U.S. patent application number 12/422035 was filed with the patent office on 2009-08-13 for commercial production of polysaccharide degrading enzymes in plants and methods of using same.
This patent application is currently assigned to APPLIED BIOTECHNOLOGY INSTITUTE. Invention is credited to Elizabeth E. Hood, John A. Howard.
Application Number | 20090205086 12/422035 |
Document ID | / |
Family ID | 46322579 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090205086 |
Kind Code |
A1 |
Hood; Elizabeth E. ; et
al. |
August 13, 2009 |
Commercial production of polysaccharide degrading enzymes in plants
and methods of using same
Abstract
Expression of recombinant polysaccharide degrading enzymes in
plants is described. In one embodiment, expression of the enzyme is
preferentially directed to the seed of the plant. Expression may
also be preferentially targeted to specific locations within the
plant cell. Expression of cellulases in corn is shown. The result
is the capacity to produce polysaccharide degrading enzymes in
plants at commercially acceptable levels in a reliable manner.
Methods of using same in production of ethanol is also described,
including use of the plant-produced enzymes in the ethanol
production process.
Inventors: |
Hood; Elizabeth E.;
(Jonesboro, AR) ; Howard; John A.; (Caycos,
CA) |
Correspondence
Address: |
PATRICIA A. SWEENEY
1835 PLEASANT ST.
WEST DES MOINES
IA
50265
US
|
Assignee: |
APPLIED BIOTECHNOLOGY
INSTITUTE
San Luis Obispo
CA
|
Family ID: |
46322579 |
Appl. No.: |
12/422035 |
Filed: |
April 10, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11219180 |
Sep 2, 2005 |
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12422035 |
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10310292 |
Dec 6, 2002 |
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11219180 |
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60607098 |
Sep 3, 2004 |
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60340035 |
Dec 6, 2001 |
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Current U.S.
Class: |
800/298 |
Current CPC
Class: |
C12N 15/8242 20130101;
C12N 15/8257 20130101; C12N 15/8246 20130101 |
Class at
Publication: |
800/298 |
International
Class: |
A01H 5/00 20060101
A01H005/00 |
Goverment Interests
GOVERNMENT INTEREST
[0002] The work of this invention was funded in part by a grant
from the USDA and the government has certain rights therein.
Claims
1. A transgenic plant comprising a nucleic acid encoding a
.beta.-1,4-endoglucanase (EC 3.2.1.4), and a targeting sequence,
wherein said nucleic acid is stably integrated into the nuclear
genome of a cell of said plant and is operably linked to a promoter
and wherein said target sequence targets the
.beta.-1,4-endoglucanase to the cell wall.
2. A transgenic plant comprising a nucleic acid encoding a
.beta.-1,4-endoglucanase (EC 3.2.1.4), and a targeting sequence,
wherein said nucleic acid is stably integrated into the nuclear
genome of a cell of said plant and is operably linked to a promoter
and wherein said target sequence targets the
.beta.-1,4-endoglucanase to the endoplasmic reticulum.
3. A transgenic seed comprising a nucleic acid encoding a
.beta.-1,4-endoglucanase (EC 3.2.1.4), and a targeting sequence,
wherein said nucleic acid is stably integrated into the nuclear
genome of a cell of said seed and is operably linked to a promoter
and wherein said target sequence targets the
.beta.-1,4-endoglucanase to the cell wall.
4. A transgenic seed comprising a nucleic acid encoding a
.beta.-1,4-endoglucanase (EC 3.2.1.4), and a targeting sequence,
wherein said nucleic acid is stably integrated into the nuclear
genome of a cell of said seed and is operably linked to a promoter
and wherein said target sequence targets the
.beta.-1,4-endoglucanase to the endoplasmic reticulum.
Description
[0001] This application is a continuation of previously filed and
co-pending application U.S. Ser. No. 11/219,180, filed Sep. 2,
2005, which claims benefit under 35 U.S.C..sctn.119(e) to
previously filed and co-pending application U.S. Ser. No.
60/607,098, filed Sep. 3, 2004 and which application U.S. Ser. No.
11/219,180 is also a continuation-in-part of U.S. Ser. No.
10/310,292, filed Dec. 5, 2002, which claims benefit under 35
U.S.C..sctn. 119(e) to U.S. Ser. No. 60/340,035, filed Dec. 6,
2001, the contents of all such prior filings are incorporated
herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to commercial production of
heterologous proteins in plants. More specifically, the invention
is to novel methods of expressing a heterologous polysaccharide
degrading enzyme in plants, particularly in grains, and to methods
of targeting expression to cell organelles and the cell wall to
achieve high levels of expression. Methods of using such enzymes in
saccharification methods and in production of ethanol from crop
residues are also provided.
BACKGROUND OF THE INVENTION
[0004] Polysaccharide degrading enzymes are useful in a variety of
applications, such as in animal feed, industrial applications, and,
in particular, in ethanol production.
[0005] Fossilized hydrocarbon-based energy sources, such as coal,
petroleum and natural gas, provide a limited, non-renewable
resource pool. Because of the world's increasing population and
increasing dependence on energy sources for electricity and
heating, transportation fuels, and manufacturing processes, energy
consumption is rising at an accelerating rate. The US
transportation sector alone consumes over 100 billion gallons of
gasoline per year. Most (.about.60%) of the oil used in the US
today is imported, creating a somewhat precarious situation in
today's political climate because supply disruptions are highly
likely and would cripple the ability of the economy to function.
Fossil petroleum resources, on which our standard of living
currently depends, will likely be severely limited within the next
50-100 years.
[0006] The production of ethanol from lignocellulosic biomass can
utilize large volumes of agricultural resources that are untapped
today. Ethanol is key to partially replacing petroleum resources,
which are limited. Ethanol fuels burn cleanly and because of this,
ethanol replacement of petroleum fuels at any ratio will have a
positive impact on the environment. Production of ethanol from
domestic, renewable sources also ensures a continuing supply. For
these reasons, the production of ethanol fuels from lignocellulosic
biomass are being developed into a viable industry. High yields of
glucose from cellulose (using cellulase enzymes) are required for
any economically viable biomass utilization strategy to be
realized. The US is one country involved in ethanol production and
currently manufactures approximately over three billion gallons of
ethanol from corn grain-derived starch. (American Coalition of
Ethanol Production, www.ethanol.org; also, Sheehan, J. "The road to
bioethanol: A strategic perspective of the US Department of
Energy's National Ethanol Program" Himmel M E, Baker J O, Saddler J
N eds., Glycosyl Hydrolases for Biomass Conversion, 2-25). Ethanol
that is produced from corn starch, however, has not been
cost-effective alternative to fossil fuels.
[0007] Unharvested residues from agricultural crops are estimated
at a mass approximately equal to the harvested portion of the
crops. Specifically for the corn crop, if half of the residue could
be used as a feedstock for the manufacture of ethanol, then about
120 million tons of corn stover would be available annually for
biomass conversion processes (Walsh, Marie E. Biomass Feedstock
Availability in the United States. State Level Analysis. 1999).
Assuming that mature, dry corn stover is approximately 40%
cellulose on a dry weight basis then 48 million tons of
cellulose/year would be available for hydrolysis to glucose. Using
today's technology, a ton of cellulose will yield approximately 100
gallons of ethanol.
[0008] Because known technologies for ethanol production from plant
biomass have been more costly than the market price for ethanol,
ethanol will not become an important alternative to fossil fuels,
unless the price of fossil fuels rises substantially. If, however,
the cost of the production of ethanol from plant biomass could be
reduced, then ethanol might become a cost-effective alternative to
fossil fuels even at today's prices for fossil fuels.
[0009] Plant biomass is a complex matrix of polymers comprising the
polysaccharides cellulose and hemicellulose, and a polyphenolic
complex, lignin, as the major structural components. Any strategy
designed to substitute lignocellulosic feedstocks for petroleum in
the manufacture of fuels and chemicals must include the ability to
efficiently convert the polysaccharide components of plant cell
walls to soluble, monomeric sugar streams. Cellulose, the most
abundant biopolymer on earth, is a simple, linear polymer of
glucose. However, its semi-crystalline structure is notoriously
resistant to hydrolysis by both enzymatic and chemical means. Yet,
high yields of glucose from cellulose are critical to any
economically viable biomass utilization strategy.
[0010] Nature has developed effective cellulose hydrolytic
machinery, mostly microbial in origin, for recycling carbon from
plant biomass in the environment. Without it, the global carbon
cycle would not function. To date, many cellulase genes have been
cloned and sequenced from a wide variety of bacteria, fungi and
plants, and many more certainly await discovery and
characterization (Schulein, M, 2000. Protein engineering of
cellulases. Biochim. Biophys. Acta 1543:239-252); Tomme P, et al.
1995. Cellulose Hydrolysis by Bacteria and Fungi. Advances in
Microbial Physiology 37:1-81). Cellulases are a subset of the
glycosyl hydrolase superfamily of enzymes that have been grouped
into at least 13 families based on protein sequence similarity,
enzyme reaction mechanism, and protein fold motif.
[0011] The economics of using corn stover or any other source of
lignocellulosic biomass to produce ethanol is ominous at best and
is the limiting step behind the attainment of such a goal. The
current cost of making ethanol from any source of lignocellulosic
biomass with the current enzyme production systems and the biomass
collection and pretreatment technology is in the order of about
$1.50 per gallon. This is due to the high operation costs of
collecting and transporting the lignocellulosic raw material to
destination plants, producing the polysaccharide-degrading enzymes
and the high cost of pretreating the lignocellulosic raw material
to facilitate its enzymatic degradation. To become economical, the
processes for ethanol production have to be integrated into the
cultivation of agricultural crops. In particular, the process of
producing the enzymes required for ethanol production as well as
the collection of lignocellulosic raw material have to be
integrated into the normal operations of crop cultivation. The crop
market will generate the revenues necessary to economically justify
its cultivation and the production of ethanol will be a by-product
of this operation.
[0012] At present enzyme production is primarily by submerged
culture fermentation. The scale-up of fermentation systems for the
large volumes of enzyme required for biomass conversion would be
difficult and extremely capital intensive. For purposes of
comparison, a single very large (1 million liter), aerobic
fermentation tank could produce 3,091 tons of cellulase protein/yr
in continuous culture. Currently, however, fermentation technology
is practiced commercially on a significantly smaller scale and in
batch mode, so production capacities are closer to 10% of the
theoretical 3,091 tons calculated above. Thus, using these
assumptions, current practices would yield 3000 times less than the
1.2 mM tons of enzyme needed to convert the cellulose content from
120 MM tons per year of corn stover. Capital and operating costs of
such a fermentative approach to producing cellulases are likely to
be impractical due to the huge scale and capital investment that
will be required.
[0013] Several recombinant systems are available for protein
production. Foreign proteins have been produced in animal cell
cultures and transgenic animals. However, these methods are very
expensive and time intensive, particularly in the scale-up of
cultures or herds large enough for industrial enzyme production,
making them highly impractical. Bacteria and fungi are relatively
simple systems but require a large initial investment for capital
equipment. On the other hand, crop-based production systems may
offer an attractive and cost-effective alternative for industrial
enzyme production at the scale required for biomass conversion.
Transgenic plants require the lowest capital investment (mainly for
dedicated harvesting equipment and storage) of all production
systems. The cost of producing crude recombinant protein in plants
could be three orders of magnitude lower than that of the mammalian
cell system, and 10 fold less than microbial fermentation
(Elizabeth E. Hood and Susan L. Woodard. Industrial Proteins
Produced from Plants. Molecular Farming. 2002. In: Plants as
Factories for Protein Production. E E. Hood and J A Howard, Eds.,
Kluwer Academic Publishers, Dordrecht, The Netherlands pp.
119-135). Advantages of plant systems include the low cost of
growing a large biomass, easy scale-up (increase of planted
acreage), natural storage organs (tubers, seeds), and established
practices for efficient harvesting, transporting, storing and
processing of the plant.
[0014] Plant systems have been used to express polysaccharide
degrading cellulases specifically with varying amounts of success
(Table 1). Ziegler et al. (Ziegler, M T, et al. 2000, Accumulation
of a thermostable endo-1,4-.beta.-D-glucanase in the apoplast of
Arabidopsis thaliana leaves. Molecular Breeding 6:37-46) have
expressed an endoglucanase in Arabidopsis leaves and in tobacco
tissue culture cells at high levels, but both systems are
impractical for commercialization. In addition, some preliminary
work has been done with potato (Dai Z, et al. 2000. Improved
plant-based production of E1 endoglucanase using potato: expression
optimization and tissue targeting. Molecular Breeding 6:277-285)
but expression levels were relatively low. Studies with tobacco,
alfalfa and potato leaves have shown that individual cellulase
enzymes can be expressed in these plants (Ziegelhoffer T, et al.
1999. Expression of bacterial cellulase genes in transgenic alfalfa
(Medicago sativa L.), potato (Solanum tuberosum L.) and tobacco
(Nicotiana tabacum L.). Molecular Breeding 5:309-318; and U.S. Pat.
No. 5,981,835) although not at levels that would allow economic
production of the enzymes.
TABLE-US-00001 TABLE 1 Examples of heterologous cellulase
expression in plants and production considerations. Transgenic
plant Expression Stable Enzyme Gene source system level storage
Scalability.sup.4 Endo-1,4-.beta.-D- Bacterial Arabidopsis 26% TSP
No - glucanase (Acidothermus) (cell wall in leaves.sup.1 targeted)
Endo-1,4-.beta.-D- Bacterial Potato 2.6% TSP.sup.2 No + glucanase
(Acidothermus) (cell wall or in leaves chloroplast target)
Endo-1,4-.beta.-D- Bacterial Alfalfa ~0.01% TSP.sup.3 No ++
glucanase (Thermonospora) (cytosolic in leaves cytosolic
localization) localization Tobacco 0.1% TSP.sup.3 No + (cytosolic
in leaves localization) Cellobiohydrolase Bacterial Alfalfa 0.02%
TSP.sup.3 No ++ (T. fusca) (cytosolic in leaves localization)
Tobacco 0.002% TSP.sup.3 No + (cytosolic in leaves localization)
.sup.1Zeigler et al., 2000; .sup.2Dai et al., 2000;
.sup.3Ziegelhoffer et al., 1999 and ~% TSP assumes 10% of leaf
weight is soluble protein; .sup.4Scalability defined by 2002 US
crop acreage, scale-up potential: -, unscalable; +, fair; ++,
moderate; +++, significant. TSP = Total soluble protein.
[0015] None of the expression systems to date have shown a
practical application of producing cellulases. In some of the
examples the expression level is much too low to be of any
commercial use. The highest level of expression achieved was in
Arabidopsis. However, the use of this plant is impractical for
commercial production of enzymes. It is a model organism, used
because of its ease in transformation, but grows to a height of
only three inches and could not possibly produce adequate amounts
of enzyme for commercial purposes. The volume of material needed
and the expression levels need to be such that commercial
production is practicable. In general, expression levels should be
at least about 0.1% of total soluble protein of the plant tissue
used. None of the work to date has involved expression of
cellulases in corn (Zea mays, L.). While the possibility of
expressing an enzyme to a particular organelle has been presented,
and in one instance targeted to the chloroplast (See U.S. Pat. No.
6,429,359) success in increasing expression by targeting specific
organelles in plants cells or secreting from cell wall has not been
shown. Further, for plant production of the enzymes to be
commercially viable, expression at commercial levels in a plant
that can be grown, harvested and scaled to commercial quantities
must be achieved on a reliable, consistent basis.
[0016] Combining these improvements with harvest methods that allow
the simultaneous recovery of corn stover and corn grain by a single
pass through the field reduces the cost of collecting the
lignocellulosic raw material. Such single pass (also referred to as
one-pass) harvesting cuts down on the number of times that farm
machinery are driven through the fields. This approach minimizes
soil compaction, reduces the amount of time invested in material
collection and curtails the cost of fossil fuel and labor needed
for operating the farm machinery. One-pass harvest is being
developed by several groups, for example at Iowa State University
by Dr. Graeme Quick. See records and minutes of the "Corn Stover
Harvesting Field Demonstration and Biomass Harvesting Colloquium",
Harlan, Iowa. Oct. 29, 2001.
[0017] Provided by the invention are cost-effective methods for the
saccharification of polysaccharides in crop residues. The methods
of the invention find particular use in the integration of current
practices for the cultivation of crop plants for the purpose of
obtaining a commercially desired plant material with the production
of commercial levels of polysaccharide degrading enzymes in the
tissues of the crop plants and the use of the crop plant residues
as a source of lignocellulosic biomass for the production of
fermentable sugars.
[0018] The methods of the invention find use in transforming crop
plants with a nucleotide sequence encoding at least one
polysaccharide degrading enzyme, such as those degrading cellulose,
hemicellulose or pectin. Any plant tissue expressing the enzyme can
be the source of the enzyme. In one embodiment of the invention the
same plant used to make the enzyme can be the source of the
lignocellulose. The enzymes can be produced in any part of the
plant (leaves, seed, roots, etc.) and used for subsequent treatment
in degrading polysaccharides of the plant. In an embodiment the
crop plant is a plant that produces seeds. The source of the enzyme
preferably can be seed tissue, such as one or more of whole seed,
hulls, seed coat, endosperm, or embryo (germ). More preferrably the
seeds have a germ that is capable of being fractioned from the rest
of the seed (the term degerminated is sometimes used when referring
to separation of the germ) in a commercial milling process. In a
preferred embodiment of the invention the enzyme(s) are expressed
in the germ portion of the seed. In another preferred embodiment
the level of enzymes that are produced in the germ portion of the
seed are at least about 0.1% of the dry weight of the seed.
[0019] In particular, the methods of the invention further provide
a cost-effective integrated approach to producing fermentable
sugars from corn stover that encompasses the production of
polysaccharide degrading enzymes in the seeds of genetically
engineered corn plants. A portion of or all of the seed can be the
source of the degrading enzyme with other plant parts used for
other purposes. The option is available to use a select tissue of
the seed for commercial purpose, and other tissue used as the
source of enzyme for the saccarification process. For example, the
corn endosperm can be used as a source of starch, corn stover from
the engineered plants as lignocellulosic biomass and embryo as the
enzyme source. Further economic advantages are obtained in
harvesting the seeds in a first operation and the stover in a
second operation such that both operations are carried out
concurrently by employing single-pass harvest operations.
[0020] The methods of the invention involve producing one or more
cell wall polysaccharide-degrading enzymes in a crop plant by
transforming the plant with at least one nucleotide construct
comprising a nucleotide sequence encoding a cell wall
polysaccharide-degrading enzyme operably linked to a promoter that
drives expression in the crop plant, more preferably in the crop
plant seed or a portion thereof, such that the production of the
commercially desired plant material is not forfeited by the
production of the enzymes.
[0021] The methods further involve obtaining from the transformed
plant, tissue that expresses the cell wall polysaccharide-degrading
enzyme or enzymes, contacting lignocellulosic biomass with this
plant tissue, and exposing the combination to conditions that are
favorable for the degradation of cell wall polysaccharides into
fermentable sugars. The fermentable sugars can then be utilized for
the production of ethanol or other desired molecules using
fermentation procedures that are known in the art.
[0022] The inventors have devised an integrated method for the
economic saccharification of lignocellulosic biomass and its
conversion into ethanol. It is, therefore, an object of the present
invention to provide cost-effective methods for converting
polysaccharides in lignocellulosic biomass into fermentable sugars.
It is also an object of the present invention to genetically
engineer plants to produce cell wall degrading enzymes at
commercially high levels and use such enzymes in saccharification
of polysaccharides. A still further object is to obtain both the
source of polysaccharides and source of enzymes from one crop.
Another object of the invention is to integrate efficient harvest
methods such as single pass harvest with the genetic engineering of
corn plants to cost effectively produce ethanol from corn stover. A
further object of the invention is to produce commercially
acceptable levels of polysaccharide-degrading enzymes in corn
plants. Yet another object of the invention is to target the
expression of polysaccharide-degrading enzymes to corn seeds,
preferably to the germ portion of the seed.
[0023] In one embodiment of the invention, production of
recombinant cellulases in plants is provided that improves over
prior attempts to express cellulases in plants in reliability of
enzyme production and at commercial levels.
[0024] In an embodiment of the invention cellulases are produced in
corn plants.
[0025] Another object of the invention is the application of
large-scale production of cellulases to industrial markets for
which it had previously been economically unfeasible to enter.
[0026] In yet another embodiment of the invention the cellulases
are preferentially expressed to the seed of the plant.
[0027] In an embodiment of the invention expression of cellulases
is targeted to specific locations within the plant cell in order to
increase expression levels of the enzymes in the plant.
[0028] Another embodiment of the invention is to express the E1
cellulase (endo-1,4-.beta.-D-glucanase, EC 3.2.1.4) and CBH I
(cellobiohydrolase or 1,4-.beta.-D-glucan cellobiosidase, EC
3.2.1.91) in corn. In a further embodiment, the E1 cellulase is
secreted to the cell wall, retained in the endoplasmic reticulum or
targeted to the vacuole of a plant cell. Another embodiment
provides for CBH I enzyme to be secreted to the cell wall or
retained in the endoplasmic reticulum.
[0029] Other embodiments are to further improve expression of
cellulases in plants by backcrossing transgenic plants containing
the cellulase expressing gene into plants with good agronomic
traits.
[0030] The objectives of this invention will become apparent in the
description below. All references cited are incorporated herein by
reference.
SUMMARY OF THE INVENTION
[0031] Expression of polysaccharide degrading enzymes in plants is
described. The polysaccharide degrading enzyme can be used for a
variety of applications, including in ethanol production. Use of
the crop plant as the source of enzyme to obtain fermentable
sugars, that can in turn be used in ethanol production is
described. Transgenic plants expressing commercial levels of
recombinant cellulases in plants on a reliable basis is shown.
Expression vectors are engineered to provide for preferential
expression of the enzymes to particular organelles or secreted to
the cell wall in the plant.
DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a construct map showing the construct for
expressing E1 cellulase targeted to the endoplasmic reticulum (KDEL
is disclosed as SEQ ID NO: 12).
[0033] FIG. 2 shows a sequence for the E1 cellulase encoding gene
(SEQ ID NO: 1).
[0034] FIG. 3 is a construct map showing the construct for
expressing E1 cellulase targeted to the vacuole.
[0035] FIG. 4A shows a sequence for a vacuole targeting sequence
(SEQ ID NO: 2) and FIG. 4B shows the barley alpha amylase sequence
used (SEQ ID NO: 3).
[0036] FIG. 5 is a construct map showing the construct for
expressing CBH I such that it is secreted to the cell wall.
[0037] FIG. 6 shows a sequence for CBH I encoding gene (SEQ ID NO:
4).
[0038] FIG. 7 is a construct map showing the construct for
expressing CBH I retained in the endoplasmic reticulum (KDEL is
disclosed as SEQ ID NO: 12).
[0039] FIG. 8 is a construct map showing the construct for
expressing CBH I targeted to the vacuole.
[0040] FIG. 9 is a graph depicting the results of expression of E1
cellulase, in percent of total soluble protein, retained in the
endoplasmic reticulum (KDEL is disclosed as SEQ ID NO: 12).
[0041] FIG. 10 is a graph depicting the results of expression of E1
cellulase, in percent of total soluble protein, when targeted to
the vacuole.
[0042] FIG. 11 is a graph depicting the results of expression of
CBH I, in percent of total soluble protein, when secreted to the
cell wall.
[0043] FIG. 12 is a graph depicting the results of expression of
CBH I, in percent of total soluble protein, when retained in the
endoplasmic reticulum (KDEL is disclosed as SEQ ID NO: 12).
[0044] FIG. 13 is a construct map showing the construct for
cytoplasmic expression of E1.
[0045] FIG. 14 is the construct map showing the construct for
cytoplasmic expression of CBH I
[0046] FIG. 15 shows a barley alpha amylase signal sequence (SEQ ID
NO: 5) in italics with the sequence encoding cel7D (also known as
cbh1-4) from Phanerochaete chrysosporium (SEQ ID NO: 6).
[0047] FIG. 16 is the sequence of an extended globulin-1 promoter
used in the experiments (SEQ ID NO: 7).
[0048] FIG. 17 is the construct map showing the construct for
expression of cbh1-4 secreted to the cell wall.
[0049] FIG. 18 is the sequence encoding cel5A from Phanerochaete
chrysosporium (SEQ ID NO: 8 with the BAASS sequence of SEQ ID NO: 5
in italics and a KDEL (KDEL is disclosed as SEQ ID NO: 12) sequence
(SEQ ID NO: 9) in bold).
[0050] FIG. 19 is the construct map showing the construct for
expression of cel5A retained in the endoplasmic reticulum (KDEL is
disclosed as SEQ ID NO: 12).
[0051] FIG. 20 is the sequence encoding CBH I from P. chrysosporium
C1 (SEQ ID NO: 10) with the BAASS signal sequence of SEQ ID NO: 5
in italics.
[0052] FIG. 21 is the construct map showing the construct for
expression of the P. chrysosporium C1 CBH I secreted to the cell
wall.
[0053] FIG. 22 is the sequence encoding EG5 (SEQ ID NO: 11) with
the BAASS signal sequence in italics (SEQ ID NO: 5) and the KDEL
(SEQ ID NO: 12) sequence of SEQ ID NO: 9, in bold.
[0054] FIG. 23 is the construct map showing the construct for
expressing EG5 retained in the endoplasmic reticulum (KDEL is
disclosed as SEQ ID NO: 12).
[0055] FIG. 24 is a schematic diagram which shows an embodiment of
the invention which comprises an integrated process for the
production of ethanol from corn stover.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0056] The present invention is drawn to cost-effective methods for
expressing polysaccharide degrading enzymes in plants and the use
of same, particularly for the conversion of lignocellulosic biomass
into ethanol. By "lignocellulosic biomass" is intended biomass that
is comprised predominantly of plant cell walls and the components
therein including, but not limited to, cellulose, hemicellulose,
pectin, and lignin. Current methods for the production of ethanol,
which utilize starch derived from corn grain, is a use of a food
product for a fuel, and needs to be more cost effective.
[0057] The methods of the invention involve the use of
lignocellulosic biomass that is currently under utilized for the
production of ethanol. Such lignocellulosic biomass includes, for
example, crop plant residues or other undesired plant material that
may be left behind in the field after harvest or separated from the
desired plant material. A crop refers to a collection of plants
grown in a particular cycle. By "desired plant material" is
intended the plant product that is the primary reason for
commercially growing the plant. Such desired plant material can be
any plant or plant part or plant product that has commercial value.
Corn is grown for human and animal consumption, as well as to
produce products such as industrial oils, fertilizer and many other
uses. Soybeans and wheat are used primarily in food products. There
are multitudes of purposes for which these plant materials can be
utilized. The desired plant material also includes protein produced
by a transgenic polynucleotide. In short, the desired plant
material refers to any product from the plant that is useful. The
invention allows for profitable use of what would otherwise be low
value or waste material after the desired plant is obtained. In the
invention, the enzyme used to degrade polysaccharides in a crop can
be produced by the very crop that will be degraded, thereby
providing clear advantages in eliminating or reducing the need for
an outside source of the enzyme, compacting costs with its
production by combining it with production of the cellulose
source.
[0058] By a "crop plant" is intended any plant that is cultivated
for the purpose of producing plant material that is sought after by
man for either oral consumption, or for utilization in an
industrial, pharmaceutical, or commercial process. The invention
may be applied to any of a variety of plants, including, but not
limited to maize, wheat, rice, barley, soybean, cotton, sorghum,
beans in general, rape/canola, alfalfa, flax, sunflower, safflower,
millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, cotton,
coffee, sweet potato, flax, peanut, clover; vegetables such as
lettuce, tomato, cucurbits, cassaya, potato, carrot, radish, pea,
lentils, cabbage, cauliflower, broccoli, Brussels sprouts, peppers,
and pineapple; tree fruits such as citrus, apples, pears, peaches,
apricots, walnuts, avocado, banana, and coconut; and flowers such
as orchids, carnations and roses.
[0059] The plant tissue used may be that of the original plant
transformed with the enzyme, or can be a descendant obtained by
crossing with the same plant or another plant, as described in the
methods below.
[0060] While such lignocellulosic biomass contains vast amounts of
polysaccharides, these polysaccharides are not readily fermentable
into ethanol. These polysaccharides are constituents of plant cell
walls and include, but are not limited to, cellulose,
hemicellulose, and pectin. The present invention provides
cost-effective methods that involve converting at least a portion
of these polysaccharides, particularly the portion comprising
cellulose, into a form that can be readily fermented into ethanol
by the microorganisms that are presently used for ethanol
production, namely yeasts and bacteria. The invention integrates
the economical production of the enzymes required for the
conversion of the polysaccharides in lignocellulosic biomass to
ethanol with the production of the desired plant material and the
simultaneous recovery of the desired material, the lignocellulosic
raw material and the polysaccharide-degrading enzymes in a single
harvest operation.
[0061] The methods of the invention involve the conversion of plant
cell wall polysaccharides to fermentable sugars that can then be
used in the production of ethanol or other desired molecules via
fermentation methods known in the art. The use of the term
"fermentable sugars" includes, but is not limited to,
monosaccharides and disaccharides and also encompasses sugar
derivatives such as, for example, sugar alcohols, sugar acids,
amino sugars, and the like. The fermentable sugars of the invention
encompass any sugar or sugar derivative that is capable of being
fermented into ethanol via fermentation methods known in the art.
In addition, one skilled in the art can appreciate that the enzymes
expressed in plants of the invention may be used in any commercial
polysaccharide-degrading process, such as in providing additives to
animal feed (See, for example Rode et al., "Fibrolytic enzyme
supplements for dairy cows in early lactation" J. Dairy Sci. 1999
October; 82(1):2121-6); industrial applications, (for example, in
detergent applications, see Winetzky, U.S. Pat. No. 6,565,6131; in
biofinishing of denims, see Vollmond, WO 97/25468); treatment of
genes, or, in a preferred embodiment, in the production of
ethanol.
[0062] To convert the cell wall polysaccharides to fermentable
sugars, the methods of the invention involve producing in plant
tissues one or more enzymes that are capable of degrading plant
cell wall polysaccharides. Preferably, such enzymes are produced at
high levels. Such enzymes and the sequences encoding them are known
in the art.
[0063] Current sources of cell wall polysaccharide-degrading
enzymes are fungal and microbial cultures. Producing high levels of
cell wall polysaccharide-degrading enzymes in plants, particularly
in grain crops, is less expensive and thus lowers the total cost of
producing ethanol from lignocellulosic biomass (Z. Nikolov and D.
Hammes. 2002. "Production of Recombinant Proteins from Transgenic
Crops" in Plants as Factories for Protein Production., E. E. Hood
and J. A. Howard, Eds., Kluwer Academic Publishers, Dordrecht, the
Netherlands pp. 159-174).
[0064] The methods of the invention involve transforming a plant
with at least one nucleotide construct comprising at least one
nucleotide sequence encoding an enzyme that is capable of degrading
plant cell wall polysaccharides. The nucleotide sequence is
operably linked to a promoter that drives expression in a plant.
Preferably, the promoter will preferentially direct expression to a
particular plant tissue. More preferably, the promoter will provide
high-level expression in a particular plant tissue. The plant
tissue in which the enzyme is expressed can include any plant
tissue, such as leaf, seed, root, stem, tassel, anther, pollen,
ovules, or any other tissue of the plant. In an embodiment the
tissue is leaf. Most preferably, the promoter will provide
high-level expression in a seed, or in a particular part of the
seed, such as, for example, the embryo (sometimes referred to as
the "germ"), endosperm, seed coat, bran or hull. Expression of 0.1%
total soluble protein is necessary to provide economically
practical expression. By "high-level expression" is intended that
an enzyme of the invention is present in the plant tissue at a
level of at least about 0.1% dry weight, or about 10% total soluble
protein.
[0065] The methods can involve, one, two, three, four, five, or
more of such enzymes. The enzymes are preferably produced in plant
seeds, or in a particular portion thereof, such as, for example, in
the embryo, endosperm, seed coat, bran or hull.
[0066] In one embodiment of the invention, the methods involve one
or more cell wall polysaccharide-degrading enzymes. By cell wall
"polysaccharide-degrading enzyme" is intended any enzyme that can
be utilized to promote the degradation of the plant cell wall
polysaccharides into fermentable sugars. While the methods of the
invention encompass the production of one or more cell wall
polysaccharide-degrading enzymes in a single plant, two or more
enzymes can be produced in separate plants. For example, a first
plant can be transformed with a first nucleotide construct
comprising a first promoter operably linked to a first nucleotide
sequence encoding a first polysaccharide-degrading enzyme. A second
plant can also be transformed with a second nucleotide construct
comprising a second promoter operably linked to a second nucleotide
sequence encoding a second cell wall polysaccharide-degrading
enzyme. The first and second enzymes can then be employed to
degrade cell wall polysaccharides either in combination or
sequentially.
[0067] Alternatively, the two or more enzymes can be produced in a
single plant. The enzymes may be produced in the same tissue,
expression directed to different tissue; expression may be directed
to the same organelle or different organelles. For example, one
enzyme may be expressed to the endoplasmic reticulum, and the same
or a different enzyme expressed to the vacuole. The result provides
both various options for expression of more than one enzyme, for
ease in use, and/or an increase in expression of the enzymes. This
can be accomplished by any means known in the art for breeding
plants such as, for example, cross pollination of the first and
second plants that are described above and selection for plants
from subsequent generations which express both the first and second
enzymes. The plant breeding methods used herein are well known to
one skilled in the art. For a discussion of plant breeding
techniques, see Poehlman (1987) Breeding Field Crops. AVI
Publication Co., Westport Conn. Many crop plants useful in this
method are bred through techniques that take advantage of the
plant's method of pollination. A plant is self-pollinating if
pollen from one flower is transferred to the same or another flower
of the same plant. A plant is cross-pollinated if the pollen comes
from a flower on a different plant. For example, in Brassica, the
plant is normally self sterile and can only be cross-pollinated
unless, through discovery of a mutant or through genetic
intervention, self compatibility is obtained. In self-pollinating
species, such as rice, oats, wheat, barley, peas, beans, soybeans,
tobacco and cotton, the male and female plants are anatomically
juxtaposed. During natural pollination, the male reproductive
organs of a given flower pollinate the female reproductive organs
of the same flower. Maize plants (Zea mays L.) can be bred by both
self-pollination and cross-pollination techniques. Maize has male
flowers, located on the tassel, and female flowers, located on the
ear, on the same plant. It can self or cross pollinate.
[0068] Pollination can be by any means, including but not limited
to hand, wind or insect pollination, or mechanical contact between
the male fertile and male sterile plant. For production of hybrid
seeds on a commercial scale in most plant species pollination by
wind or by insects is preferred. Stricter control of the
pollination process can be achieved by using a variety of methods
that make one plant pool male sterile, and the other the male
fertile pollen donor. This can be accomplished by hand detassling,
cytoplasmic male sterility, or control of male sterility through a
variety of methods well known to the skilled breeder. Examples of
more sophisticated male sterility systems include those described
at Brar et al., U.S. Pat. Nos. 4,654,465 and 4,727,219 and
Albertsen et al. U.S. Pat. Nos. 5,859,341 and 6,013,859.
[0069] Backcrossing methods may be used to introduce the gene into
the plants. This technique has been used for decades to introduce
traits into a plant. An example of a description of this and other
plant breeding methodologies that are well known can be found in
references such as "Plant Breeding Methodology" edit. Neal Jensen,
John Wiley & Sons, Inc. (1988). In a typical backcross
protocol, the original variety of interest (recurrent parent) is
crossed to a second variety (nonrecurrent parent) that carries the
single gene of interest to be transferred. The resulting progeny
from this cross are then crossed again to the recurrent parent and
the process is repeated until a plant is obtained wherein
essentially all of the desired morphological and physiological
characteristics of the recurrent parent are recovered in the
converted plant, in addition to the single transferred gene from
the nonrecurrent parent.
[0070] A single plant can also be transformed with both the first
and second nucleotide constructs described above or with a single
nucleotide construct comprising the first promoter operably linked
to the first nucleotide sequence and the second promoter operably
linked to the second nucleotide sequence. Furthermore, it is
recognized that both the first and second promoters can be the same
or different depending on whether or not it is desired to express
the first and second enzymes at the same level, time, and/or tissue
in a plant or in separate plants.
[0071] Furthermore, as noted, the plant can be also transformed
using such methods with another nucleotide sequence which creates a
desired plant product. Such product can provide the plant with
increased value, where the expression provides insect resistance,
disease resistance, herbicide resistance, increased yield,
increased tolerance to environmental stress, increased or decreased
starch, oil or protein content, for example. The protein expressed
in the plant can also be the desired plant product itself. By way
of example, but not limitation such products can include production
of proteases in plants (See U.S. Pat. No. 6,087,558); production of
aprotinin in plants (U.S. Pat. No. 5,824,870); production of avidin
in plants (U.S. Pat. No. 5,767,379); production of viral vaccines
in plants (U.S. Pat. No. 6,136,320); production of transmissible
gastroenteritis and hepatitis vaccines in plants (U.S. Pat. Nos.
5,914,123 and 6,034,298).
[0072] The enzymes of the invention encompass enzymes that can be
employed to degrade plant cell wall polysaccharides into
fermentable sugars. Such enzymes are known in the art and include,
but are not limited to, enzymes that can catalyze the degradation
of cellulose, hemicellulose, and/or pectin. In particular, the
methods of the invention are drawn to cellulose-degrading enzymes.
By "cellulose-degrading enzyme" is intended any enzyme that can be
utilized to promote the degradation of cellulose into fermentable
sugars including, but not limited to, cellulases and glucosidases.
By way of example, without limitation, the enzymes classified in
Enzyme Classification as 3.2.1.x are included within the scope of
the invention. An example of the many enzymes which may be employed
in the invention is presented in Table 2, a list of enzymes in the
category by the Nomenclature Committee of the International Union
of Biochemistry and Molecular Biology (NC-IUBMB).
TABLE-US-00002 TABLE 2 Polysaccharide degrading enzymes EC 3.2.1.1
.alpha.-amylase EC 3.2.1.2 .beta.-amylase EC 3.2.1.3 glucan
1,4-.alpha.-glucosidase EC 3.2.1.4 cellulase EC 3.2.1.6
endo-1,3(4)-.beta.-glucanase EC 3.2.1.7 inulinase EC 3.2.1.8
endo-1,4-.beta.-xylanase EC 3.2.1.10 oligo-1,6-glucosidase EC
3.2.1.11 dextranase EC 3.2.1.14 chitinase EC 3.2.1.15
polygalacturonase EC 3.2.1.17 lysozyme EC 3.2.1.18
exo-.alpha.-sialidase EC 3.2.1.20 .alpha.-glucosidase EC 3.2.1.21
.beta.-glucosidase EC 3.2.1.22 .alpha.-galactosidase EC 3.2.1.23
.beta.-galactosidase EC 3.2.1.24 .alpha.-mannosidase EC 3.2.1.25
.beta.-mannosidase EC 3.2.1.26 .beta.-fructofuranosidase EC
3.2.1.28 .alpha..alpha.-trehalase EC 3.2.1.31 .beta.-glucuronidase
EC 3.2.1.32 xylan endo-1,3-.beta.-xylosidase EC 3.2.1.33
amylo-1,6-glucosidase EC 3.2.1.35 hyaluronoglucosaminidase EC
3.2.1.36 hyaluronoglucuronidase EC 3.2.1.37 xylan
1,4-.beta.-xylosidase EC 3.2.1.38 .beta.-D-fucosidase EC 3.2.1.39
glucan endo-1,3-.beta.-D-glucosidase EC 3.2.1.40
.beta.-L-rhamnosidase EC 3.2.1.41 pullulanase EC 3.2.1.42
GDP-glucosidase EC 3.2.1.43 .beta.-L-rhamnosidase EC 3.2.1.44
fucoidanase EC 3.2.1.45 glucosylceramidase EC 3.2.1.46
galactosylceramidase EC 3.2.1.47
galactosylgalactosylglucosylceramidase EC 3.2.1.48 sucrose
.beta.-glucosidase EC 3.2.1.49 .alpha.-N-acetylgalactosaminidase EC
3.2.1.50 .alpha.-N-acetylglucosaminidase EC 3.2.1.51
.alpha.-L-fucosidase EC 3.2.1.52 .beta.-L-N-acetylhexosaminidase EC
3.2.1.53 .beta.-N-acetylgalactosaminidase EC 3.2.1.54
cyclomaltodextrinase EC 3.2.1.55 .alpha.-N-arabinofuranosidase EC
3.2.1.56 glucuronosyl-disulfoglucosamine glucuronidase EC 3.2.1.57
isopullulanase EC 3.2.1.58 glucan 1,3-.beta.-glucosidase EC
3.2.1.59 glucan endo-1,3-.alpha.-glucosidase EC 3.2.1.60 glucan
1,4-.alpha.-maltotetraohydrolase EC 3.2.1.61 mycodextranase EC
3.2.1.62 glycosylceramidase EC 3.2.1.63 1,2-.alpha.-L-fucosidase EC
3.2.1.64 2,6-.beta.-fructan 6-levanbiohydrolase EC 3.2.1.65
levanase EC 3.2.1.66 quercitrinase EC 3.2.1.67 galacturan
1,4-.alpha.-galacturonidase EC 3.2.1.68 isoamylase EC 3.2.1.70
glucan 1,6-.alpha.-glucosidase EC 3.2.1.71 glucan
endo-1,2-.beta.-glucosidase EC 3.2.1.72 xylan 1,3-.beta.-xylosidase
EC 3.2.1.73 licheninase EC 3.2.1.74 glucan 1,4-.beta.-glucosidase
EC 3.2.1.75 glucan endo-1,6-.beta.-glucosidase EC 3.2.1.76
L-iduronidase EC 3.2.1.77 mannan 1,2-(1,3)-.alpha.-mannosidase EC
3.2.1.78 mannan endo-1,4-.beta.-mannosidase EC 3.2.1.80 fructan
.beta.-fructosidase EC 3.2.1.81 agarase EC 3.2.1.82
exo-poly-.alpha.-galacturonosidase EC 3.2.1.83 .kappa.-carrageenase
EC 3.2.1.84 glucan 1,3-.beta.-glucosidase EC 3.2.1.85
6-phospho-.beta.-galactosidase EC 3.2.1.86
6-phospho-.beta.-glucosidase EC 3.2.1.87 capsular-polysaccharide
endo-1,3-.alpha.-galactosidase EC 3.2.1.88 .beta.-L-arabinosidase
EC 3.2.1.89 arabinogalactan endo-1,4-.beta.-galactosidase EC
3.2.1.91 cellulose 1,4-.beta.-cellobiosidase EC 3.2.1.92
peptidoglycan .beta.-N-acetylmuramidase EC 3.2.1.93
.alpha..alpha.-phosphotrehalase EC 3.2.1.94 glucan
1,6-.alpha.-isomaltosidase EC 3.2.1.95 dextran
1,6-.alpha.-isomaltotriosidase EC 3.2.1.96 mannosyl-glycoprotein
endo-.beta.-N-acetylglucosaminidase EC 3.2.1.97 glycopeptide
.alpha.-N-acetylgalactosaminidase EC 3.2.1.98 glucan
1,4-.alpha.-maltohexaosidase EC 3.2.1.99 arabinan
endo-1,5-.alpha.-L-arabinosidase EC 3.2.1.100 mannan
1,4-mannobiosidase EC 3.2.1.101 mannan endo-1,6-.alpha.-mannosidase
EC 3.2.1.102 blood-group-substance endo-1,4-.beta.-galactosidase EC
3.2.1.103 keratan-sulfate endo-1,4-.beta.-galactosidase EC
3.2.1.104 steryl-.beta.-glucosidase EC 3.2.1.105 strictosidine
.beta.-glucosidase EC 3.2.1.106 mannosyl-oligosaccharide
glucosidase EC 3.2.1.107 protein-glucosylgalactosylhydroxylysine
glucosidase EC 3.2.1.108 lactase EC 3.2.1.109 endogalactosaminidase
EC 3.2.1.110 mucinaminylserine mucinaminidase EC 3.2.1.111
1,3-.alpha.-L-fucosidase EC 3.2.1.112 2-deoxyglucosidase EC
3.2.1.113 mannosyl-oligosaccharide 1,2-.alpha.-mannosidase EC
3.2.1.114 mannosyl-oligosaccharide 1,3-1,6-.alpha.-mannosidase EC
3.2.1.115 branched-dextran exo-1,2-.alpha.-glucosidase EC 3.2.1.116
glucan 1,4-.alpha.-maltotriohydrolase EC 3.2.1.117 amygdalin
.beta.-glucosidase EC 3.2.1.118 prunasin .beta.-glucosidase EC
3.2.1.119 vicianin .beta.-glucosidase EC 3.2.1.120 oligoxyloglucan
.beta.-glycosidase EC 3.2.1.121 polymannuronate hydrolase EC
3.2.1.122 maltose-6'-phosphate glucosidase EC 3.2.1.123
endoglycosylceramidase EC 3.2.1.124 3-deoxy-2-octulosonidase EC
3.2.1.125 raucaffricine .beta.-glucosidase EC 3.2.1.126 coniferin
.beta.-glucosidase EC 3.2.1.127 1,6-.alpha.-L-fucosidase EC
3.2.1.128 glycyrrhizinate .beta.-glucuronidase EC 3.2.1.129
endo-.alpha.-sialidase EC 3.2.1.130 glycoprotein
endo-.alpha.-1,2-mannosidase EC 3.2.1.131 xylan
.alpha.-1,2-glucuronosidase EC 3.2.1.132 chitosanase EC 3.2.1.133
glucan 1,4-.alpha.-maltohydrolase EC 3.2.1.134 difructose-anhydride
synthase EC 3.2.1.135 neopullulanase EC 3.2.1.136
glucuronoarabinoxylan endo-1,4-.beta.-xylanase EC 3.2.1.137 mannan
exo-1,2-1,6-.beta.-mannosidase EC 3.2.1.139 .alpha.-glucuronidase
EC 3.2.1.140 lacto-N-biosidase EC 3.2.1.141
4-.alpha.-D-{(1.fwdarw.4)-.alpha.-D-glucano}trehalose
trehalohydrolase EC 3.2.1.142 limit dextrinase EC 3.2.1.143
poly(ADP-ribose) glycohydrolase EC 3.2.1.144 3-deoxyoctulosonase EC
3.2.1.145 galactan 1,3-.beta.-galactosidase EC 3.2.1.146
.beta.-galactofuranosidase EC 3.2.1.147 thioglucosidase EC
3.2.1.149 .beta.-primeverosidase EC 3.2.1.150 oligoxyloglucan
reducing-end-specific cellobiohydrolase EC 3.2.1.151
xyloglucan-specific endo-.beta.-1,4-glucanase EC 3.2.1.152
mannosylglycoprotein endo-.beta.-mannosidase EC 3.2.1.153 fructan
.beta.-(2,1)-fructosidase EC 3.2.1.154 fructan
.beta.-(2,6)-fructosidase EC 3.2.1.156 oligosaccharide reducing-end
xylanase
[0073] For the degradation of cellulose, for example, two general
types of cellulase enzymes can be employed. Cellulase enzymes which
cleave the cellulose chain internally are referred to as
endo-.beta.-1,4-glucanases (E.C. 3.2.1.4) and serve to provide new
reducing and non-reducing chain termini on which
exo-.beta.-1,4-glucanases (cellobiohydrolase, CBH; E.C. 3.2.1.91)
can operate (Tomme et al. (1995) Microbial Physiology 37:1-81). Two
types of exoglucanase have been described that differ in their
approach to the cellulose chain. One type attacks the non-reducing
end and the other attacks the reducing end. The product of the
exoglucanase reaction is typically cellobiose, so a third activity,
.beta.-D-glucosidase (E.C. 3.2.1.21), is required to cleave
cellobiose to glucose. The exoglucanase can also yield longer
glucose chains (up to 6 glucose units) that will require a
.beta.-D-glucosidase activity to reduce their size. Relative to the
other enzyme activities needed for degradation of cellulose into
fermentable sugars, only a minor amount of the .beta.-D-glucosidase
activity is required. Therefore, while the methods of the invention
encompass the production of such a glucosidase in a plant, the
necessary glucosidase activity could be supplied by a downstream
fermentative organism or from .beta.-D-glucosidase enzyme that is
added during saccharification and/or fermentation.
[0074] Nucleotide sequences encoding endo-.beta.-1,4-glucanases,
exo-.beta.-1,4-glucanases, and .beta.-D-glucosidases are known in
the art. Nucleotide sequences encoding endo-.beta.-1,4-glucanases
include, but are not limited to, the nucleotide sequence having
Accession No. U33212. Nucleotide sequences encoding
exo-.beta.-1,4-glucanases include, but are not limited to, the
nucleotide sequence having Accession No. X69976. Nucleotide
sequences encoding .beta.-D-glucosidases include, but are not
limited to, the nucleotide sequence having Accession No.
U13672.
[0075] Expression of cellulases in plants has several advantages.
Plants are more economical to grow and can be far more readily
produced in large quantities than fungi. In addition, recombinant
protein targeted to seeds allows for stable storage of the
recombinant proteins for extended periods. The inventors have
determined that expression of cellulases in plants at commercial
levels on a reliable basis is feasible and provides substantial
advantages over prior attempts of producing the enzyme in
microorganisms.
[0076] One reason that cellulose utilization has not yet been
commercially realized is due to the high cost of the large
quantities of cellulase enzymes required for its complete
hydrolysis. Approximately 1.3 million tons/yr of cellulase would be
required to convert the 48 million tons of stover-derived cellulose
to glucose. While the development of superior enzymes for
processing of plant polymers is important, superior enzymes are of
little value unless the means to produce them economically on a
large scale are also available. The methods of the instant
invention provide for the cost-effective production of cellulases
and other polysaccharide-degrading enzymes in plants, particularly
transgenic maize.
[0077] The inventors have discovered that it is possible to obtain
commercial level expression of a recombinant nucleic acid sequence
encoding cellulases in plants, with improved enzyme production when
expression is directed to the seed of the plant, to particular
organelles and/or cell wall, and that expression is possible and
preferable in corn. The result is consistent, reliable production
in plants of commercial levels of cellulases.
[0078] With today's specific activity, 1.2 million tons of
cellulase are required to convert 48 million tons of cellulose
(from 120 million tons of corn stover) to a sugar stream. This
would require 120 million tons of grain assuming the enzymes
showing synergy in cellulose digestion were present at expression
levels of 1% of dry weight of seed. US production of corn grain is
estimated at 200 MM tons per year. Therefore at these expression
levels, 60% of corn production would be required for the cellulase
enzymes. However, with improved enzymes and expression technology,
a much lower amount of the corn crop would be required to produce
enough enzymes to convert all the available cellulose in corn
stover to glucose. While Ziegler, supra, showed high expression
levels in Arabidopsis, the plant is impractical for reliable
commercial production of the enzymes. The expression levels in both
Ziegler and Dai, supra, is several orders of magnitude below
commercially practical levels.
[0079] Further, expression of cellulases in corn has been
demonstrated for the first time. Corn has considerable advantages
over other plants as bioreactors. In comparison with other plants,
it produces seed which is easily stored and transported, has low
production costs, the plant parts have use in a variety of
processes and products, thereby reducing costs by the sale of
coproducts, and it is the largest crop in North America in terms of
both acreage and total value. Thus production of the enzymes in
corn is desirable.
[0080] Further, according to the present invention, the
preferential direction of expression of the cellulases to internal
organelles and/or cell wall of the plant is a preferred method of
expressing the enzymes at high levels. The inventors have
determined that targeting the expression of E1
endo-1,4-.beta.-D-glucanase (E1 cellulase) to the cell wall results
in expression levels of more than 1% total soluble protein, and
when targeted to the endoplasmic reticulum (ER) results in levels
of expression over 15% of the total soluble protein (TSP) using
extraction methods as described in Example 3. High levels of
expression were also achieved when the enzyme was targeted to the
vacuole. In this instance, seeds had levels of expression in excess
of 10% TSP. When cellobiohydrolase I (CBH I) was targeted to the
cell wall, high levels of expression were obtained, and improved
expression, (in excess of 15% TSP) was obtained when targeted to
the ER. However, vacuole expression resulted in no expression for
CBH I. Thus, targeting to either the cell wall or specific
organelles can improve expression. As discussed supra, more than
one enzyme can be expressed in crop plants. For example, the E1
cellulase expression can be preferentially directed to the
endoplasmic reticulum, to the vacuole, or cell wall, and CBH I
targeted to the cell wall or to the endoplasmic reticulum, the
person skilled in the art selecting the targeted tissue so that the
each enzyme expresses at optimum levels, and both enzymes available
in one plant or one crop.
[0081] In addition to cellulose-degrading enzymes, enzymes that
degrade hemicellulose and pectin can also be employed in the
methods of the invention. While it is recognized that the soluble
sugars can be liberated from the hemicellulose portion of
lignocellulosic biomass by incubation in dilute acid at high
temperatures, enzymes can be also be employed in the methods of the
instant invention to convert hemicellulose into fermentable sugars.
Such enzymes that can be used to the convert the polysaccharides of
the hemicellulose portion into fermentable sugars are known in the
art and include, but are not limited to, endo-.beta.-1,4-xylanases,
endo-.beta.-1,4-mannanases, endo-.beta.-1,4-galactanases,
endoxylanases, .alpha.-glucuronidases,
.alpha.-arabinofuranosidases, and .alpha.-arabinosidases.
Nucleotide sequences encoding such enzymes are also known in the
art. See http://us.expasy.org/cgi-bin/lists?glycosid.txt.
Furthermore, additional fermentable sugars can be liberated from
the pectin portion via the use of enzymes such as, for example,
pectinases. Nucleotide sequences encoding such enzymes are also
known in the art. See, Fry, S. C. 1985. Primary cell wall
metabolism. Oxford Surveys of Plant Molecular and Cell Biology, ed.
B. J. Miflin. 2:1-42. Oxford: Clarendon.
[0082] In accordance with the present invention, a DNA molecule
comprising a transformation/expression vector is engineered to
incorporate a polysaccaride degrading-encoding cDNA. Such enzymes
can then be used in any process employing polysaccharide degrading
enzymes, such as in feed additives, treatment of genes, or ethanol
production. In one embodiment of the invention, when cellulase
enzymes are used in ethanol production, it is preferable to use the
following criteria to select the cellulases for expression in
plants. Such enzymes will be those stable at temperatures and at a
pH that is higher or lower than the temperature or pH at which the
plant expressing the enzyme grows, thereby reducing the possibility
the enzyme will have an adverse impact on the plant cell. Further,
when selecting more than one enzyme for expression in a plant the
pH and temperature stability requirements of the enzymes will be
such that one enzyme does not require an environment hostile to the
other enzyme in order to remain stable. It is also preferable that
the enzymes when combined in the polysaccharide degrading process
have a synergistic effect on the substrate. In one embodiment these
cellulase enzymes are thermostable to at least 45.degree. C., have
pH optima that are similar, exhibit synergistic activity on
lignocellulosic substrates, and the genes encoding these enzymes
have been cloned.
[0083] Using these criteria, in one embodiment, the E1
.beta.-1,4-endoglucanase from Acidothermus celluloliticus
(Mohagheghi et al. (1986) Isolation and Characterization of
Acidothermus cellulolyticus gen. Nov., sp. Nov., a new genus of
thermophillic, acidophillic, cellulolytic bacteria. Int. J. Syst.
Bacteriol. 36:435-443; Nieves et al. (1995) Appl. Biochem.
Biotechnol. 51/52:211-223; U.S. Pat. No. 5,536,655),
cellobiohydrolase I (CBH I) from Trichoderma reesii (Shoemaker et
al. (1983). Molecular Cloning of Exo-Cellobiohydrolase I Derived
From Trichoderma Reesei Strain L27. Bio/Technology 691-696) and the
.beta.-D-glucosidase from Candida wickerhamii (Skory and Freer
(1995) Appl. Environ. Microbiol. 61:518-525; Freer (1993) J. Biol.
Chem. 268:9337-9342) have been selected. This latter enzyme is a
preferred glucosidase because it is resistant to feedback
inhibition by glucose and cellobiose--an important consideration if
one separates the process of saccharification from fermentation. If
saccharification is performed separately from fermentation, the
glucosidase should be selected which will not be feedback inhibited
by their products. The first two enzymes--E1 and CBHI--have been
shown to exhibit synergistic activity on lignocellulosic substrates
that have been pretreated with dilute acid and steam (Baker et al.
(1994) Appl. Biochem. Biotechnol. 45/46:245-256). E1 has optimal
activity at 81.degree. C. (Table 3) but is compatible at
45-50.degree. C. with the CBHI enzyme which shows optimal and
sustained activity at 50.degree. C. Thermostable enzymes with high
temperature optima are less likely to produce detrimental affects
on plants during their growth and development at ambient
temperatures. Some physical characteristics of the selected enzymes
for this embodiment of the invention are presented in Table 3.
TABLE-US-00003 TABLE 3 Characteristics of Selected
Cellulose-Degrading Enzymes E1 cellulase CBH I .beta.-glucosidase
Family 5-3.2.1.4 7-3.2.1.91 1-3.2.1.21 Calculated MW 521 aa; 56,500
Da 496 aa; 52,500 Da 94,000 Da native 116,000 Da in yeast Native
source Bacterial Fungal Fungal (catalytic 363 aa; 40,610 Da domain)
MW by SDS PAGE 72,000 Da 65,000 Da 94,000 Da (catalytic 60,000 Da
72,000 non-glycosyl domain) Glycosylated native No Yes, primarily
Yes, 30% protein linker region pI 5.2 (holo) 4.51 3.89.sup.2 4.87
(cat domain) pH optimum 5-6 5 4.75 Temperature 81.degree. C.
45-50.degree. C. <45.degree. C. optimum Bond cleaved
.beta.-1,4-glycosidic .beta.-1,4-glycosidic .beta.1,4-glycosidic
Mechanism Retained anomeric Retained anomeric Retained anomeric
configuration.sup.1 configuration.sup.1 configuration.sup.1
Substrates Cellulose fibrils; Cellulose fibrils; Cellobiose (and
purified cellulose purified cellulose other water-soluble
preparations (Solka- preparations cello-oligomers up floc,
Sigmacell, (Solka-floc, to dp 6); other .beta.-1,4- Avicel); para-
Sigmacell, Avicel) glycosides (para- nitrophenyl-.beta.-1,4-
nitrophenyl-.beta.-1,4- D-cellobiose D-glucose (pNPG); (pNPC);
methylumbelliferyl- methylumbelliferyl- .beta.-1,4-D-glucose
.beta.-1,4-D-cellobioside (MUG) (MUC) Primary reaction Decreased
degree of Cellobiose (and Glucose products polymerization (dp),
other water-soluble long-chain, water- short chain cello- insoluble
cellulose oligomers) .sup.1Schulein (2000) Biochim. Biophys. Acta
1543: 239-252. .sup.2Freer (1993) J. Biol. Chem. 268:
9337-9342.
[0084] There are numerous cellulase genes cloned and sequenced from
a wide variety of bacteria, fungi and plants. For example, see,
Schulein M, 2000. Protein engineering of cellulases. Biochim.
Biophys. Acta 1543:239-252; Tomme P, et al., 1995. Cellulose
Hydrolysis by Bacteria and Fungi. Advances in Microbial Physiology
37:1-81; Zeigler et al, supra, Dai et al, supra, Ziegelhoffer,
supra, Jensen supra; Henrissat B. A, Classification of glycosyl
hydrolases based on amino-acid sequence similarities Biochem. J.
280:309-316 (1991); Henrissat B., Bairoch A., New families in the
classification of glycosyl hydrolases based on amino-acid sequence
similarities, Biochem. J. 293:781-788 (1993); Henrissat B., Bairoch
A. Updating the sequence-based classification of glycosyl
hydrolases, Biochem. J. 316:695-696 (1996); Davies G., Henrissat B,
Structures and mechanisms of glycosyl hydrolases, Structure
3:853-859 (1995); Jang. S. J. et al, New integration vector using a
cellulase gene as a screening marker for Lactobacillus, FEMS
Microbiol Lett. 2003 Jul. 29; 224(2):191-5; Rees, H. C. et al.
Detecting cellulase and esterase enzyme activities encoded by novel
genes present in environmental DNA libraries. Extremophiles. 2003
Jul. 5; Moriya, T. et al. Cloning and overexpression of the avi2
gene encoding a major cellulase produced by Humicola insolens FERM
BP-5977. Biosci Biotechnol Biochem. 2003 June; 67(6): 1434-7;
Sanchez, M M et al., Exo-mode of action of cellobiohydrolase Cel48C
from Paenibacillus sp. BP-23. A unique type of cellulase among
Bacillales. Eur J. Biochem. 2003 July; 270(13):2913-9; Abdeev, R.
M. et al, Expression of a thermostable bacterial cellulase in
transgenic tobacco plants Genetika. March; 39(3):376-82.; PMID:
12722638; Qin Q et al., Characterization of a tomato protein that
inhibits a xyloglucan-specific endoglucanase. Plant J. 2003 May;
34(3):327-38.; Murray P. G. et al., Molecular cloning,
transcriptional, and expression analysis of the first cellulase
gene (cbh2), encoding cellobiohydrolase II, from the moderately
thermophilic fungus Talaromyces emersonii and structure prediction
of the gene product. Biochem Biophys Res Commun. 2003 Feb. 7;
301(2):280-6; Nakashima, K. I. et al., Cellulase genes from the
parabasalian symbiont Pseudotrichonympha grassii in the hindgut of
the wood-feeding termite Coptotermes formosanus. Cell Mol Life Sci.
2002 September; 59(9):1554-60. The above is a small sampling of the
myriad of cellulase encoding genes available to one skilled in the
art.
[0085] The use of the term "nucleotide constructs" and "nucleic
acids" herein is not intended to limit the present invention to
nucleotide constructs comprising DNA. Those of ordinary skill in
the art will recognize that nucleic acid molecules, particularly
polynucleotides and oligonucleotides, comprised of ribonucleotides
and combinations of ribonucleotides and deoxyribonucleotides may
also be employed in the methods disclosed herein. Thus, the
nucleotide constructs of the present invention encompass all
nucleotide constructs that can be employed in the methods of the
present invention for transforming plants including, but not
limited to, those comprised of deoxyribonucleotides,
ribonucleotides, and combinations thereof. Such
deoxyribonucleotides and ribonucleotides include both naturally
occurring molecules and synthetic analogues. The nucleotide
constructs of the invention also encompass all forms of nucleotide
constructs including, but not limited to, single-stranded forms,
double-stranded forms, hairpins, stem-and-loop structures, and the
like. By referring to a "heterologous" nucleic acid is meant that
the nucleic acid has been introduced in to the plant by human
intervention, such as by transformation with a nucleotide sequence,
crossing or backcrossing with another plant transformed with the
nucleotide sequence, infection of the plant through bacterial or
viral methodology, or the like.
[0086] The expression vector can optionally also contain a signal
sequence located between the promoter and the gene of interest
and/or after the gene of interest. A signal sequence is a
nucleotide sequence, translated to give an amino acid sequence,
which is used by a cell to direct the protein or polypeptide of
interest to be placed in a particular place within or outside the
eukaryotic cell. Many signal sequences are known in the art. See,
for example Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P.
S., Master's Thesis, Iowa State University (1993), Knox, C., et
al., "Structure and Organization of Two Divergent Alpha-Amylase
Genes from Barley", Plant Mol. Biol. 9:3-17 (1987), Lerner et al.,
Plant Physiol. 91:124-129 (1989), Fontes et al., Plant Cell
3:483-496 (1991), Matsuoka et al., Proc. Natl. Acad. Sci. 88:834
(1991), Gould et al., J. Cell. Biol. 108:1657 (1989), Creissen et
al., Plant J. 2:129 (1991), Kalderon, et al., A short amino acid
sequence able to specify nuclear location, Cell 39:499-509 (1984),
Steifel, et al., Expression of a maize cell wall
hydroxyproline-rich glycoprotein gene in early leaf and root
vascular differentiation, Plant Cell 2:785-793 (1990). When
targeting the enzyme to the cell wall use of a signal sequence is
necessary. One example is the barley alpha-amylase signal sequence
(Rogers, J. C. 1985. Two barley alpha-amylase gene families are
regulated differently in aleurone cells. J. Biol. Chem. 260:
3731-3738).
[0087] In a preferred embodiment, the enzyme production is retained
in the endoplasmic reticulum of the plant cell. This may be
accomplished by use of a localization sequence, such as KDEL (KDEL
is disclosed as SEQ ID NO: 12). This sequence (Lys-Asp-Glu-Leu)
(SEQ ID NO: 12) contains the binding site for a receptor in the
endoplasmic reticulum. (Munro, S, and Pelham, H. R. B. 1987 "A
C-terminal signal prevents secretion of luminal ER proteins" Cell
48:899-907. The use of such a localization sequence will increase
expression over levels obtained when the enzyme is otherwise
expressed in the cytoplasm.
[0088] Targeting the enzyme to the vacuole is another preferred
embodiment. Signal sequences to accomplish this are well known. For
example, Raikhel U.S. Pat. No. 5,360,726 shows a vacuole signal
sequence as does Warren et al at U.S. Pat. No. 5,889,174. Vacuolar
targeting signals may be present either at the amino-terminal
portion, (Holwerda et al., The Plant Cell, 4:307-318 (1992),
Nakamura et al., Plant Physiol., 101: 1-5 (1993)), carboxy-terminal
portion, or in the internal sequence of the targeted protein.
(Tague et al., The Plant Cell, 4:307-318 (1992), Saalbach et al.
The Plant Cell, 3:695-708 (1991)). Additionally, amino-terminal
sequences in conjunction with carboxy-terminal sequences are
responsible for vacuolar targeting of gene products (Shinshi et al.
Plant Molec. Biol. 14:357-368 (1990)).
[0089] The nucleotide constructs of the invention encompass
expression cassettes for expression in the plant of interest. The
cassette will include 5' and 3' regulatory sequences operably
linked to a nucleotide sequence encoding a polysaccharide-degrading
enzyme of the invention. By "operably linked" is intended a
functional linkage between a promoter and a second sequence,
wherein the promoter sequence initiates and mediates transcription
of the nucleotide sequence corresponding to the second sequence.
Generally, operably linked means that the nucleotide sequences
being linked are contiguous and, where necessary to join two
protein coding regions, contiguous and in the same reading frame.
Promoter elements employed to control expression of cellulases and
the selection gene, respectively, can be any plant-compatible
promoter.
[0090] In the methods of the invention, a number of promoters that
direct expression of a gene in a plant can be employed. Such
promoters can be selected from constitutive, chemically-regulated,
inducible, and tissue-preferred promoters. Constitutive promoters
include, for example, the core CaMV 35S promoter (Odell et al.
(1985) Nature 313:810-812); ubiquitin promoters (Quail et al.,
5,510,474; ubiquitin-like promoters (Jilka et al. US Publication
20030066108); rice actin (McElroy et al. (1990) Plant Cell
2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.
12:619-632 and Christensen et al. (1992) Plant Mol. Biol.
18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.
81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730), and
the like. Other constitutive promoters include, for example, those
described at U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121;
5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.
[0091] Chemically-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Chemically-inducible promoters are
known in the art and include, but are not limited to, the maize
In2-2 promoter, which is activated by benzenesulfonamide herbicide
safeners, the maize GST promoter, which is activated by hydrophobic
electrophilic compounds that are used as pre-emergent herbicides,
and the tobacco PR-1a promoter, which is activated by salicylic
acid. Other chemical-regulated promoters of interest include
steroid-responsive promoters (see, for example, the
glucocorticoid-inducible promoter in Schena et al. (1991) Proc.
Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998)
Plant J. 14(2):247-257) and tetracycline-inducible and
tetracycline-repressible promoters (see, for example, Gatz et al.
(1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618
and 5,789,156).
[0092] In an embodiment of the invention the promoter is a
seed-preferred promoter that is active during seed development. For
dicots, seed-preferred promoters include, but are not limited to,
bean .beta.-phaseolin, napin, .beta.-conglycinin, soybean lectin,
cruciferin, and the like. For monocots, seed-preferred promoters
include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27
kDa zein, .gamma.-zein, waxy, shrunken 1, shrunken 2, globulin 1,
etc. Seed-preferred promoters of particular interest are those
promoters that direct gene expression predominantly to specific
tissues within the seed such as, for example, the
endosperm-preferred promoter of .gamma.-zein, the cryptic promoter
from tobacco (Fobert et al. 1994. T-DNA tagging of a seed
coat-specific cryptic promoter in tobacco. Plant J. 4: 567-577),
the P-gene promoter from corn (Chopra et al. 1996. Alleles of the
maize P gene with distinct tissue specificities encode
Myb-homologous proteins with C-terminal replacements. Plant Cell
7:1149-1158, Erratum in Plant Cell.1997, 1:109), the globulin-1
promoter from corn (Belanger and Kriz. 1991. Molecular basis for
Allelic Polymorphism of the maize Globulin-1 gene. Genetics 129:
863-972), and promoters that direct expression to the seed coat or
hull of corn kernels, for example the pericarp-specific glutamine
synthetase promoter (Muhitch et al., 2002. Isolation of a Promoter
Sequence From the Glutamine Synthetase.sub.1-2 Gene Capable of
Conferring Tissue-Specific Gene Expression in Transgenic Maize.
Plant Science 163:865-872); Genbank accession number AF359511.
[0093] In a preferred embodiment, the globulin promoter (PGNpr2) is
used. This is the promoter of the maize globulin-1 gene, described
by Belanger, F. C. and Kriz, A. L. 1991. Molecular Basis for
Allelic Polymorphism of the maize Globulin-1 gene. Genetics 129:
863-972. It also can be found as accession number L22344 in the
Genbank database. Another example is the phaseolin promoter. See,
Bustos et al. 1989. Regulation of B-glucuronidase expression in
transgenic tobacco plants by an A/T-rich cis-acting sequence found
upstream of a french bean B-phaseolin gene. The Plant Cell. (1):
839-853.
[0094] In a preferred embodiment, the expression vector also
contains a gene encoding a selection marker that is functionally
linked to a promoter that controls transcription initiation. For a
general description of plant expression vectors and reporter genes,
see Gruber et al. 1993. "Vectors for Plant Transformation" in
Methods of Plant Molecular Biology and Biotechnology. CRC Press. p
89-119. In a preferred embodiment, the selective gene is a
glufosinate-resistance encoding DNA and in another preferred
embodiment can be the phosphinothricin acetyl transferase ("PAT")
or maize optimized PAT gene under the control of the CaMV 35S
promoter. The gene confers resistance to bialaphos (Gordon-Kamm.
1990. The Plant Cell 2: 603; Uchimiya et al. 1993. Bio/Technology
11: 835; and Anzai et al, 1989. Mol. Gen. Gen. 219: 492).
[0095] In addition to a promoter, the expression cassette can
include one or more enhancers. By "enhancer" is intended a
cis-acting sequence that increases the utilization of a promoter.
Such enhancers can be native to a gene or from a heterologous gene.
Further, it is recognized that some promoters can contain one or
more native, enhancers or enhancer-like elements.
[0096] The termination region can be native with the
transcriptional initiation region, can be native with the operably
linked DNA sequence of interest, or can be derived from another
source. Convenient termination regions are available from the
Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase termination regions. In one embodiment of the
invention the pin II terminator from the protease inhibitor II gene
from potato (An et al., 1989. Functional analysis of the 3' control
region of the potato wound-inducible proteinase inhibitor II gene.
Plant Cell 1:115-122) is used. See also, Guerineau et al. (1991)
Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674;
Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990)
Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;
Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et
al. (1987) Nucleic Acid Res. 15:9627-9639.
[0097] Where appropriate, the gene(s) may be optimized for
increased expression in the transformed plant. That is, the genes
can be synthesized using plant-preferred codons for improved
expression. See, for example, Campbell and Gowri (1990) Plant
Physiol. 92:1-11 for a discussion of host-preferred codon usage.
Methods are available in the art for synthesizing plant-preferred
genes. See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and
Murray et al. (1989) Nucleic Acids Res. 17:477-498.
[0098] Additional sequence modifications are known to enhance gene
expression in a plant. These include elimination of sequences
encoding spurious polyadenylation signals, exon-intron splice site
signals, transposon-like repeats, and other such well-characterized
sequences that may be deleterious to gene expression. The G-C
content of the sequence may be adjusted to levels average for a
given cellular host, as calculated by reference to known genes
expressed in the host cell. When possible, the sequence is modified
to avoid predicted hairpin secondary mRNA structures.
[0099] The expression cassettes can additionally contain 5'-leader
sequences in the expression cassette construct. Such leader
sequences can act to enhance translation. Translation leaders are
known in the art and include but are not limited to: picornavirus
leaders, for example, potyvirus leaders such as the TEV leader
(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize
Dwarf Mosaic Virus); Virology 154:9-20), untranslated leader from
the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling
et al. (1987) Nature 325:622-625); tobacco mosaic virus leader
(TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Czech
(Liss, New York), pp. 237-256); and maize chlorotic mottle virus
leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). Other
methods known to enhance translation can also be utilized, for
example, introns, and the like.
[0100] In preparing the nucleotide construct, the various DNA
fragments can be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers can be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0101] Obviously, many variations on the promoters, selectable
markers, signal sequences and other components of the construct are
available to one skilled in the art.
[0102] The methods available for construction of recombinant genes
comprising various modifications for improved expression described
above can differ in detail. However, the methods generally include
the designing and synthesis of overlapping, complementary synthetic
oligonucleotides which are annealed and ligated together to yield a
gene with convenient restriction sites for cloning. The methods
involved are standard methods for a molecular biologist.
[0103] Once the gene is engineered to contain desired features,
such as the desired localization sequences, it is placed into an
expression vector by standard methods. The selection of an
appropriate expression vector will depend upon the method of
introducing the expression vector into host cells. A typical
expression vector contains prokaryotic DNA elements coding for a
bacterial replication origin and an antibiotic resistance gene to
provide for the growth and selection of the expression vector in
the bacterial host; a cloning site for insertion of an exogenous
DNA sequence, which in this context will encode a polysaccharide
degrading enzyme, such as E1 or CBH I; eukaryotic DNA elements that
control initiation of transcription of the exogenous gene, such as
a promoter; and DNA elements that control the processing of
transcripts, such as transcription termination/polyadenylation
sequences. It also can contain such sequences as are needed for the
eventual integration of the vector into the plant chromosome.
[0104] In accordance with the present invention, a transgenic plant
is produced that contains a DNA molecule, comprised of elements as
described above, integrated into its genome so that the plant
expresses a heterologous cellulase-encoding DNA sequence. In order
to create such a transgenic plant, the expression vectors
containing the gene can be introduced into protoplasts, into intact
tissues, such as immature embryos and meristems, into callus
cultures, or into isolated cells. Preferably, expression vectors
are introduced into intact tissues. General methods of culturing
plant tissues are provided, for example, by Miki et al. 1993.
"Procedures for Introducing Foreign DNA into Plants" in Methods in
Plant Molecular Biology and Biotechnology, Glick et al (eds) CRC
Press pp. 67-68 and by Phillips et al. 1988 "Cell/Tissue Culture
and In Vitro Manipulation" in Corn and Corn Improvement 3d Edit.
Sprague et al (eds) American Soc. of Agronomy pp. 345-387. The
selectable marker incorporated in the DNA molecule allows for
selection of transformants.
[0105] Methods for introducing expression vectors into plant tissue
available to one skilled in the art are varied and will depend on
the plant selected. Procedures for transforming a wide variety of
plant species are well known and described throughout the
literature. See, for example, Miki et al, supra; Klein et al. 1992.
Bio/Technology 10:26; and Weisinger et al. 1988. Ann. Rev. Genet.
22: 421-477. For example, the DNA construct may be introduced into
the genomic DNA of the plant cell using techniques such as
microprojectile-mediated delivery (Klein et al. 1987. Nature 327:
70-73); electroporation (Fromm et al. 1985. Proc. Natl. Acad. Sci.
82: 5824); polyethylene glycol (PEG) precipitation (Paszkowski et
al. 1984. Embo J. 3: 2717-272); direct gene transfer (WO 85/01856
and EP No. 0 275 069); in vitro protoplast transformation (U.S.
Pat. No. 4,684,611) and microinjection of plant cell protoplasts or
embryogenic callus (Crossway, 1985. Mol. Gen. Genetics
202:179-185). Co-cultivation of plant tissue with Agrobacterium
tumefaciens is another option, where the DNA constructs are placed
into a binary vector system (Ishida et al. 1996. "High Efficiency
Transformation of Maize (Zea mays L.) Mediated by Agrobacterium
tumefaciens". Nature Biotechnology 14:745-750). The virulence
functions of the Agrobacterium tumefaciens host will direct the
insertion of the construct into the plant cell DNA when the cell is
infected by the bacteria. See, for example Horsch et al. 1984.
Science 233: 496-498, and Fraley et al. 1983. Proc. Natl. Acad.
Sci. 80: 4803.
[0106] Standard methods for transformation of canola are described
by Moloney et al. 1989. "High Efficiency Transformation of Brassica
napus Using Agrobacterium Vectors" Plant Cell Reports 8:238-242.
Corn transformation is described by Fromm et al, 1990.
Bio/Technology 8:833 and Gordon-Kamm et al, supra. Agrobacterium is
primarily used in dicots, but certain monocots such as maize can be
transformed by Agrobacterium. U.S. Pat. No. 5,550,318. Rice
transformation is described by Hiei et al. 1994. "Efficient
Transformation of Rice (Oryza sativs L.) Mediated by Agrobacterium
and Sequence Analysis of the Boundaries of the T-DNA" The Plant
Journal 6(2): 271-282, Christou et al. 1992. Trends in
Biotechnology 10:239 and Lee et al. 1991. Proc. Nat'l Acad. Sci.
USA 88:6389. Wheat can be transformed by techniques similar to
those used for transforming corn or rice. Sorghum transformation is
described by Casas et al., 1997. Transgenic sorghum plants obtained
after microprojectile bombardment of immature inflorescences. In
vitro cellular and developmental biology, Plant. 33:92-100 and by
Wan et al. 1994. Plant Physiology. 104:37. Soybean transformation
is described in a number of publications, including U.S. Pat. No.
5,015,580.
[0107] In one preferred method, the Agrobacterium transformation
methods of Ishida supra and also described in U.S. Pat. No.
5,591,616, are generally followed, with modifications that the
inventors have found improve the number of transformants obtained.
The Ishida method uses the A188 variety of maize that produces Type
I callus in culture. In one preferred embodiment the Hi-II maize
line is used which initiates Type II embryogenic callus in culture.
While Ishida recommends selection on phosphinothricin when using
the bar or PAT gene for selection, another preferred embodiment
provides for use of bialaphos instead.
[0108] The bacterial strain used in the Ishida protocol is LBA4404
with the 40 kb super binary plasmid containing three vir loci from
the hypervirulent A281 strain. The plasmid has resistance to
tetracycline. The cloning vector cointegrates with the super binary
plasmid. Since the cloning vector has an E. coli specific
replication origin, but not an Agrobacterium replication origin, it
cannot survive in Agrobacterium without cointegrating with the
super binary plasmid. Since the LBA4404 strain is not highly
virulent, and has limited application without the super binary
plasmid, the inventors have found in yet another embodiment that
the EHA 101 strain is preferred. It is a disarmed helper strain
derived from the hypervirulent A281 strain. The cointegrated super
binary/cloning vector from the LBA4404 parent is isolated and
electroporated into EHA101, selecting for spectinomycin resistance.
The plasmid is isolated to assure that the EHA101 contains the
plasmid.
[0109] Further, the Ishida protocol as described provides for
growing fresh culture of the Agrobacterium on plates, scraping the
bacteria from the plates, and resuspending in the co-culture medium
as stated in the '616 patent for incubation with the maize embryos.
This medium includes 4.3 g MS salts, 0.5 mg nicotinic acid, 0.5 mg
pyridoxine hydrochloride, 1.0 ml thiamine hydrochloride, casamino
acids, 1.5 mg 2,4-D, 68.5 g sucrose and 36 g glucose, all at a pH
of 5.8. In a further preferred method, the bacteria are grown
overnight in a 1 ml culture, then a fresh 10 ml culture
re-inoculated the next day when transformation is to occur. The
bacteria grow into log phase, and are harvested at a density of no
more than OD600=0.5 and is preferably between 0.2 and 0.5. The
bacteria are then centrifuged to remove the media and resuspended
in the co-culture medium. Since Hi-II is used, medium preferred for
Hi-II is used. This medium is described in considerable detail by
Armstrong, C.I. and Green C. E. 1985. Establishment and maintenance
of friable, embryogenic maize callus and involvement of L-proline.
Planta 154:207-214. The resuspension medium is the same as that
described above. All further Hi-II media are as described in
Armstrong et al. The result is redifferentiation of the plant cells
and regeneration into a plant. Redifferentiation is sometimes
referred to as dedifferentiation, but the former term more
accurately describes the process where the cell begins with a form
and identity, is placed on a medium in which it loses that
identity, and becomes "reprogrammed" to have a new identity. Thus
the scutellum cells become embryogenic callus.
[0110] It is preferred to select the highest level of expression of
polysaccharide degrading enzymes, and it is thus useful to
ascertain expression levels in transformed plant cells, transgenic
plants and tissue specific expression. One such method is to
measure the expression of the target protein as a percentage of
total soluble protein. One standard assay is the Bradford assay
which is well known to those skilled in the art (Bradford, M. 1976.
Anal. Biochem. 72:248). The biochemical activity of the recombinant
protein should also be measured and compared with a wildtype
standard. The activity of polysaccharide degrading enzymes can be
determined by the methods described in Dai et al, supra.
[0111] A variety of assays for endo-.beta.-1,4-glucanase,
cellobiohydrolase and .beta.-D-glucosidase are known in the art
which can be used to detect enzyme activity in extracts prepared
from maize callus and seeds. See, Coughlan et al. ((1988) J. Biol.
Chem. 263:16631-16636) and Freer ((1993) J. Biol. Chem.
268:9337-9342). In addition, western analysis and ELISAs can be
used to assess protein integrity and expression levels. Individual
T.sub.1 seeds are screened by the assay of choice for expression of
the target protein, in this case the cellulases or
.beta.-glucosidase. The individual plants expressing the highest
levels of active enzyme are chosen for field studies, which include
back-crosses (See "Plant Breeding Methodology" edit. Neal Jensen,
John Wile & Sons, Inc. 1988), selection for increased
expression and increased seed amounts. A Western analysis is a
variation of the Southern analysis technique. With a Southern
analysis, DNA is cut with restriction endonucleases and
fractionated on an agarose gel to separate the DNA by molecular
weight and then transferring to nylon membranes. It is then
hybridized with the probe fragment which was radioactively labeled
with .sup.32P and washed in an SDS solution. In the Western
analysis, instead of isolating DNA, the protein of interest is
extracted and placed on an acrylamide gel. The protein is then
blotted onto a membrane and contacted with a labeling substance.
See e.g., Hood et al., "Commercial Production of Avidin from
Transgenic Maize; Characterization of Transformants, Production,
Processing, Extraction and Purification" Molecular Breeding
3:291-306 (1997).
[0112] The ELISA or enzyme linked immunoassay has been known since
1971. In general, antigens solubilised in a buffer are coated on a
plastic surface. When serum is added, antibodies can attach to the
antigen on the solid phase. The presence or absence of these
antibodies can be demonstrated when conjugated to an enzyme. Adding
the appropriate substrate will detect the amount of bound conjugate
which can be quantified. A common ELISA assay is one which uses
biotinylated anti-(protein) polyclonal antibodies and an alkaline
phosphatase conjugate. For example, an ELISA used for quantitative
determination of laccase levels can be an antibody sandwich assay,
which utilizes polyclonal rabbit antibodies obtained commercially.
The antibody is conjugated to alkaline phosphatases for detection.
In another example, an ELISA assay to detect trypsin or trypsinogen
uses biotinylated anti-trypsin or anti-trypsinogen polyclonal
antibodies and a streptavidin-alkaline phosphatase conjugate
[0113] An initial test of enzyme function is performed with lines
of processed corn seed containing single enzymes. For
saccharification of cellulose, seed tissue from these lines are
mixed in the appropriate ratio to produce a high specific activity
for degradation of crystalline cellulose. According to Baker et al.
((1995) "Synergism between purified bacterial and fungal
cellulases", in Enzymatic Degradation of Insoluble Carbohydrates.
ACS Series 618, American Chemical Society, Washington, D.C., pp.
113-141.), maximum synergism for saccharification of cellulose is
with a composite that is about 80% of the Trichoderma reesei CBHI
(exo-.beta.-1,4-glucanase) and about 20% of the Acidothermus
cellulolyticus endo-.beta.-1,4-glucanase. The addition of about
0.1% of the Candida wickerhamii .beta.-D-glucosidase facilitates
the degradation of short glucose oligomers (dp=2-6) to yield
glucose. Later, cross pollination of the selected lines is used to
produce lines that express all three of the cellulase-degrading
enzymes.
[0114] The levels of expression of the gene of interest can be
enhanced by the stable maintenance of a polysaccharide degrading
enzyme encoding gene on a chromosome of the transgenic plant. Use
of linked genes, with herbicide resistance in physical proximity to
the cellulase gene, would allow for maintaining selective pressure
on the transgenic plant population and for those plants where the
genes of interest are not lost.
[0115] In a preferred embodiment of the invention and as also
described at Methods for Cost-Effective Saccharification of
Lignocelluosic Biomass, US publication no. 2003-0109011, both corn
seeds and corn stover are harvested by a single harvesting
operation. Such a procedure allows for the cost-effective recovery
of both the seeds and the stover in one pass through the field.
Using this procedure the seeds are collected in a first container
and the corn stover in a second container and the collection of
both the seeds and the stover is carried out concurrently in a
single step. Single pass harvest integrates the collection of the
lignocellulosic biomass with normal crop harvest operations. With
this procedure the crop residues are collected without incurring a
significant additional cost to the cost of harvesting the corn crop
and without causing any additional soil compaction to cultivated
fields from the passage of farm machinery, with decreased time and
overall costs. Such a process has been demonstrated by Quick, G. R.
(Oct. 29, 2001) Corn Stover Harvesting Field Demonstration and
Biomass Harvesting Colloquium, Harlan, Iowa (record and minutes of
program). In this particular process an IH 1460 with a John Deere
653A row crop head was coupled to a Hesston Stakhand wagon. The
machines were modified by the Iowa State Agriculture Engineering
department so that two crop streams were provided. Grain was taken
up into the combine bin, and whole stover with cobs collected out
the back of the machine and conveyed into the Stakhand wagon. This
is just one example of the type of machine that can be used in such
single pass harvesting.
[0116] Following harvest, the kernels can be milled either by the
wet or dry milling methods that are known in the art. When the germ
is to be separated from the seed, to be practical in this process,
the germ should be capable of being separated in a commercial
milling process, that is a process which does not require hand
separation, but can be carried out in a commercial operation. Corn
seed, for example, is readily separated from the germ or embryo,
where soybean embryos are of a size that the only option for
separation is by hand. In instances where the only means of
separation of germ is by hand, the process would not provide the
cost effective advantages as provided here.
[0117] There are two major milling processes for corn. Dry milling
of corn separates the germ from the endosperm. The endosperm is
recovered in the form of coarse grit and corn flakes, or it may be
passed through fine rollers and reduced to corn flour.
[0118] The bulk of the corn starch produced in the United States is
prepared by the wet-milling process. The first step in the
wet-milling process is to steep the corn kernels in an aqueous
solution. Steeping the kernels serves two main purposes. First it
softens the kernels for subsequent milling, and second, it allows
undesired soluble proteins, peptides, minerals and other components
to be extracted from the kernels. After steeping, the kernels are
separated from the steep water and then wet milled. The steep water
is typically concentrated by evaporation to yield a solution
referred to as a corn steep liquor. Corn steep liquor typically
contains about 3.5 pounds dry solids per bushel of corn kernels
with a nitrogen content between 45-48% (Blanchard (1992) Technology
of Corn Wet Milling and Associated Processes, Elsevier, New York).
Protein content in corn steep liquor has been estimated at about
one pound per bushel of steeped corn which amounts to approximately
15-20% (w/w) of total corn kernel protein (Blanchard (1992)
Technology of Corn Wet Milling and Associated Processes, Elsevier,
New York).
[0119] While typical corn wet-milling processes employ a steeping
that ranges from 12 to 48 hours, other wet-milling processes such
as, for example, those known as the dry-grind process and the
intermittent-milling-and-dynamic-steeping process involve an
initial steeping of shorter duration and can additionally involve
steeping at a higher temperature. Typically, the dry-grind and
intermittent-milling-and-dynamic-steeping processes involve a
steeping of whole kernels for about 12 hours or less at
temperatures of about 60.degree. C. The main objective of such a
short initial steeping is to hydrate the embryo or germ. Breaking
open the kernel after such a short initial steeping reduces the
damage to the germ as compared to dry milling. The hydrated germ
can then be recovered by methods typically utilized in the
wet-milling process. The degerminated kernel fraction can then be
subjected to a second steeping with additional grinding or milling
to facilitate removal of soluble material from the kernel
particles. See, Singh and Eckhoff (1996) Cereal Chem. 73:716-720
and Lopes-Filho et al. (1997) Cereal Chem. 74:633-638.
[0120] Dry milling does not use the steeping process. The procedure
can include, for example, tempering cleaned corn kernels with water
or steam to bring them up to 20 to 22% moisture and the corn is
then held for about one to three hours. A degerminator or impact
mill is used to break open the corn. Discharge from the
degerminator is dried to about 15% to 18% moisture. The germ and
endosperm are separated by size and/or density, resulting in an
enriched fraction for germ or endosperm. See, e.g., Watson, S.,
Chapter 15, "Corn Marketing, Processing and Utilityzation" pp.
918-923, Corn and Corn Improvement, Eds. G. F. Sprague and J. W.
Dudley, American Soc. of Agronomy, Crop Society of America, Soil
Society of America, Madison, Wis. (1988).
[0121] While the invention does not depend on the use of either dry
or wet milling, it is recognized that either milling method can be
used to separate the germ from the endosperm. By expressing the
cell wall polysaccharide-degrading enzymes of the invention under
the control of an embryo-preferred promoter, these enzymes can be
preferentially produced in the corn germ. Thus, the isolated germ
can be used as a source of enzymes for cell wall polysaccharide
degradation, and the starch-laden endosperm can be utilized for
other purposes. If desired, oil can also be extracted from the
germ, using solvents such as, for example, hexane, before the germ
is contacted with corn stover. Methods for extracting oil from corn
germ are known in the art.
[0122] With dry or wet-milling, the desired
polysaccharide-degrading enzymes can be separated from the starch.
As described above, a promoter that drives expression in an embryo,
particularly a promoter that preferentially drives expression in
the corn germ, can be operably linked to a nucleotide sequence
encoding a polysaccharide-degrading enzyme of the invention.
Because the germ is separated from the starch during wet milling,
the germ, in the substantial absence of kernel starch, can be used
as the enzyme source for degradation of cell wall polysaccharides
in the corn stover. While the corn starch can be used for any
purpose or in any process known in the art, the starch can also be
used for the production of ethanol by methods known in the art. If
desired, the starch can be used for ethanol production together
with the corn stover. Thus, the starch can be recombined with the
germ or combined with the stover or the stover-germ mixture.
Starch-degrading enzymes are then utilized to degrade the starch
into glucose for fermentation into ethanol.
[0123] Although the methods of the invention can be used for the
saccharification of plant cell wall polysaccharides in any of the
processes in which saccharification is desired, such as animal feed
additives, gene treatment, and preferably, in the subsequent
fermentation into ethanol, the invention does not depend on the
production of ethanol. The invention encompasses any fermentative
method known in the art that can utilize the fermentable sugars
that are produced as disclosed herein. Such fermentative methods
also include, but are not limited to those methods that can be used
to produce lactic acid, malonic acid and succinic acid. Such
organic acids can be used as precursors for the synthesis of a
variety of chemical products that can be used as replacements for
similar products that are currently produced by petroleum-based
methods. See, United States Department of Energy Fact Sheets
DOE99-IOFC17 (1999), DOE99-IOFC21 (1999), and DOE/GO-102001-1458
(2001).
[0124] With transgenic plants according to the present invention,
polysaccharide degrading enzymes can be produced in commercial
quantities. Thus, the selection and propagation techniques
described above yield a plurality of transgenic plants that are
harvested in a conventional manner. The plant seed expressing the
recombinant polysaccharide degrading enzymes can be used in a
commercial process, or the polysaccharide degrading enzymes
extracted. When using the seed itself, it can, for example, be made
into flour and then applied in the commercial process.
Polysaccharide degrading enzyme extraction from biomass can be
accomplished by known methods. Downstream processing for any
production system refers to all unit operations after product
synthesis, in this case protein production in transgenic seed
(Kusnadi, A. R., Nikolov, Z. L., Howard, J. A., 1997. Biotechnology
and Bioengineering. 56:473-484). Seed is processed either as whole
seed ground into flour, or fractionated and the germ separated from
the hulls and endosperm. If germ is used, it is usually defatted
using a hexane extraction and the remaining crushed germ ground
into a meal or flour. In some cases the germ is used directly in
the industrial process or the protein can be extracted (See, e.g.
WO 98/39461). Extraction is generally made into aqueous buffers at
specific pH to enhance recombinant protein extraction and minimize
native seed protein extraction. Subsequent protein concentration or
purification can follow. In the case of industrial enzymes,
concentration through membrane filtration is usually
sufficient.
[0125] Following the degradation or saccharification of cell wall
polysaccharides, the fermentable sugars that result therefrom can
be converted into ethanol via fermentation methods employing
microorganisms, particularly yeasts and/or bacteria. Such
microorganisms and methods of their use in ethanol production are
known in the art. See, Sheehan 2001. "The road to Bioethanol: A
strategic Perspective of the US Department of Energy's National
Ethanol Program" In: Glucosyl Hydrolases For Biomass Conversion.
ACS Symposium Series 769. American Chemical Society, Washington,
D.C. Existing ethanol production methods that utilize corn grain as
the biomass typically involve the use of yeast, particularly
strains of Saccharomyces cerevisiae. Such strains can be utilized
in the methods of the invention. While such strains may be
preferred for the production of ethanol from glucose that is
derived from the degradation of cellulose and/or starch, the
methods of the present invention do not depend on the use of a
particular microorganism, or of a strain thereof, or of any
particular combination of said microorganisms and said strains.
[0126] Furthermore, it is recognized that the strains of
Saccharomyces cerevisiae that are typically utilized in
fermentative ethanol production from corn starch might not be able
to utilize galacturonic acid and pentose sugars such as, for
example, xylose and arabinose. However, strains of microorganisms
are known in the art that are capable of fermenting these molecules
into ethanol. For example, recombinant Saccharomyces strains have
been produced that are capable of simultaneously fermenting glucose
and xylose to ethanol. See, U.S. Pat. No. 5,789,210, herein
incorporated by reference. Similarly, a recombinant Zymomonas
mobilis strain has been produced that is capable of simultaneously
fermenting glucose, xylose and arabinose to produce ethanol. See,
U.S. Pat. No. 5,843,760; herein incorporated by reference. See,
also U.S. Pat. Nos. 4,731,329, 4,812,410, 4,816,399, and 4,876,196,
all of which are herein incorporated by reference. These patents
disclose the use of Z. mobilis for the production of industrial
ethanol from glucose-based feedstocks. Finally, a recombinant
Escherichia coli strain has been disclosed that is able to convert
pure galacturonic acid to ethanol with minimal acetate production.
See, Doran et al. ((2000) Appl. Biochem. Biotechnol.
84-86:141-152); herein incorporated by reference.
[0127] The methods of the invention involve obtaining plant tissue
that expresses at least one of the cell
wall-polysaccharide-degrading enzymes of the invention and
lignocellulosic biomass. Any plant tissue where the enzyme
expresses can be used in the invention, including, for example,
leaf, stem, root, tassel, anther, pollen, seed, ovules, or any
other tissue of the plant. In an embodiment the plant tissue may be
leaf. In another embodiment, the plant tissue is a seed or part
thereof. The plant tissue may be in another embodiment a grain seed
or part thereof. In yet another embodiment, the plant tissue is a
corn kernel or part thereof, such as, for example, an embryo that
is also referred to as the germ. More than one plant tissue may be
the source of one or more enzymes. The lignocellulosic biomass can
originate from the same plants as the plant tissue or from
different plants. Preferably, the lignocellulosic biomass comprises
plant residues. More preferably, the lignocellulosic biomass
comprises crop residues left in the field after the harvest of corn
grain, which is also known as corn stover. Most preferably, the
lignocellulosic biomass comprises corn stover that is from the same
plants as the cell wall polysaccharide-degrading enzymes for
increased cost efficiency.
[0128] The lignocellulosic biomass is contacted with the plant
tissue and exposed to conditions favorable for the degradation of
the polysaccharides in the lignocellulosic biomass. Prior to
contacting the lignocellulosic biomass with the plant tissue, the
plant tissue, the lignocellulosic biomass, or both, can be
pretreated or processed in any manner known in the art that would
enhance the degradation of the polysaccharides. For example, the
lignocellulosic biomass can be processed by being chopped, sliced,
minced, ground, pulverized, crushed, mashed or soaked. The plant
tissue, such as the seed, containing the enzymes can be treated
with dry or wet-milling processes. Such processing can also include
incubating the plant tissue and/or lignocellulosic biomass in a
solution, particularly an aqueous solution. If desired, the
solution can be agitated, mixed, or stirred. The solution can
comprise any components known in the art that would favor
extraction of an active enzyme from the plant tissue and/or enhance
the degradation of cell wall polysaccharides in the lignocellulosic
biomass. Such components include, but are not limited to, salts,
acids, bases, chelators, detergents, antioxidants,
polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP), and
SO.sub.2. Furthermore, specific environmental conditions, such as,
for example, temperature, pressure, pH, O.sub.2 concentration,
CO.sub.2 concentration, and ionic strength, can be controlled
during any processing and/or subsequent steps to enhance
polysaccharide degradation and/or ethanol production.
[0129] In certain embodiments of the invention, it may be desired
to process the plant tissue so as to produce an extract comprising
the polysaccharide-degrading enzyme and then contacting the
lignocellulosic biomass with the extract. The processing of the
plant tissue to prepare such an extract can be accomplished as
described supra, or by any method known in the art for the
extraction of an enzyme from plant tissue. In other embodiments of
the invention, the plant tissue and the lignocellulosic biomass may
be combined and then processed as described supra. See, e.g., Henry
& Orit (1989) anal. Biochem. 114:92-96.
[0130] In yet another embodiment of the invention, prior to
contacting the lignocellulosic biomass with the plant tissue or
extract thereof, the lignocellulosic biomass can be prepared by
pretreating the lignocellulosic biomass by methods known in the art
(Nguyen et al. 1996. NREL/DOE Ethanol Pilot Plant: Current Status
and Capabilities. Bioresource Technology 58:189-196). In the
pretreatment step, the hemicellulosic fraction of the feedstock is
hydrolyzed to soluble sugars. This step also increases the
enzymes's ability to convert the major fraction of the feedstock
(cellulose) to soluble glucose. The pretreatment step mixes the
feedstock with sulfuric acid and water (approximately 1% acid in
the final solution), then raises the slurry (20-25% solids) to
reaction temperature (160-200.degree. C.) with steam. The mixture
is held at the reaction temperature for a predetermined time (2-20
min) then flashed into a tank maintained at near atmospheric
pressure. Because of the sudden pressure drop, a fraction of the
steam condensate and volatile compounds formed during the heating
is evaporated and removed as flash tank overhead, which is
condensed and sent to waste treatment. Lime is added to the
remaining slurry to adjust the pH to 4.5.
[0131] While the cell wall polysaccharides are degraded prior to
utilization of the fermentable sugars by microorganisms, the
methods are not limited to a saccharification step which precedes
the fermentation step. In certain embodiments of the invention, a
single combined saccharification/fermentation step can be employed
in the methods of the invention. In other embodiments,
saccharification is initiated before fermentation and can be fully
or partially complete prior to the initiation of the
fermentation.
[0132] The methods of the invention find use with any plant species
capable of producing a polysaccharide-degrading enzyme of the
invention. Preferably, the plant species are crop plant species.
More preferably, the plant species are selected from the grain and
oilseed plants. Most preferably, the plant species is corn.
[0133] The following illustrates, but is not intended to limit the
scope of the invention. It will be evident to one skilled in the
art that variations and modifications are possible and fall within
the scope and spirit of the invention.
Example 1
Preparation of Plasmids
[0134] FIG. 1 shows the E1 vector, having the E1 cellulase sequence
(FIG. 2, SEQ ID NO: 1), the seed-preferred promoter PGNpr2 (supra),
the KDEL (SEQ ID NO: 12) endoplasmic reticulum retention sequence
shown in FIG. 4A (SEQ ID NO: 2); the barley alpha-amylase signal
sequence, (BAASS), which was optimized and is shown in FIG. 4B (SEQ
ID NO: 3), and a pin II terminator, supra. The 35S promoter, supra,
drives the selectable marker, the maize optimized PAT gene. The
gene confers resistance to bialaphos. See, Gordon-Kamm et al, The
Plant Cell 2:603 (1990); Uchimiya et al, Bio/Technology 11:835
(1993), and Anzai et al, Mol. Gen. Gen. 219:492 (1989). The E1
cellulase gene from Acidothermus cellulolyticus was received from
NREL. For expression in maize, the first 40 amino acids were
optimized to maize preferred codons. The BAASS and KDEL (SEQ ID NO:
12) sequences were added to the gene by PCR using the NREL clone as
template. The PCR product moved to a PCR-ready cloning vector, then
moved to an intermediate vector to add the pin II terminator
sequence, and then shuttled into the plant expression vector as a
complete unit. PGNpr2 is just upstream of the E1 gene.
[0135] FIG. 3 shows the E1 construct where the vacuole signal
sequence is substituted for the BAASS sequence. The vacuole
targeted version of the E1 cellulase gene was constructed by adding
the vacuole leader to the codon preferred optimized E1 gene
generated in a previous construct (BAASS:E1) using PCR. This PCR
product was cloned into the intermediate vector to add the pin II
terminator and then transferred to the plant expression vector
behind promoter PGNpr2. The vacuole signal sequence is shown in
FIG. 4A (SEQ ID NO:2).
[0136] FIG. 5 shows the CBH I gene construct, similar to the E1
construct but in this case having the BAASS sequence only, such
that the enzyme is secreted to the cell wall. The starting CBH I
clone was received from NREL. This gene most closely matches the
CBH I gene from Trichoderma koningii at the nucleic acid level. The
gene was maize optimized for the first 40 amino acids using a PCR
based mutagenesis approach--this includes the 24 amino acid BAASS
sequence. Codons D346 and D386 were also maize codon optimized to
remove the potentially destabilizing sequences at those positions.
The CBH I sequence used is shown in FIG. 6 (SEQ ID NO: 4). The
BAASS sequence was added to the optimized CBH I gene by PCR. The
PCR product was moved to an PCR-ready cloning vector to add the pin
II terminator, and then the whole unit was transferred to the
transformation vector. The promoter PGNpr2 is used to drive the
transcription of CBH I coding sequence.
[0137] FIG. 7 shows the CBH I vector, which is similar to the E1
vector targeted to the endoplasmic reticulum. FIG. 8 shows the CBH
I vector, which is similar to the E1 vector targeted to the
vacuole.
Example 2
Transformation of Maize
[0138] Fresh immature zygotic embryos were harvested from Hi-II
maize kernels at 1-2 mm in length. The general methods of
Agrobacterium transformation were used as described by Japan
Tobacco, at Ishida et al. 1996. "High efficiency transformation of
maize (Zea mays L.) mediated by Agrobacterium tumefaciens" Nature
Biotechnology 14:745-750 with the modifications described supra.
Fresh embryos were treated with 0.5 ml log phase Agrobacterium
strains EHA101. Bacteria were grown overnight in a rich medium with
kanamycin and spectinomycin to an optical density of 0.5 at 600 nm,
pelleted, then re-inoculated in a fresh 10 ml culture. The bacteria
were allowed to grow into log phase and were harvested at no more
dense than OD600=0.5. The bacterial culture is resuspended in a
co-culture medium.
[0139] For stable transformations, embryos were transferred to a
bialaphos selective agent on embryogenic callus medium and
transferred thereafter every two weeks to allow growth of
transformed type II callus. Plants were regenerated from the
callus.
Example 3
Enzyme Analysis
[0140] Six single seed from each plant (up to 10 plants per event)
were assayed separately. Each seed was pulverized in an automatic
seed pounder and extracted in a high-speed shaker in 1 ml of 50 mM
sodium acetate, pH 5. Cell debris was pelleted and the supernatant
recovered for analysis of 1) total soluble protein using the
Bradford assay (Bradford, M. 1976. Anal. Biochem. 72:248) and 2)
the concentration of the target protein using the assay described
below.
[0141] The E1 enzyme concentration was determined through the
following activity assay. The assay is performed in a microtiter
plate format. An appropriate amount of extract from transgenic seed
containing 1 ug of TSP is transferred to a well of a 96-well
microtiter plate. The total sample volume is brought to 0.1 ml with
the addition of extraction/reaction buffer. The reaction is started
with the addition of 0.025 mL of 5 mM
4-methylumbelliferyl-m-D-cellobioside (MUC). The reaction is
incubated at 50.degree. C. for 30-45 minutes. At each reading time,
0.025 mL of the reaction mix is pipetted into 0.175 mL of stop
buffer (0.2 M Na.sub.2CO.sub.3), then the amount of fluorescence is
read at 460 nm with excitation of 360 nm, and enzyme concentration
determined in relation to a standard curve generated with purified
enzyme spiked into corn seed extract.
[0142] The CBH I enzyme concentration is determined through exactly
the same procedure except that the incubation time is extended to
two hours before reading the fluorescence on the plate.
Example 4
Increasing Expression Levels and Agronomic Yield Through
Breeding
[0143] The Hi-II maize line that is used in tissue culture for
plant transformation shows poor agronomic characteristics and is
not high-yielding in the field. However, one of the most important
goals for industrial protein production is yield near that of
commercial corn lines. Thus, agronomic quality of early transgenic
material can be improved through breeding the transgenic plant into
plants with improved agronomic characteristics and/or which have
characteristics that provide for improved expression of the enzyme.
To accomplish this, T.sub.1 seed from selected
high-cellulase-expressing independent lines was planted in
nurseries and crossed to elite inbreds. The goal is to develop
high-yielding hybrids with good agronomic qualities. Improved
expression levels are expected by breeding into elite varieties
using the backcrossing methods described, supra.
[0144] Crossing the Hi-II events with Stiff Stalk elite germplasm
in particular can also increase event recovery. (See U.S. Ser. No.
10/349,392, to be published; Horn, Michael E.; Harkey, Robin L.;
Vinas, Amanda K.; Drees, Carol F.; Barker, Donna K.; and Lane,
Jeffrey R., "Use of Hill-Elite Hybrids in Agrobacterium-based
Transformation of Maize" In Vitro Cell. Dev. Biol.-Plant. (In
press)). Stiff Stalk inbreds have been available since at least
about the 1950s and are derived from the Iowa Stiff Stalk synthetic
population. Sprague, G. F. "Early testing of inbred lines of maize"
J. Amer. Soc. Agron. (1946)38:108-117; for examples see PI
accession no. 550481 and discussion of Stiff Stalk germplasm at
U.S. Pat. Nos. 5,706,603; 6,252,148; 5,245,975; 6,344,599;
5,134,074; and Neuhausen, S. "A survey of Iowa Stiff Stalk parents
derived inbreds and BSS(HT)C5 using RFLP analysis" MNL
(1989)63:110-111.
[0145] In this instance, the transgenic plant was crossed into
elite Stiff Stalk elite plants, SP122. Improved expression of
cellulases of ten times levels achieved in Hi-II is expected. In
each generation, the highest expressing ears showing agronomic
promise are selected and seed replanted from those ears in
subsequent nurseries. After pollination, maturation and harvest, 50
seed from each progeny ear are combined, ground and analyzed for
expression levels of extractable cellulase. Only those showing
improvement in the amount of cellulases are selected for
replanting. At each generation, approximately the top 10% of lines
are replanted for the breeding program.
Example 5
Expression of Cellulases in Plants
[0146] The results of expression of the E1 cellulase, when targeted
to the ER are shown in FIG. 9. The numbers on the x-axis represent
an ear of corn from an event. The ears are grouped by the event
which produced the ear, as shown by the number above each group.
For each ear of corn, six individual seeds were assayed for total
soluble protein.
[0147] Expression levels were impressive with values greater than
15% TSP, however a few events did not express detectable amounts of
E1 cellulase. Even better expression was obtained when the E1
cellulase was retained in the vacuole, as shown in FIG. 10. While
fewer events were recovered, all lines showed expression of E1
cellulase, with the best line in each event ranging from 8% TSP to
more than 15% TSP.
[0148] Expression of the CBH I enzyme, where secreted to the cell
wall is graphed in FIG. 11. In this graph and in FIG. 12, the
numbers on the x-axis represent a selected ear of corn produced
from an event. The ears are grouped by the event which produced the
ear, as shown by the number above each group In this instance, the
highest expressing seed was assayed for total soluble protein.
Overall, high expression levels were obtained, with the top line
containing 23% TSP as CBH I. Even better results were obtained by
targeting CBH I to the ER. FIG. 12 shows that a greater fraction of
events contained lines expressing CBH I at levels greater than both
5% and 10% TSP. These high expression results are extremely
significant because the CBH I enzyme has not been recovered
previously at high expression levels in any plant or fungal system.
The highest expression published to date is 0.02% TSP in tobacco
leaves (Ziegelhoffer et al, supra). Thus, the highest single seed
levels at 23% TSP are 1000 fold higher than the next best system.
However, use of a vacuole retention sequence resulted in plants
with no enzyme expressed.
[0149] Levels of enzymatically active cellulases that are produced
in transgenic plants are commercially very attractive. Levels of
10% TSP are considerably higher than those obtained by conventional
means and are higher than other attempts at expression, other than
the commercially unfeasible Arabidposis. Table 4 summarizes the
potential of using corn to produce cellulases. High expression
combined with the significant production scalability and storage of
enzyme in grain demonstrates the advantages of the maize
system.
TABLE-US-00004 TABLE 4 Heterologous cellulase expression in corn
and production potential. Transgenic plant Expression Stable Enzyme
Gene source system level storage Scalability.sup.1
Endo-1,4-.beta.-D- Bacterial Corn 16% TSP in Yes +++ glucanase
(Acidothermus) (vacuole seed targeted) Endo-1,4-.beta.-D- Bacterial
Corn 18% TSP Yes +++ glucanase (Acidothermus) (ER in seed targeted)
Cellobiohydrolase Fungal Corn 23% TSP Yes +++ (Trichoderma (cell
wall in seed reesei) targeted) Cellobiohydrolase Fungal Corn 16%
TSP Yes +++ (Trichoderma (ER in seed reesei) targeted)
.sup.1Scalability defined by 2002 US crop acreage, scale-up
potential: -, unscalable; +, fair; ++, moderate; +++,
significant.
Example 6
Transformation with Exocellulase and Endocellulase Sequences
[0150] In further exemplification of the invention, additional
exocellulase and endocellulase encoding sequences were transformed
into plants.
[0151] Two vectors were prepared expressing the E1 and CBH I
cellulases described supra in the cytoplasm. The vector for
expression of E1 is shown in FIG. 13, driven by the globulin-1
promoter PGNpr2, supra. The vector for cytoplasmic expression of
CBH I is shown in FIG. 14.
[0152] A BAASS signal sequence (in italics in FIG. 15, SEQ ID NO:
5) was used with the exocellulase gene cel7D (also known as cbh1-4
from Phaneorchaete chrysosporium (the genomic is shown in Gen Bank
accession L22656) lacking the native signal sequence, the sequence
used in this instance was received from Dan Cullen of Forest
Products and is set forth in FIG. 15 (SEQ ID NO: 6). In this
instance an extended globulin-1 promoter as represented in FIG. 16
(SEQ ID NO: 7) was used to drive expression in the cell wall
targeted construct. The final vector for plant transformation,
pAB19159 is shown in FIG. 17.
[0153] The endocellulase gene cel5A from Phaneorchaete
chrysosporium (the genomic is shown in GenBank accession AY682743)
lacking the native signal sequence, was also received from Forest
Products and is SEQ ID NO: 8, shown in FIG. 18. It was used with a
BAASS sequence of SEQ ID NO: 5 (here in italics) and with a KDEL
sequence (SEQ ID NO: 12), in bold (SEQ ID NO: 9). The final vector
for plant transformation, pAB19160, shown in FIG. 19, contains the
extended globulin-1 promoter of SEQ ID NO: 7, in this vector
driving expression of an endoplasmic reticulim targeted version of
the cel5A gene product.
[0154] The exocellulase gene from Phanerochaete chrysosporium C1
encoding CBH I was received from Dyadic (See U.S. Pat. No.
6,573,086) and the sequence shown in FIG. 20 (SEQ ID NO: 10) along
with the BAASS sequence of SEQ ID NO: 5. The final vector for plant
transformation, shown in FIG. 21, contains the extended globulin-1
promoter, supra, driving expression of a cell wall targeted version
of CBH I lacking the native signal sequence.
[0155] The endocellulase gene from Phanerochaete chrysosporium C1
encoding EG5, shown in FIG. 22, was received from Dyadic (See '086
patent; SEQ ID NO: 11) along with the BAASS sequence (in italics)
of SEQ ID NO: 5 and the KDEL (SEQ ID NO: 12) sequence (in bold) of
SEQ ID NO: 9. The final vector for plant transformation, shown in
FIG. 23, contains the extended globulin-1 promoter, supra, driving
expression of an endoplasmic reticulum targeted version of EG5
lacking the native signal sequence.
Example 7
Use of the Enzyme in Ethanol Production
[0156] In an embodiment of the invention, maize plants are
genetically engineered to produce large amounts (beginning at 0.1%
of whole seed or embryo dry weight) of active bacterial or fungal
polysaccharide degrading enzymes in grain. Corn grain that
expresses the desired cellulases is grown and harvested. The corn
grain can be economically transported (low water content) and
fractionated using either a wet or dry milling process to produce a
enzyme-rich fraction that can be employed in conversion of a
variety of lignocellulosic feedstocks. The paradigm illustrated in
FIG. 24 is even more cost--effective if a single pass harvesting of
stover--the lignocellullosic biomass feedstock--and grain--the
enzyme source--can be implemented.
[0157] Therefore, this invention allows the production of
polysaccharide degrading enzymes in amounts that far exceed the
current capacity of traditional recombinant protein sources such as
filamentous fungi or bacteria. Thus it is evident that the
invention accomplishes at least all of its objectives.
Sequence CWU 1
1
1211566DNAAcidothermus cellulolyticus 1gccggcggtg gctactggca
caccagcggc agggagatcc tggacgccaa caatgtgccg 60gtgaggatcg ccggcatcaa
ctggtttggg ttcgaaacct gcaattacgt cgtgcacggt 120ctctggtcac
gcgactaccg cagcatgctc gaccagataa agtcgctcgg ctacaacaca
180atccggctgc cgtactctga cgacattctc aagccgggca ccatgccgaa
cagcatcaat 240ttttaccaga tgaatcagga cctgcagggt ctgacgtcct
tgcaggtcat ggacaaaatc 300gtcgcgtacg ccggtcagat cggcctgcgc
atcattcttg accgccaccg accggattgc 360agcgggcagt cggcgctgtg
gtacacgagc agcgtctcgg aggctacgtg gatttccgac 420ctgcaagcgc
tggcgcagcg ctacaaggga aacccgacgg tcgtcggctt tgacttgcac
480aacgagccgc atgacccggc ctgctggggc tgcggcgatc cgagcatcga
ctggcgattg 540gccgccgagc gggccggaaa cgccgtgctc tcggtgaatc
cgaacctgct cattttcgtc 600gaaggtgtgc agagctacaa cggagactcc
tactggtggg gcggcaacct gcaaggagcc 660ggccagtacc cggtcgtgct
gaacgtgccg aaccgcctgg tgtactcggc gcacgactac 720gcgacgagcg
tctacccgca gacgtggttc agcgatccga ccttccccaa caacatgccc
780ggcatctgga acaagaactg gggatacctc ttcaatcaga acattgcacc
ggtatggctg 840ggcgaattcg gtacgacact gcaatccacg accgaccaga
cgtggctgaa gacgctcgtc 900cagtacctac ggccgaccgc gcaatacggt
gcggacagct tccagtggac cttctggtcc 960tggaaccccg attccggcga
cacaggagga attctcaagg atgactggca gacggtcgac 1020acagtaaaag
acggctatct cgcgccgatc aagtcgtcga ttttcgatcc tgtcggcgcg
1080tctgcatcgc ctagcagtca accgtccccg tcggtgtcgc cgtctccgtc
gccgagcccg 1140tcggcgagtc ggacgccgac gcctactccg acgccgacag
ccagcccgac gccaacgctg 1200acccctactg ctacgcccac gcccacggca
agcccgacgc cgtcaccgac ggcagcctcc 1260ggagcccgct gcaccgcgag
ttaccaggtc aacagcgatt ggggcaatgg cttcacggta 1320acggtggccg
tgacaaattc cggatccgtc gcgaccaaga catggacggt cagttggaca
1380ttcggcggaa atcagacgat taccaattcg tggaatgcag cggtcacgca
gaacggtcag 1440tcggtaacgg ctcggaatat gagttataac aacgtgattc
agcctggtca gaacaccacg 1500ttcggattcc aggcgagcta taccggaagc
aacgcggcac cgacagtcgc ctgcgcagca 1560agttaa 15662129DNAHordeum
vulgare 2atggcccacg cccgcgtcct cctcctggcg ctcgccgtcc tggccacggc
cgccgtcgcc 60gtcgcctcct cctcctcctt cgccgactcc aacccgatcc ggccggtcac
cgaccgcgcc 120gcgtccacc 129372DNAHordeum vulgare 3atggcgaaca
agcacctgag ccttagcctc ttcctcgtgc tcctgggcct ctccgcctcc 60ctcgcctccg
gc 7241494DNATrichodesmium sp. 4cagagcgcct gcaccctgca gagcgagacc
cacccgccac tgacctggca gaaatgctcg 60tctggtggca cgtgcactca acagacaggc
tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag
cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca
acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc
240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac
ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg
acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat
gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt
gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg
gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg
540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa
caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg
atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc
acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta
ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga
acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt
840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc
gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc
ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgactac
tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg
cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca
tgagtctgtg ggatgactac tacgccaaca tgctgtggct ggactccacc
1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg
ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg
ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc
aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac
cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt
ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc
1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct gtaa
1494572DNAHordeum vulgare 5atggcgaaca agcacctctc cctgagcctc
ttcctggtgc tcctgggcct ctccgcgagc 60ctggcctccg gg
7261548DNAArtificial SequenceDescription of Artificial Sequence
Synthetic nucleotide construct 6atggcgaaca agcacctctc cctgagcctc
ttcctggtgc tcctgggcct ctccgcgagc 60ctggcctccg ggcaacaggc tggcaccaac
acggcggaga accaccccca gctccagtcg 120cagcagtgca cgacgagcgg
cggctgcaag ccgttgagca cgaaggtcgt cctcgactcg 180aactggcgct
gggtccacag cacctcgggc tacaccaact gctacaccgg caacgagtgg
240gacacctcgc tctgccccga cggcaagaca tgcgccgcga actgcgcgct
cgacggtgcg 300gactactctg gcacctacgg tatcacctcc accggcaccg
cgctcacgct caagtttgtc 360acgggctcca atgtcggctc ccgcgtctac
ctcatggcgg atgatacgca ctaccagctg 420ctcaagctcc tgaaccagga
gttcaccttt gacgtcgaca tgtccaacct cccctgcggt 480ctcaacggcg
cgctctacct ctccgcgatg gacgccgacg gtggcatgtc gaagtacccc
540ggaaacaagg ctggtgccaa gtacggaact ggttactgcg actcgcagtg
cccgaaggac 600atcaagttca ttaacggcga ggctaatgtc ggcaactgga
ccgagaccgg cagcaacacc 660ggtacgggca gctacggtac ctgctgcagc
gagatggaca tatgggaggc caacaacgat 720gccgctgctt tcactcccca
cccttgcacc accaccggtc agacccgttg ctctggggat 780gactgcgcgc
gtaacaccgg tctttgcgac ggtgacggct gcgatttcaa ctcgttccgc
840atgggtgaca agaccttcct cggcaagggg atgaccgtcg acacctccaa
gcccttcacc 900gtcgtcaccc agttcctgac caacgacaac acctccaccg
gcacgctctc tgagatccgc 960cgcatctaca ttcagaacgg caaggtcatc
cagaactcgg tcgcgaacat ccccggtgtc 1020gaccccgtca acagcatcac
cgacaacttc tgcgcgcagc agaagaccgc gttcggcgac 1080accaactggt
tcgcgcagaa gggcggcctg aagcagatgg gcgaggccct cggcaacggc
1140atggtcctcg ctctctcgat ctgggacgac cacgccgcga acatgctctg
gctcgactcc 1200gactacccga ccgacaagga cccgtccgcc cccggtgtcg
cgcgcggcac gtgcgcgacc 1260acctcgggtg tcccctccga cgtcgagtcc
caggtgccca actcccaggt cgtcttctcc 1320aacatcaagt tcggcgacat
cggcagcacc ttcagcggca cctcctcccc caacccgcca 1380ggcggctcca
ccacctcctc gcccgtcacc accagcccta cgcccccgcc cacaggcccg
1440accgtccctc agtggggtca gtgcggtggt attggctact ctggctcgac
tacctgcgcc 1500agcccgtaca cttgccacgt cctcaaccct tactactcgc agtgctac
154873006DNAZea mays 7cggtatgaat ttggaaacaa attcagtact tttaaaaaaa
tttgttgtag ggagcaaata 60atacataaaa taatttatgc attattttat tttttatttg
taataatatg cttgaaacga 120taattcagta tgcatgttgt gccagtgtac
tacacgggcg gggggagggg attgagtggg 180ccagcgcggt gcgtagggta
gatgggctga aattgataac tcaagtccga ctaggttctc 240tttttatttc
ccttcctttt ctattttcct ttcttttaat tttcatgctt tcaaactaaa
300ttcaaattcg agttttgaat ttcagcttct aaattgtaca ctaaaattat
atgataaggt 360aacccctact attactttta atttttttat tctaccccat
attgtttact taggggagaa 420taattgactt aatcacattc ttcctaggtt
tcaattctca atctttcaaa tccacatttt 480tagatttcta ttttgaattt
aaataccagt ttggatttag agttcaattt caaaatacac 540aaccaaaata
ccagcatgaa tgcaaatata ttttatgttt atgtatttac ttttctttta
600tactttgctc aaaatagtta ttttcatgta tgaaactcaa taagcaagga
actcacgtta 660ttatataacc taataggaat aatttaggta acataattta
tcatcctctt gatttaaaag 720agatatgcct ccagaataag acacatacta
aaaataactc taatattgaa taactaaagt 780cgtacaaatc tctactatta
ttcctataaa ataataaaga actagctaca acttctttaa 840ggcattattc
agggtttaca gcttgagagg catgaaccca tcctgtatac tcctggactt
900ggaagacaaa atgtcaacca aagtgaaagg ttttcttatg gttgctgcta
agagatagat 960tgaacactag atctctccta agacgtcagg gcatgcgttt
agactcctac acatgcgaaa 1020actgcatctt acagttggaa gaaactatat
ctcaccactt cctgcggtgt aactttgccc 1080aaagatgttg gctcactgtt
ggaatcactc cgccccgaac tttggatcta acgcttgcag 1140tgctacatat
tagagcaaga ctaacaatgc cgtggagaat ggaaggtatt ataaccatgt
1200catggtgcat atggaaatgt cgaaataact ggatattcga aaacataccg
ccaacggtgg 1260cggcctgcaa ggaaatgttc aagactgaaa tgaactacat
ctgctaccaa gttaagctcg 1320agacaggagc taaaagtaga aactggatac
aacactttgt aacatagtga cactcccctt 1380ttcctttctt ttaccttaga
actatacata caatccacat tcaataaaaa tttgtaggta 1440cgccatacac
actaccggaa tccggctctt tgccgagtgt gaggcgcttt gtcgagtgct
1500ttttgtccag cactcggcaa aaaagtcttt gccatgtgcc gcactcggca
aagtcctgct 1560ctcggtaacg accgcgttta ccgagagcag gactctcgac
acagaaatac actcgacaaa 1620gaaatctttg ccgagagcca aacactcggc
gaacggcagc gctcggcaaa gggtcgtcag 1680ccgccgtcta aagctgacgg
tcgttatctt tgtcgagtgc cccctcgtcc gacactcagt 1740agagcaagct
tgccgagtgc catccttgga cactcgataa agtatatttt attttttttt
1800attttgccaa ccaaactttt tgtggtatgt tcctacacta tgtagatcta
catgtaccat 1860tttggcacaa ttacaaaaat gttttctata actattagat
ttagttcgtt tatttgaatt 1920tcttcggaaa attcacatat gaactgcaag
tcactcgaaa catgaaaaac cgtgcatgca 1980aaataaatga tatgcatgtt
atctagcaca agttacgacc gaattcagaa gcagaccaga 2040atcttcaagc
accatgctca ctaaacatga ccgtgaactt gttatccagt tgtttaaaaa
2100ttgtataaaa cacaaataaa gtcagaaatt aatgaaactt gtccacatgt
catgatatca 2160tatatagagg ttgtgataaa aatttgataa tgtttcggta
aagttgtgac gtactatgtg 2220tagaaaccta agtgacctac acataaaatc
atagagtttc aatgtagttc actcgacaaa 2280gactttgtca agtgtccgat
aaaaagtatt cagcaaagaa gccgttgtcg atttactgtt 2340cgtcgagatc
tctttgccga gtgtcacact aggcaaagtc tttacggagt gtttttcagg
2400ctttgacact cggcaaagcg ctcgattcca gtagtgacag taatttgcat
caaaaatagc 2460cgagagattt aaaatgagtc aactaataga ccaactaatt
attagctatt agtcgttagc 2520ttctttaatc taagctaaaa ccaactaata
gcttatttgt tgaattacaa ttagctcaac 2580ggaattctct gttttttcta
taaaaaaaag ggaaactgcc cctcatttac agcaaactgt 2640ccgctgcctg
tcgtccagat acaatgaacg tacctagtag gaactctttt acacgctcgg
2700tcgctcgccg cggatcggag tcccaggaac acgacaccac tgtggaacac
gacaaagtct 2760gctcagaggc ggccacaccc tggcgtgcac cgagccggag
cccggataag cacggtaagg 2820agagtacggc gggacgtggc gacccgtgtg
tctgctgcca cgcagccttc ctccacgtag 2880ccgcgcggcc gcgccacgta
ccagggcccg gcgctggtat aaatgcgcgc cacctccgct 2940ttagttctgc
atacagccaa cccaacacac acccgagcat atcacagtga cagacactac 3000acgatg
300681191DNAArtificial SequenceDescription of Artificial Sequence
Synthetic nucleotide construct 8atggcgaaca agcacctctc cctgagcctc
ttcctggtgc tcctgggcct ctccgcgagc 60ctggcctccg ggcagcagca acaatggggt
caatgtggtg gtattggatg gactggcgcc 120acgacttgcg tagctggctc
cgtctgctcc gtcttgaacc cttactactc ccagtgcatc 180cctggcgctg
ccacggtcac ctcttcaagc gcgccgtcca ctccaactcc ccccgctggt
240gctcttcctc gtcttggagg tgtgaacacg gctggctatg acttcagcgt
tgctacagat 300ggtagcttca caggcaccgg tgtctcccct ccagtctctc
aattctccca cttctcgtct 360cagggcgcga acctgtatcg tattcctttc
gcctggcagc tcatgactcc taccctcggc 420ggtaccatca gccaaagttt
cctgtctcgc tatgaccaga ccgtccaagc cgccttgaac 480tccggtccca
acgtcttcgt catcatcgac ctgcacaact acgcgcgctg gaacgggggc
540atcattgctc agggtggtcc caccgacgcc cagttccaga gcatctggac
tcagctcgct 600cagaagtatg gcagcaacca gcgcgtcatt ttcggcatca
tgaacgagcc gcacgatatt 660ccttctatct cgacctgggt caactccgtg
caaggagctg tcaacgctat ccgcgccgcc 720ggagctacga actacctcct
tcttccaggc agcagctggt cgtctgcaca agcgttcccc 780accgaggccg
gccccctcct cgttaaggtt acggatcctc tcggcggcac cagcaagttg
840atctttgatg ttcacaagta cctggacagc gataacagtg gcactcaccc
tgactgcacc 900accgacaacg tccaggtcct ccagaccctt gtccaattct
tgcaggccaa cggcaatagg 960caggccatcc tcagtgaaac cggaggaggc
aacacctcta gctgcgagtc tctccttgca 1020aatgaactcg cctacgtcaa
gtctgcttac cccactcttg ctggtttctc cgtctgggcc 1080gctggtgcct
ttgataccac ctacgttctc actgttaccc cgaacgctga cggttctgac
1140caacctctct gggttgacgc tgtaaagccc aaccttccta aggacgagct c
1191912DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 9aaggacgagc tc 12101593DNAArtificial
SequenceDescription of Artificial Sequence Synthetic nucleotide
construct 10atggcgaaca agcacctctc cctgagcctc ttcctggtgc tcctgggcct
ctccgcgagc 60ctggcctccg gggcctgcac tctgaccgct gagaaccacc cctcgctgac
gtggtccaag 120tgcacgtctg gcggcagctg caccagcgtc cagggttcca
tcaccatcga cgccaactgg 180cggtggactc accggaccga tagcgccacc
aactgctacg agggcaacaa gtgggatact 240tcgtactgca gcgatggtcc
ttcttgcgcc tccaagtgct gcatcgacgg cgctgactac 300tcgagcacct
atggcatcac cacgagcggt aactccctga acctcaagtt cgtcaccaag
360ggccagtact cgaccaacat cggctcgcgt acctacctga tggagagcga
caccaagtac 420cagatgttcc agctcctcgg caacgagttc accttcgatg
tcgacgtctc caacctcggc 480tgcggcctca atggcgccct ctacttcgtg
tccatggatg ccgatggtgg catgtccaag 540tactcgggca acaaggcagg
tgccaagtac ggtaccggct actgtgattc tcagtgcccc 600cgcgacctca
agttcatcaa cggcgaggcc aacgtagaga actggcagag ctcgaccaac
660gatgccaacg ccggcacggg caagtacggc agctgctgct ccgagatgga
cgtctgggag 720gccaacaaca tggccgccgc cttcactccc cacccttgcn
ccgtgatcgg ccagtcgcgc 780tgcgagggcg actcgtgcgg cggtacctac
agcaccgacc gctatgccgg catctgcgac 840cccgacggat gcgacttcaa
ctcgtaccgc cagggcaaca agaccttcta cggcaagggc 900atgacggtcg
acacgaccaa gaagatcacg gtcgtcaccc agttcctcaa gaactcggcc
960ggcgagctct ccgagatcaa gcggttctac gtccagaacg gcaaggtcat
ccccaactcc 1020gagtccacca tcccgggcgt cgagggcaac tccatcaccc
aggactggtg cgaccgccag 1080aaggccgcct tcggcgacgt gaccgacttn
caggacaagg gcggcatggt ccagatgggc 1140aaggccctcg cggggcccat
ggtcctcgtc atgtccatct gggacgacca cgccgtcaac 1200atgctctggc
tcgactccac ctggcccatc gacggcgccg gcaagccggg cgccgagcgc
1260ggtgcctgcc ccaccacctc gggcgtcccc gctgaggtcg aggccgaggc
ccccaactcc 1320aacgtcatct tctccaacat ccgcttcggc cccatcggct
ccaccgtctc cggcctgccc 1380gacggcggca gcggcaaccc caacccgccc
gtcagctcgt ccaccccggt cccctcctcg 1440tccaccacat cctccggttc
ctccggcccg actggcggca cgggtgtcgc taagcactat 1500gagcaatgcg
gaggaatcgg gttcactggc cctacccagt gcgagagccc ctacacttgc
1560accaagctga atgactggta ctcgcagtgc ctg 159311705DNAArtificial
SequenceDescription of Artificial Sequence Synthetic nucleotide
construct 11atggcgaaca agcacctctc cctgagcctc ttcctggtgc tcctgggcct
ctccgcgagc 60ctggcctccg ggcagctctc gggcagcggc cagacgaccc ggtactggga
ctgctgcaag 120ccgagctgcg cctggcccgg caagggcccc tcgtctccgg
tgcaggcctg cgacaagaac 180gacaacccgc tcaacgacgg cggctccacc
cggtccggct gcgacgcggg cggcagcgcc 240tacatgtgct cctcccagag
cccctgggcc gtcagcgacg agctgtcgta cggctgggcg 300gccgtcaagc
tcgccggcag ctccgagtcg cagtggtgct gcgcctgcta cgagctgacc
360ttcaccagcg ggccggtcgc gggcaagaag atgattgtgc aggcgaccaa
caccggtggc 420gacctgggcg acaaccactt tgacctggcc atccccggtg
gcggtgtcgg tattttcaac 480gcctgcaccg accagtacgg cgctcccccg
aacggctggg gcgaccgcta cggcggcatc 540cattccaagg aagagtgcga
atccttcccg gaggccctca agcccggctg caactggcgc 600ttcgactggt
tccaaaacgc cgacaacccg tcggtcacct tccaggaggt ggcctgcccg
660tcggagctca cgtccaagag cggctgctcc cgtaaggacg agctc
705124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Lys Asp Glu Leu1
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References