generically herein. Each of the narrower species and subgeneric
groupings falling within the generic disclosure also form part(s)
of the invention. The invention described herein suitably may be
practiced in the absence of any element or elements, limitation or
limitations which is/are not specifically disclosed herein. The
terms and expressions which have been employed are used as terms of
description and not of limitation. There is no intention that in
the use of such terms and expressions, of excluding any equivalents
of the features described and/or shown or portions thereof, but it
is recognized that various modifications are possible within the
scope of the claimed invention. Thus, it should be understood that
although the present invention has been specifically disclosed by
some preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be utilized by those
skilled in the art, and that such modifications and variations are
considered to be within the scope of the present invention.
Sequence CWU 1
1
11960DNAPichia stipitis 1atgccttcta ttaagttgaa ctctggttac
gacatgccag ccgtcggttt cggctgttgg 60aaagtcgacg tcgacacctg ttctgaacag
atctaccgtg ctatcaagac cggttacaga 120ttgttcgacg gtgccgaaga
ttacgccaac gaaaagttag ttggtgccgg tgtcaagaag 180gccattgacg
aaggtatcgt caagcgtgaa gacttgttcc ttacctccaa gttgtggaac
240aactaccacc acccagacaa cgtcgaaaag gccttgaaca gaaccctttc
tgacttgcaa 300gttgactacg ttgacttgtt cttgatccac ttcccagtca
ccttcaagtt cgttccatta 360gaagaaaagt acccaccagg attctactgt
ggtaagggtg acaacttcga ctacgaagat 420gttccaattt tagagacctg
gaaggctctt gaaaagttgg tcaaggccgg taagatcaga 480tctatcggtg
tttctaactt cccaggtgct ttgctcttgg acttgttgag aggtgctacc
540atcaagccat ctgtcttgca agttgaacac cacccatact tgcaacaacc
aagattgatc 600gaattcgctc aatcccgtgg tattgctgtc accgcttact
cttcgttcgg tcctcaatct 660ttcgttgaat tgaaccaagg tagagctttg
aacacttctc cattgttcga gaacgaaact 720atcaaggcta tcgctgctaa
gcacggtaag tctccagctc aagtcttgtt gagatggtct 780tcccaaagag
gcattgccat cattccaaag tccaacactg tcccaagatt gttggaaaac
840aaggacgtca acagcttcga cttggacgaa caagatttcg ctgacattgc
caagttggac 900atcaacttga gattcaacga cccatgggac tgggacaaga
ttcctatctt cgtctaataa 960
* * * * *