U.S. patent application number 12/172852 was filed with the patent office on 2008-11-27 for methods for degrading or converting plant cell wall polysaccharides.
This patent application is currently assigned to Novozymes, Inc.. Invention is credited to Randy Berka, Joel Cherry.
Application Number | 20080293109 12/172852 |
Document ID | / |
Family ID | 35150556 |
Filed Date | 2008-11-27 |
United States Patent
Application |
20080293109 |
Kind Code |
A1 |
Berka; Randy ; et
al. |
November 27, 2008 |
Methods for degrading or converting plant cell wall
polysaccharides
Abstract
The present invention relates to methods for converting plant
cell wall polysaccharides into one or more products, comprising:
treating the plant cell wall polysaccharides with an effective
amount of a spent whole fermentation broth of a recombinant
microorganism, wherein the recombinant microorganism expresses one
or more heterologous genes encoding enzymes which degrade or
convert the plant cell wall polysaccharides into the one or more
products. The present invention also relates to methods for
producing an organic substance, comprising: (a) saccharifying plant
cell wall polysaccharides with an effective amount of a spent whole
fermentation broth of a recombinant microorganism, wherein the
recombinant microorganism expresses one or more heterologous genes
encoding enzymes which degrade or convert the plant cell wall
polysaccharides into saccharified material; (b) fermenting the
saccharified material of step (a) with one or more fermenting
microoganisms; and (c) recovering the organic substance from the
fermentation.
Inventors: |
Berka; Randy; (Davis,
CA) ; Cherry; Joel; (Davis, CA) |
Correspondence
Address: |
NOVOZYMES, INC.
1445 DREW AVE
DAVIS
CA
95616
US
|
Assignee: |
Novozymes, Inc.
Davis
CA
|
Family ID: |
35150556 |
Appl. No.: |
12/172852 |
Filed: |
July 14, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11078921 |
Mar 10, 2005 |
7413882 |
|
|
12172852 |
|
|
|
|
60556779 |
Mar 25, 2004 |
|
|
|
Current U.S.
Class: |
435/107 ;
435/106; 435/108; 435/109; 435/110; 435/111; 435/112; 435/113;
435/114; 435/115; 435/116; 435/132; 435/157; 435/160; 435/165;
435/41 |
Current CPC
Class: |
C12P 19/02 20130101;
C12P 5/023 20130101; C12P 19/14 20130101; C12P 7/20 20130101; C12P
7/28 20130101; C12P 7/40 20130101; C12P 7/16 20130101; C12P 7/02
20130101; C12P 7/26 20130101; C12P 7/06 20130101; Y02E 50/30
20130101; C12P 13/04 20130101; C12P 7/00 20130101; C12P 7/10
20130101; C12P 17/04 20130101; C12P 7/04 20130101; C12P 7/18
20130101; Y02E 50/10 20130101 |
Class at
Publication: |
435/107 ; 435/41;
435/157; 435/132; 435/106; 435/160; 435/165; 435/108; 435/109;
435/110; 435/111; 435/112; 435/113; 435/114; 435/115; 435/116 |
International
Class: |
C12P 13/24 20060101
C12P013/24; C12P 1/00 20060101 C12P001/00; C12P 7/04 20060101
C12P007/04; C12P 7/00 20060101 C12P007/00; C12P 13/04 20060101
C12P013/04; C12P 7/16 20060101 C12P007/16; C12P 7/10 20060101
C12P007/10; C12P 13/06 20060101 C12P013/06; C12P 13/08 20060101
C12P013/08; C12P 13/10 20060101 C12P013/10; C12P 13/12 20060101
C12P013/12; C12P 13/14 20060101 C12P013/14; C12P 13/16 20060101
C12P013/16; C12P 13/18 20060101 C12P013/18; C12P 13/20 20060101
C12P013/20 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with Government support under NREL
Subcontract No. ZCO-30017-02, Prime Contract DE-AC36-98GO10337
awarded by the Department of Energy. The government has certain
rights in this invention.
Claims
1. A method for producing one or more organic substances,
comprising: (a) saccharifying plant cell wall polysaccharides with
an effective amount of a spent whole fermentation broth of a
recombinant microorganism, wherein the recombinant microorganism
expresses one or more heterologous genes encoding enzymes which
degrade or convert the plant cell wall polysaccharides into
saccharified material; (b) fermenting the saccharified material of
step (a) with one or more fermenting microoganisms; and (c)
recovering the one or more organic substances from the
fermentation.
2. The method of claim 1, wherein the plant cell wall
polysaccharides are obtained from a source selected from the group
consisting of herbaceous material, agricultural residue, forestry
residue, municipal solid waste, waste paper, and pulp and paper
mill residue.
3. The method of claim 1, wherein the plant cell wall
polysaccharide source is corn stover.
4. The method of claim 1, wherein the one or more heterologous
genes encode enzymes selected from the group consisting of a
cellulase, endoglucanase, cellobiohydrolase, and
beta-glucosidase.
5. The method of claim 1, wherein the heterologous gene encodes a
cellulase.
6. The method of claim 1, wherein the heterologous gene encodes an
endoglucanase.
7. The method of claim 1, wherein the heterologous gene encodes a
cellobiohydrolase.
8. The method of claim 1, wherein the heterologous gene encodes a
beta-glucosidase.
9. The method of claim 1, wherein the one or more heterologous
genes encode enzymes further selected from the group consisting of
a glucohydrolase, xyloglucanase, xylanase, xylosidase,
alpha-arabinofuranosidase, alpha-glucuronidase, acetyl xylan
esterase, mannanase, mannosidase, alpha-galactosidase, mannan
acetyl esterase, galactanase, arabinanase, pectate lyase, pectin
lyase, pectate lyase, polygalacturonase, pectin acetyl esterase,
pectin methyl esterase, alpha-arabinofuranosidase,
beta-galactosidase, galactanase, arabinanase,
alpha-arabinofuranosidase, rhamnogalacturonase, rhamnogalacturonan
lyase, rhamnogalacturonan acetyl esterase, xylogalacturonosidase,
xylogalacturonase, rhamnogalacturonan lyase, lignin peroxidase,
manganese-dependent peroxidase, hybrid peroxidase, and laccase.
10. The method of claim 1, wherein the one or more heterologous
genes encode enzymes even further selected from the group
consisting of an esterase, lipase, oxidase, phospholipase, phytase,
protease, and peroxidase.
11. The method of claim 1, wherein the spent whole fermentation
broth of the recombinant microorganism is supplemented by the
addition of one or more enzymes selected from the group consisting
of a cellulase, endoglucanase, cellobiohydrolase, and
beta-glucosidase.
12. The method of claim 1, wherein the spent whole fermentation
broth of the recombinant microorganism is supplemented by the
further addition of one or more enzymes selected from the group
consisting of a xyloglucanase, xylanase, xylosidase,
alpha-arabinofuranosidase, alpha-glucuronidase, acetyl xylan
esterase, mannanase, mannosidase, alpha-galactosidase, mannan
acetyl esterase, galactanase, arabinanase, pectate lyase, pectin
lyase, pectate lyase, polygalacturonase, pectin acetyl esterase,
pectin methyl esterase, alpha-arabinofuranosidase,
beta-galactosidase, galactanase, arabinanase,
alpha-arabinofuranosidase, rhamnogalacturonase, rhamnogalacturonan
lyase, rhamnogalacturonan, acetyl esterase, xylogalacturonosidase,
xylogalacturonase, rhamnogalacturonan lyase, lignin peroxidase,
manganese-dependent peroxidase, hybrid peroxidase, and laccase.
13. The method of claim 1, wherein the spent whole fermentation
broth of the recombinant microorganism is supplemented by the even
further addition of one or more enzymes selected from the group
consisting of an esterase, lipase, oxidase, phospholipase, phytase,
protease, and peroxidase.
14. The method of claim 1, wherein steps (a) and (b) are performed
simultaneously in a simultaneous saccharification and
fermentation.
15. The method of claim 1, wherein the one or more organic
substances are selected from the group consisting of an alcohol,
organic acid, ketone, aldehyde, amino acid, gas, and a combination
thereof.
16. The method of claim 15, wherein the alcohol is arabinitol,
butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, or
xylitol.
17. The method of claim 15, wherein the organic acid is acetic
acid, adipic acid, ascorbic acid, citric acid,
2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric
acid, gluconic acid, glucuronic acid, glutaric acid,
3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,
malonic acid, oxalic acid, propionic acid, succinic acid, or
xylonic acid.
18. The method of claim 15, wherein the ketone is acetone.
19. The method of claim 15, wherein the aldehyde is furfural.
20. The method of claim 15, wherein the amino acid is aspartic
acid, alanine, arginine, asparagine, glutamine, glutamic acid,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or
valine.
21. The method of claim 15, wherein the gas is methane, hydrogen,
carbon dioxide, and carbon monoxide.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 11/078,921, filed Mar. 10, 2005, which claims the benefit of
U.S. Provisional Application No. 60/556,779, filed Mar. 25, 2004,
which applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to methods for degrading or
converting plant cell wall polysaccharides and to products obtained
by such methods.
[0005] 2. Description of the Related Art
[0006] Plant cell walls are composed of a mixture of
polysaccharides interlocked in a complex structure (Carpita et al.,
2001, Plant Physiology 127: 551-565). The mixture of
polysaccharides include cellulose, xyloglycan (hemicellulose), and
pectic polymers, which are primarily composed of hexoses, e.g.,
glucose, galactose, and mannose; pentoses, e.g., xylose and
arabinose; uronic acids, e.g., galacturonic acid and glucuronic
acid; and deoxyhexoses, e.g., rhamnose and fucose.
[0007] Plant cell wall polysaccharides can be enzymatically
degraded to glucose, xylose, mannose, galactose, and arabinose,
which can then be converted to other organic substances, for
example, glucose is easily fermented by yeast into ethanol. Wood,
agricultural residues, herbaceous crops, and municipal solid wastes
can be used as sources of plant cell wall polysaccharides.
[0008] Cellulose is a primary component of plant cell walls. Many
microorganisms produce enzymes that degrade cellulose. These
enzymes include, for example, endoglucanases, cellobiohydrolases,
and beta-glucosidases. Endoglucanases digest the cellulose polymer
at random locations, opening it to attack by cellobiohydrolases.
Cellobiohydrolases sequentially release molecules of cellobiose
from the ends of the cellulose polymer. Cellobiose is a
water-soluble beta-1,4-linked dimer of glucose. Beta-glucosidases
hydrolyze cellobiose to glucose.
[0009] Natural microorganisms that degrade cellulose and other cell
wall polysaccharides may not be ideal for large-scale conversion of
cellulosic materials because (a) the full complement of enzymes may
be lacking, (b) one or more enzyme components perform poorly, are
labile, or their kinetic behavior fails to meet the specification
of the intended use, (c) the conversion and/or degradation could be
improved by expression of a heterologous enzyme gene that enhances
the conversion/degradation, or (d) the full complement of enzymes
may be in insufficient amounts to be economically viable. It would
be an advantage to the art to improve the degradation and
conversion of plant cell wall polysaccharides by using whole
fermentation broth from recombinant microorganisms to circumvent
expensive cell removal and enzyme formulation steps.
[0010] It is an object of the present invention to provide new
methods for degrading or converting plant cell wall polysaccharides
into various products using spent whole fermentation broths from
recombinant microorganisms.
SUMMARY OF THE INVENTION
[0011] The present invention relates to methods for degrading or
converting plant cell wall polysaccharides into one or more
products, comprising: treating the plant cell wall polysaccharides
with an effective amount of a spent whole fermentation broth of a
recombinant microorganism, wherein the recombinant microorganism
expresses one or more heterologous genes encoding enzymes which
degrade or convert the plant cell wall polysaccharides into the one
or more products.
[0012] The present invention also relates to methods for producing
one or more organic substances, comprising:
[0013] (a) saccharifying plant cell wall polysaccharides with an
effective amount of a spent whole fermentation broth of a
recombinant microorganism, wherein the recombinant microorganism
expresses one or more heterologous genes encoding enzymes which
degrade or convert the plant cell wall polysaccharides into
saccharified material;
[0014] (b) fermenting the saccharified material of step (a) with
one or more fermenting microoganisms; and
[0015] (c) recovering the one or more organic substances from the
fermentation.
[0016] The present invention further relates to products or organic
substances obtained by such methods. In a preferred aspect, the
organic substance is alcohol.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows a restriction map of pAILo01.
[0018] FIG. 2 shows a restriction map of pMJ04.
[0019] FIG. 3 shows a restriction map of pCaHj527.
[0020] FIG. 4 shows a restriction map of pMT2188.
[0021] FIG. 5 shows a restriction map of pCaHj568.
[0022] FIG. 6 shows a restriction map of pMJ05.
[0023] FIG. 7 shows a restriction map of pSMai130.
[0024] FIG. 8 shows the DNA sequence (SEQ ID NO: 32) and deduced
amino acid sequence (SEQ ID NO: 33) of the secretion signal
sequence of an Aspergillus oryzae beta-glucosidase.
[0025] FIG. 9 shows the DNA sequence (SEQ ID NO: 36) and deduced
amino acid sequence (SEQ ID NO: 37) of the secretion signal
sequence of a Humicola insolens endoglucanase V.
[0026] FIG. 10 shows a restriction map of pSMai135.
[0027] FIG. 11 shows the PCS hydrolysis profiles of whole
fermentation broth (WB) (panel A) and cell-free broth (CB) (panel
B) at enzyme doses ranging from 2.5 to 20 mg/g of PCS (noted in the
lower right of each panel).
[0028] FIG. 12 shows the PCS hydrolysis curves for WB and CB
samples derived from freshly harvested Trichoderma reesei RutC30
fermentation material. Each profile is plotted as % RS yield (% of
theoretical maximum reducing sugar based on the glucan composition
of 10 mg of PCS per ml) as a function of hydrolysis time (1-120
hours). Enzyme doses are noted in the upper right of each
panel.
[0029] FIG. 13 shows a comparison of total reducing sugar (RS) and
glucose liberated during PCS hydrolysis reactions using WB and CB
samples from Trichoderma reesei RutC30. Enzyme doses are noted at
the top of each panel. The sample numbers noted on the X-axis
correspond to hydrolysis times spanning 1 to 120 hours.
[0030] FIG. 14 shows the PCS hydrolysis curves for WB and CB
samples derived from freshly harvested Trichoderma reesei SMA135-04
fermentation broth. Trichoderma reesei strain SMA135-04 expresses
recombinant Aspergillus oryzae beta-glucosidase. Each profile is
plotted as % RS yield (% of theoretical maximum reducing sugar
based on the glucan composition of 10 mg of PCS per ml) as a
function of hydrolysis time (1-120 hours). Enzyme doses are noted
in the upper right of each panel.
[0031] FIG. 15 shows a comparison of total reducing sugar (RS) and
glucose liberated during PCS hydrolysis reactions using WB and CB
samples from Trichoderma reesei SMA135-04 that harbors an
expression vector directing synthesis and secretion of Aspergillus
oryzae beta-glucosidase. Enzyme doses are noted at the top of each
panel. The sample numbers noted on the X-axis correspond to
hydrolysis times spanning 1 to 120 hours.
[0032] FIG. 16 shows the PCS hydrolysis curves for WB and CB
samples derived from Trichoderma reesei RutC30 fermentation broth
stored two weeks at 4.degree. C. Each profile is plotted as % RS
yield (% of theoretical maximum reducing sugar based on the glucan
composition of 10 mg of PCS per ml) as a function of hydrolysis
time (1-120 hours). Enzyme doses are noted in the upper right of
each panel.
[0033] FIG. 17 shows the PCS hydrolysis curves for WB and CB
samples derived from Trichoderma reesei SMA135-04 fermentation
broth stored two weeks at 4.degree. C. Each profile is plotted as %
RS yield (% of theoretical maximum based on the glucan composition
of 10 mg/ml PCS) as a function of hydrolysis time (1-120 hours).
Enzyme doses are noted in the upper right of each panel.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention relates to methods for degrading or
converting plant cell wall polysaccharides into one or more
products, comprising: treating the plant cell wall polysaccharides
with an effective amount of a spent whole fermentation broth of a
recombinant microorganism, wherein the recombinant microorganism
expresses one or more heterologous genes encoding enzymes which
degrade or convert the plant cell wall polysaccharides into the one
or more products. The present invention also relates to methods for
producing one or more organic substances, comprising: (a)
saccharifying plant cell wall polysaccharides with an effective
amount of a spent whole fermentation broth of a recombinant
microorganism, wherein the recombinant microorganism expresses one
or more heterologous genes encoding enzymes which degrade or
convert the plant cell wall polysaccharides into one or more
products; (b) fermenting the saccharified material of step (a) with
one or more fermenting microoganisms; and (c) recovering the one or
more organic substances from the fermentation.
Plant Cell Wall Polysaccharides
[0035] In the methods of the present invention, the source of the
plant cell wall polysaccharides can be any plant biomass containing
cell wall polysaccharides. Such sources include, but are not
limited to, herbaceous material, agricultural residues, forestry
residues, municipal solid waste, waste paper, and pulp and paper
mill residues.
[0036] In a preferred aspect, the plant cell wall biomass is corn
stover. In another preferred aspect, the plant cell wall biomass is
corn fiber. In another preferred aspect, the plant cell wall
biomass is rice straw. In another preferred aspect, the plant cell
wall biomass is paper and pulp processing waste. In another
preferred aspect, the plant cell wall biomass is woody or
herbaceous plants. In another preferred aspect, the plant cell wall
biomass is fruit pulp. In another preferred aspect, the plant cell
wall biomass is vegetable pulp. In another preferred aspect, the
plant cell wall biomass is pumice. In another preferred aspect, the
plant cell wall biomass is distillers grain.
[0037] The plant cell wall biomass may be used as is or may be
subjected to pretreatment using conventional methods known in the
art. Such pretreatments includes physical, chemical, and biological
pretreatment. For example, physical pretreatment techniques can
include various types of milling, crushing, irradiation,
steaming/steam explosion, and hydrothermolysis. Chemical
pretreatment techniques can include dilute acid, alkaline, organic
solvent, ammonia, sulfur dioxide, carbon dioxide, and pH-controlled
hydrothermolysis. Biological pretreatment techniques can involve
applying lignin-solubilizing microorganisms (see, for example, Hsu,
T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:
Production and Utilization, Wyman, C. E., ed., Taylor &
Francis, Washington, D.C., 179-212; Ghosh, P., Singh, A., 1993,
Physicochemical and biological treatments for enzymatic/microbial
conversion of lignocellulosic biomass, Adv. Appl. Microbiol., 39:
295-333; McMillan, J. D., 1994, Pretreating lignocellulosic
biomass: a review, in Enzymatic Conversion of Biomass for Fuels
Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds.,
ACS Symposium Series 566, American Chemical Society, Washington,
D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,
1999, Ethanol production from renewable resources, in Advances in
Biochemical Engineering/Biotechnology, Scheper, T., ed.,
Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson,
L., and Hahn-Hagerdal, B., 1996, Fermentation of lignocellulosic
hydrolysates for ethanol production, Enz. Microb. Tech, 18:
312-331; and Vallander, L., and Eriksson, K.-E. L., 1990,
Production of ethanol from lignocellulosic materials: State of the
art, Adv. Biochem. Eng./Biotechnol., 42: 63-95).
[0038] In the present invention, the plant cell wall
polysaccharides include, but are not limited to, cellulose,
hemicellulose, and pectic substances.
[0039] Cellulose is composed of beta-1,4-glucan. Hemicellulose is
composed of beta-1,3-1,4-glucan, xyloglucan, xylan (arabinoxylan),
mannan (galactomannan), galactan (arabinogalactan), and arabinan.
Pectic substances are composed of homogalacturonan (pectin),
rhamnogalacturonan, and xylogalacturonan.
[0040] Beta-1,4-glucan is composed of beta-1,4-linked glucose.
Enzymes that degrade beta-1,4-glucan include endoglucanase,
cellobiohydrolase, and beta-glucosidase.
[0041] Beta-1,3-1,4-glucan is composed of beta-1,4-linked glucose
interrupted by beta-1,3-linked glucose. Enzymes that degrade
beta-1,3-1,4-glucan include endo-beta-1,3(4)-glucanase,
endoglucanase (beta-glucanase, cellulase), and
beta-glucosidase.
[0042] Xyloglucans are composed of beta-1,4-linked glucose with
alpha-1,6-linked xylose substituents. Enzymes that degrade
xyloglucans include xyloglucanase, endoglucanase, and
cellulase.
[0043] Xylan (arabinoxylan) is composed of beta-1,4-linked xylose,
with alpha-1,2 or alpha-1,3 linked arabinoses. The xylose can be
acetylated. Glucuronic acid is also present. Enzymes that degrade
xylan include xylanase, xylosidase, alpha-arabinofuranosidase,
alpha-glucuronidase, and acetyl xylan esterase.
[0044] Mannan (galactomannan) is composed of beta-1,4-linked
mannose with alpha-1,6-linked galactose substituents. The mannose
substituents can also be acetylated. Enzymes that degrade mannan
include mannanase, mannosidase, alpha-galactosidase, and mannan
acetyl esterase.
[0045] Galactan (arabinogalactan) is composed of D-galactose and
3,6-anhydrogalactose linked by beta-1,3-linkages. Enzymes that
degrade galactan include galactanases.
[0046] Arabinan is composed of 1,3-1,5-linked L-arabinose. Enzymes
that degrade arabinan include arabinanases.
[0047] Homogalacturonan is composed of alpha-1,4-linked
galacturonic acid. The galacturonic acid substituents may be
acetylated and/or methylated. Enzymes that degrade homogalacturonan
include pectate lyase, pectin lyase, pectate lyase,
polygalacturonase, pectin acetyl esterase, and pectin methyl
esterase.
[0048] Rhamnogalacturonan is composed of alternating
alpha-1,4-rhamnose and alpha-1,2-linked galacturonic acid, with
side chains linked 1,4 to rhamnose. The side chains include Type I
galactan, which is beta-1,4-linked galactose with alpha-1,3-linked
arabinose substituents; Type II galactan, which is
beta-1,3-1,6-linked galactoses (very branched) with arabinose
substituents; and arabinan, which is alpha-1,5-linked arabinose
with alpha-1,3-linked arabinose branches. The galacturonic acid
substituents may be acetylated and/or methylated. Enzymes that
degrade rhamnogalacturonan include alpha-arabinofuranosidase,
beta-galactosidase, galactanase, arabinanase,
alpha-arabinofuranosidase, rhamnogalacturonase, rhamnogalacturonan
lyase, and rhamnogalacturonan acetyl esterase.
[0049] Xylogalacturonan is composed of alpha-1,4-linked
galacturonic acid with side chains of xylose. Galactose and fucose
may be linked to the xylose substituents. Rhamnose is also present.
The galacturonic acid substituents may be acetylated and/or
methylated. Enzymes that degrade xylogalacturonan include
xylogalacturonosidase, xylogalacturonase, and rhamnogalacturonan
lyase.
[0050] Cellulose may also be present as lignocellulose. Lignin is
composed of methoxylated phenyl-propane units linked by ether
linkages and C--C bonds. The chemical composition of lignin differs
according to the plant species. Such components include guaiacyl,
4-hydroxyphenyl, and syringyl groups. Enzymes that degrade the
lignin component of lignocellulose include lignin peroxidases,
manganese-dependent peroxidases, hybrid peroxidases, with combined
properties of lignin peroxidases and manganese-dependent
peroxidases, and laccases (Vicuna, 2000, Molecular Biotechnology
14: 173-176; Broda et al., 1996, Molecular Microbiology 19:
923-932).
Recombinant Microorganisms
[0051] In the methods of the present invention, the recombinant
microorganism can be any microorganism that is useful as a host for
the recombinant production of enzymes useful in the conversion or
degradation of plant cell wall polysaccharides. The microorganism
chosen as a host for recombinant production may already contain one
or more native genes encoding enzymes that degrade or convert plant
cell wall polysaccharides. However, the host may be deficient in
the full complement of enzymes necessary to degrade or convert
plant cell wall polysaccharides, i.e., the host may lack one or
more genes. Alternatively, the host may contain the full complement
of enzymes, but one or more enzymes may be poorly expressed.
Moreover, the host may lack one or more genes required to produce
the full complement of enzymes and one or more enzymes the host
does produce may be poorly expressed. It will be understood in the
present invention that a gene native to the host that has undergone
manipulation, as described herein, will be considered a
heterologous gene.
[0052] The host is preferably a fungal strain. "Fungi" as used
herein includes the phyla Ascomycota, Basidiomycota,
Chytridiomycota, and Zygomycota (as defined by Hawksworth et al.,
In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,
1995, CAB International, University Press, Cambridge, UK) as well
as the Oomycota (as cited in Hawksworth et al., 1995, supra, page
171) and all mitosporic fungi (Hawksworth et al., 1995, supra).
[0053] In a preferred aspect, the fungal host is a yeast strain.
"Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Biology and Activities of
Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0054] In a more preferred aspect, the yeast host is a Candida,
Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia strain.
[0055] In a most preferred aspect, the yeast host is a
Saccharomyces carisbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis
strain. In another most preferred aspect, the yeast host is a
Kluyveromyces lactis strain. In another most preferred aspect, the
yeast host is a Yarrowia lipolytica strain.
[0056] In another preferred aspect, the fungal host is a
filamentous fungal strain. "Filamentous fungi" include all
filamentous forms of the subdivision Eumycota and Oomycota (as
defined by Hawksworth et al., 1995, supra). The filamentous fungi
are generally characterized by a mycelial wall composed of chitin,
cellulose, glucan, chitosan, mannan, and other complex
polysaccharides. Vegetative growth is by hyphal elongaton and
carbon catabolism is obligately aerobic. In contrast, vegetative
growth by yeasts such as Saccharomyces cerevisiae is by budding of
a unicellular thallus and carbon catabolism may be
fermentative.
[0057] In a more preferred aspect, the filamentous fungal host is,
but not limited to, an Acremonium, Aspergillus, Fusarium, Humicola,
Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium,
Thielavia, Tolypocladium, or Trichoderma strain.
[0058] In an even more preferred aspect, the filamentous fungal
host is an Aspergillus awamori, Aspergillus foetidus, Aspergillus
japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus
oryzae strain. In another even more preferred aspect, the
filamentous fungal host is a Fusarium bactridioides, Fusarium
cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium
graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium
negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum,
Fusarium sambucinum, Fusarium sarcochroum, Fusarium
sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium
trichothecioides, or Fusarium venenatum strain. In another even
more preferred aspect, the filamentous fungal host is a Humicola
insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium purpurogenum,
Scytalidium thermophilum, or Thielavia terrestris strain. In a
further even more preferred aspect, the filamentous fungal host is
a Trichoderma harzianum, Trichoderma koningii, Trichoderma
longibrachiatum, Trichodermaa reesei, or Trichoderma viride
strain.
[0059] In a most preferred aspect, the filamentous fungal host is
Trichoderma reesei RutC30, which is available from the American
Type Culture Collection as Trichoderma reesei ATCC 56765.
[0060] In a preferred aspect, the host or recombinant microorganism
comprises one or more heterologous genes encoding enzymes selected
from the group consisting of endoglucanase (cellulase),
cellobiohydrolase, and beta-glucosidase.
[0061] In a more preferred aspect, the recombinant microorganism
comprises a heterologous gene encoding an endoglucanase. In another
more preferred aspect, the recombinant microorganism comprises a
heterologous gene encoding a cellobiohydrolase gene. In another
more preferred aspect, the recombinant microorganism comprises a
heterologous gene encoding a beta-glucosidase.
[0062] In a most preferred aspect, the recombinant microorganism
comprises heterologous genes encoding an endoglucanase and a
cellobiohydrolase. In another most preferred aspect, the
recombinant microorganism comprises heterologous genes encoding an
endoglucanase and a beta-glucosidase.
[0063] In another most preferred aspect, the recombinant
microorganism comprises heterologous genes encoding an
endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[0064] In another preferred aspect, the recombinant microorganism
further comprises a glucohydrolase.
[0065] In another preferred aspect, the recombinant microorganism
further comprises one or more heterologous genes encoding enzymes
selected from the group consisting of xyloglucanase, xylanase,
xylosidase, alpha-arabinofuranosidase, alpha-glucuronidase, and
acetyl xylan esterase.
[0066] In another preferred aspect, the recombinant microorganism
further comprises one or more heterologous genes encoding enzymes
selected from the group consisting of mannanase, mannosidase,
alpha-galactosidase, mannan acetyl esterase, galactanase, and
arabinanase.
[0067] In another preferred aspect, the recombinant microorganism
further comprises one or more heterologous genes encoding enzymes
selected from the group consisting of pectate lyase, pectin lyase,
polygalacturonase, pectin acetyl esterase, pectin methyl esterase,
alpha-arabinofuranosidase, beta-galactosidase, galactanase,
arabinanase, alpha-arabinofuranosidase, rhamnogalacturonase,
rhamnogalacturonan lyase, rhamnogalacturonan acetyl esterase,
xylogalacturonosidase, xylogalacturonase, and rhamnogalacturonan
lyase.
[0068] In another preferred aspect, the recombinant microorganism
further comprises one or more heterologous genes encoding enzymes
selected from the group consisting of a lignin peroxidase,
manganese-dependent peroxidase, and hybrid peroxidase.
[0069] In another preferred aspect, the recombinant microorganism
even further comprises one or more heterologous genes encoding
enzymes selected from the group consisting of an esterase, lipase,
oxidase, phospholipase, phytase, protease, and peroxidase.
[0070] A gene encoding a plant cell wall degrading or converting
enzyme may be of fungal or bacterial origin, e.g., species of
Humicola, Coprinus, Thielavia, Fusarium, Myceliophthora,
Acremonium, Cephalosporium, Scytalidium, Penicillium or Aspergillus
(see, for example, EP 458162), especially those selected from the
species Humicola insolens (reclassified as Scytalidium
thermophilum, see for example, U.S. Pat. No. 4,435,307), Coprinus
cinereus, Fusarium oxysporum, Myceliophthora thermophila, Meripilus
giganteus, Thielavia terrestris, Acremonium sp., Acremonium
persicinum, Acremonium acremonium, Acremonium brachypenium,
Acremonium dichromosporum, Acremonium obclavatum, Acremonium
pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, and
Acremonium furatum; preferably from the species Humicola insolens
DSM 1800, Fusarium oxysporum DSM 2672, Myceliophthora thermophila
CBS 117.65, Cephalosporium sp. RYM-202, Acremonium sp. CBS 478.94,
Acremonium sp. CBS 265.95, Acremonium persicinum CBS 169.65,
Acremonium acremonium AHU 9519, Cephalosporium sp. CBS 535.71,
Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS
683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertoniae
CBS 157.70, Acremonium roseogriseum CBS 134.56, Acremonium
incoloratum CBS 146.62, and Acremonium furatum CBS 299.70H. Plant
cell wall hydrolytic enzyme genes may also be obtained from
Trichoderma (particularly Trichoderma viride, Trichoderma reesei,
and Trichoderma koningii), alkalophilic Bacillus (see, for example,
U.S. Pat. No. 3,844,890 and EP 458162), and Streptomyces (see, for
example, EP 458162).
[0071] The enzymes and genes thereof referenced herein may be
obtained from any suitable origin, including, bacterial, fungal,
yeast or mammalian origin. The term "obtained" as used herein in
connection with a given source shall mean that the polypeptide
encoded by a nucleotide sequence is produced by the source or by a
strain in which the nucleotide sequence from the source has been
inserted. Encompassed within the meaning of a native enzyme are
natural variants or variants obtained, for example, by
site-directed mutagenesis or shuffling.
[0072] Techniques used to isolate or clone a gene encoding an
enzyme are known in the art and include isolation from genomic DNA,
preparation from cDNA, or a combination thereof. The cloning of a
gene from such genomic DNA can be effected, e.g., by using the well
known polymerase chain reaction (PCR) or antibody screening of
expression libraries to detect cloned DNA fragments with shared
structural features. See, e.g., Innis et al., 1990, PCR: A Guide to
Methods and Application, Academic Press, New York. Other nucleic
acid amplification procedures such as ligase chain reaction (LCR),
ligated activated transcription (LAT) and nucleotide sequence-based
amplification (NASBA) may be used.
[0073] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus and Trichoderma host
strains are described in EP 238 023 and Yelton et al., 1984,
Proceedings of the National Academy of Sciences USA 81: 1470-1474.
Suitable methods for transforming Fusarium species are described by
Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast
may be transformed using the procedures described by Becker and
Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to
Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume
194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983,
Journal of Bacteriology 153: 163; and Hinnen et al., 1978,
Proceedings of the National Academy of Sciences USA 75:1920.
Enzymes Having Plant Cell Wall Hydrolytic Activity and Genes
Thereof
[0074] In the methods of the present invention, the recombinant
microorganism comprises one or more genes which are heterologous or
foreign to the microorganism, wherein the one or more genes encode
enzymes involved in the degradation or conversion of plant cell
wall polysaccharides.
[0075] The heterologous genes may encode enzymes that degrade
beta-1,4-glucan such as endoglucanase (cellulase),
cellobiohydrolase, glucohydrolase, and beta-glucosidase; degrade
beta-1,3-1,4-glucan such as endo-beta-1,3(4)-glucanase,
endoglucanase (beta-glucanase, cellulase), and beta-glucosidase;
degrade xyloglucans such as xyloglucanase, endoglucanase, and
cellulase; degrade xylan such as xylanase, xylosidase,
alpha-arabinofuranosidase, alpha-glucuronidase, and acetyl xylan
esterase; degrade mannan such as mannanase, mannosidase,
alpha-galactosidase, and mannan acetyl esterase; degrade galactan
such as galactanase; degrade arabinan such as arabinanase; degrade
homogalacturonan such as pectate lyase, pectin lyase, pectate
lyase, polygalacturonase, pectin acetyl esterase, and pectin methyl
esterase; degrade rhamnogalacturonan such as
alpha-arabinofuranosidase, beta-galactosidase, galactanase,
arabinanase, alpha-arabinofuranosidase, rhamnogalacturonase,
rhamnogalacturonan lyase, and rhamnogalacturonan acetyl esterase;
degrade xylogalacturonan such as xylogalacturonosidase,
xylogalacturonase, and rhamnogalacturonan lyase; and degrade lignin
such as lignin peroxidases, manganese-dependent peroxidases, hybrid
peroxidases, with combined properties of lignin peroxidases and
manganese-dependent peroxidases, and laccases.
[0076] Genes encoding polysaccharide-degrading enzymes may be
obtained from sources as described by B. Henrissat, 1991, A
classification of glycosyl hydrolases based on amino-acid sequence
similarities, Biochem. J. 280: 309-316, and Henrissat B., and
Bairoch A., 1996, Updating the sequence-based classification of
glycosyl hydrolases, Biochem. J. 316: 695-696., which is
incorporated herein by reference.
[0077] The recombinant microorganism may further comprise one or
more heterologous genes encoding enzymes such as esterases,
lipases, oxidases, phospholipases, phytases, proteases, and
peroxidases.
[0078] The enzymes may have activity either in the acid, neutral,
or alkaline pH-range. In a preferred aspect, the enzymes have
activity in the pH range of about 2 to about 7.
[0079] Endoglucanases
[0080] The term "endoglucanase" is defined herein as an
endo-1,4(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. No.
3.2.1.4) which catalyses endohydrolysis of 1,4-beta-D-glycosidic
linkages in cellulose, cellulose derivatives (such as carboxymethyl
cellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in
mixed beta-1,3 glucans such as cereal beta-D-glucans or
xyloglucans, and other plant material containing cellulosic
components. For purposes of the present invention, endoglucanase
activity is determined using carboxymethyl cellulose (CMC)
hydrolysis according to the procedure of Ghose, 1987, Pure and
Appl. Chem. 59: 257-268.
[0081] In a preferred aspect, an endoglucanase gene is obtained
from a Trichoderma reesei strain. In another preferred aspect, an
endoglucanase gene is obtained from an Aspergillus oryzae strain.
In another preferred aspect, an endoglucanase gene is obtained from
an Aspergillus aculeatus strain. In another preferred aspect, an
endoglucanase gene is obtained from a Humicola insolens strain.
[0082] Preferred examples of endoglucanase genes that can be used
in the invention are obtained from Aspergillus aculeatus (U.S. Pat.
No. 6,623,949; WO 94/14953), Aspergillus kawachii (U.S. Pat. No.
6,623,949), Aspergillus oryzae (Kitamoto et al., 1996, Appl.
Microbiol. Biotechnol. 46: 538-544; U.S. Pat. No. 6,635,465),
Aspergillus nidulans (Lockington et al., 2002, Fungal Genet. Biol.
37: 190-196), Cellulomonas fimi (Wong et al., 1986, Gene 44:
315-324), Bacillus subtilis (MacKay et al., 1986, Nucleic Acids
Res. 14: 9159-9170), Cellulomonas pachnodae (Cazemier et al., 1999,
Appl. Microbiol. Biotechnol. 52: 232-239), Fusarium equiseti
(Goedegebuur et al., 2002, Curr. Genet. 41: 89-98), Fusarium
oxysporum (Hagen et al., 1994, Gene 150: 163-167; Sheppard et al.,
1994, Gene 150: 163-167), Humicola insolens (U.S. Pat. No.
5,912,157; Davies et al., 2000, Biochem J. 348: 201-207), Hypocrea
jecorina (Penttila et al., 1986, Gene 45: 253-263), Humicola grisea
(Goedegebuur et al., 2002, Curr. Genet. 41: 89-98), Micromonospora
cellulolyticum (Lin et al., 1994, J. Ind. Microbiol. 13: 344-350),
Myceliophthora thermophila (U.S. Pat. No. 5,912,157), Rhizopus
oryzae (Moriya et al., 2003, J. Bacteriol. 185: 1749-1756),
Trichoderma reesei (Saloheimo et al., 1994, Mol. Microbiol. 13:
219-228), and Trichoderma viride (Kwon et al., 1999, Biosci.
Biotechnol. Biochem. 63: 1714-1720; Goedegebuur et al., 2002, Curr.
Genet. 41: 8998).
[0083] Cellobiohydrolases
[0084] Cellobiohydrolase, an exo-1,4-beta-D-glucan
cellobiohydrolase (E.C. 3.2.1.91), catalyzes the hydrolysis of
1,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides,
or any beta-1,4-linked glucose containing polymer, releasing
cellobiose from the reducing or non-reducing ends of the chain. For
purposes of the present invention, cellobiohydrolase activity is
determined according to the procedures described by Lever et al.,
1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS
Letters, 149: 152-156; and van Tilbeurgh and Claeyssens, 1985, FEBS
Letters, 187: 283-288. In the present invention, the Lever et al.
method is employed to assess hydrolysis of cellulose in corn
stover, while the methods of van Tilbeurgh et al. are used to
determine the cellobiohydrolase activity on a fluorescent
disaccharide derivative.
[0085] In a preferred aspect, a cellobiohydrolase gene is obtained
from a Trichoderma reesei strain. In another preferred aspect, a
cellobiohydrolase gene is obtained from an Aspergillus aculeatus
strain. In another preferred aspect, a cellobiohydrolase gene is
obtained from an Aspergillus niger strain. In another preferred
aspect, a cellobiohydrolase gene is obtained from an Aspergillus
oryzae strain. In another preferred aspect, a cellobiohydrolase
gene is obtained from an Emericella nidulans strain.
[0086] Preferred examples of cellobiohydrolase genes that can be
used in the invention are obtained from Acremonium cellulolyticus
(U.S. Pat. No. 6,127,160), Agaricus bisporus (Chow et al., 1994,
Appl. Environ. Microbiol. 60: 2779-2785; Yague et al., 1997,
Microbiology (Reading, Engl.) 143: 239-244), Aspergillus aculeatus
(Takada et al., 1998, J. Ferment. Bioeng. 85: 1-9), Aspergillus
niger (Gielkens et al., 1999, Appl. Environ. Microbiol. 65:
4340-4345), Aspergillus oryzae (Kitamoto et al., 1996, Appl.
Microbiol. Biotechnol. 46: 538-544), Athelia rolfsii (EMBL
accession number AB103461), Chaetomium thermophilum (EMBL accession
numbers AX657571 and CQ838150), Cullulomonas fimi (Meinke et al.,
1994, Mol. Microbiol. 12: 413-422), Emericella nidulans (Lockington
et al., 2002, Fungal Genet. Biol. 37: 190-196), Fusarium oxysporum
(Hagen et al., 1994, Gene 150: 163-167), Geotrichum sp. 128 (EMBL
accession number AB089343), Humicola grisea (de Oliviera and
Radford, 1990, Nucleic Acids Res. 18: 668; Takashima et al., 1998,
J. Biochem. 124: 717-725), Humicola nigrescens (EMBL accession
number AX657571), Hypocrea koningii (Teeri et al., 1987, Gene 51:
43-52), Mycelioptera thermophila (EMBL accession numbers AX657599),
Neocallimastix patriciarum (Denman et al., 1996, Appl. Environ.
Microbiol. 62 (6), 1889-1896), Phanerochaete chrysosporium
(Tempelaars et al., 1994, Appl. Environ. Microbiol. 60: 4387-4393),
Thermobifida fusca (Zhang, 1995, Biochemistry 34: 3386-3395),
Trichoderma reesei (Terri et al., 1983, Bio/Technology 1: 696-699;
Chen et al., 1987, Bio/Technology 5: 274-278), and Trichoderma
viride (EMBL accession numbers A4368686 and A4368688).
[0087] Beta-Glucosidase
[0088] Beta-glucosidase, a beta-D-glucoside glucohydrolase (E.C.
3.2.1.21), catalyzes the hydrolysis of terminal non-reducing
beta-D-glucose residues with the release of beta-D-glucose. For
purposes of the present invention, beta-glucosidase activity is
determined according to the basic procedure described by Venturi et
al., 2002, J. Basic Microbiol. 42: 55-66, except different
conditions were employed as described herein. One unit of
beta-glucosidase activity is defined as 1.0 .mu.mole of
p-nitrophenol produced per minute at 50.degree. C., pH 5 from 4 mM
p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium
citrate, 0.01% Tween-20.
[0089] Encompassed within the definition of beta-glucosidases are
cellobiases. Cellobiases hydrolyze cellobiose to glucose.
[0090] In a preferred aspect, a beta-glucosidase gene is obtained
from an Aspergillus aculeatus strain. In another preferred aspect,
a beta-glucosidase gene is obtained from an Aspergillus kawachi
strain. In another preferred aspect, a beta-glucosidase gene is
obtained from a Trichoderma reesei strain.
[0091] Preferred examples of beta-glucosidase genes that can be
used in the invention are obtained from Aspergillus aculeatus
(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus kawachi
(Iwashita et al., 1999, Appl. Environ. Microbiol. 65: 5546-5553),
Aspergillus oryzae (WO 2002/095014), Cellulomonas biazotea (Wong et
al., 1998, Gene 207: 79-86), Penicillium funiculosum (WO
200478919), Saccharomycopsis fibuligera (Machida et al., 1988,
Appl. Environ. Microbiol. 54: 3147-3155), Schizosaccharomyces pombe
(Wood et al., 2002, Nature 415: 871-880), and Trichoderma reesei
(Barnett et al., 1991, Bio/Technology 9: 562-567).
[0092] Glucohydrolases
[0093] Glucohydrolase, an exo-1,4-beta-D-glucan glucohydrolase
(E.C. 3.2.1.74), catalyzes the hydrolysis of 1,4-linkages
(O-glycosyl bonds) in 1,4-beta-D-glucans so as to remove successive
glucose units. For purposes of the present invention, exoglucanase
activity is determined according to the procedure described by
Himmel et al., 1986, J. Biol. Chem. 261: 12948-12955.
[0094] In a preferred aspect, a glucohydrolase gene is obtained
from a Trichoderma reesei strain. In another preferred aspect, a
glucohydrolase gene is obtained from a Humicola insolens strain. In
another preferred aspect, a glucohydrolase gene is obtained from an
Aspergillus niger strain. In another preferred aspect, a
cellobiohydrolase gene is obtained from a Chaetomium thermophilum
strain. In another preferred aspect, a glucohydrolase gene is
obtained from a Thermoascus aurantiacus strain. In another
preferred aspect, a glucohydrolase gene is obtained from a
Thielavia terrestris strain.
[0095] Hemicellulases
[0096] Enzymatic hydrolysis of hemicellulose can be performed by a
wide variety of fungi and bacteria (Saha, 2003, J. Ind. Microbiol.
Biotechnol. 30: 279-291). Similar to cellulose degradation,
hemicellulose hydrolysis requires coordinated action of several
enzymes. Hemicellulases can be placed into three general
categories: the endo-acting enzymes that attack internal bonds
within the polysaccharide chain, the exo-acting enzymes that act
processively from either the reducing or nonreducing end of
polysaccharide chain, and the accessory enzymes, acetylesterases
and esterases that hydrolyze lignin glycoside bonds, such as
coumaric acid esterase and ferulic acid esterase (Wong, K. K .Y.,
Tan, L. U. L., and Saddler, J. N., 1988, Multiplicity of
.beta.-1,4-xylanase in microorganisms: Functions and applications,
Microbiol. Rev., 52: 305-317; Tenkanen, M., and Poutanen, K., 1992,
Significance of esterases in the degradation of xylans, in Xylans
and Xylanases, Visser, J., Beldman, G., Kuster-van Someren, M. A.,
and Voragen, A. G. J., eds., Elsevier, New York, N.Y., 203-212;
Coughlan, M. P., and Hazlewood, G. P., 1993, Hemicellulose and
hemicellulases, Portland, London, UK; Brigham, J. S., Adney, W. S.,
and Himmel, M. E., 1996, Hemicellulases: Diversity and
applications, in Handbook on Bioethanol: Production and
Utilization, Wyman, C. E., ed., Taylor & Francis, Washington,
D.C., 119-141).
[0097] Examples of endo-acting hemicellulases and accessory enzymes
include endoarabinanase, endoarabinogalactanase, endoglucanase,
endomannanase, endoxylanase, and feraxan endoxylanase. Examples of
exo-acting hemicellulases and accessory enzymes include
.alpha.-L-arabinosidase, .beta.-L-arabinosidase,
.alpha.-1,2-L-fucosidase, .alpha.-D-galactosidase,
.beta.-D-galactosidase, .beta.-D-glucosidase,
.beta.-D-glucuronidase, .beta.-D-mannosidase, .beta.-D-xylosidase,
exo-glucosidase, exo-cellobiohydrolase, exo-mannobiohydrolase,
exo-mannanase, exo-xylanase, xylan .alpha.-glucuronidase, and
coniferin .beta.-glucosidase. Examples of esterases include acetyl
esterases (acetylgalactan esterase, acetylmannan esterase, and
acetylxylan esterase), and aryl esterases (coumaric acid esterase
and ferulic acid esterase).
[0098] Hemicellulases include xylanases, arabinofuranosidases,
acetyl xylan esterases, glucuronidases, endo-galactanases,
mannanases, endo- or exo-arabinases, exo-galactanases, and mixtures
thereof. Preferably, the hemicellulase is an exo-acting
hemicellulase, and more preferably, an exo-acting hemicellulase
which has the ability to hydrolyze hemicellulose preferably in the
pH range of about 2 to about 7.
[0099] A hemicellulase, such as a xylanase, arabinofuranosidase,
acetyl xylan esterase, glucuronidase, endo-galactanase, mannanase,
endo- or exo-arabinase, or exo-galactanase, or genes thereof, may
be obtained from any suitable source, including fungal and
bacterial organisms, such as Aspergillus, Disporotnchum,
Penicillium, Neurospora, Fusarium, Trichoderma, Humicola,
Thermomyces, and Bacillus.
[0100] Preferred examples of hemicellulase genes that can be used
in the invention are obtained from Acidobacterium capsulatum
(Inagaki et al., 1998, Biosci. Biotechnol. Biochem. 62: 1061-1067),
Agaricus bisporus (De Groot et al., 1998, J. Mol. Biol. 277:
273-284), Aspergillus aculeatus (U.S. Pat. No. 6,197,564; U.S. Pat.
No. 5,693,518), Aspergillus kawachii (Ito et al., 1992, Biosci.
Biotechnol. Biochem. 56: 906-912), Aspergillus niger (EMBL
accession number AF108944), Magnaporthe grisea (Wu et al., 1995,
Mol. Plant Microbe Interact. 8: 506-514), Penicillium chrysogenum
(Haas et al., 1993, Gene 126: 237-242), Talaromyces emersonii (WO
02/24926), and Trichoderma reesei (EMBL accession numbers X69573,
X69574, and AY281369).
[0101] Lignin-Degrading Enzymes
[0102] Lignin is an aromatic polymer occurring in the woody tissue
of higher plants. Due to its hydrophobicity and complex random
structure lacking regular hydrolyzable bonds, lignin is poorly
degraded by most organisms. The best degraders of lignin are white
rot fungi that produce extracellular peroxidases and laccases,
which are involved in the initial attack of lignin.
[0103] Lignin-degrading enzymes include, but are not limited to,
lignin peroxidases, manganese-dependent peroxidases, hybrid
peroxidases, with combined properties of lignin peroxidases and
manganese-dependent peroxidases, and laccases (Vicuna, 2000, supra;
Broda et al., 1996, supra). Hydrogen peroxide, required as a
co-substrate by the peroxidases, can be generated by glucose
oxidase, aryl alcohol oxidase, and/or lignin peroxidase-activated
glyoxal oxidase.
[0104] Manganese-dependent peroxidase is a frequently encountered
peroxidase produced by white rot fungi. The peroxidase has a
catalytic cycle involving a 2-electron oxidation of the heme by
hydrogen peroxide and subsequent oxidation of compound I via
compound II in two 1-electron steps to the native enzyme. The best
reducing substrate for compounds I and II is Mn(II), a metal
naturally present in wood. The Mn(III) formed oxidizes other
substrates.
[0105] Organic acids such as oxalate, glyoxylate, and lactate are
known to have an important role in the mechanism of
manganese-dependent peroxidase and lignin degradation. Mn(III) is
stripped from the enzyme by organic acids, and the produced
Mn(III)-organic acid complex acts as a diffusible mediator in the
oxidation of lignin by manganese-dependent peroxidase. Mn(III) can
also oxidize organic acids, yielding radicals. The organic acids
may also be supplied from the degradation of lignin and by
microorganisms.
[0106] Lignin-degrading enzymes and genes thereof may be obtained
from a Bjerkandera adusta, Ceriporiopsis subvermispora (see WO
02/079400), Coprinus cinereus, Coriolus hirsutus, Humicola
insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium purpurogenum,
Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,
Thielavia terrestris, Trametes villosa, Trametes versicolor,
Trichoderma harzianum, Trichoderma koningii, Trichoderma
longibrachiatum, Trichoderma reesei, or Trichoderma viride
strain.
[0107] Preferred examples of genes encoding lignin-degrading
enzymes that can be used in the invention are obtained from
Bjerkandera adusta (WO 2001/098469), Ceriporiopsis subvermispora
(Conesa et al., 2002, Journal of Biotechnology 93: 143-158),
Cantharellus cibariusi (Ng et al., 2004, Biochemical and
Biophysical Research Communications 313: 37-41), Coprinus cinereus
(WO 97/008325; Conesa et al., 2002, supra), Lentinula edodes (Nagai
et al., 2002, Applied Microbiology and Biotechnology 60: 327-335,
2002), Melanocarpus albomyces (Kiiskinen et al., 2004, FEBS Letters
576: 251-255, 2004), Myceliophthora thermophila (WO 95/006815),
Phanerochaete chrysosporium (Conesa et al., 2002, supra; Martinez,
2002, Enzyme and Microbial Technology 30: 425-444, 2002), Phlebia
radiata (Conesa et al., 2002, supra), Pleurotus eryngii (Conesa et
al., 2002, supra), Polyporus pinsitus (WO 96/000290), Rigidoporus
lignosus (Garavaglia et al., 2004, Journal of Molecular Biology
342: 1519-1531), Rhizoctonia solani (WO 96/007988), Scytalidium
thermophilum (WO 95/033837), Tricholoma giganteum (Wang et al.,
2004, Biochemical and Biophysical Research Communications 315:
450-454), and Trametes versicolor (Conesa et al., 2002, supra).
[0108] Esterases
[0109] Esterase, a carboxylic ester hydrolase (EC 3.1.1), catalyzes
the hydrolysis of ester bonds. Esterases useful in the degradation
or conversion of plant cell wall polysaccharides include acetyl
esterases such as acetylgalactan esterase, acetylmannan esterase,
and acetylxylan esterase, and esterases that hydrolyze lignin
glycoside bonds, such as coumaric acid esterase and ferulic acid
esterase.
[0110] Non-limiting examples of esterases include arylesterase,
triacylglycerol lipase, acetylesterase, acetylcholinesterase,
cholinesterase, tropinesterase, pectinesterase, sterol esterase,
chlorophyllase, L-arabinonolactonase, gluconolactonase,
uronolactonase, tannase, retinyl-palmitate esterase,
hydroxybutyrate-dimer hydrolase, acylglycerol lipase, 3-oxoadipate
enol-lactonase, 1,4-lactonase, galactolipase, 4-pyridoxolactonase,
acylcarnitine hydrolase, aminoacyl-tRNA hydrolase,
D-arabinonolactonase, 6-phosphogluconolactonase, phospholipase A1,
6-acetylglucose deacetylase, lipoprotein lipase, dihydrocoumarin
lipase, limonin-D-ring-lactonase, steroid-lactonase,
triacetate-lactonase, actinomycin lactonase, orsellinate-depside
hydrolase, cephalosporin-C deacetylase, chlorogenate hydrolase,
alpha-amino-acid esterase, 4-methyloxaloacetate esterase,
carboxymethylenebutenolidase, deoxylimonate A-ring-lactonase,
2-acetyl-1-alkylglycerophosphocholine esterase, fusarinine-C
ornithinesterase, sinapine esterase, wax-ester hydrolase,
phorbol-diester hydrolase, phosphatidylinositol deacylase, sialate
O-acetylesterase, acetoxybutynylbithiophene deacetylase,
acetylsalicylate deacetylase, methylumbelliferyl-acetate
deacetylase, 2-pyrone-4,6-dicarboxylate lactonase,
N-acetylgalactosaminoglycan deacetylase, juvenile-hormone esterase,
bis(2-ethylhexyl)phthalate esterase, protein-glutamate
methylesterase, 11-cis-retinyl-palmitate hydrolase,
all-trans-retinyl-palmitate hydrolase, L-rhamnono-1,4-lactonase,
5-(3,4-diacetoxybut-1-ynyl)-2,2'-bithiophene deacetylase,
fatty-acyl-ethyl-ester synthase, xylono-1,4-lactonase,
N-acetylglucosaminylphosphatidylinositol deacetylase, cetraxate
benzylesterase, acetylalkylglycerol acetylhydrolase, and
acetylxylan esterase.
[0111] Preferred esterases for use in the present invention are
lipolytic enzymes, such as, lipases (EC 3.1.1.3, EC 3.1.1.23 and/or
EC 3.1.1.26) and phospholipases (EC 3.1.1.4 and/or EC 3.1.1.32,
including lysophospholipases classified by EC 3.1.1.5). Other
preferred esterases are cutinases (EC 3.1.1.74). Further preferred
esterases are acetylxylan esterase and pectin methylesterase.
[0112] The esterase may be added in an amount effective to obtain
the desired benefit to improve the performance of the spent whole
broth or a fermenting microorganism, e.g., to change the lipid
composition/concentration inside and/or outside of the fermenting
microorganism or in the cell membrane of the fermenting
microorganism, to result in an improvement in the movement of
solutes into and/or out of the fermenting microorganisms during
fermentation and/or to provide more metabolizable energy sources
(such as, e.g., by converting components, such as, oil from the
corn substrate, to components useful the fermenting microorganism,
e.g., unsaturated fatty acids and glycerol), to increase ethanol
yield. Examples of effective amounts of esterase are from 0.01 to
400 LU/g DS (Dry Solids). Preferably, the esterase is used in an
amount of 0.1 to 100 LU/g DS, more preferably 0.5 to 50 LU/g DS,
and even more preferably 1 to 20 LU/g DS. Further optimization of
the amount of esterase can hereafter be obtained using standard
procedures known in the art.
[0113] One Lipase Unit (LU) is the amount of enzyme which liberates
1.0 .mu.mol of titratable fatty acid per minute with tributyrin as
substrate and gum arabic as an emulsifier at 30.degree. C., pH 7.0
(phosphate buffer).
[0114] In a preferred aspect the esterase is a lipolytic enzyme,
more preferably, a lipase. As used herein, a "lipolytic enzyme"
refers to lipases and phospholipases (including
lyso-phospholipases). In a more preferred aspect, the lipolytic
enzyme is a lipase. Lipases may be applied herein for their ability
to modify the structure and composition of triglyceride oils and
fats in the fermentation media (including fermentation yeast), for
example, resulting from a corn substrate. Lipases catalyze
different types of triglyceride conversions, such as hydrolysis,
esterification and transesterification. Suitable lipases include
acidic, neutral and basic lipases, as are well-known in the art,
although acidic lipases (such as, e.g., the lipase G AMANO 50,
available from Amano) appear to be more effective at lower
concentrations of lipase as compared to either neutral or basic
lipases. Preferred lipases for use in the present invention
included Candida antarctica lipase and Candida cylindracea lipase.
More preferred lipases are purified lipases such as Candida
antarctica lipase (lipase A), Candida antarctica lipase (lipase B),
Candida cylindracea lipase, and Penicillium camembertii lipase.
[0115] The lipase may be the lipase disclosed in EP 258,068-A or
may be a lipase variant such as a variant disclosed in WO 00/60063
or WO 00/32758, hereby incorporated by reference.
[0116] Lipases are preferably present in amounts from about 1 to
400 LU/g DS, preferably 1 to 10 LU/g DS, and more preferably 1 to 5
LU/g DS.
[0117] The lipolytic enzyme is preferably of microbial origin, in
particular, of bacterial, fungal or yeast origin. The lipolytic
enzyme or gene thereof used may be obtained from any source,
including, for example, a strain of Absidia, in particular Absidia
blakesleena and Absidia corymbifera, a strain of Achromobacter, in
particular Achromobacter iophagus, a strain of Aeromonas, a strain
of Alternaia, in particular Alternaria brassiciola, a strain of
Aspergillus, in particular Aspergillus niger and Aspergillus
flavus, a strain of Achromobacter, in particular Achromobacter
iophagus, a strain of Aureobasidium, in particular Aureobasidium
pullulans, a strain of Bacillus, in particular Bacillus pumilus,
Bacillus strearothermophilus, and Bacillus subtilis, a strain of
Beauveria, a strain of Brochothrix, in particular Brochothrix
thernosohata, a strain of Candida, in particular Candida
cylindracea (Candida rugosa), Candida paralipolytica, and Candida
antarctica, a strain of Chromobacter, in particular Chromobacter
viscosum, a strain of Coprinus, in particular Coprinus cinerius, a
strain of Fusarium, in particular Fusarium oxysporum, Fusarium
solani, Fusarium solani pisi, Fusarium roseum culmorum, and
Fusarium venenatum, a strain of Geotricum, in particular Geotricum
penicillatum, a strain of Hansenula, in particular Hansenula
anomala, a strain of Humicola, in particular Humicola brevispora,
Humicola brevis var. thermoidea, and Humicola insolens, a strain of
Hyphozyma, a strain of Lactobacillus, in particular Lactobacillus
curvatus, a strain of Metarhizium, a strain of Mucor, a strain of
Paecilomyces, a strain of Penicillium, in particular Penicillium
cyclopium, Penicillium crustosum and Penicillium expansum, a strain
of Pseudomonas in particular Pseudomonas aeruginosa, Pseudomonas
alcaligenes, Pseudomonas cepacia (syn. Burkholderia cepacia),
Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas
maltophilia, Pseudomonas mendocina, Pseudomonas mephitica
lipolytica, Pseudomonas alcaligenes, Pseudomonas plantari,
Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas
stutzeri, and Pseudomonas wisconsinensis, a strain of Rhizoctonia,
in particular Rhizoctonia solani, a strain of Rhizomucor, in
particular Rhizomucor miehei, a strain of Rhizopus, in particular
Rhizopus japonicus, Rhizopus microsporus, and Rhizopus nodosus, a
strain of Rhodosporidium, in particular Rhodosporidium toruloides,
a strain of Rhodotorula, in particular Rhodotorula glutinis, a
strain of Sporobolomyces, in particular Sporobolomyces shibatanus,
a strain of Thermomyces, in particular Thermomyces lanuginosus
(formerly Humicola lanuginosa), a strain of Thiarosporella, in
particular Thiarosporella phaseolina, a strain of Trichoderma, in
particular, Trichoderma harzianum and Trichoderma reesei, and/or a
strain of Verticillium.
[0118] In a preferred aspect, the lipolytic enzyme or gene thereof
is obtained from a strain of Aspergillus, Achromobacter, Bacillus,
Candida, Chromobacter, Fusarium, Humicola, Hyphozyma, Pseudomonas,
Rhizomucor, Rhizopus, or Thermomyces.
[0119] Preferred examples of lipase genes that can be used in the
invention are obtained from Absidia sp. (WO 97/027276), Candida
antarctica (EMBL accession number Z30645), Candida cylindracea
(EMBL accession numbers X64703, X64704, X66006, X66007, and
X66008), Fusarium oxysporum (WO 98/26057), Penicillium camembertii
(Yamaguchi et al., 1991, Gene 103: 61-67), and Thermomyces
lanuginosus (EMBL accession number AF054513).
[0120] In another preferred aspect, at least one esterase is a
cutinase. Cutinases are enzymes which are able to degrade cutin.
The cutinase or gene thereof may be obtained from any source. In a
preferred aspect, the cutinase or gene thereof is obtained from a
strain of Aspergillus, in particular Aspergillus oryzae, a strain
of Alternaria, in particular Alternaria brassiciola, a strain of
Fusarium, in particular Fusarium solani, Fusarium solani pisi,
Fusarium roseum culmorum, or Fusarium roseum sambucium, a strain of
Helminthosporum, in particular Helminthosporum sativum, a strain of
Humicola, in particular Humicola insolens, a strain of Pseudomonas,
in particular Pseudomonas mendocina or Pseudomonas putida, a strain
of Rhizoctonia, in particular Rhizoctonia solani, a strain of
Streptomyces, in particular Streptomyces scabies, or a strain of
Ulocladium, in particular Ulocladium consortiale.
[0121] In a most preferred aspect, the cutinase or gene thereof is
obtained from a strain of Humicola insolens, in particular Humicola
insolens DSM 1800. Humicola insolens cutinase is described in WO
96/13580 which is hereby incorporated by reference. The cutinase
gene may encode a variant such as one of the variants disclosed in
WO 00/34450 and WO 01/92502, hereby incorporated by reference.
Preferred cutinase variants include variants listed in Example 2 of
WO 01/92502 which are hereby specifically incorporated by
reference. An effective amount of cutinase is between 0.01 and 400
LU/g DS, preferably from about 0.1 to 100 LU/g DS, more preferably,
1 to 50 LU/g DS.
[0122] Preferred examples of cutinase genes that can be used in the
invention are obtained from Fusarium solani (WO 90/09446; U.S. Pat.
No. 5,827,719; WO 00/34450; and WO 01/92502) and Humicola insolens
(WO 96/13580), and variants thereof.
[0123] In another preferred aspect, at least one esterase is a
phospholipase. As used herein, the term "phospholipase" is an
enzyme which has activity towards phospholipids. Phospholipids,
such as lecithin or phosphatidylcholine, consist of glycerol
esterified with two fatty acids in an outer (sn-1) and the middle
(sn-2) positions and esterified with phosphoric acid in the third
position. The phosphoric acid, in turn, may be esterified to an
amino-alcohol. Several types of phospholipase activity can be
distinguished, including phospholipases A.sub.1 and A.sub.2 which
hydrolyze one fatty acyl group (in the sn-1 and sn-2 position,
respectively) to form lysophospholipid; and lysophospholipase (or
phospholipase B), which hydrolyze the remaining fatty acyl group in
lysophospholipid. Phospholipase C and phospholipase D
(phosphodiesterases) release diacyl glycerol or phosphatidic acid,
respectively.
[0124] The term "phospholipase" includes enzymes with phospholipase
activity, e.g., phospholipase A (A.sub.1 or A.sub.2), phospholipase
B activity, phospholipase C activity, or phospholipase D activity.
The phospholipase activity may be provided by enzymes having other
activities as well, such as, e.g., a lipase with phospholipase
activity. In other aspects of the invention, phospholipase activity
is provided by an enzyme having essentially only phospholipase
activity and wherein the phospholipase enzyme activity is not a
side activity.
[0125] The phospholipase, or gene thereof may be of any origin,
e.g., of animal origin (e.g., mammalian such as from bovine or
porcine pancreas), or snake venom or bee venom. Alternatively, the
phospholipase may be of microbial origin, e.g., from filamentous
fungi, yeast, or bacteria, such as Aspergillus, e.g., Aspergillus
fumigatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus
japonicus, Aspergillus niger, and Aspergillus oryzae,
Dictyostelium, e.g., Dictyostelium discoideum; Fusarium, e.g.,
Fusarium culmorum, Fusarium heterosporum, Fusarium oxysporum,
Fusarium solani, and Fusarium venenatum; Mucor, e.g., Mucor
javanicus, Mucor mucedo, and Mucor subtilissimus; Neurospora, e.g.,
Neurospora crassa; Rhizomucor, e.g., Rhizomucor pusillus; Rhizopus,
e.g., Rhizopus arrhizus, Rhizopus japonicus, and Rhizopus
stolonifer, Sclerotinia, e.g., Sclerotinia libertiana;
Trichophyton, e.g., Trichophyton rubrum; Whetzelinia, e.g.,
Whetzelinia sclerotiorum; Bacillus, e.g., Bacillus megaterium and
Bacillus subtilis; Citrobacter, e.g., Citrobacter freundii;
Enterobacter, e.g., Enterobacter aerogenes and Enterobacter
cloacae; Edwardsiella, Edwardsiella tarda; Erwinia, e.g., Erwinia
herbicola; Escherichia, e.g., E. coli; Klebsiella, e.g., Klebsiella
pneumoniae; Proteus, e.g., Proteus vulgaris; Providencia, e.g.,
Providencia stuartii; Salmonella, e.g., Salmonella typhimurium;
Serratia, e.g., Serratia liquefasciens and Serratia marcescens;
Shigella, e.g., Shigella flexneri; Streptomyces, e.g., Streptomyces
violeceoruber; and Yersinia, e.g., Yersinia enterocolitica.
Preferred commercial phospholipases include LECITASE.TM. and
LECITASE.TM. ULTRA (available from Novozymes A/S, Denmark).
[0126] An effective amount of phospholipase is between 0.01 and 400
LU/g DS, preferably from about 0.1 to 100 LU/g DS, more preferably,
1 to 50 LU/g DS. Further optimization of the amount of
phospholipase can hereafter be obtained using standard procedures
known in the art.
[0127] Enzyme assays for phospholipases are well known in the art
(see, for example, Kim et al., 1997, Anal. Biochem. 250: 109-116;
Wu and Cho, 1994, Anal. Biochem. 221: 152-159; Hirashima et al.,
1983, Brain and Nerve 35: 811-817; and Chen et al., 1997, Infection
and Immun. 65: 405-411).
[0128] Preferred examples of phospholipase genes that can be used
in the invention are obtained from Fusarium venenatum (WO
00/028044), Aspergillus oryzae (WO 01/029222), Fusarium oxysporum
(WO 98/26057), Penicillum notatum (Masuda et al., 1991, European
Journal of Biochemistry 202: 783-787), Torulaspora delbrueckii
(Watanabe et al., 1994, FEMS Microbiology Letters 124: 29-34),
Saccharomyces cerevisiae (Lee at al., 1994, Journal of Biological
Chemistry 269: 19725-19730), Aspergillus (JP 10155493), Neurospora
crassa (EMBL O42791), and Schizosaccharomyces pombe (EMBL
O13857).
[0129] Proteases
[0130] In another preferred aspect, a protease may be useful in the
degradation of plant cell wall polysaccharides into one or more
products. The protease may be used, for example, to digest protein
to produce free amino nitrogen (FAN), where such free amino acids
function as nutrients for yeast, thereby enhancing the growth of
the yeast and, consequently, the production of ethanol. Proteases
may also liberate bound polysaccharide material.
[0131] The propagation of a fermenting microorganism with an
effective amount of at least one protease may reduce the lag time
of the fermenting microorganism. The action of the protease in the
propagation process is believed to directly or indirectly result in
the suppression or expression of genes which are detrimental or
beneficial, respectively, to the fermenting microorganism during
fermentation, thereby decreasing lag time and resulting in a faster
fermentation cycle.
[0132] Proteases are well known in the art and refer to enzymes
that catalyze the cleavage of peptide bonds. Suitable proteases
include fungal and bacterial proteases. Preferred proteases are
acidic proteases, i.e., proteases characterized by the ability to
hydrolyze proteins under acidic conditions below pH 7. Acid fungal
proteases or genes thereof can be obtained from Aspergillus, Mucor,
Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex,
Penicillium, Sclerotium, and Torulopsis. In a preferred aspect, a
protease or gene thereof is obtained from
[0133] Preferably, the protease is an aspartic acid protease, as
described, for example, in Handbook of Proteolytic Enzymes, Edited
by A. J. Barrett, N. D. Rawlings and J. F. Woessner, Academic
Press, San Diego, 1998, Chapter 270).
[0134] Enzyme assays for acid proteases, e.g., aspartic acid
proteases, are well known in the art (see, for example, Litvinov et
al., 1998, Bioorg. Khim. 24: 175-178).
[0135] Preferred examples of acid protease genes that can be used
in the invention are obtained from Aspergillus awamori (Berka et
al., 1990, Gene 86: 153-162), Aspergillus niger (Koaze et al.,
1964, Agr. Biol. Chem. Japan 28: 216), Aspergillus saitoi (Yoshida,
1954, J. Agr. Chem. Soc. Japan 28: 66), Aspergillus awamori
(Hayashida et al., 1977, Agric. Biol. Chem. 42: 927-933),
Aspergillus aculeatus (WO 95/02044), and Aspergillus oryzae (Berka
et al., 1993, Gene 125: 195-198).
[0136] Peroxidases
[0137] A peroxidase may be any peroxidase (e.g., EC 1.11.1.7), or
any fragment obtained therefrom, exhibiting peroxidase
activity.
[0138] The peroxidase or gene thereof can be obtained from plants
(e.g., horseradish or soybean peroxidase) or microorganisms (e.g.,
fungi or bacteria).
[0139] Some preferred fungi include strains belonging to the
subdivision Deuteromycotina, class Hyphomycetes, e.g., Fusarium,
Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces,
Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera,
in particular Fusarium oxysporum (DSM 2672), Humicola insolens,
Trichoderma resii, Myrothecium verrucaria (IFO 6113), Verticillum
alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754),
Caldariomyces fumago, Ulocladium chartarum, Embellisia alli, and
Dreschlera halodes.
[0140] Other preferred fungi include strains belonging to the
subdivision Basidiomycotina, class Basidiomycetes, e.g., Coprinus,
Phanerochaete, Coriolus or Trametes, in particular Coprinus
cinereus f. microsporus (IFO 8371), Coprinus macrorhizus,
Phanerochaete chrysosporium (e.g. NA-12), or Trametes (previously
called Polyporus), e.g., T versicolor (e.g. PR4 28-A).
[0141] Further preferred fungi include strains belonging to the
subdivision Zygomycotina, class Mycoraceae, e.g., Rhizopus or
Mucor, in particular Mucor hiemalis.
[0142] Some preferred bacteria include strains of the order
Actinomycetales, e.g. Streptomyces spheroides (ATTC 23965),
Streptomyces thermoviolaceus (IFO 12382), and Streptoverticillum
verticillium ssp. verticillium.
[0143] Other preferred bacteria include Rhodobacter sphaeroides,
Rhodomonas palustri, Streptococcus lactis, Pseudomonas purrocinia
(ATCC 15958), Pseudomonas fluorescens (NRRL B-11), and Bacillus
strains, e.g., Bacillus pumilus (ATCC 12905) and Bacillus
stearothermophilus.
[0144] Further preferred bacteria include strains belonging to
Myxococcus, e.g., M. virescens.
[0145] In a preferred aspect, a gene encoding a peroxidase is
obtained from a Coprinus sp., in particular, Coprinus macrorhizus
or Coprinus cinereus according to WO 92/16634.
[0146] In the present invention, genes encoding a peroxidase
include peroxidases and peroxidase active fragments obtained from
cytochromes, haemoglobin, or peroxidase enzymes.
[0147] One peroxidase unit (POXU) is the amount of enzyme which
under the following conditions catalyzes the conversion of 1
.mu.mole hydrogen peroxide per minute: 0.1 M phosphate buffer pH
7.0, 0.88 mM hydrogen peroxide, and 1.67 mM
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) at
30.degree. C. The reaction is followed for 60 seconds (15 seconds
after mixing) by the change in absorbance at 418 nm, which should
be in the range 0.15 to 0.30. For calculation of activity is used
an absorption coefficient of oxidized ABTS of 36 mM.sup.-1
cm.sup.-1 and a stoichiometry of one .mu.mole H.sub.2O.sub.2
converted per two .mu.mole ABTS oxidized.
[0148] Preferred examples of peroxidase genes that can be used in
the invention are obtained from Bjerkandera adusta (WO
2001/098469), Ceriporiopsis subvermispora (Conesa et al., 2002,
Journal of Biotechnology 93: 143-158), Coprinus cinereus (Conesa et
al., 2002, supra), Phanerochaete chrysosporium (Conesa et al.,
2002, supra), Phlebia radiata (Conesa et al., 2002, supra),
Pleurotus eryngii (Conesa et al., 2002, supra), and Trametes
versicolor (Conesa et al., 2002, supra).
[0149] Laccases
[0150] In the present invention, the laccase may be any laccase or
laccase-related enzyme including any laccase (EC 1.10.3.2), any
catechol oxidase (EC 1.10.3.1), any bilirubin oxidase (EC 1.3.3.5),
or any monophenol monooxygenase (EC 1.14.18.1).
[0151] The above-mentioned enzymes or genes thereof may be obtained
from a microorganism, i.e., bacteria or fungi (including
filamentous fungi and yeasts), or they may be obtained from
plants.
[0152] Suitable fungal sources include Aspergillus, Neurospora,
e.g., Neurospora crassa, Podospora, Botrytis, Collybia, Fomes,
Lentinus, Pleurotus, Trametes, e.g., Trametes villosa and Trametes
versicolor, Rhizoctonia, e.g., Rhizoctonia solani, Coprinus, e.g.,
Coprinus cinereus, Coprinus comatus, Coprinus friesii, and Coprinus
plicatilis, Psathyrella, e.g., Psathyrella condelleana, Panaeolus,
e.g., Panaeolus papilionaceus, Myceliophthora, e.g., Myceliophthora
thermophila, Scytalidium, e.g., Scytalidium thermophilum,
Polyporus, e.g., Polyporus pinsitus, Pycnoporus, e.g., Pycnoporus
cinnabarinus, Phlebia, e.g., Phlebia radita (WO 92/01046), or
Coriolus, e.g., Coriolus hirsutus (JP 2-238885). Suitable bacteria
sources are Bacillus.
[0153] A laccase or gene thereof is preferably obtained from
Coprinus, Myceliophthora, Polyporus, Pycnoporus, Scytalidium or
Rhizoctonia; in particular Coprinus cinereus, Myceliophthora
thermophila, Polyporus pinsitus, Pycnoporus cinnabarinus,
Scytalidium thermophilum, or Rhizoctonia solani.
[0154] Laccase activity (LACU) is determined from the oxidation of
syringaldazine under aerobic conditions. The violet colour produced
is photometered at 530 nm. The analytical conditions are 19 mM
syringaldazine, 23 mM acetate buffer, pH 5.5, 30.degree. C., 1
minute reaction time. One laccase unit (LACU) is the amount of
enzyme that catalyses the conversion of 1.0 .mu.mole syringaldazine
per minute at these conditions.
[0155] Laccase activity (LAMU) is determined from the oxidation of
syringaldazine under aerobic conditions. The violet colour produced
is photometered at 530 nm. The analytical conditions are 19 mM
syringaldazine, 23 mM Tris/maleate pH 7.5, 30.degree. C., 1 minute
reaction time. One laccase unit (LAMU) is the amount of enzyme that
catalyses the conversion of 1.0 .mu.mole syringaldazine per minute
at these conditions.
[0156] Preferred examples of laccase genes that can be used in the
invention are obtained from Cantharellus cibariusi (Ng et al.,
2004, Biochemical and Biophysical Research Communications 313:
37-41), Coprinus cinereus (WO 97/008325), Lentinula edodes (Nagai
et al., 2002, Applied Microbiology and Biotechnology 60: 327-335,
2002), Melanocarpus albomyces (Kiiskinen et al., 2004, FEBS Letters
576: 251-255, 2004), Myceliophthora thermophila (WO 95/006815),
Polyporus pinsitus (WO 96/000290), Rigidoporus lignosus (Garavaglia
et al., 2004, Journal of Molecular Biology 342: 1519-1531),
Rhizoctonia solani (WO 96/007988), Scytalidium thermophilum (WO
95/033837), and Tricholoma giganteum (Wang et al., 2004,
Biochemical and Biophysical Research Communications 315:
450-454).
Nucleic Acid Constructs
[0157] An isolated gene encoding a plant cell wall polysaccharide
degrading or converting enzyme, e.g., a cellulose-degrading enzyme,
hemicellulase, esterase, laccase, ligninase, protease, or
peroxidase may be manipulated in a variety of ways to provide for
expression of the enzyme. Manipulation of the gene prior to its
insertion into a vector may be desirable or necessary depending on
the expression vector. The techniques for modifying nucleotide
sequences utilizing recombinant DNA methods are well known in the
art.
[0158] The term "nucleic acid construct" as used herein refers to a
nucleic acid molecule, either single- or double-stranded, which is
isolated from a naturally occurring gene or which has been modified
to contain segments of nucleic acids in a manner that would not
otherwise exist in nature. The term nucleic acid construct is
synonymous with the term "expression cassette" when the nucleic
acid construct contains the control sequences required for
expression of a coding sequence of the present invention.
[0159] The term "control sequences" is defined herein to include
all components, which are necessary or advantageous for the
expression of a polypeptide having an enzyme activity of interest.
Each control sequence may be native or foreign to the nucleotide
sequence encoding the polypeptide. Such control sequences include,
but are not limited to, a leader, polyadenylation sequence,
propeptide sequence, promoter, signal peptide sequence, and
transcription terminator. At a minimum, the control sequences
include a promoter, and transcriptional and translational stop
signals. The control sequences may be provided with linkers for the
purpose of introducing specific restriction sites facilitating
ligation of the control sequences with the coding region of the
nucleotide sequence encoding a polypeptide.
[0160] The term "operably linked" as used herein refers to a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of the DNA
sequence such that the control sequence directs the expression of a
polypeptide.
[0161] When used herein the term "coding sequence" is intended to
cover a nucleotide sequence, which directly specifies the amino
acid sequence of its protein product. The boundaries of the coding
sequence are generally determined by an open reading frame, which
usually begins with the ATG start codon or alternative start codons
such as GTG and TTG. The coding sequence typically include DNA,
cDNA, and recombinant nucleotide sequences.
[0162] The term "expression" includes any step involved in the
production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0163] The control sequence may be an appropriate promoter
sequence, a nucleotide sequence which is recognized by a host for
expression of the gene. The promoter sequence contains
transcriptional control sequences which mediate the expression of
the polypeptide. The promoter may be any nucleotide sequence which
shows transcriptional activity in the host of choice including
mutant, truncated, and hybrid promoters, and may be obtained from
genes encoding extracellular or intracellular polypeptides either
homologous or heterologous to the host.
[0164] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900),
Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn
(WO 00/56900), Fusarium oxysporum trypsin-like protease (WO
96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma
reesei endoglucanase IV, Trichoderma reesei endoglucanase V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,
Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter
(a hybrid of the promoters from the genes for Aspergillus niger
neutral alpha-amylase and Aspergillus oryzae triose phosphate
isomerase); and mutant, truncated, and hybrid promoters
thereof.
[0165] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH1,ADH2/GAP), Saccharomyces cerevisiae triose phosphate
isomerase (TPI), Saccharomyces cerevisiae metallothionine (CUP1),
and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other
useful promoters for yeast hosts are described by Romanos et al.,
1992, Yeast 8: 423-488.
[0166] In the case of the degradation or conversion of plant cell
wall polysaccharides, the choice of the promoter necessarily
requires that it be induced by growth of the host on the
polysaccharide biomass.
[0167] The control sequence may also be a suitable transcription
terminator sequence, a sequence recognized by a host to terminate
transcription. The terminator sequence is operably linked to the 3'
terminus of the gene encoding an enzyme. Any terminator which is
functional in the host of choice may be used in the present
invention.
[0168] Preferred terminators for filamentous fungal hosts are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Trichoderma reesei CBHI, Aspergillus niger
alpha-glucosidase, and Fusarium oxysporum trypsin-like
protease.
[0169] Preferred terminators for yeast hosts are obtained from the
genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C(CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast hosts are described by Romanos et al., 1992, supra.
[0170] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA which is important for
translation by the host. The leader sequence is operably linked to
the 5' terminus of a gene. Any leader sequence that is functional
in the host of choice may be used in the present invention.
[0171] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0172] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0173] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3' terminus of a gene and which,
when transcribed, is recognized by the host as a signal to add
polyadenosine residues to transcribed mRNA. Any polyadenylation
sequence which is functional in the host of choice may be used in
the present invention.
[0174] Preferred polyadenylation sequences for filamentous fungal
hosts are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0175] Useful polyadenylation sequences for yeast hosts are
described by Guo and Sherman, 1995, Molecular Cellular Biology 15:
5983-5990.
[0176] The control sequence may also be a signal peptide coding
region that codes for an amino acid sequence linked to the amino
terminus of an enzyme and directs the encoded enzyme into the
cell's secretory pathway. The 5' end of the coding sequence of the
gene may inherently contain a signal peptide coding region
naturally linked in translation reading frame with the segment of
the coding region which encodes the secreted polypeptide.
Alternatively, the 5' end of the coding sequence may contain a
signal peptide coding region which is foreign to the coding
sequence. The foreign signal peptide coding region may be required
where the coding sequence does not naturally contain a signal
peptide coding region. Alternatively, the foreign signal peptide
coding region may simply replace the natural signal peptide coding
region in order to enhance secretion of the enzyme. However, any
signal peptide coding region which directs the expressed
polypeptide into the secretory pathway of a host cell of choice,
i.e., secreted into a culture medium, may be used in the present
invention.
[0177] Effective signal peptide coding regions for filamentous
fungal hosts are the signal peptide coding regions obtained from
the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger
neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei
aspartic proteinase, Humicola insolens cellulase, Humicola
lanuginosa lipase, Trichoderma reesei CBHI, Trichoderma reesei
CBHII, Trichoderma reesei EGI, and Trichoderma reesei CBHII.
[0178] Useful signal peptides for yeast hosts are obtained from the
genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces
cerevisiae invertase. Other useful signal peptide coding regions
are described by Romanos et al., 1992, supra.
[0179] The control sequence may also be a propeptide coding region
that codes for an amino acid sequence positioned at the amino
terminus of an enzyme. The resultant polypeptide is known as a
proenzyme or propolypeptide (or a zymogen in some cases). A
propolypeptide is generally inactive and can be converted to a
mature active enzyme by catalytic or autocatalytic cleavage of the
propeptide from the propolypeptide. The propeptide coding region
may be obtained from genes for Saccharomyces cerevisiae
alpha-factor, Rhizomucor miehei aspartic proteinase, and
Myceliophthora thermophila laccase (WO 95/33836).
[0180] Where both signal peptide and propeptide regions are present
at the amino terminus of an enzyme, the propeptide region is
positioned next to the amino terminus of the enzyme and the signal
peptide region is positioned next to the amino terminus of the
propeptide region.
[0181] It may also be desirable to add regulatory sequences which
allow the regulation of the expression of an enzyme relative to the
growth of the host. Examples of regulatory systems are those which
cause the expression of a gene to be turned on or off in response
to a chemical or physical stimulus, including the presence of a
regulatory compound. In yeast, the ADH2 system or GAL1 system may
be used. In filamentous fungi, the TAKA alpha-amylase promoter,
Aspergillus niger glucoamylase promoter, and Aspergillus oryzae
glucoamylase promoter may be used as regulatory sequences. Other
examples of regulatory sequences are those which allow for gene
amplification. In eukaryotic systems, these include the
dihydrofolate reductase gene which is amplified in the presence of
methotrexate, and the metallothionein genes which are amplified
with heavy metals. In these cases, the gene would be operably
linked with the regulatory sequence.
Expression Vectors
[0182] The various nucleic acids and control sequences described
above may be joined together to produce a recombinant expression
vector which may include one or more convenient restriction sites
to allow for insertion or substitution of a gene at such sites.
Alternatively, a gene may be expressed by inserting the nucleotide
sequence or a nucleic acid construct comprising the sequence into
an appropriate vector for expression. In creating the expression
vector, the coding sequence is located in the vector so that the
coding sequence is operably linked with the appropriate control
sequences for expression.
[0183] The term "expression vector" encompasses a DNA molecule,
linear or circular, that comprises a segment encoding an enzyme,
and which is operably linked to additional segments that provide
for its transcription.
[0184] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) which can be conveniently subjected to
recombinant DNA procedures and can bring about the expression of a
gene of interest. The choice of the vector will typically depend on
the compatibility of the vector with the host into which the vector
is to be introduced. The vectors may be linear or closed circular
plasmids.
[0185] The vector may be an autonomously replicating vector, i.e.,
a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the host, is integrated into the genome
and replicated together with the chromosome(s) into which it has
been integrated. Furthermore, a single vector or plasmid or two or
more vectors or plasmids which together contain the total DNA to be
introduced into the genome of the host, or a transposon may be
used.
[0186] The vectors preferably contain one or more selectable
markers which permit easy selection of transformed hosts. A
selectable marker is a gene the product of which provides for
biocide or viral resistance, resistance to heavy metals,
prototrophy to auxotrophs, and the like.
[0187] Suitable markers for yeast hosts are ADE2, HIS3, LEU2, LYS2,
MET3, TRP1, and URA3. Selectable markers for use in a filamentous
fungal host include, but are not limited to, amdS (acetamidase),
argB (ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Preferred for use in Aspergillus
are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus
oryzae and the bar gene of Streptomyces hygroscopicus. Preferred
for use in Trichoderma are bar and amdS.
[0188] The vectors preferably contain an element(s) that permits
integration of the vector into the hosts genome or autonomous
replication of the vector in the cell independent of the
genome.
[0189] For integration into the host genome, the vector may rely on
the gene's sequence or any other element of the vector for
integration of the vector into the genome by homologous or
nonhomologous recombination. Alternatively, the vector may contain
additional nucleotide sequences for directing integration by
homologous recombination into the genome of the host. The
additional nucleotide sequences enable the vector to be integrated
into the host genome at a precise location(s) in the chromosome(s).
To increase the likelihood of integration at a precise location,
the integrational elements should preferably contain a sufficient
number of nucleic acids, such as 100 to 10,000 base pairs,
preferably 400 to 10,000 base pairs, and most preferably 800 to
10,000 base pairs, which are highly homologous with the
corresponding target sequence to enhance the probability of
homologous recombination. The integrational elements may be any
sequence that is homologous with the target sequence in the genome
of the host. Furthermore, the integrational elements may be
non-encoding or encoding nucleotide sequences. On the other hand,
the vector may be integrated into the genome of the host by
non-homologous recombination.
[0190] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host in question. The origin of replication may
be any plasmid replicator mediating autonomous replication which
functions in a cell. The term "origin of replication" or "plasmid
replicator" is defined herein as a sequence that enables a plasmid
or vector to replicate in vivo. Examples of origins of replication
for use in a yeast host are the 2 micron origin of replication,
ARS1, ARS4, the combination of ARS1 and CEN3, and the combination
of ARS4 and CEN6. Examples of origins of replication useful in a
filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene
98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15:
9163-9175; WO 00/24883). Isolation of the AMA1 gene and
construction of plasmids or vectors comprising the gene can be
accomplished according to the methods disclosed in WO 00/24883.
[0191] More than one copy of a gene may be inserted into the host
to increase production of the gene product. An increase in the copy
number of the gene can be obtained by integrating at least one
additional copy of the gene into the host genome or by including an
amplifiable selectable marker gene with the nucleotide sequence
where cells containing amplified copies of the selectable marker
gene, and thereby additional copies of the gene, can be selected
for by cultivating the cells in the presence of the appropriate
selectable agent.
[0192] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Preparation of Spent Whole Fermentation Broth
[0193] In the methods of the present invention, the preparation of
a spent whole fermentation broth of a recombinant microorganism can
be achieved using any cultivation method known in the art resulting
in the expression of a plant cell wall polysaccharide degrading or
converting enzyme. Fermentation may, therefore, be understood as
comprising shake flask cultivation, small- or large-scale
fermentation (including continuous, batch, fed-batch, or solid
state fermentations) in laboratory or industrial fermenters
performed in a suitable medium and under conditions allowing the
cellulase to be expressed or isolated. The term "spent whole
fermentation broth" is defined herein as unfractionated contents of
fermentation material that includes culture medium, extracellular
proteins (e.g., enzymes), and cellular biomass. It is understood
that the term "spent whole fermentation broth" also encompasses
cellular biomass that has been lysed or permeabilized using methods
well known in the art.
[0194] Generally, the recombinant microorganism is cultivated in a
nutrient medium suitable for production of enzymes having plant
cell wall degrading or converting activity. The cultivation takes
place in a suitable nutrient medium comprising carbon and nitrogen
sources and inorganic salts, using procedures known in the art.
Suitable media are available from commercial suppliers or may be
prepared according to published compositions (e.g., in catalogues
of the American Type Culture Collection). Temperature ranges and
other conditions suitable for growth and cellulase production are
known in the art (see, e.g., Bailey, J. E., and Ollis, D. F.,
Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY,
1986).
[0195] The enzymes may be detected using methods known in the art
that are specific for the polypeptides, for example, as described
supra.
[0196] In the methods of the present invention, the spent whole
fermentation broth is preferably used "as is" without any
processing or minimal treatment such as refrigeration to preserve
activity, heat treatment to prevent or decrease organism viability,
or addition of chemical agents that prevent or decrease organism
viability.
[0197] The cellulose-degrading activity of the spent whole
fermentation broth may be determined using carboxymethyl cellulose
(CMC) as a substrate. Hydrolysis of carboxymethyl cellulose (CMC)
decreases the viscosity of the assay mixture, which may be
determined by a vibration viscosimeter (e.g., MIVI 3000 from
Sofraser, France). Determination of cellulose-degrading activity,
measured in terms of Cellulase Viscosity Unit (CEVU), quantifies
the amount of catalytic activity present in the spent whole
fermentation broth by measuring the ability of the sample to reduce
the viscosity of a solution of carboxymethyl cellulose (CMC). The
assay is carried out at 40.degree. C.; pH 9.0; 0.1M phosphate
buffer; time 30 minutes; CMC substrate (33.3 g/L carboxymethyl
cellulose Hercules 7 LFD); enzyme concentration approx. 3.3-4.2
CEVU/ml. The CEVU activity is calculated relative to a declared
enzyme standard, such as Celluzyme.TM. Standard 17-1194 (obtained
from Novozymes A/S, Bagsvaerd, Denmark).
[0198] Other enzyme activities can be measured as described
herein.
Supplements
[0199] In the methods of the present invention, the spent whole
fermentation broth may be supplemented with one or more enzyme
activities not expressed by the recombinant microorganism to
improve the degradation or conversion of plant cell wall
polysaccharides.
[0200] Preferred additional enzymes include, but are not limited
to, endoglucanase (cellulase), cellobiohydrolase, beta-glucosidase,
endo-beta-1,3(4)-glucanase, glucohydrolase, xyloglucanase,
xylanase, xylosidase, alpha-arabinofuranosidase,
alpha-glucuronidase, acetyl xylan esterase, mannanase, mannosidase,
alpha-galactosidase, mannan acetyl esterase, galactanase,
arabinanase, pectate lyase, pectin lyase, pectate lyase,
polygalacturonase, pectin acetyl esterase, pectin methyl esterase,
alpha-arabinofuranosidase, beta-galactosidase, galactanase,
arabinanase, alpha-arabinofuranosidase, rhamnogalacturonase,
rhamnogalacturonan lyase, rhamnogalacturonan acetyl esterase,
xylogalacturonosidase, xylogalacturonase, rhamnogalacturonan lyase,
lignin peroxidases, manganese-dependent peroxidases, hybrid
peroxidases, with combined properties of lignin peroxidases and
manganese-dependent peroxidases, and laccases.
[0201] The enzymes may be obtained from a suitable microbial or
plant source or by recombinant means as described herein or may be
obtained from commercial sources.
[0202] The additional enzyme(s) added as a supplement to the spent
whole broth may be used "as is" or may be purified. The term "as
is" as used herein refers to an enzyme preparation produced by
fermentation that undergoes no or minimal recovery and/or
purification. The term "purified" as used herein covers enzymes
free from other components from the organism from which it is
obtained. The term "purified" also covers enzymes free from
components from the native organism from which it is obtained. The
enzymes may be purified, with only minor amounts of other proteins
being present. The term "purified" as used herein also refers to
removal of other components, particularly other proteins and most
particularly other enzymes present in the cell of origin of the
enzyme. The enzyme may be "substantially pure," that is, free from
other components from the organism in which it is produced, that
is, for example, a host organism for enzymes produced by
recombinant means. In preferred aspect, the enzymes are at least
20% pure, preferably at least 40% pure, more preferably at least
60% pure, more preferably at least 80% pure, even more preferably
at least 90% pure, most preferably at least 95% pure, and even most
preferably at least 99% pure, as determined by SDS-PAGE.
[0203] Where the enzyme(s) is obtained from a suitable microbial or
plant source or by recombinant means, the enzyme may be recovered
using recovery methods well known in the art. For example, the
enzyme may be recovered from a nutrient medium by conventional
procedures including, but not limited to, centrifugation,
filtration, extraction, spray-drying, evaporation, or
precipitation.
[0204] The enzyme(s) may be purified by a variety of procedures
known in the art including, but not limited to, chromatography
(e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and
size exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing), differential solubility (e.g., ammonium
sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein
Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989).
[0205] The enzymes may also be obtained from commercial
sources.
[0206] Examples of cellulases suitable for use in the present
invention include, for example, CELLUCLAST.TM. (available from
Novozymes A/S), NOVOZYM.TM. 188 (available from Novozymes A/S).
Other commercially available preparations comprising cellulase
which may be used include CELLUZYME.TM., CEREFLO.TM. and
ULTRAFLO.TM. (Novozymes A/S), LAMINEX.TM. and SPEZYME.TM. CP
(Genencor Int.) and ROHAMENT.TM. 7069 W (Rohm GmbH). The cellulase
enzymes are added in amounts effective from about 0.001 to 5.0% wt.
of solids, more preferably from about 0.025% to 4.0% wt. of solids,
and most preferably from about 0.005% to 2.0% wt. of solids.
[0207] Preferred commercially available preparations comprising
xylanase include SHEARZYME.RTM., BIOFEED WHEAT.RTM., BIO-FEED
Plus.RTM. L, CELLUCLAST.RTM.i, ULTRAFLO.RTM., VISCOZYME.RTM.,
PENTOPAN MONO.RTM. BG, PULPZYME.RTM. HC (Novozymes A/S);
LAMINEX.RTM., SPEZYME.RTM. CP (Genencor Int.). The hemicellulase is
preferably added in an amount effective of from about 0.001 to 5.0%
wt. of solids, more preferably from about 0.025 to 4.0% wt. of
solids, and most preferably from about 0.005 to 2.0% wt. of
solids.
[0208] A preferred commercially available preparation comprising
hemicellulase includes VISCOZYME.TM. (Novozymes A/S). The
hemicellulase enzymes are added in amounts effective from about
0.001 to 5.0% wt. of solids, more preferably from about 0.025% to
4.0% wt. of solids, and most preferably from about 0.005% to 2.0%
wt. of solids.
[0209] Preferred commercial lipases include LECITASE.TM.,
LIPOLASE.TM. and LIPEX.TM. (Novozymes A/S, Denmark) and G AMANO.TM.
50 (Amano). Lipases are preferably added or present in amounts from
about 1 to 400 LU/g DS, preferably 1 to 10 LU/g DS, and more
preferably 1 to 5 LU/g DS.
[0210] Preferred commercial phospholipases include LECITASE.TM. and
LECITASE.TM. ULTRA (Novozymes A/S, Denmark).
[0211] Preferred commercial proteases include ALCALASE.TM.,
SAVINASE.TM., and NEUTRASE.TM. (Novozymes A/S), GC106 (Genencor
Int, Inc.), and NOVOZYM.TM. 50006 (Novozymes A/S).
[0212] The additional enzyme(s) used in the present invention may
be in any form suitable for use in the processes described herein,
such as, e.g., in the form of a dry powder or granulate, a
non-dusting granulate, a liquid, a stabilized liquid, or a
protected enzyme. Granulates may be produced, e.g., as disclosed in
U.S. Pat. Nos. 4,106,991 and 4,661,452, and may optionally be
coated by process known in the art. Liquid enzyme preparations may,
for instance, be stabilized by adding stabilizers such as a sugar,
a sugar alcohol or another polyol, lactic acid or another organic
acid according to established process. Protected enzymes may be
prepared according to the process disclosed in EP 238,216.
Processing of Plant Cell Wall Polysaccharides
[0213] The methods of the present invention may be used in the
production of monosaccharides, disaccharides, and polysaccharides
as chemical or fermentation feedstocks from biomass for the
production of organic products, chemicals and fuels, plastics, and
other products or intermediates. In particular, the value of
processing residues (dried distillers grain, spent grains from
brewing, sugarcane bagasse, etc.) can be increased by partial or
complete solubilization of cellulose or hemicellulose. In addition
to ethanol, some commodity and specialty chemicals that can be
produced from cellulose and hemicellulose include xylose, acetone,
acetate, glycine, lysine, organic acids (e.g., lactic acid),
1,3-propanediol, butanediol, glycerol, ethylene glycol, furfural,
polyhydroxyalkanoates, cis, cis-muconic acid, and animal feed
(Lynd, L. R., Wyman, C. E., and Gerngross, T. U., 1999,
Biocommodity engineering, Biotechnol. Prog., 15: 777-793;
Philippidis, G. P., 1996, Cellulose bioconversion technology, in
Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,
ed., Taylor & Francis; Washington, D.C., 179-212; and Ryu, D.
D. Y., and Mandels, M., 1980, Cellulases: biosynthesis and
applications, Enz. Microb. Technol., 2: 91-102). Potential
coproduction benefits extend beyond the synthesis of multiple
organic products from fermentable carbohydrate. Lignin-rich
residues remaining after biological processing of a plant cell wall
polysaccharide can be converted to lignin-obtained chemicals, or
used for power production (Lynd et al., 1999, supra; Philippidis,
1996, supra; Ryu and Mandels, 1980, supra).
[0214] Conventional methods used to process the plant cell wall
polysaccharides in accordance with the methods of the present
invention are well understood to those skilled in the art. The
methods of the present invention may be implemented using any
conventional biomass processing apparatus configured to operate in
accordance with the invention.
[0215] Such an apparatus may include, but is not limited to, a
batch-stirred reactor, a continuous flow stirred reactor with
ultrafiltration, a continuous plug-flow column reactor (Gusakov, A.
V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysis
of cellulose: 1. A mathematical model for a batch reactor process,
Enz. Microb. Technol., 7: 346-352), an attrition reactor (Ryu, S.
K., and Lee, J. M., 1983, Bioconversion of waste cellulose by using
an attrition bioreactor, Biotechnol. Bioeng., 25: 53-65), or a
reactor with intensive stirring induced by electromagnetic field
(Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y.,
Protas, O. V., 1996, Enhancement of enzymatic cellulose hydrolysis
using a novel type of bioreactor with intensive stirring induced by
electromagnetic field, Appl. Biochem. Biotechnol., 56:141-153).
[0216] The conventional methods include, but are not limited to,
saccharification, fermentation, separate hydrolysis and
fermentation (SHF), simultaneous saccharification and fermentation
(SSF), simultaneous saccharification and cofermentation (SSCF),
hybrid hydrolysis and fermentation (HHF), and direct microbial
conversion (DMC).
[0217] SHF uses separate process steps to first enzymatically
hydrolyze cellulose to glucose and then ferment glucose to ethanol.
In SSF, the enzymatic hydrolysis of cellulose and the fermentation
of glucose to ethanol are combined in one step (Philippidis, G. P.,
1996, Cellulose bioconversion technology, in Handbook on
Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor
& Francis, Washington, D.C., 179-212). SSCF includes the
coferementation of multiple sugars (Sheehan, J., and Himmel, M.,
1999, Enzymes, energy and the environment: A strategic perspective
on the U.S. Department of Energy's research and development
activities for bioethanol, Biotechnol. Prog., 15: 817-827). Hybrid
hydrolysis and fermentation (HHF) process includes two separate
steps carried out in the same reactor but at different
temperatures, high temperature enzymatic saccharification followed
by SSF at a lower temperature that the fermentation strain can
tolerate. DMC combines all three processes (cellulase production,
cellulose hydrolysis, and fermentation) in one step (Lynd, L. R.,
Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002,
Microbial cellulose utilization: Fundamentals and biotechnology,
Microbiol. Mol. Biol. Reviews, 66: 506-577).
[0218] "Fermentation" or "fermentation process" refers to any
fermentation process or any process comprising a fermentation step.
A fermentation process includes, without limitation, fermentation
processes used to produce fermentation products including alcohols
(e.g., arabinitol, butanol, ethanol, glycerol, methanol,
1,3-propanediol, sorbitol, and xylitol); organic acids (e.g.,
acetic acid, adipic acid, ascorbic acid, citric acid,
2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric
acid, gluconic acid, glucuronic acid, glutaric acid,
3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,
malonic acid, oxalic acid, propionic acid, succinic acid, and
xylonic acid); ketones (e.g., acetone); amino acids (e.g., aspartic
acid, glutamic acid, glycine, lysine, serine, and threonine);
and/or gases (e.g., methane, hydrogen (H.sub.2), carbon dioxide
(CO.sub.2), and carbon monoxide (CO).sub.j. Fermentation processes
also include fermentation processes used in the consumable alcohol
industry (e.g., beer and wine), dairy industry (e.g., fermented
dairy products), leather industry, and tobacco industry.
[0219] The present invention also relates to methods for producing
one or more organic substances, comprising: (a) saccharifying plant
cell wall polysaccharides with an effective amount of a spent whole
fermentation broth of a recombinant microorganism, wherein the
recombinant microorganism expresses one or more heterologous genes
encoding enzymes which degrade or convert the plant cell wall
polysaccharides into saccharified material; (b) fermenting the
saccharified material of step (a) with one or more fermenting
microoganisms; and (c) recovering the one or more organic
substances from the fermentation.
[0220] The organic substance can be any substance derived from the
fermentation. In a preferred aspect, the organic substance is an
alcohol. It will be understood that the term "alcohol" encompasses
an organic substance that contains one or more hydroxyl moieties.
In a more preferred aspect, the alcohol is arabinitol. In another
more preferred aspect, the alcohol is butanol. In another more
preferred aspect, the alcohol is ethanol. In another more preferred
aspect, the alcohol is glycerol. In another more preferred aspect,
the alcohol is methanol. In another more preferred aspect, the
alcohol is 1,3-propanediol. In another more preferred aspect, the
alcohol is sorbitol. In another more preferred aspect, the alcohol
is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and
Tsao, G. T., 1999, Ethanol production from renewable resources, in
Advances in Biochemical Engineering/Biotechnology, Scheper, T.,
ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241;
Silveira, M. M., and Jonas, R., 2002, The biotechnological
production of sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408;
Nigam, P., and Singh, D., 1995, Processes for fermentative
production of xylitol--a sugar substitute, Process Biochemistry 30
(2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H. P., 2003,
Production of acetone, butanol and ethanol by Clostridium
beijerinckii BA101 and in situ recovery by gas stripping, World
Journal of Microbiology and Biotechnology 19 (6): 595-603.
[0221] In another preferred aspect, the organic substance is an
organic acid. In another more preferred aspect, the organic acid is
acetic acid. In another more preferred aspect, the organic acid is
adipic acid. In another more preferred aspect, the organic acid is
ascorbic acid. In another more preferred aspect, the organic acid
is citric acid. In another more preferred aspect, the organic acid
is 2,5-diketo-D-gluconic acid. In another more preferred aspect,
the organic acid is formic acid. In another more preferred aspect,
the organic acid is fumaric acid. In another more preferred aspect,
the organic acid is glucaric acid. In another more preferred
aspect, the organic acid is gluconic acid. In another more
preferred aspect, the organic acid is glucuronic acid. In another
more preferred aspect, the organic acid is glutaric acid. In
another preferred aspect, the organic acid is 3-hydroxypropionic
acid. In another more preferred aspect, the organic acid is
itaconic acid. In another more preferred aspect, the organic acid
is lactic acid. In another more preferred aspect, the organic acid
is malic acid. In another more preferred aspect, the organic acid
is malonic acid. In another more preferred aspect, the organic acid
is oxalic acid. In another more preferred aspect, the organic acid
is propionic acid. In another more preferred aspect, the organic
acid is succinic acid. In another more preferred aspect, the
organic acid is xylonic acid. See, for example, Chen, R., and Lee,
Y. Y., 1997, Membrane-mediated extractive fermentation for lactic
acid production from cellulosic biomass, Appl. Biochem. Biotechnol.
63-65: 435-448.
[0222] In another preferred aspect, the organic substance is a
ketone. It will be understood that the term "ketone" encompasses an
organic substance that contains one or more ketone moieties. In
another more preferred aspect, the ketone is acetone. See, for
example, Qureshi and Blaschek, 2003, supra.
[0223] In another preferred aspect, the organic substance is an
aldehyde. In another more preferred aspect, the aldehyde is a
furfural.
[0224] In another preferred aspect, the organic substance is an
amino acid. In another more preferred aspect, the organic acid is
aspartic acid. In another more preferred aspect, the amino acid is
alanine. In another more preferred aspect, the amino acid is
arginine. In another more preferred aspect, the amino acid is
asparagine. In another more preferred aspect, the amino acid is
glutamine. In another more preferred aspect, the amino acid is
glutamic acid. In another more preferred aspect, the amino acid is
glycine. In another more preferred aspect, the amino acid is
histidine. In another more preferred aspect, the amino acid is
isoleucine. In another more preferred aspect, the amino acid is
leucine. In another more preferred aspect, the amino acid is
lysine. In another more preferred aspect, the amino acid is
methionine. In another more preferred aspect, the amino acid is
phenylalanine. In another more preferred aspect, the amino acid is
proline. In another more preferred aspect, the amino acid is
serine. In another more preferred aspect, the amino acid is
threonine. In another more preferred aspect, the amino acid is
tryptophan. In another more preferred aspect, the amino acid is
tyrosine. In another more preferred aspect, the amino acid is
valine. See, for example, Richard, A., and Margaritis, A., 2004,
Empirical modeling of batch fermentation kinetics for poly(glutamic
acid) production and other microbial biopolymers, Biotechnology and
Bioengineering 87 (4): 501-515.
[0225] In another preferred aspect, the organic substance is a gas.
In another more preferred aspect, the gas is methane (CH.sub.4). In
another more preferred aspect, the gas is hydrogen (H.sub.2). In
another more preferred aspect, the gas is carbon dioxide
(CO.sub.2). In another more preferred aspect, the gas is carbon
monoxide (CO). See, for example, Kataoka, N., A. Miya, and K.
Kiriyama, 1997, Studies on hydrogen production by continuous
culture system of hydrogen-producing anaerobic bacteria, Water
Science and Technology 36 (6-7): 41-47; and Gunaseelan V. N. in
Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic
digestion of biomass for methane production: A review.
[0226] Production of an organic substance from polysaccharides,
such as cellulose, typically requires four major steps. These four
steps are pretreatment, enzymatic hydrolysis, fermentation, and
recovery. Exemplified below is a process for producing ethanol, but
it will be understood that similar processes can be used to produce
other organic substances, for example, the substances described
above.
[0227] Pretreatment. In the pretreatment or pre-hydrolysis step,
the cellulosic material is heated to break down the lignin and
carbohydrate structure to make the cellulose fraction accessible to
cellulolytic enzymes. The heating is performed either directly with
steam or in slurry where a catalyst may also be added to the
material to speed up the reactions. Catalysts include strong acids,
such as sulfuric acid and SO.sub.2, or alkali, such as sodium
hydroxide. The purpose of the pre-treatment stage is to facilitate
the penetration of the enzymes and microorganisms. Cellulosic
biomass may also be subject to a hydrothermal steam explosion
pre-treatment (See U.S. Patent Application No. 20020164730).
[0228] Saccharification. In the enzymatic hydrolysis step, also
known as saccharification, enzymes as described herein are added to
the pretreated material to convert the cellulose fraction to
glucose and/or other sugars. The saccharification is generally
performed in stirred-tank reactors or fermentors under controlled
pH, temperature, and mixing conditions. A saccharification step may
last up to 200 hours. Saccharification may be carried out at
temperatures from about 30.degree. C. to about 65.degree. C., in
particular around 50.degree. C., and at a pH in the range between
about 4 and about 5, especially around pH 4.5. To produce glucose
that can be metabolized by yeast, the hydrolysis is typically
performed in the presence of a beta-glucosidase.
[0229] Fermentation. In the fermentation step, sugars, released
from the plant cell wall polysaccharides as a result of the
pretreatment and enzymatic hydrolysis steps, are fermented to one
or more organic substances, e.g., ethanol, by a fermenting
organism, such as yeast, or fermenting organisms. The fermentation
can also be carried out simultaneously with the enzymatic
hydrolysis in the same vessels, again under controlled pH,
temperature and mixing conditions. When saccharification and
fermentation are performed simultaneously in the same vessel, the
process is generally termed simultaneous saccharification and
fermentation or SSF.
[0230] Any suitable plant cell wall biomass may be used in a
fermentation process of the present invention. The plant cell wall
biomass is generally selected based on the desired fermentation
product(s) and the process employed, as is well known in the art.
Examples of substrates suitable for use in the methods of the
present invention, include cellulose-containing materials, such as
wood or plant residues or low molecular sugars DP.sub.1-3 obtained
from processed plant cell wall polysaccharides that can be
metabolized by the fermenting microorganism, and which may be
supplied by direct addition to the fermentation media.
[0231] The term "fermentation medium" will be understood to refer
to a medium before the fermenting microorganism(s) is(are) added,
such as, a medium resulting from a saccharification process, as
well as a medium used in a simultaneous saccharification and
fermentation process (SSF).
[0232] "Fermenting microorganism" refers to any microorganism
suitable for use in a desired fermentation process. Suitable
fermenting microorganisms according to the invention are able to
ferment, i.e., convert, sugars, such as glucose, xylose, arabinose,
mannose, galactose, or oligosaccharides, directly or indirectly
into the desired fermentation product(s). Examples of fermenting
microorganisms include fungal organisms, such as yeast. Preferred
yeast include strains of Saccharomyces spp., and in particular,
Saccharomyces cerevisiae. Commercially available yeast include,
e.g., Red Star.RTM./Lesaffre Ethanol Red (available from Red
Star/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a
division of Burns Philp Food Inc., USA), SUPERSTART (available from
Alltech), GERT STRAND (available from Gert Strand AB, Sweden) and
FERMIOL (available from DSM Specialties). Other microorganisms may
also be used depending the fermentation product(s) desired. These
other microorganisms include Gram positive bacteria, e.g.,
Lactobacillus such as Lactobacillus lactis, Propionibacterium such
as Propionibacterium freudenreichii; Clostridium sp. such as
Clostridium butyricum, Clostridium beijerinckii, Clostridium
diolis, Clostridium acetobutylicum, and Clostridium thermocellum;
Gram negative bacteria, e.g., Zymomonas such as Zymomonas mobilis;
and filamentous fungi, e.g., Rhizopus oryzae.
[0233] In a preferred aspect, the yeast is a Saccharomyces sp. In a
more preferred aspect, the yeast is Saccharomyces cerevisiae. In
another more preferred aspect, the yeast is Saccharomyces
distaticus. In another more preferred aspect, the yeast is
Saccharomyces uvarum. In another preferred aspect, the yeast is a
Kluyveromyces. In another more preferred aspect, the yeast is
Kluyveromyces marxianus. In another more preferred aspect, the
yeast is Kluyveromyces fragilis. In another preferred aspect, the
yeast is a Candida. In another more preferred aspect, the yeast is
Candida pseudotropicalis. In another more preferred aspect, the
yeast is Candida brassicae. In another preferred aspect, the yeast
is a Clavispora. In another more preferred aspect, the yeast is
Clavispora lusitaniae. In another more preferred aspect, the yeast
is Clavispora opuntiae. In another preferred aspect, the yeast is a
Pachysolen. In another more preferred aspect, the yeast is
Pachysolen tannophilus. In another preferred aspect, the yeast is a
Bretannomyces. In another more preferred aspect, the yeast is
Bretannomyces clausenii (Philippidis, G. P., 1996, Cellulose
bioconversion technology, in Handbook on Bioethanol: Production and
Utilization, Wyman, C. E., ed., Taylor & Francis, Washington,
D.C., 179-212).
[0234] Bacteria that can efficiently ferment glucose to ethanol
include, for example, Zymomonas mobilis and Clostridium
thermocellum (Philippidis, 1996, supra).
[0235] It is well known in the art that the organisms described
above can also be used to produce other organic substances, as
described herein.
[0236] The cloning of heterologous genes into Saccharomyces
cerevisiae (Chen, Z., Ho, N. W. Y., 1993, Cloning and improving the
expression of Pichia stipitis xylose reductase gene in
Saccharomyces cerevisiae, Appl. Biochem. Biotechnol., 39-40:
135-147; Ho, N. W. Y., Chen, Z, Brainard, A. P., 1998, Genetically
engineered Saccharomyces yeast capable of effectively cofermenting
glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859), or in
bacteria such as Escherichia coli (Beall, D. S., Ohta, K., Ingram,
L. O., 1991, Parametric studies of ethanol production from xylose
and other sugars by recombinant Escherichia coli, Biotech. Bioeng.
38: 296-303), Klebsiella oxytoca (Ingram, L. O., Gomes, P. F., Lai,
X., Moniruzzaman, M., Wood, B. E., Yomano, L. P., York, S. W.,
1998, Metabolic engineering of bacteria for ethanol production,
Biotechnol. Bioeng., 58: 204-214), and Zymomonas mobilis (Zhang,
M., Eddy, C., Deanda, K., Finkelstein, M., and Picataggio, S.,
1995, Metabolic engineering of a pentose metabolism pathway in
ethanologenic Zymomonas mobilis, Science, 267: 240-243; Deanda, K.,
Zhang, M., Eddy, C., and Picataggio, S., 1996, Development of an
arabinose-fermenting Zymomonas mobilis strain by metabolic pathway
engineering, Appl. Environ. Microbiol, 62: 4465-4470) has led to
the construction of organisms capable of converting hexoses and
pentoses to ethanol (cofermentation).
[0237] Yeast or other microorganisms are typically added to the
hydrolysate and the fermentation is allowed to proceed for 24-96
hours, such as 35-60 hours. The temperature is typically between
26-40.degree. C., in particular at about 32.degree. C., and at pH
3-6, in particular about pH 4-5.
[0238] In a preferred aspect, yeast is applied to the hydrolysate
and the fermentation proceeds for 24-96 hours, such as typically
35-60 hours. In another preferred aspect, the temperature is
generally between 26-40.degree. C., in particular about 32.degree.
C., and the pH is generally from pH 3 to 6, preferably about pH
4-5. Yeast cells are preferably applied in amounts of 10.sup.5 to
10.sup.12, preferably from 10.sup.7 to 10.sup.10, especially
5.times.10.sup.7 viable yeast count per ml of fermentation broth.
During the ethanol producing phase the yeast cell count should
preferably be in the range from 10.sup.7 to 10.sup.10, especially
around 2.times.10.sup.8. Further guidance in respect of using yeast
for fermentation can be found in, e.g., "The Alcohol Textbook"
(Editors K. Jacques, T. P. Lyons and D. R. Kelsall, Nottingham
University Press, United Kingdom 1999), which is hereby
incorporated by reference.
[0239] The most widely used process in the art is the simultaneous
saccharification and fermentation (SSF) process where there is no
holding stage for the saccharification, meaning that the fermenting
microorganism and enzyme are added together.
[0240] For ethanol production, following the fermentation the mash
is distilled to extract the ethanol. The ethanol obtained according
to the process of the invention may be used as, e.g., fuel ethanol;
drinking ethanol, i.e., potable neutral spirits, or industrial
ethanol.
[0241] A fermentation stimulator may be used in combination with
any of the enzymatic processes described herein to further improve
the fermentation process, and in particular, the performance of the
fermenting microorganism, such as, rate enhancement and ethanol
yield. A "fermentation stimulator" refers to stimulators for growth
of the fermenting microorganisms, in particular, yeast. Preferred
fermentation stimulators for growth include vitamins and minerals.
Examples of vitamins include multivitamins, biotin, pantothenate,
nicotinic acid, meso-inositol, thiamine, pyridoxine,
para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B,
C, D, and E. See, e.g., Alfenore et al., Improving ethanol
production and viability of Saccharomyces cerevisiae by a vitamin
feeding strategy during fed-batch process," Springer-Verlag (2002),
which is hereby incorporated by reference. Examples of minerals
include minerals and mineral salts that can supply nutrients
comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
[0242] Recovery. Following the fermentation, the organic substance
of interest is recovered from the mash by any method known in the
art. Such methods include, but are not limited to, chromatography
(e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and
size exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing), differential solubility (e.g., ammonium
sulfate precipitation), SDS-PAGE, distillation, or extraction. For
example, in an ethanol fermentation, the alcohol is separated from
the fermented plant cell wall polysaccharides and purified by
conventional methods of distillation. Ethanol with a purity of up
to about 96 vol. % ethanol can be obtained, which can be used as,
e.g., fuel ethanol; drinking ethanol, i.e., potable neutral
spirits; or industrial ethanol.
[0243] The present invention is further described by the following
examples which should not be construed as limiting the scope of the
invention.
EXAMPLES
Materials
[0244] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
Strains
[0245] Trichoderma reesei (synonym Hypocrea jecorina) RutC30 was
used as the source for cellulase. Trichoderma reesei RutC30 is
available from the American Type Culture Collection (ATCC 56765).
Trichoderma reesei SMA135-04 is a recombinant derivative of
Trichoderma reesei RutC30 that harbors multiple copies of the
Aspergillus oryzae beta-glucosidase gene expressed under the
transcriptional control of the Trichoderma reesei cbh1 gene
promoter.
Example 1
Construction of pAlLo01 Expression Vector
[0246] Expression vector pAlL01 was constructed by modifying pBANe6
(U.S. Pat. No. 6,461,837), which comprises a hybrid of the
promoters from the genes for Aspergillus niger neutral
alpha-amylase and Aspergillus oryzae triose phosphate isomerase
(NA2-tpi promoter), Aspergillus niger amyloglucosidase terminator
sequence (AMG terminator), and Aspergillus nidulans acetamidase
gene (amdS). All mutagenesis steps were verified by sequencing
using Big-Dye.TM. terminator chemistry (Applied Biosystems, Inc.,
Foster City, Calif.). Modification of pBANe6 was performed by first
eliminating three Nco I restriction sites at positions 2051, 2722,
and 3397 bp from the amdS selection marker by site-directed
mutagenesis. All changes were designed to be "silent" leaving the
actual protein sequence of the amdS gene product unchanged. Removal
of these three sites was performed simultaneously with a
GeneEditor.TM. in vitro Site-Directed Mutagenesis Kit (Promega,
Madison, Wis.) according to the manufacturer's instructions using
the following primers (underlined nucleotide represents the changed
base):
TABLE-US-00001 AMDS3NcoMut (2050): 5'-GTGCCCCATGATACGCCTCCGG-3'
(SEQ ID NO: 1) AMDS2NcoMut (2721): 5'-GAGTCGTATTTCCAAGGCTCCTGACC-3'
(SEQ ID NO: 2) AMDS1NcoMut (3396): 5'-GGAGGCCATGAAGTGGACCAACGG-3'
(SEQ ID NO: 3)
[0247] A plasmid comprising all three expected sequence changes was
then submitted to site-directed mutagenesis, using a
QuickChange.TM. Site-Directed Mutagenesis Kit (Stratagene, La
Jolla, Calif.), to eliminate the Nco I restriction site at the end
of the AMG terminator at position 1643. The following primers
(underlined nucleotide represents the changed base) were used for
mutagenesis:
TABLE-US-00002 Upper Primer to mutagenize the AMG terminator
sequence: (SEQ ID NO: 4)
5'-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACAG- 3' Lower Primer
to mutagenize the AMG terminator sequence: (SEQ ID NO: 5)
5'-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCTG- 3'
[0248] The last step in the modification of pBANe6 was the addition
of a new Nco I restriction site at the beginning of the polylinker
using a QuickChange.TM. Site-Directed Mutagenesis Kit and the
following primers (underlined nucleotides represent the changed
bases) to yield pAlL01 (FIG. 6).
TABLE-US-00003 Upper Primer to mutagenize the NA2-tpi promoter:
(SEQ ID NO: 6) 5'-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3'
Lower Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 7)
5'-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3'
Example 2
Construction of pMJ04 Expression Vector
[0249] Expression vector pMJ04 was constructed by PCR amplification
of the Trichoderma reesei exocellobiohydrolase 1 gene (cbh 1)
terminator from Trichoderma reesei RutC30 genomic DNA using primers
993429 (antisense) and 993428 (sense) shown below. The antisense
primer was engineered to have a PacI site at the 5'-end and a SpeI
site at the 3'-end of the sense primer.
TABLE-US-00004 Primer 993429 (antisense):
5'-AACGTTAATTAAGGAATCGTTTTGTGTTT-3' (SEQ ID NO: 8) Primer 993428
(sense): 5'-AGTACTAGTAGCTCCGTGGCGAAAGCCTG-3' (SEQ ID NO: 9)
[0250] Trichoderma reesei RutC30 genomic DNA was isolated using a
DNeasy Plant Maxi Kit (QIAGEN Inc., Valencia, Calif.).
[0251] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer (New England BioLabs, Beverly,
Mass.), 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic
DNA, 0.3 .mu.M primer 993429, 0.3 .mu.M primer 993428, and 2 units
of Vent polymerase (New England BioLabs, Beverly, Mass.). The
reactions were incubated in an Eppendorf Mastercycler 5333
programmed as follows: 30 cycles, each for 30 seconds at 94.degree.
C., 30 seconds at 55.degree. C., and 30 seconds at 72.degree. C.
(15 minute final extension).
[0252] The reaction products were isolated on a 1.0% agarose gel
using 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE)
buffer where a 229 bp product band was excised from the gel and
purified using a QIAGEN QIAquick Gel Extraction Kit according to
the manufacturer's instructions.
[0253] The resulting PCR fragment was digested with Pac I and Spe I
and ligated into pAlL01 digested with the same restriction enzymes
using a Rapid Ligaton Kit (Roche, Indianapolis, Ind.), to generate
pMJ04 (FIG. 2).
Example 3
Construction of pCaHj568 Expression Vector
[0254] Expression plasmid pCaHj568 was constructed from pCaHj170
(U.S. Pat. No. 5,763,254) and pMT2188. Plasmid pCaHj170 comprises
the Humicola insolens endoglucanase V (EGV) coding region. Plasmid
pMT2188 was constructed as follows: The pUC19 origin of replication
was PCR amplified from pCaHj483 (WO 98/00529) with primers 142779
and 142780 shown below. Primer 142780 introduces a Bbu I site in
the PCR fragment.
TABLE-US-00005 142779: (SEQ ID NO: 10)
5'-TTGAATTGAAAATAGATTGATTTAAAACTTC-3' 142780: (SEQ ID NO: 11)
5'-TTGCATGCGTAATCATGGTCATAGC-3'
[0255] The Expand PCR System (Roche Molecular Biochemicals, Basel,
Switserland) was used for the amplification following the
manufacturer's instructions for this and the subsequent PCR
amplifications. PCR products were separated on an agarose gel and
an 1160 bp fragment was isolated and purified using a Jetquick Gel
Extraction Spin Kit (Genomed, Wielandstr, Germany).
[0256] The URA3 gene was amplified from the general Saccharomyces
cerevisiae cloning vector pYES2 (Invitrogen, Carlsbad, Calif.)
using primers 140288 and 142778 below. Primer 140288 introduces an
Eco RI site in the PCR fragment.
TABLE-US-00006 140288: (SEQ ID NO: 12)
5'-TTGAATTCATGGGTAATAACTGATAT-3' 142778: (SEQ ID NO: 13)
5'-AAATCAATCTATTTTCAATTCAATTCATCATT-3'
[0257] PCR products were separated on an agarose gel and an 1126 bp
fragment was isolated and purified using a Jetquick Gel Extraction
Spin Kit.
[0258] The two PCR fragments were fused by mixing and amplified
using primers 142780 and 140288 shown above by overlap method
splicing (Horton et al., 1989, Gene 77: 61-68). PCR products were
separated on an agarose gel and a 2263 bp fragment was isolated and
purified using a Jetquick Gel Extraction Spin Kit.
[0259] The resulting fragment was digested with Eco RI and Bbu I
and ligated to the largest fragment of pCaHj483 digested with the
same enzymes. The ligation mixture was used to transform pyrF E.
coli strain DB6507 (ATCC 35673) made competent by the method of
Mandel and Higa, 1970, J. Mol. Biol. 45: 154. Transformants were
selected on solid M9 medium (Sambrook et al., 1989, Molecular
Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor
Laboratory Press) supplemented per liter with 1 g of casamino
acids, 500 .mu.g of thiamine, and 10 mg of kanamycin. A plasmid
from one transformant was isolated and designated pCaHj527 (FIG.
3).
[0260] The NA2/tpi promoter present on pCaHj527 was subjected to
site-directed mutagenesis by a simple PCR approach. Nucleotides
134-144 were converted from GTACTAAAACC to CCGTTAAATTT using
mutagenic primer 141223:
TABLE-US-00007 Primer 141223: (SEQ ID NO: 14)
5'-GGATGCTGTTGACTCCGGAAATTTAACGGTTTGGTCTTGCATCCC- 3'
Nucleotides 423-436 were converted from ATGCAATTTAAACT to
CGGCAATTTAACGG using mutagenic primer 141222:
TABLE-US-00008 Primer 141222: (SEQ ID NO: 15)
5'-GGTATTGTCCTGCAGACGGCAATTTAACGGCTTCTGCGAATCGC-3'
[0261] The resulting plasmid was designated pMT2188 (FIG. 4).
[0262] The Humicola insolens endoglucanase V coding region was
transferred from pCaHj170 as a Bam HI-Sal I fragment into pMT2188
digested with Bam HI and Xho I to generate pCaHj568 (FIG. 5).
Example 4
Construction of pMJ05 Expression Vector
[0263] Expression vector pMJ05 was constructed by PCR amplifying
the 915 bp Humicola insolens endoglucanase V coding region from
pCaHj568 using primers HiEGV-F and HiEGV-R shown below.
TABLE-US-00009 HiEGV-F (sense): (SEQ ID NO: 16)
5'-AAGCTTAAGCATGCGTTCCTCCCCCCTCC-3' HiEGV-R (antisense): (SEQ ID
NO: 17) 5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3'
[0264] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 10 ng/.mu.l
pCaHj568 plasmid, 0.3 .mu.M HiEGV-F primer, 0.3 .mu.M HiEGV-R
primer, and 2 U of Vent polymerase. The reactions were incubated in
an Eppendorf Mastercycler 5333 programmed as follows: 5 cycles each
for 30 seconds at 94.degree. C., 30 seconds at 50.degree. C., and
60 seconds at 72.degree. C., followed by 25 cycles each for 30
seconds at 94.degree. C., 30 seconds at 65.degree. C., and 120
seconds at 72.degree. C. (5 minute final extension). The reaction
products were isolated on a 1.0% agarose gel using TAE buffer where
a 937 bp product band was excised from the gel and purified using a
QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0265] This 937 bp purified fragment was used as template DNA for
subsequent amplifications using the following primers:
TABLE-US-00010 HiEGV-R (antisense): (SEQ ID NO: 18)
5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3' HIEGV-F-overlap (sense):
(SEQ ID NO: 19) 5'-ACCGCGGACTGCGCATCATGCGTTCCTCCCCCCTCC-3'
Primer sequences in italics are homologous to 17 bp of the
Trichoderma reesei cbh1 promoter and underlined primer sequences
are homologous to 29 bp of the Humicola insolens endoglucanase V
coding region. The 36 bp overlap between the promoter and the
coding sequence allowed precise fusion of the 994 bp fragment
comprising the Trichoderma reesei cbh1 promoter to the 918 bp
fragment comprising the Humicola insolens endoglucanase V open
reading frame.
[0266] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 1 ul of 937 bp
purified PCR fragment, 0.3 .mu.M HiEGV-F-overlap primer, 0.3 .mu.M
HiEGV-R primer, and 2 U of Vent polymerase. The reactions were
incubated in an Eppendorf Mastercycler 5333 programmed as follows:
5 cycles each for 30 seconds at 94.degree. C., 30 seconds at
50.degree. C., and 60 seconds at 72.degree. C., followed by 25
cycles each for 30 seconds at 94.degree. C., 30 seconds at
65.degree. C., and 120 seconds at 72.degree. C. (5 minute final
extension). The reaction products were isolated on a 1.0% agarose
gel using TAE buffer where a 945 bp product band was excised from
the gel and purified using a QIAquick Gel Extraction Kit according
to the manufacturer's instructions.
[0267] A separate PCR was performed to amplify the Trichoderma
reesei cbh1 promoter sequence extending from 994 bp upstream of the
ATG start codon of the gene from Trichoderma reesei RutC30 genomic
DNA using the following primers (sense primer was engineered to
have a Sal I restriction site at the 5'-end):
TABLE-US-00011 TrCBHIpro-F (sense): (SEQ ID NO: 20)
5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' TrCBHIpro-R (antisense): (SEQ
ID NO: 21) 5'-GATGCGCAGTCCGCGGT-3'
[0268] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 100 ng of
Trichoderma reesei RutC30 genomic DNA, 0.3 .mu.M TrCBH1pro-F
primer, 0.3 .mu.M TrCBHIpro-R primer, and 2 U of Vent polymerase.
The reactions were incubated in an Eppendorf Mastercycler 5333
programmed as follows: 30 cycles each for 30 seconds at 94.degree.
C., 30 seconds at 55.degree. C., and 120 seconds at 72.degree. C.
(5 minute final extension). The reaction products were isolated on
a 1.0% agarose gel using TAE buffer where a 998 bp product band was
excised from the gel and purified using a QIAquick Gel Extraction
Kit according to the manufacturer's instructions.
[0269] The 998 bp purified PCR fragment was used as template DNA
for subsequent amplifications using the following primers:
TABLE-US-00012 TrCBHIpro-F: (SEQ ID NO: 22)
5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' TrCBHIpro-R-overlap: (SEQ ID
NO: 23) 5'-GGAGGGGGGAGGAACGCATGATGCGCAGTCCGCGGT-3'
[0270] Sequences in italics are homologous to 17 bp of the
Trichoderma reesei cbh1 promoter and underlined sequences are
homologous to 29 bp of the Humicola insolens endoglucanase V coding
region. The 36 bp overlap between the promoter and the coding
sequence allowed precise fusion of the 994 bp fragment comprising
the Trichoderma reesei cbh1 promoter to the 918 bp fragment
comprising the Humicola insolens endoglucanase V open reading
frame.
[0271] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 1 .mu.l of 998 bp
purified PCR fragment, 0.3 .mu.M TrCBH1 pro-F primer, 0.3 .mu.M
TrCBH1 pro-R-overlap primer, and 2 U of Vent polymerase. The
reactions were incubated in an Eppendorf Mastercycler 5333
programmed as follows: 5 cycles each for 30 seconds at 94.degree.
C., 30 seconds at 50.degree. C., and 60 seconds at 72.degree. C.,
followed by 25 cycles each for 30 seconds at 94.degree. C., 30
seconds at 65.degree. C., and 120 seconds at 72.degree. C. (5
minute final extension). The reaction products were isolated on a
1.0% agarose gel using TAE buffer where a 1017 bp product band was
excised from the gel and purified using a QIAquick Gel Extraction
Kit according to the manufacturer's instructions.
[0272] The 1017 bp Trichoderma reesei cbh1 promoter PCR fragment
and the 945 bp Humicola insolens endoglucanase V PCR fragments were
used as template DNA for subsequent amplification using the
following primers to precisely fuse the 994 bp Trichoderma reesei
cbh1 promoter to the 918 bp Humicola insolens endoglucanase V
coding region using overlapping PCR:
TABLE-US-00013 TrCBHIpro-F: (SEQ ID NO: 24)
5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' HiEGV-R: (SEQ ID NO: 25)
5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3'
[0273] The amplification reactions (50 ul) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 0.3 .mu.M
TrCBH1pro-F primer, 0.3 .mu.M HiEGV-R primer, and 2 U of Vent
polymerase. The reactions were incubated in an Eppendorf
Mastercycler 5333 programmed as follows: 5 cycles each for 30
seconds at 94.degree. C., 30 seconds at 50.degree. C., and 60
seconds at 72.degree. C., followed by 25 cycles each for 30 seconds
at 94.degree. C., 30 seconds at 65.degree. C., and 120 seconds at
72.degree. C. (5 minute final extension). The reaction products
were isolated on a 1.0% agarose gel using TAE buffer where a 1926
bp product band was excised from the gel and purified using a
QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0274] The resulting 1926 bp fragment was cloned into
pCR-Blunt-II-TOPO (Invitrogen, Carlsbad, Calif.) using a ZeroBlunt
TOPO PCR Cloning Kit following the manufacturer's protocol. The
resulting plasmid was digested with Not I and Sal I and the 1926 bp
fragment was purified and ligated into pMJ04, which was also
digested with the same two restriction enzymes, to generate pMJ05
(FIG. 6).
Example 5
Construction of pSMai130 Expression Vector
[0275] A 2586 bp DNA fragment spanning from the ATG start codon to
the TAA stop codon of the Aspergillus oryzae beta-glucosidase
coding sequence (SEQ ID NO: 26 for cDNA sequence and SEQ ID NO: 27
for the deduced amino acid sequence; E. coli DSM 14240) was
amplified by PCR from pJaL660 (WO 2002/095014) as template with
primers 993467 (sense) and 993456 (antisense) shown below. A Spe I
site was engineered at the 5' end of the antisense primer to
facilitate ligation. Primer sequences in italics are homologous to
24 bp of the Trichoderma reesei cbh1 promoter and underlined
sequences are homologous to 22 bp of the Aspergillus oryzae
beta-glucosidase coding region.
TABLE-US-00014 Primer 993467: (SEQ ID NO: 28)
5'-ATAGTCAACCGCGGACTGCGCATCATGAAGCTTGGTTGGATCGAGG- 3' Primer
993456: (SEQ ID NO: 29) 5'- ACTAGTTTACTGGGCCTTAGGCAGCG-3'
[0276] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer (Invitrogen, Carlsbad, Calif.), 0.25 mM dNTPs,
10 ng of pJaL660 plasmid, 6.4 .mu.M primer 993467, 3.2 .mu.M primer
993456, 1 mM MgCl.sub.2, and 2.5 U of Pfx polymerase (Invitrogen,
Carlsbad, Calif.). The reactions were incubated in an Eppendorf
Mastercycler 5333 programmed as follows: 30 cycles each for 60
seconds at 94.degree. C., 60 seconds at 55.degree. C., and 180
seconds at 72.degree. C. (15 minute final extension). The reaction
products were isolated on a 1.0% agarose gel using TAE buffer where
a 2586 bp product band was excised from the gel and purified using
a QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0277] A separate PCR was performed to amplify the Trichoderma
reesei cbh1 promoter sequence extending from 1000 bp upstream of
the ATG start codon of the gene, using primer 993453 (sense) and
primer 993463 (antisense) shown below to generate a 1000 bp PCR
fragment. Primer sequences in italics are homologous to 24 bp of
the Trichoderma reesei cbh1 promoter and underlined primer
sequences are homologous to 22 bp of the Aspergillus oryzae
beta-glucosidase coding region. The 46 bp overlap between the
promoter and the coding sequence allows precise fusion of the 1000
bp fragment comprising the Trichoderma reesei cbh1 promoter to the
2586 bp fragment comprising the Aspergillus oryzae beta-glucosidase
open reading frame.
TABLE-US-00015 Primer 993453: (SEQ ID NO: 30)
5'-GTCGACTCGAAGCCCGAATGTAGGAT-3' Primer 993463: (SEQ ID NO: 31)
5'-CCTCGATCCAACCAAGCTTCATGATGCGCAGTCCGCGGTTGACTA- 3'
[0278] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer, 0.25 mM dNTPs, 100 ng of Trichoderma reesei
RutC30 genomic DNA, 6.4 .mu.M primer 993453, 3.2 .mu.M primer
993463, 1 mM MgCl.sub.2, and 2.5 U of Pfx polymerase The reactions
were incubated in an Eppendorf Mastercycler 5333 programmed as
follows: 30 cycles each for 60 seconds at 94.degree. C., 60 seconds
at 55.degree. C., and 180 seconds at 72.degree. C. (15 minute final
extension). The reaction products were isolated on a 1.0% agarose
gel using TAE buffer where a 1000 bp product band was excised from
the gel and purified using a QIAquick Gel Extraction Kit according
to the manufacturer's instructions.
[0279] The purified fragments were used as template DNA for
subsequent amplification using primer 993453 (sense) and primer
993456 (antisense) shown above to precisely fuse the 1000 bp
Trichoderma reesei cbh1 promoter to the 2586 bp Aspergillus oryzae
beta-glucosidase fragment by overlapping PCR.
[0280] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer, 0.25 mM dNTPs, 6.4 .mu.M primer 99353, 3.2
.mu.M primer 993456, 1 mM MgCl.sub.2, and 2.5 U of Pfx polymerase.
The reactions were incubated in an Eppendorf Mastercycler 5333
programmed as follows: 30 cycles each for 60 seconds at 94.degree.
C., 60 seconds at 60.degree. C., and 240 seconds at 72.degree. C.
(15 minute final extension).
[0281] The resulting 3586 bp fragment was digested with SalI and
SpeI and ligated into pMJ04, digested with the same two restriction
enzymes, to generate pSMai130 (FIG. 7).
Example 6
Construction of pSMai135
[0282] The Aspergillus oryzae beta-glucosidase coding region (WO
2002/095014, E. coli DSM 14240, minus the signal sequence, see FIG.
8, DNA sequence (SEQ ID NO: 32) and deduced amino acid sequence
(SEQ ID NO: 33)) from Lys-20 to the TAA stop codon was PCR
amplified from pJaL660 (WO 2002/095014) as template with primer
993728 (sense) and primer 993727 (antisense) shown below. Sequences
in italics are homologous to 20 bp of the Humicola insolens
endoglucanase V signal sequence and sequences underlined are
homologous to 22 bp of the Aspergillus oryzae beta-glucosidase
coding region. A Spe I site was engineered into the 5' end of the
antisense primer.
TABLE-US-00016 Primer 993728: (SEQ ID NO: 34)
5'-TGCCGGTGTTGGCCCTTGCCAAGGATGATCTCGCGTACTCCC-3' Primer 993727:
(SEQ ID NO: 35) 5'-GACTAGTCTTACTGGGCCTTAGGCAGCG-3'
[0283] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer, 0.25 mM dNTPs, 10 ng/.mu.l Jal660, 6.4 .mu.M
primer 993728, 3.2 .mu.M primer 993727, 1 mM MgCl.sub.2, and 2.5 U
of Pfx polymerase. The reactions were incubated in an Eppendorf
Mastercycler 5333 programmed as follows: 30 cycles each for 60
seconds at 94.degree. C., 60 seconds at 55.degree. C., and 180
seconds at 72.degree. C. (15 minute final extension). The reaction
products were isolated on a 1.0% agarose gel using TAE buffer where
a 2523 bp product band was excised from the gel and purified using
a QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0284] A separate PCR amplification was performed to amplify 1000
bp of the Trichoderma reesei Cel7A cellobiohydrolase 1 promoter and
63 bp of the putative Humicola insolens endoglucanase V signal
sequence (ATG start codon to Ala-21, FIG. 9, SEQ ID NOs: 36 (DNA
sequence) and 37 (deduced amino acid sequence; accession no.
AAB03660 for DNA sequence), using primer 993724 (sense) and primer
993729 (antisense) shown below. Primer sequences in italics are
homologous to 20 bp of the Humicola insolens endoglucanase V signal
sequence and underlined primer sequences are homologous to 22 bp of
the Aspergillus oryzae beta-glucosidase coding region. Plasmid
pMJ05, which comprises the Humicola insolens endoglucanase V coding
region under the control of the cbh1 promoter, was used as a
template to generate a 1063 bp fragment comprising the Trichoderma
reesei cbh1 promoter/Humicola insolens endoglucanase V signal
sequence fragment. A 42 bp of overlap was shared between the
Trichoderma reesei cbh1 promoter/Humicola insolens endoglucanase V
signal sequence and the Aspergillus oryzae coding sequence to
provide a perfect linkage between the promoter and the ATG start
codon of the 2523 bp Aspergillus oryzae beta-glucosidase
fragment.
TABLE-US-00017 Primer 993724: (SEQ ID NO: 38)
5'-ACGCGTCGACCGAATGTAGGATTGTTATCC-3' Primer 993729: (SEQ ID NO: 39)
5'-GGGAGTACGCGAGATCATCCTTGGCAAGGGCCAACACCGGCA-3'
[0285] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer, 0.25 mM dNTPs, 10 ng/.mu.l pMJ05, 6.4 .mu.M
primer 993728, 3.2 .mu.M primer 993727, 1 mM MgCl.sub.2, and 2.5 U
of Pfx polymerase. The reactions were incubated in an Eppendorf
Mastercycler 5333 programmed as follows: 30 cycles each for 60
seconds at 94.degree. C., 60 seconds at 60.degree. C., and 240
seconds at 72.degree. C. (15 minute final extension). The reaction
products were isolated on a 1.0% agarose gel using TAE buffer where
a 1063 bp product band was excised from the gel and purified using
a QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0286] The purified overlapping fragments were used as a template
for amplification using primer 993724 (sense) and primer 993727
(antisense) described above to precisely fuse the 1063 bp
Trichoderma reesei cbh1 promoter/Humicola insolens endoglucanase V
signal sequence fragment to the 2523 bp of Aspergillus oryzae
beta-glucosidase fragment by overlapping PCR.
[0287] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer, 0.25 mM dNTPs, 6.4 .mu.M primer 993724, 3.2
.mu.M primer 993727, 1 mM MgCl.sub.2, and 2.5 U of Pfx polymerase.
The reactions were incubated in an Eppendorf Mastercycler 5333
programmed as follows: 30 cycles each for 60 seconds at 94.degree.
C., 60 seconds at 60.degree. C., and 240 seconds at 72.degree. C.
(15 minute final extension). The reaction products were isolated on
a 1.0% agarose gel using TAE buffer where a 3591 bp product band
was excised from the gel and purified using a QIAquick Gel
Extraction Kit according to the manufacturer's instructions.
[0288] The resulting 3591 bp fragment was digested with Sal I and
Spe I and ligated into pMJ04 digested with the same restriction
enzymes to generate pSMai135 (FIG. 10).
Example 7
Expression of Aspergillus oryzae beta-glucosidase in Trichoderma
reesei
[0289] Plasmid pSMai130, in which the Aspergillus oryzae
beta-glucosidase is expressed from the cbh1 promoter and native
secretion signal (FIG. 8), or pSMai135 encoding the mature
Aspergillus oryzae beta-glucosidase enzyme linked to the Humicola
insolens endoglucanase V secretion signal (FIG. 9), was introduced
into Trichoderma reesei RutC30 by PEG-mediated transformation as
described below. Both plasmids contain the Aspergillus nidulans
amdS gene to enable transformants to grow on acetamide as the sole
nitrogen source.
[0290] Trichoderma reesei RutC30 was cultivated at 27.degree. C.
and 90 rpm in 25 ml of YP medium (composed per liter of 10 g of
yeast extract and 20 g of Bactopeptone) supplemented with 2% (w/v)
glucose and 10 mM uridine for 17 hours. Mycelia were collected by
filtration using Millipore's Vacuum Driven Disposable Filtration
System (Millipore, Bedford, Mass.) and washed twice with deionized
water and twice with 1.2 M sorbitol. Protoplasts were generated by
suspending the washed mycelia in 20 ml of 1.2 M sorbitol containing
15 mg of Glucanex (Novozymes A/S, Bagsvaerd, Denmark) per ml and
0.36 units of chitinase (Sigma Chemical Co., St. Louis, Mo.) per ml
and incubating for 15-25 minutes at 34.degree. C. with gentle
shaking at 90 rpm. Protoplasts were collected by centrifuging for 7
minutes at 400.times.g and washed twice with cold 1.2 M sorbitol.
The protoplasts were counted using a haemocytometer and
re-suspended in STC (1M sorbitol, 10 mM Tris-HCl, pH 6.5, 10 mM
CaCl.sub.2) to a final concentration of 1.times.10.sup.8
protoplasts per ml. Excess protoplasts were stored in a Cryo
1.degree. C. Freezing Container (Nalgene, Rochester, N.Y.) at
-80.degree. C.
[0291] Approximately 7 .mu.g of Pme I digested expression plasmid
(pSMai130 or pSMai135) was added to 100 .mu.l of protoplast
solution and mixed gently, followed by 260 .mu.l of PEG buffer (60%
PEG-4000, 10 mM Tris-HCl, pH 6.5, 10 mM CaCl.sub.2), mixed, and
incubated at room temperature for 30 minutes. STC (3 ml) was then
added and mixed and then the transformation solution was plated
onto COVE plates (composed per liter of 342.3 g of sucrose, 10 ml
of 1 M acetamide solution, 10 ml of 1.5 M CsCl solution, 25 g of
agar, and 20 ml of Cove salts solution; Cove salts solution was
composed per liter of 26 g of KCl, 26 g of MgSO.sub.4.7H.sub.2O, 76
g of KH.sub.2PO.sub.4, and 50 ml of Cove trace metals solution;
Cove trace metals solution was composed per liter of 0.04 g of
Na.sub.2B.sub.4O.sub.7.10H.sub.2O, 0.4 g of CuSO.sub.4.5H.sub.2O,
1.2 g of FeSO.sub.4.7H.sub.2O, 0.7 g of MnSO.sub.4.H.sub.2O, 0.8 g
of Na.sub.7MoO.sub.7.2H.sub.2O, and 10 g of ZnSO.sub.4.7H.sub.2O).
The plates were incubated at 28.degree. C. for 5-7 days.
Transformants were subcultured onto COVE2 plates (composed per
liter of 30 g of sucrose, 10 ml of 1 M acetamide solution, 20 ml of
Cove salts solution, and 25 g of agar) and grown at 28.degree.
C.
[0292] One hundred and ten amdS positive transformants were
obtained with pSMai130 and 65 transformants with pSMai135. Twenty
pSMai130 (native secretion signal) and 67 pSMai135 (heterologous
secretion signal) transformants were subcultured onto fresh plates
containing acetamide and allowed to sporulate for 7 days at
28.degree. C.
[0293] The 20 pSMA130 and 67 pSMA135 Trichoderma reesei
transformants were cultivated in 125 ml baffled shake flasks
containing 25 ml of cellulase-inducing medium at pH 6.0 inoculated
with spores of the transformants and incubated at 28.degree. C. and
200 rpm for 7 days. Trichoderma reesei RutC30 was run as a control.
Culture broth samples were removed at day 7. One ml of each culture
broth was centrifuged at 15,700.times.g for 5 minutes in a
micro-centrifuge and the supernatants transferred to new tubes.
Samples were stored at 4.degree. C. until enzyme assay. The
supernatants were assayed for beta-glucosidase activity using
p-nitrophenyl-beta-D-glucopyranoside as substrate, as described
below.
[0294] Beta-glucosidase activity was determined at ambient
temperature using 25 .mu.l aliquots of culture supernatants,
diluted 1:10 in 50 mM succinate pH 5.0, using 200 .mu.l of 0.5
mg/ml p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM
succinate pH 5.0. After 15 minutes incubation the reaction was
stopped by adding 100 .mu.l of 1 M Tris-HCl pH 8.0 and the
absorbance was read spectrophotometrically at 405 nm.
[0295] One unit of beta-glucosidase activity corresponded to
production of 1 .mu.mol of p nitrophenyl per minute per liter at pH
5.0, ambient temperature. Aspergillus niger beta-glucosidase
(Novozyme 188, Novozymes A/S, Bagsvaerd, Denmark) was used as an
enzyme standard.
[0296] All 20 SMA130 transformants exhibited equivalent
beta-glucosidase activity to that of the host strain, Trichoderma
reesei RutC30. In contrast, a number of SMA135 transformants showed
beta-glucosidase activities several fold more than that of
Trichoderma reesei RutC30. Transformant SMA135-04 produced the
highest beta-glucosidase activity, having seven times greater
beta-glucosidase activity than produced by Trichoderma reesei
RutC30 as a control.
[0297] SDS polyacrylamide electrophoresis was carried out using
Criterion Tris-HCl (5% resolving) gels (BioRad, Hercules, Calif.)
with The Criterion System (BioRad, Hercules, Calif.). Five .mu.l of
day 7 supernatants (see above) were suspended in 2.times.
concentration of Laemmli Sample Buffer (BioRad, Hercules, Calif.)
and boiled for 3 minutes in the presence of 5%
beta-mercaptoethanol. The supernatant samples were loaded onto a
polyacrylamide gel and subjected to electrophoresis with 1.times.
Tris/Glycine/SDS as running buffer (BioRad, Hercules, Calif.). The
resulting gel was stained with BioRad's Bio-Safe Coomassie
Stain.
[0298] No beta-glucosidase protein was visible by SDS-PAGE for the
Trichoderma reesei SMA130 transformant culture broth supernatants.
In contrast, 26 of the 38 Trichoderma reesei SMA135 transformants
produced a protein of approximately 110 kDa that was not visible in
Trichoderma reesei RutC30 as control. Transformant Trichoderma
reesei SMA135-04 produced the highest level of
beta-glucosidase.
Example 8
Fermentation of Trichoderma reesei SMA135-04
[0299] Fermentations of Trichoderma reesei SMA135-04 were performed
to determine the production level of beta-glucosidase activity.
Trichoderma reesei RutC30 (host strain) was run as a control.
Spores of Trichoderma reesei SMA135-04 were inoculated into 500 ml
shake flasks, containing 100 ml of inoculum medium composed per
liter of 20 g of glucose, 10 g of corn steep solids, 1.45 g of
(NH.sub.4).sub.2SO.sub.4, 2.08 g of KH.sub.2PO.sub.4, 0.36 g of
CaCl.sub.2.2H.sub.2O, 0.42 g of MgSO.sub.4.7H.sub.2O, and 0.2 ml of
trace metals solution. The trace metals solution was composed per
liter of 216 g of FeCl.sub.3.6H.sub.2O, 58 g of
ZnSO.sub.4.7H.sub.2O, 27 g of MnSO.sub.4.H.sub.2O, 10 g of
CuSO.sub.4.5H.sub.2O, 2.4 g of H.sub.3BO.sub.3, and 336 g of citric
acid. The flasks were placed into an orbital shaker at 28.degree.
C. for approximately 48 hours at which time 50 ml of the culture
was inoculated into 1.8 liters of fermentation medium composed per
liter of 4 g of glucose, 10 g of corn steep solids, 30 g of
cellulose, 2.64 g of CaCl.sub.2.2H.sub.2O, 3.8 g of
(NH.sub.4).sub.2SO.sub.4, 2.8 g of KH.sub.2PO.sub.4, 1.63 g of
MgSO.sub.4.7H.sub.2O, 0.75 ml of trace metals solution (described
above) in a 2 liter fermentation vessel. The fermentations were run
at a pH of 5.0, 28.degree. C., with minimum dissolved oxygen at a
25% at a 1.0 VVM air flow and an agitation of 1100. Feed medium was
delivered into the fermentation vessel at 18 hours with a feed rate
of 3.6 g/hour for 33 hours and then 7.2 g/hour. The fermentations
ran for 165 hours at which time the final fermentation broths were
centrifuged and the supernatants stored at -20.degree. C. until
beta-glucosidase activity assay using the procedure described in
Example 7.
[0300] Beta-glucosidase activity on the Trichoderma reesei
SMA135-04 fermentation sample was determined to be approximately
eight times greater than that produced by Trichoderma reesei
RutC30.
Example 9
PCS Hydrolysis Using Fresh Fermentation Samples
[0301] PCS hydrolysis reactions were formulated using washed and
milled corn stover that was pretreated with dilute sulfuric acid at
elevated temperature and pressure. The following conditions were
used for the pretreatment: acid concentration--1.4 wt %;
temperature---165.degree. C.; pressure 107 psi; time--8 minutes.
Prior to enzymatic hydrolysis, the pretreated corn stover (PCS) was
washed with a large volume of distilled-deionized (DDI) water on a
glass filter. The dry weight of the water-washed PCS was found to
be 24.54%. The water-insoluble solids in PCS contained 56.5%
cellulose, 4.6% hemicellulose, and 28.4% lignin.
[0302] Prior to enzymatic hydrolysis, a suspension of milled PCS in
DDI water was prepared as follows: DDI water-washed PCS was
additionally washed with 95% ethanol on a 22 .mu.m Millipore Filter
(6P Express Membrane, Stericup), and then milled using a
coffee-grinder to reduce the particle size. Dry weight of the
milled PCS was found to be 41.8%. Milled PCS was washed with DDI
water three times in order to remove the ethanol. After each
washing, the suspension was centrifuged at 17,000.times.g for 10
minutes at 4.degree. C. to separate the solids. Finally, DDI water
was added to the milled water-washed solids to make 20 mg/ml
suspension. The suspension was stored at 4.degree. C. and used for
1 ml scale PCS hydrolysis at final concentration of 10 mg/ml.
[0303] Trichoderma reesei strains were grown in two-liter Applikon
laboratory fermentors using a cellulase producing medium at
28.degree. C., pH 4.5, and a growth time of approximately 120
hours. The cellulose producing medium was composed per liter of 5 g
of glucose, 10 g of corn steep solids, 2.08 g of CaCl.sub.2, 3.87 g
of (NH.sub.4).sub.2SO.sub.4, 2.8 g of KH.sub.2PO.sub.4, 1.63 g of
MgSO.sub.4.7H.sub.2O, 0.75 ml of trace metals solution, and 1.8 ml
of pluronic with a feed of 20 g of cellulose per liter. The trace
metals solution was composed per liter of 216 g of
FeCl.sub.3.6H.sub.2O, 58 g of ZnSO.sub.4.7H.sub.2O, 27 g of
MnSO.sub.4H.sub.2O, 10 g of CuSO.sub.4.5H.sub.2O, 2.4 g of
H.sub.3BO.sub.3, and 336 g of citric acid.
[0304] The procedure for preparation of whole fermentation broth
(WB) and cell-free broth (CB) samples is outlined as follows.
Briefly, two 50 ml aliquots of Trichoderma reesei culture were
harvested aseptically in conical centrifuge tubes. One of these
tubes was designated as the WB enzyme preparation without further
treatment, and the other was centrifuged twice to remove cells and
insoluble material to yield a CB enzyme sample. The first
centrifugation was at low speed (1800.times.g for 10 minutes), and
the second was at higher speed (12,000.times.g for 15 minutes).
[0305] PCS (10 mg/ml, 56.5% cellulose) was enzymatically hydrolyzed
at 50.degree. C. in 0.05 M sodium acetate buffer (pH 5.0) with
intermittent mixing. Two types of enzyme preparations were used in
these experiments: (a) Whole fermentation broth (WB) and (b)
centrifuged fermentation broth (CB) as defined above. In one series
of experiments CB that was centrifuged prior to storage (CB-A) for
two weeks at 4.degree. C. was compared with broth that was
centrifuged after storage (CB-B). Four enzyme doses were tested:
2.5, 5.0, 10, and 20 mg/g of PCS. These doses were based on
estimated protein concentrations of 60 g/L for standard lab-scale
fermentations. The volume of each reaction was 1 ml in
MicroWell96.TM. deep well plates (Fisher Scientific, Pittsburgh,
Pa.). At specified time points (1, 3, 6, 9, 12, 24, 48, 72, 96, and
120 hours) 20 .mu.l aliquots were removed from the microplates
using an 8-channel pipettor, and added to 180 .mu.l of alkaline
mixture (0.102 M Na.sub.2CO.sub.3+0.058 M NaHCO.sub.3) in a 96-well
flat-bottomed plate (Millipore, Billerica, Mass.) to terminate the
reaction. The samples were centrifuged at 1800.times.g for 15
minutes to remove unreacted PCS residue. After appropriate
dilutions, the filtrates were analyzed for reducing sugars (RS)
using a microplate assay (see below).
[0306] The concentrations of reducing sugars (RS) in hydrolyzed PCS
samples were measured using a .rho.-hydroxybenzoic acid hydrazide
(PHBAH) assay (Lever, 1972, Anal. Biochem, 47: 273-279), which was
modified and adapted to a 96-well microplate format. Before the
assay, the analyzed samples were diluted in water to bring the RS
concentration into the 0.005-0.200 mg/ml range.
[0307] A 90 .mu.l aliquot of each diluted reaction sample was
placed in a 96-well conical-bottomed microplate (Corning Inc.,
Costar, clear polycarbonate). The reactions were started by
addition of 60 .mu.l of 1.25% PHBAH in 2% sodium hydroxide. Each
assay plate was heated on a custom-made heating block for 10
minutes at 95.degree. C., and allowed to cool at room temperature.
After cooling, 60 .mu.l of water was added to each well. A 100
.mu.l aliquot was removed and transferred to a flat-bottomed
96-well plate (Corning Inc., Costar, medium binding polystyrene),
and the absorbance at 405 nm (A.sub.405) was measured using an
UltraMark Microplate Reader (Bio-Rad, Hercules, Calif.). The
A.sub.405 values were translated into glucose equivalents using a
standard curve. In order to increase the statistical precision of
the assays, 32 replicates were done for each time point at each
enzyme dose.
[0308] Standard curves were generated with eight glucose standards
(0.000, 0.005, 0.010, 0.020, 0.030, 0.050, 0.075, and 0.100 mg/ml),
which were treated similarly to the samples. Glucose standards were
prepared by diluting a 10 mg/ml stock glucose solution with sodium
carbonate/bicarbonate mixture (0.102 M Na.sub.2CO.sub.3+0.058 M
NaHCO.sub.3). Eight replicates of each standard were done to
increase precision of the assays. The average correlation
coefficient for the standard curves was greater than 0.99.
[0309] Glucose concentrations in each hydrolyzed sample were
measured using an enzyme-linked assay method in which 50 .mu.l of
each diluted PCS hydrolysate were mixed with 100 .mu.l of assay
buffer (100 mM MOPS, pH 7, 0.01% Tween-20) and 150 .mu.l of glucose
assay reagent. The assay reagent contained the following
ingredients (per liter): 0.5511 g of ATP, 0.9951 g of NAD, 0.5176 g
of MgSO.sub.4.7H.sub.2O, 1000 Units/L hexokinase Type 300 (Sigma
Chemical Co., St. Louis, Mo.), 1000 Units/L of glucose-6-phosphate
dehydrogenase (Sigma Chemical Co., St. Louis, Mo.), 0.1 g of
Tween-20, and 20.9 g of MOPS, pH 7.0. The reactions were incubated
for 30 minutes at ambient temperature, and the absorbance was
measured at 340 nm. Background absorbance was subtracted based on a
zero glucose control, and the glucose concentrations were
determined with respect to a standard curve generated with glucose
concentrations ranging from 0.00 to 0.25 mg/ml.
[0310] The mean RS yield was calculated using data from all
replicates at a particular enzyme dose and incubation time.
Standard error of the mean (SEM) was calculated as the standard
deviation divided by the square-root n, the number of replicates.
The degree of cellulose conversion to reducing sugar (RS yield,
percent) was calculated using the following equation:
RS
Yield.sub.(%)=RS.sub.(mg/ml).times.100.times.162/(5.65.sub.(mg/ml).ti-
mes.180)=RS.sub.(mg/ml).times.100/(5.65.sub.(mg/ml).times.1.111)
In this equation, RS is the concentration of reducing sugar in
solution measured in glucose equivalents (mg/ml), 5.65 mg/ml is the
initial concentration of cellulose, and the factor 1.111 reflects
the weight gain in converting cellulose to glucose.
[0311] The probability that WB and CB data points represented
statistically different populations was estimated using a Student
t-test (with unequal variance) at each time point.
[0312] As shown in FIG. 11, the use of freshly harvested enzyme
samples (WB and CB) produced PCS hydrolysis profiles that were
nearly identical. These profiles could not be differentiated with a
Student t-test suggesting that they were statistically
indistinguishable. Using enzyme samples from Trichoderma reesei
RutC30, the final RS yields ranged from approximately 30%
conversion of the total glucan at the lowest enzyme dose to about
50% at the highest dose (FIG. 11). When the concentration of
glucose was measured instead of reducing sugars, a similar picture
emerged in that the glucose yields were comparable regardless of
whether WB or CB was used (FIG. 12). However, it may be noteworthy
that the glucose yields were approximately 20 to 25% lower than the
reducing sugar concentrations suggesting that beta-glucosidase
might be a limiting enzyme activity under these conditions.
[0313] In an effort to convert a higher percentage of RS to
glucose, enzyme samples were deployed from the recombinant
Trichoderma reesei strain SMA135-04 which expresses an Aspergillus
oryzae beta-glucosidase gene. When these preparations with elevated
.alpha.-glucosidase activity were employed, several differences
were observed based on a comparison to the results from Trichoderma
reesei RutC30 enzyme samples. First, within the limits of
systematic and experimental errors the PCS hydrolysis curves for WB
and CB were very similar (FIG. 13). Second, at the lowest enzyme
dose (2.5 mg/g of PCS) the final RS yields obtained from
Trichoderma reesei SAM135-04 enzyme samples were approximately 40%
of the total glucan hydrolyzed compared to 30% for Trichoderma
reesei RutC30 enzyme. Third, the final RS titers at higher enzyme
doses were essentially unchanged compared to those obtained when
using WB and CB preparations derived from Trichoderma reesei RutC30
(FIG. 13). The reasons for this phenomenon are unclear, but it may
reflect either thermal inactivation of endoglucanases and
cellobiohydrolases during prolonged incubation at 50.degree. C. or
end product inhibition of the Aspergillus oryzae
.beta.-glucosidase. On the basis of these comparisons the data
consistently suggested that there is little difference between WB
and CB hydrolysis profiles. Both the reaction kinetics and final RS
titers appeared to be similar.
[0314] When the RS and glucose yields generated from WB and CB
samples of Trichoderma reesei SMA135-04 were compared to those
obtained from Trichoderma reesei RutC30 preparations, we observed
that a higher percentage of RS was converted to glucose by
SMA135-04 enzyme samples (FIG. 14). This was not unexpected since
Trichoderma reesei SMA135-04 produces higher levels of
.beta.-glucosidase than Trichoderma reesei RutC30. Interestingly,
at early time points (up to 24 hours), the RS and glucose levels
generated from the Trichoderma reesei SMA135-04 enzyme preparations
differed by only a few percent (FIG. 14). However, during later
stages of the reactions, the RS and glucose curves diverge
perceptibly, suggesting that the beta-glucosidase activity may be
declining in the later stages of PCS hydrolysis under these
conditions. This was particularly apparent at later times for the
highest enzyme dose (20 mg/g of PCS). In addition, it appeared that
WB was outperforming CB in generation of both RS and glucose over
this same time period. A Student t-test predicted that the variance
in RS values was statistically significant (P<0.05) over the
time frame of 48-120 hours. Whether the observed difference in
performance can be attributed to specific enzyme(s) or non-specific
effects attributed to the presence of the mycelia is unknown.
However, it should be noted that the phenomenon was not observed
when using WB and CB enzyme samples from Trichoderma reesei RutC30
(FIG. 15), suggesting instability of the heterologous Aspergillus
oryzae beta-glucosidase expressed by Trichoderma reesei SMA135-04
during prolonged incubation.
Example 10
PCS Hydrolysis Using Fermentation Broth Stored for Two Weeks at
4.degree. C.
[0315] A biomass-to-ethanol process scheme involving on-site enzyme
manufacturing should incorporate enough flexibility to allow for
finite storage of enzyme preparations without significant loss of
potency. Therefore, whether prolonged cold storage of enzyme
samples affected their performance in microtiter-scale PCS
hydrolysis reactions was investigated. In addition to WB, two types
of CB preparations were tested. CB-A samples were centrifuged at
time of harvest and stored at 4.degree. C. as cell-free
supernatant; CB-B preparations were stored at 4.degree. C. as whole
broth, then centrifuged to remove cells at the time of the
assay.
[0316] The PCS hydrolysis reactions were performed as described in
Example 9.
[0317] FIGS. 16 and 17 shows that the hydrolysis curves for WB,
CB-A, and CB-B were principally similar. Statistical analyses using
Student t-tests supported that these data points were not
appreciably different. Furthermore, the final RS yields obtained
using enzyme samples that were stored for two weeks were
essentially the same as those obtained from the use of fresh
fermentation broth. As was observed with fresh broth material, the
lowest dose of Trichoderma reesei SMA135-04 enzyme (2.5 mg/g of
PCS) gave slightly higher RS yields than the same dose of Tv10
material, ostensibly because of higher beta-glucosidase levels
produced by Trichoderma reesei SMA135-04.
[0318] These results can be summarized as follows:
[0319] 1. WB appears to perform as well as CB for hydrolysis of PCS
under the assay conditions described in this series of
experiments.
[0320] 2. It is possible to store WB and CB enzyme samples from
Trichoderma reesei fermentations for at least two weeks at
4.degree. C. without significant loss of potency in our hydrolysis
assay.
[0321] 3. CB may be processed from WB that has been stored for two
weeks at 4.degree. C. without appreciable loss of activity in our
hydrolysis assay.
[0322] Collectively, the results suggested that it is possible to
achieve similar PCS hydrolysis results using WB instead of
fractionated or formulated culture filtrates. It should be
highlighted that the hydrolysis experiments were dosed on an equal
volume basis, and they were not normalized on the basis of enzyme
activity or protein concentration. Consequently, it was surprising
to have observed equivalent performance of WB and CB dosed in this
manner, because the fungal cell mass accounted for approximately
20-30% of the volume in WB. This implied that the effective dose of
extracellular enzyme in the WB preparations was about 20-30% lower
than that of CB.
[0323] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
[0324] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
39122DNAAspergillus nidulens 1gtgccccatg atacgcctcc gg
22226DNAAspergillus nidulens 2gagtcgtatt tccaaggctc ctgacc
26324DNAAspergillus nidulens 3ggaggccatg aagtggacca acgg
24445DNAAspergillus niger 4caccgtgaaa gccatgctct ttccttcgtg
tagaagacca gacag 45545DNAAspergillus niger 5ctggtcttct acacgaagga
aagagcatgg ctttcacggt gtctg 45644DNAAspergillus niger 6ctatatacac
aactggattt accatgggcc cgcggccgca gatc 44744DNAAspergillus niger
7gatctgcggc cgcgggccca tggtaaatcc agttgtgtat atag
44829DNATrichoderma reesei 8aacgttaatt aaggaatcgt tttgtgttt
29929DNATrichoderma reesei 9agtactagta gctccgtggc gaaagcctg
291031DNAHumicola insolens 10ttgaattgaa aatagattga tttaaaactt c
311125DNAHumicola insolens 11ttgcatgcgt aatcatggtc atagc
251226DNASaccharomyces cerevisiae 12ttgaattcat gggtaataac tgatat
261332DNASaccharomyces cerevisiae 13aaatcaatct attttcaatt
caattcatca tt 321445DNAAspergillus nidulens 14ggatgctgtt gactccggaa
atttaacggt ttggtcttgc atccc 451544DNAAspergillus nidulens
15ggtattgtcc tgcagacggc aatttaacgg cttctgcgaa tcgc
441629DNAHumicola insolens 16aagcttaagc atgcgttcct cccccctcc
291732DNAHumicola insolens 17ctgcagaatt ctacaggcac tgatggtacc ag
321832DNAHumicola insolens 18ctgcagaatt ctacaggcac tgatggtacc ag
321936DNAHumicola insolens 19accgcggact gcgcatcatg cgttcctccc
ccctcc 362029DNATrichoderma reesei 20aaacgtcgac cgaatgtagg
attgttatc 292117DNATrichoderma reesei 21gatgcgcagt ccgcggt
172229DNATrichoderma reesei 22aaacgtcgac cgaatgtagg attgttatc
292336DNATrichoderma reesei 23ggagggggga ggaacgcatg atgcgcagtc
cgcggt 362429DNATrichoderma reesei 24aaacgtcgac cgaatgtagg
attgttatc 292532DNAHumicola insolens 25ctgcagaatt ctacaggcac
tgatggtacc ag 32262771DNAAspergillus
oryzaeCDS(30)..(2612)mat_peptide(87)..() 26ctgttctgct ggttacctgc
cacgttatc atg aag ctt ggt tgg atc gag gtg 53Met Lys Leu Gly Trp Ile
Glu Val-15gcc gca ttg gcg gct gcc tca gta gtc agt gcc aag gat gat
ctc gcg 101Ala Ala Leu Ala Ala Ala Ser Val Val Ser Ala Lys Asp Asp
Leu Ala-10 -5 -1 1 5tac tcc cct cct ttc tac cct tcc cca tgg gca gat
ggt cag ggt gaa 149Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp
Gly Gln Gly Glu10 15 20tgg gcg gaa gta tac aaa cgc gct gta gac ata
gtt tcc cag atg acg 197Trp Ala Glu Val Tyr Lys Arg Ala Val Asp Ile
Val Ser Gln Met Thr25 30 35ttg aca gag aaa gtc aac tta acg act gga
aca gga tgg caa cta gag 245Leu Thr Glu Lys Val Asn Leu Thr Thr Gly
Thr Gly Trp Gln Leu Glu40 45 50agg tgt gtt gga caa act ggc agt gtt
ccc aga ctc aac atc ccc agc 293Arg Cys Val Gly Gln Thr Gly Ser Val
Pro Arg Leu Asn Ile Pro Ser55 60 65ttg tgt ttg cag gat agt cct ctt
ggt att cgt ttc tcg gac tac aat 341Leu Cys Leu Gln Asp Ser Pro Leu
Gly Ile Arg Phe Ser Asp Tyr Asn70 75 80 85tca gct ttc cct gcg ggt
gtt aat gtc gct gcc acc tgg gac aag acg 389Ser Ala Phe Pro Ala Gly
Val Asn Val Ala Ala Thr Trp Asp Lys Thr90 95 100ctc gcc tac ctt cgt
ggt cag gca atg ggt gag gag ttc agt gat aag 437Leu Ala Tyr Leu Arg
Gly Gln Ala Met Gly Glu Glu Phe Ser Asp Lys105 110 115ggt att gac
gtt cag ctg ggt cct gct gct ggc cct ctc ggt gct cat 485Gly Ile Asp
Val Gln Leu Gly Pro Ala Ala Gly Pro Leu Gly Ala His120 125 130ccg
gat ggc ggt aga aac tgg gaa ggt ttc tca cca gat cca gcc ctc 533Pro
Asp Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro Asp Pro Ala Leu135 140
145acc ggt gta ctt ttt gcg gag acg att aag ggt att caa gat gct ggt
581Thr Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile Gln Asp Ala
Gly150 155 160 165gtc att gcg aca gct aag cat tat atc atg aac gaa
caa gag cat ttc 629Val Ile Ala Thr Ala Lys His Tyr Ile Met Asn Glu
Gln Glu His Phe170 175 180cgc caa caa ccc gag gct gcg ggt tac gga
ttc aac gta agc gac agt 677Arg Gln Gln Pro Glu Ala Ala Gly Tyr Gly
Phe Asn Val Ser Asp Ser185 190 195ttg agt tcc aac gtt gat gac aag
act atg cat gaa ttg tac ctc tgg 725Leu Ser Ser Asn Val Asp Asp Lys
Thr Met His Glu Leu Tyr Leu Trp200 205 210ccc ttc gcg gat gca gta
cgc gct gga gtc ggt gct gtc atg tgc tct 773Pro Phe Ala Asp Ala Val
Arg Ala Gly Val Gly Ala Val Met Cys Ser215 220 225tac aac caa atc
aac aac agc tac ggt tgc gag aat agc gaa act ctg 821Tyr Asn Gln Ile
Asn Asn Ser Tyr Gly Cys Glu Asn Ser Glu Thr Leu230 235 240 245aac
aag ctt ttg aag gcg gag ctt ggt ttc caa ggc ttc gtc atg agt 869Asn
Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly Phe Val Met Ser250 255
260gat tgg acc gct cat cac agc ggc gta ggc gct gct tta gca ggt ctg
917Asp Trp Thr Ala His His Ser Gly Val Gly Ala Ala Leu Ala Gly
Leu265 270 275gat atg tcg atg ccc ggt gat gtt acc ttc gat agt ggt
acg tct ttc 965Asp Met Ser Met Pro Gly Asp Val Thr Phe Asp Ser Gly
Thr Ser Phe280 285 290tgg ggt gca aac ttg acg gtc ggt gtc ctt aac
ggt aca atc ccc caa 1013Trp Gly Ala Asn Leu Thr Val Gly Val Leu Asn
Gly Thr Ile Pro Gln295 300 305tgg cgt gtt gat gac atg gct gtc cgt
atc atg gcc gct tat tac aag 1061Trp Arg Val Asp Asp Met Ala Val Arg
Ile Met Ala Ala Tyr Tyr Lys310 315 320 325gtt ggc cgc gac acc aaa
tac acc cct ccc aac ttc agc tcg tgg acc 1109Val Gly Arg Asp Thr Lys
Tyr Thr Pro Pro Asn Phe Ser Ser Trp Thr330 335 340agg gac gaa tat
ggt ttc gcg cat aac cat gtt tcg gaa ggt gct tac 1157Arg Asp Glu Tyr
Gly Phe Ala His Asn His Val Ser Glu Gly Ala Tyr345 350 355gag agg
gtc aac gaa ttc gtg gac gtg caa cgc gat cat gcc gac cta 1205Glu Arg
Val Asn Glu Phe Val Asp Val Gln Arg Asp His Ala Asp Leu360 365
370atc cgt cgc atc ggc gcg cag agc act gtt ctg ctg aag aac aag ggt
1253Ile Arg Arg Ile Gly Ala Gln Ser Thr Val Leu Leu Lys Asn Lys
Gly375 380 385gcc ttg ccc ttg agc cgc aag gaa aag ctg gtc gcc ctt
ctg gga gag 1301Ala Leu Pro Leu Ser Arg Lys Glu Lys Leu Val Ala Leu
Leu Gly Glu390 395 400 405gat gcg ggt tcc aac tcg tgg ggc gct aac
ggc tgt gat gac cgt ggt 1349Asp Ala Gly Ser Asn Ser Trp Gly Ala Asn
Gly Cys Asp Asp Arg Gly410 415 420tgc gat aac ggt acc ctt gcc atg
gcc tgg ggt agc ggt act gcg aat 1397Cys Asp Asn Gly Thr Leu Ala Met
Ala Trp Gly Ser Gly Thr Ala Asn425 430 435ttc cca tac ctc gtg aca
cca gag cag gcg att cag aac gaa gtt ctt 1445Phe Pro Tyr Leu Val Thr
Pro Glu Gln Ala Ile Gln Asn Glu Val Leu440 445 450cag ggc cgt ggt
aat gtc ttc gcc gtg acc gac agt tgg gcg ctc gac 1493Gln Gly Arg Gly
Asn Val Phe Ala Val Thr Asp Ser Trp Ala Leu Asp455 460 465aag atc
gct gcg gct gcc cgc cag gcc agc gta tct ctc gtg ttc gtc 1541Lys Ile
Ala Ala Ala Ala Arg Gln Ala Ser Val Ser Leu Val Phe Val470 475 480
485aac tcc gac tca gga gaa agc tat ctt agt gtg gat gga aat gag ggc
1589Asn Ser Asp Ser Gly Glu Ser Tyr Leu Ser Val Asp Gly Asn Glu
Gly490 495 500gat cgt aac aac atc act ctg tgg aag aac ggc gac aat
gtg gtc aag 1637Asp Arg Asn Asn Ile Thr Leu Trp Lys Asn Gly Asp Asn
Val Val Lys505 510 515acc gca gcg aat aac tgt aac aac acc gtg gtc
atc atc cac tcc gtc 1685Thr Ala Ala Asn Asn Cys Asn Asn Thr Val Val
Ile Ile His Ser Val520 525 530gga cca gtt ttg atc gat gaa tgg tat
gac cac ccc aat gtc act ggt 1733Gly Pro Val Leu Ile Asp Glu Trp Tyr
Asp His Pro Asn Val Thr Gly535 540 545att ctc tgg gct ggt ctg cca
ggc cag gag tct ggt aac tcc atc gcc 1781Ile Leu Trp Ala Gly Leu Pro
Gly Gln Glu Ser Gly Asn Ser Ile Ala550 555 560 565gat gtg ctg tac
ggt cgt gtc aac cct ggc gcc aag tct cct ttc act 1829Asp Val Leu Tyr
Gly Arg Val Asn Pro Gly Ala Lys Ser Pro Phe Thr570 575 580tgg ggc
aag acc cgg gag tcg tat ggt tct ccc ttg gtc aag gat gcc 1877Trp Gly
Lys Thr Arg Glu Ser Tyr Gly Ser Pro Leu Val Lys Asp Ala585 590
595aac aat ggc aac gga gcg ccc cag tct gat ttc acc cag ggt gtt ttc
1925Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp Phe Thr Gln Gly Val
Phe600 605 610atc gat tac cgc cat ttc gat aag ttc aat gag acc cct
atc tac gag 1973Ile Asp Tyr Arg His Phe Asp Lys Phe Asn Glu Thr Pro
Ile Tyr Glu615 620 625ttt ggc tac ggc ttg agc tac acc acc ttc gag
ctc tcc gac ctc cat 2021Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu
Leu Ser Asp Leu His630 635 640 645gtt cag ccc ctg aac gcg tcc cga
tac act ccc acc agt ggc atg act 2069Val Gln Pro Leu Asn Ala Ser Arg
Tyr Thr Pro Thr Ser Gly Met Thr650 655 660gaa gct gca aag aac ttt
ggt gaa att ggc gat gcg tcg gag tac gtg 2117Glu Ala Ala Lys Asn Phe
Gly Glu Ile Gly Asp Ala Ser Glu Tyr Val665 670 675tat ccg gag ggg
ctg gaa agg atc cat gag ttt atc tat ccc tgg atc 2165Tyr Pro Glu Gly
Leu Glu Arg Ile His Glu Phe Ile Tyr Pro Trp Ile680 685 690aac tct
acc gac ctg aag gca tcg tct gac gat tct aac tac ggc tgg 2213Asn Ser
Thr Asp Leu Lys Ala Ser Ser Asp Asp Ser Asn Tyr Gly Trp695 700
705gaa gac tcc aag tat att ccc gaa ggc gcc acg gat ggg tct gcc cag
2261Glu Asp Ser Lys Tyr Ile Pro Glu Gly Ala Thr Asp Gly Ser Ala
Gln710 715 720 725ccc cgt ttg ccc gct agt ggt ggt gcc gga gga aac
ccc ggt ctg tac 2309Pro Arg Leu Pro Ala Ser Gly Gly Ala Gly Gly Asn
Pro Gly Leu Tyr730 735 740gag gat ctt ttc cgc gtc tct gtg aag gtc
aag aac acg ggc aat gtc 2357Glu Asp Leu Phe Arg Val Ser Val Lys Val
Lys Asn Thr Gly Asn Val745 750 755gcc ggt gat gaa gtt cct cag ctg
tac gtt tcc cta ggc ggc ccg aat 2405Ala Gly Asp Glu Val Pro Gln Leu
Tyr Val Ser Leu Gly Gly Pro Asn760 765 770gag ccc aag gtg gta ctg
cgc aag ttt gag cgt att cac ttg gcc cct 2453Glu Pro Lys Val Val Leu
Arg Lys Phe Glu Arg Ile His Leu Ala Pro775 780 785tcg cag gag gcc
gtg tgg aca acg acc ctt acc cgt cgt gac ctt gca 2501Ser Gln Glu Ala
Val Trp Thr Thr Thr Leu Thr Arg Arg Asp Leu Ala790 795 800 805aac
tgg gac gtt tcg gct cag gac tgg acc gtc act cct tac ccc aag 2549Asn
Trp Asp Val Ser Ala Gln Asp Trp Thr Val Thr Pro Tyr Pro Lys810 815
820acg atc tac gtt gga aac tcc tca cgg aaa ctg ccg ctc cag gcc tcg
2597Thr Ile Tyr Val Gly Asn Ser Ser Arg Lys Leu Pro Leu Gln Ala
Ser825 830 835ctg cct aag gcc cag taaggggcaa gtcctgattg tacagagcat
ttcgagattt 2652Leu Pro Lys Ala Gln840atgatgtaca tgtttatgaa
tgacctaggg tagggtaata cttagtaggg ttagttctaa 2712ttcttggagt
caagtattga ctcactgggc cgataaaaaa aaaaaaaaaa aaaaaaaaa
277127861PRTAspergillus oryzae 27Met Lys Leu Gly Trp Ile Glu Val
Ala Ala Leu Ala Ala Ala Ser Val-15 -10 -5Val Ser Ala Lys Asp Asp
Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser-1 1 5 10Pro Trp Ala Asp Gly
Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala15 20 25Val Asp Ile Val
Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr30 35 40 45Thr Gly
Thr Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser50 55 60Val
Pro Arg Leu Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu65 70
75Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn80
85 90Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln
Ala95 100 105Met Gly Glu Glu Phe Ser Asp Lys Gly Ile Asp Val Gln
Leu Gly Pro110 115 120 125Ala Ala Gly Pro Leu Gly Ala His Pro Asp
Gly Gly Arg Asn Trp Glu130 135 140Gly Phe Ser Pro Asp Pro Ala Leu
Thr Gly Val Leu Phe Ala Glu Thr145 150 155Ile Lys Gly Ile Gln Asp
Ala Gly Val Ile Ala Thr Ala Lys His Tyr160 165 170Ile Met Asn Glu
Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly175 180 185Tyr Gly
Phe Asn Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys190 195 200
205Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg
Ala210 215 220Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn
Asn Ser Tyr225 230 235Gly Cys Glu Asn Ser Glu Thr Leu Asn Lys Leu
Leu Lys Ala Glu Leu240 245 250Gly Phe Gln Gly Phe Val Met Ser Asp
Trp Thr Ala His His Ser Gly255 260 265Val Gly Ala Ala Leu Ala Gly
Leu Asp Met Ser Met Pro Gly Asp Val270 275 280 285Thr Phe Asp Ser
Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly290 295 300Val Leu
Asn Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val305 310
315Arg Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr
Thr320 325 330Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly
Phe Ala His335 340 345Asn His Val Ser Glu Gly Ala Tyr Glu Arg Val
Asn Glu Phe Val Asp350 355 360 365Val Gln Arg Asp His Ala Asp Leu
Ile Arg Arg Ile Gly Ala Gln Ser370 375 380Thr Val Leu Leu Lys Asn
Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu385 390 395Lys Leu Val Ala
Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly400 405 410Ala Asn
Gly Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met415 420
425Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro
Glu430 435 440 445Gln Ala Ile Gln Asn Glu Val Leu Gln Gly Arg Gly
Asn Val Phe Ala450 455 460Val Thr Asp Ser Trp Ala Leu Asp Lys Ile
Ala Ala Ala Ala Arg Gln465 470 475Ala Ser Val Ser Leu Val Phe Val
Asn Ser Asp Ser Gly Glu Ser Tyr480 485 490Leu Ser Val Asp Gly Asn
Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp495 500 505Lys Asn Gly Asp
Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn510 515 520 525Thr
Val Val Ile Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp530 535
540Tyr Asp His Pro Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro
Gly545 550 555Gln Glu Ser Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly
Arg Val Asn560 565 570Pro Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys
Thr Arg Glu Ser Tyr575 580 585Gly Ser Pro Leu Val Lys Asp Ala Asn
Asn Gly Asn Gly Ala Pro Gln590 595 600 605Ser Asp Phe Thr Gln Gly
Val Phe Ile Asp Tyr Arg His Phe Asp Lys610 615 620Phe Asn Glu Thr
Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr625 630 635Thr Phe
Glu Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg640 645
650Tyr Thr Pro Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly
Glu655 660 665Ile Gly Asp Ala Ser Glu Tyr Val Tyr Pro Glu Gly Leu
Glu Arg Ile670 675 680 685His Glu Phe Ile Tyr Pro Trp Ile Asn Ser
Thr Asp Leu Lys Ala Ser690 695 700Ser Asp Asp Ser Asn Tyr Gly Trp
Glu Asp Ser Lys Tyr Ile Pro Glu705 710 715Gly Ala Thr Asp Gly Ser
Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly720 725 730Ala Gly Gly Asn
Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val735 740 745Lys Val
Lys Asn Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu750 755 760
765Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Lys
Val Val Leu Arg Lys770 775 780Phe Glu Arg Ile His Leu Ala Pro Ser
Gln Glu Ala Val Trp Thr Thr785 790 795Thr Leu Thr Arg Arg Asp Leu
Ala Asn Trp Asp Val Ser Ala Gln Asp800 805 810Trp Thr Val Thr Pro
Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser Ser815 820 825Arg Lys Leu
Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln830 835
8402846DNAAspergillus oryzae 28atagtcaacc gcggactgcg catcatgaag
cttggttgga tcgagg 462926DNAAspergillus oryzae 29actagtttac
tgggccttag gcagcg 263026DNAAspergillus oryzae 30gtcgactcga
agcccgaatg taggat 263145DNAAspergillus oryzae 31cctcgatcca
accaagcttc atgatgcgca gtccgcggtt gacta 453257DNAAspergillus oryzae
32atgaagcttg gttggatcga ggtggccgca ttggcggctg cccctcagta gcagtgc
573319PRTAspergillus oryzae 33Met Lys Leu Gly Trp Ile Glu Val Ala
Ala Leu Ala Ala Ala Ser Val1 5 10 15Val Ser Ala3442DNAAspergillus
oryzae 34tgccggtgtt ggcccttgcc aaggatgatc tcgcgtactc cc
423528DNAAspergillus oryzae 35gactagtctt actgggcctt aggcagcg
283663DNAHumicola insolens 36atgcgttcct cccccctcct ccgctccgcc
gttgtggccg ccctgccggt gttggccctt 60gcc 633721PRTHumicola insolens
37Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro1
5 10 15Val Leu Ala Leu Ala203830DNAAspergillus oryzae 38acgcgtcgac
cgaatgtagg attgttatcc 303942DNAAspergillus oryzae 39gggagtacgc
gagatcatcc ttggcaaggg ccaacaccgg ca 42
* * * * *