U.S. patent application number 12/835661 was filed with the patent office on 2011-10-13 for transgenic plants used as a bioreactor system.
This patent application is currently assigned to The University of Queensland. Invention is credited to Stevens Michael Brumbley, Barrie Fong Chong, Richard Bruce McQualter, Lars Keld Nielsen, Lars Arved Petrasovits, Matthew Peter Purnell.
Application Number | 20110252507 12/835661 |
Document ID | / |
Family ID | 30115783 |
Filed Date | 2011-10-13 |
United States Patent
Application |
20110252507 |
Kind Code |
A1 |
Brumbley; Stevens Michael ;
et al. |
October 13, 2011 |
TRANSGENIC PLANTS USED AS A BIOREACTOR SYSTEM
Abstract
The present invention relates generally to the use of plants as
bioreactors for the production of molecules having useful
properties such as inter alia polymers, metabolites, proteins,
pharmaceuticals and nutraceuticals. More particularly, the present
invention contemplates the use of grasses, and even more
particularly C4 grasses, such as sugarcane, for the production of a
range of compounds such as, for example, polyhydroxyalkanoates,
pHBA, vanillin, indigo, adipic acid, 2-phenylethanol,
1,3-propanediol, sorbitol, fructan polymers and lactic acid as well
as other products including, inter alia, other plastics, silks,
carbohydrates, therapeutic and nutraceutic proteins and antibodies.
The present invention further extends to transgenic plants and, in
particular, transgenic C4 grass plants, capable of producing the
compounds noted above and other products, and to methods for
generating such plants. The ability to utilize the high growth rate
and efficient carbon fixation of C4 grasses is advantageous, in
that it obviates the significant growth penalties observed in other
plants, and results in high yields of desired product without
necessarily causing concomitant deleterious effects on individual
plants. In addition, the C4 grass, sugarcane, is particularly
advantageous, as in addition to the features common to all C4
grasses, this plant accumulates sucrose. This sucrose store
provides a ready supply of carbon based compounds and energy which
may further obviate any deleterious effects on the growth of the
plant associated with the production of the product. The present
invention provides, therefore, a bioreactor system comprising a
genetically modified plant designed to produce particular metabolic
or biosynthetic products of interest.
Inventors: |
Brumbley; Stevens Michael;
(Redbank Plains, AU) ; Purnell; Matthew Peter;
(Tarragindi, AU) ; Chong; Barrie Fong; (Wynnum
West, AU) ; Petrasovits; Lars Arved; (West End,
AU) ; Nielsen; Lars Keld; (Brisbane, AU) ;
McQualter; Richard Bruce; (Kallangur, AU) |
Assignee: |
The University of
Queensland
St. Lucia
AU
BSES Limited
Indooroopilly
AU
|
Family ID: |
30115783 |
Appl. No.: |
12/835661 |
Filed: |
July 13, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10520882 |
Nov 16, 2005 |
7754943 |
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PCT/AU03/00903 |
Jul 11, 2003 |
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12835661 |
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60394869 |
Jul 11, 2002 |
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Current U.S.
Class: |
800/288 ;
435/320.1; 435/419; 536/123.1; 548/457; 562/475; 562/589; 562/590;
568/442; 568/715; 568/852; 800/320 |
Current CPC
Class: |
C08G 63/06 20130101;
C12N 15/8243 20130101; C12N 15/8257 20130101 |
Class at
Publication: |
800/288 ;
435/320.1; 435/419; 800/320; 568/715; 568/852; 562/590; 562/589;
536/123.1; 548/457; 568/442; 562/475 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12N 5/10 20060101 C12N005/10; A01H 5/10 20060101
A01H005/10; C07C 33/22 20060101 C07C033/22; C07C 31/20 20060101
C07C031/20; C07C 65/03 20060101 C07C065/03; C07C 59/08 20060101
C07C059/08; C08B 37/00 20060101 C08B037/00; C07D 403/04 20060101
C07D403/04; C07C 31/26 20060101 C07C031/26; C07C 47/58 20060101
C07C047/58; C12N 15/82 20060101 C12N015/82; C07C 55/14 20060101
C07C055/14 |
Claims
1-54. (canceled)
55. A method of producing a bioproduct in a genetically modified C4
grass which C4 grass substantially does not produce the bioproduct
prior to genetic modification, said method comprising introducing a
genetic sequence encoding an enzyme required for synthesis of the
bioproduct or a precursor of said bioproduct into a cell or group
of cells of said C4 grass, regenerating a C4 grass from said cell
or group of cells and growing the C4 grass or genetically modified
progeny therefrom under conditions sufficient to produce the
bioproduct.
56. The method of claim 55 wherein the bioproduct is selected from
the list comprising a polyhydroxyalkanoate (PHA), p-hydroxybenzoic
acid (pHBA), vanillin, sorbitol and fructan.
57. The method of claim 56 wherein the biproduct is
polyhydroxyalkanoate (PHA) and the genetic sequence comprises one
or more genetic sequences selected from the list comprising: (i) a
nucleotide sequence encoding a phaA; (ii) a nucleotide sequence
encoding phaB; (iii) a nucleotide sequence encoding phaC; (iv) a
nucleotide sequence encoding phaC1; (v) a nucleotide sequence
encoding phaG; (vi) a nucleotide sequence encoding phaJ; (vii) SEQ
ID NO:1 or SEQ ID NO:3 or SEQ ID NO:10 or SEQ ID NO:12 or a
nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to SEQ ID NO:1 or SEQ
ID NO:3 or SEQ ID NO:10 or SEQ ID NO:12 or a complementary form
thereof under low stringency conditions; (viii) SEQ ID NO:4 or SEQ
ID NO:6 or SEQ ID NO:13 or SEQ ID NO:15 or a nucleotide sequence
having at least 60% identity thereto after optimal alignment, or
capable of hybridizing to SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID
NO:13 or SEQ ID NO:15 or a complementary form thereof under low
stringency conditions; (ix) SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID
NO:16 or SEQ ID NO:18 or a nucleotide sequence having at least 60%
identity thereto after optimal alignment, or capable of hybridizing
to SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18 or a
complementary form thereof under low stringency conditions; (x) SEQ
ID NO:19 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID
NO:25 or SEQ ID NO:27 or a nucleotide sequence having at least 60%
identity thereto after optimal alignment, or capable of hybridizing
to SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:24 or
SEQ ID NO:25 or SEQ ID NO:27 or a complementary form thereof under
low stringency conditions; (xi) SEQ ID NO:28 or SEQ ID NO:30 or a
nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to SEQ ID NO:28 or SEQ
ID NO:30 or a complementary form thereof under low stringency
conditions; (xii) SEQ ID NO:31 or SEQ ID NO:33 or a nucleotide
sequence having at least 60% identity thereto after optimal
alignment, or capable of hybridizing to SEQ ID NO:31 or SEQ ID
NO:33 or a complementary form thereof under low stringency
conditions;
58. The method of claim 56 wherein the bioproduct is
p-hydroxybenzoic acid (pHBA) and the genetic sequence comprises one
or more genetic sequences selected from the list comprising a
nucleotide sequence encoding hydroxycinnamoyl-CoA hydratase/lysase,
a nucleotide sequence encoding chorismate pyruvate lyase, a
nucleotide sequence encoding the ubiC gene from E. coli, a
nucleotide sequence encoding the HCHL gene from Pseudomonas
fluorescens
59. The method of claim 56 wherein the bioproduct is vanillin and
the enzyme encoded by the genetic sequence is selected from the
list comprising 3-dehydroshikimate dehyhratase,
catechol-O-methyltransferase, aryl aldehyde dehybrogenase,
feruloyl-CoA synthetase, enoyl-CoA hydratase and enoyl-CoA
aldolase.
60. The method of claim 56 wherein the bioproduct is sorbitol and
the enzyme encoded by the genetic sequence is glucose-fructose
oxidoreductase.
61. The method of claim 60 wherein the glucose-fructose
oxidoreductases is encoded by the polynucleotide sequence set forth
in GenBank Accession number Z80356, or a homolog thereof having at
least 60% identity thereto after optimal alignment, or capable of
hybridizing to GenBank Accession number Z80356 or a complementary
form thereof under low stringency conditions.
62. The method of claim 60 wherein the glucose-fructose
oxidoreductase is encoded by the polynucleotide sequence set forth
in GenBank Accession number M97379, or a homolog thereof having at
least 60% identity thereto after optimal alignment, or capable of
hybridizing to GenBank Accession number M97379 or a complementary
form thereof under low stringency conditions.
63. The method of claim 56 wherein the bioproduct is fructan and
the enzyme encoded by the genetic sequence is selected from the
list comprising fructosyltransferase and levan sucrase.
64. The method of claim 63 wherein the fructosyltransferase is
encoded by the polynucleotide sequence set forth in GenBank
Accession number AY150365, or a homolog thereof having at least 60%
identity thereto after optimal alignment, or capable of hybridizing
to GenBank Accession number AY150365 or a complementary form
thereof under low stringency conditions.
65. A vector comprising a genetic sequence encoding an enzyme of
claim 55.
66. The vector of claim 65 wherein the vector is an expression
vector.
67. A genetically modified C4 grass cell or group of cells
comprising an introduced genetic sequence encoding an enzyme of
claim 55.
68. A genetically modified C4 grass cell or group of cells
comprising an introduced genetic sequence encoding a vector of
claim 65.
69. A genetically modified C4 grass plant comprising a cell or
group of cells of claim 67 or genetically modified progeny
thereof.
70. Seeds or other reproductive material from the plant of claim
68.
71. A product produced in a genetically modified plant or
genetically modified cells or parts of a plant by the method of
claim 55.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to the use of plants
as bioreactors for the production of molecules having useful
properties such as inter alia polymers, metabolites, proteins,
pharmaceuticals and nutraceuticals. More particularly, the present
invention contemplates the use of grasses, and even more
particularly C4 grasses, such as sugarcane, for the production of a
range of compounds such as, for example, polyhydroxyalkanoates,
pHBA, vanillin, indigo, adipic acid, 2-phenylethanol,
1,3-propanediol, sorbitol, fructan polymers and lactic acid as well
as other products including, inter alia, other plastics, silks,
carbohydrates, therapeutic and nutraceutic proteins and antibodies.
The present invention further extends to transgenic plants and, in
particular, transgenic C4 grass plants, capable of producing the
compounds noted above and other products, and to methods for
generating such plants. The ability to utilize the high growth rate
and efficient carbon fixation of C4 grasses is advantageous, in
that it obviates the significant growth penalties observed in other
plants, and results in high yields of desired product without
necessarily causing concomitant deleterious effects on individual
plants. In addition, the C4 grass, sugarcane, is particularly
advantageous, as in addition to the features common to all C4
grasses, this plant accumulates sucrose. This sucrose store
provides a ready supply of carbon based compounds and energy which
may further obviate any deleterious effects on the growth of the
plant associated with the production of the product. The present
invention provides, therefore, a bioreactor system comprising a
genetically modified plant designed to produce particular metabolic
or biosynthetic products of interest.
[0003] 2. Description of the Prior Art
[0004] Reference to any prior art in this specification is not, and
should not be taken as, an acknowledgment or any form of suggestion
that this prior art forms part of the common general knowledge in
any country.
[0005] Modern techniques of biotechnology are driving a new
revolution that promises both scientific and financial gains for a
range of industries. One difficulty, however, is the large
financial cost of establishing sufficient infrastructure to
generate recombinant products or to generate the products resulting
from recombinant processes. Alternative, more cost effective
systems are required to assist the generation of large amounts of
product resulting from recombinant processes.
[0006] For agricultural industries, the generation of genetically
engineered plants enables plants to be quickly developed with
desired traits such as resistance to pathogen infestation. However,
plants can also be used to produce a wide range of compounds not
normally produced within the plant, thereby providing a source of
renewable raw materials for the manufacturing, energy and
pharmaceutical industries.
[0007] This endeavour is aided by the fact that plants, animals,
insects, bacteria, fungi and even viruses have evolved in a wide
range of different habitats and, hence, produce a remarkable array
of compounds which allow them to survive and thrive under very
varied environmental conditions. It is estimated that up to 100,000
unique compounds exist in the plant kingdom alone. In the future,
genes and even entire genetic pathways may become available from
different sources to assist in the manufacture of a wide range of
commercial products.
[0008] Traditional chemical industries are increasingly looking
towards biological systems for the production of bulk and fine
chemicals. Biological processes offer numerous advantages over
chemical processes, including the elimination of complicated and
difficult high pressure and high temperature reactions, the use of
aqueous systems rather than organic solvents, high degrees of
product stereo-specificity, a capacity for highly complex synthesis
and comparatively simple scale-up. The use of biological processes
is not a new phenomenon, as many fine chemicals (e.g. enzymes,
antibiotics) and bulk chemicals (e.g. ethanol, amino acids, citric
acid, lactic acid) are produced effectively in microbial systems.
Advances in molecular biology and genomics have enabled an
expansion of the available product range, the transfer of
production systems to microbes with desirable production traits,
and significantly increased yields. Nevertheless, inherent
limitations remain, in that the raw materials (e.g. molasses,
sucrose, or high fructose corn syrup) and scaled up fermentation
processes are relatively expensive.
[0009] By contrast, genetically modified plants should not require
any more raw materials than are already required by their
non-transformed counterparts and have the potential to provide a
low cost of production per tonne of biomass, when compared with
fermentation methods. Thus, if reasonable product yields could be
achieved in plants, and if these products could be extracted at
reasonable cost, the potential for chemical production in plants
would be extremely high.
[0010] The polyhydroxyalkanoate (PHA), poly-(D-3-hydroxybutyrate)
(PHB), is a thermoplastic with physical properties akin to
polypropylene. Both PHB and polypropylene are water insoluble,
exhibit good gas-barrier properties and possess similar melting
points, degrees of crystallinity and glass-rubber transition
temperatures (De Koning, Can. J. Micro. 41(1): 303-309, 1995),
although PHB is more resistant to UV radiation. Moreover, unlike
polypropylene, PHB is rapidly degraded by numerous bacteria and
fungi under composting conditions (Jendrossek et al., App. Micro.
Biotech. 46: 451-463, 1996; Mergaert and Swings, Indust. Micro.
Biotech. 17: 463-469, 1996).
[0011] Cost is the major reason why PHA produced by bacterial
fermentation cannot compete with conventional plastics production
methodologies. Major contributors to the cost of PHA are substrate
cost, energy consumption during fermentation, disposal of waste
product, and the cost of constructing and maintaining plant and
machinery. The use of transgenic plants for PHA production,
however, has the potential to either eliminate or drastically
reduce these costs since atmospheric carbon dioxide would be the
substrate and energy would be derived from sunlight. Operating
costs would be no more than what is incurred in ordinary
agricultural practices. Waste products are the same as for a
non-transgenic crop. This makes plants an attractive potential
alternative to bacterial fermentation.
[0012] The production of PHB has been most closely studied in the
bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus),
which accumulates PHB at up to 80% of its cell dry-weight
(Steinbuchel and Schlegel, Mol. Micro. 5: 535-542, 1991). The PHB
biosynthetic pathway within R. eutropha is well known and consists
of three steps catalyzed by the three enzymes 3-ketothiolase,
acetoacetyl-CoA reductase and PHB synthase, respectively. For
large-scale industrial production of PHB, bacterial fermentation is
economically and environmentally less favourable than the
corresponding production of petrochemically derived plastics like
polypropylene (Lee, Trends Biotech. 14: 431-438, 1996; Gerngross,
Nature Biotech. 17: 541-544, 1999). Hence, since the glucose
supplied to PHB-producing bacteria is derived from plants, it would
be advantageous to be able to produce PHB in plants directly.
[0013] Attempts to achieve this goal have been unsuccessful, due
largely to the significant added burden placed upon individual
plants by requiring them to produce this macromolecule, resulting
in a severe reduction in plant growth and infertility (Poirier et
al., Science 256: 520-523, 1992; Bohmert et al., Planta 211:
841-845, 2000).
[0014] In accordance with the present invention, an efficient
bioreactor system is developed in plants and in particular
Saccharum sp. such as sugarcane.
SUMMARY OF THE INVENTION
[0015] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element or integer or group of elements or integers but not
the exclusion of any other element or integer or group of elements
or integers.
[0016] Nucleotide and amino acid sequences are referred to by a
sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond
numerically to the sequence identifiers <400>1 (SEQ ID NO:1),
<400>2 (SEQ ID NO:2), etc. A summary of the sequence
identifiers is provided in Table 1. A sequence listing is provided
after the claims.
[0017] The present invention provides a plant-derived bioreactor
system. Although previous attempts to effect the manufacture of
useful industrial and other products in plants have not been overly
successful, it has been determined in accordance with the present
invention that this was due to the extra load placed on individual
plants which resulted in deleterious growth effects. C4 grasses,
which have particularly efficient mechanisms for the assimilation
of carbon, are identified as having a high growth rate and high
accumulation of biomass making them useful as bioreactors for the
production of a wide range of products. Furthermore, the C4 grass,
sugarcane, is particularly useful as this plant stores sugars in
dimeric and/or polymeric forms. These stores may be utilized when
needed such as, for example, for rapid vegetative growth, or for
energy during times of significant environmental stress. The store
of carbohydrate is identified in accordance with the present
invention as providing a ready supply of precursor for many
metabolic pathways, and utilisation of this store does not stress
the producing plant. Therefore, the present invention is
predicated, in part, on the identification of a subset of plants,
namely the C4 grasses (particularly sugarcane), as useful
bioreactors on the basis of their high carbon assimilation rate,
rapid growth, high biomass production and large carbohydrate store,
such as is found in the stem.
[0018] The present inventors have capitalized on the potential of a
crop that has a highly efficient C4 carbon assimilation mechanism,
a rapid growth rate and naturally harbours large quantities of
sucrose in its stems, thereby having the ideal properties of a
bioreactor. The instant inventors have developed a means to
engineer this crop so as to effectively accumulate significant
quantities of a product without significant decreases in biomass or
growth rate. In so doing, the plants contemplated herein permit the
manufacture of products such as biodegradable plastics, vanillin,
indigo, adipic acid, 2-phenylethanol, 1,3-propanediol, sorbitol,
fructan polymers and lactic acid as well as therapeutic,
nutraceutic and diagnostic agents without incurring the previously
observed deleterious effects on growth and viability.
[0019] Accordingly, one aspect of the present invention provides a
method for generating a plant-based bioreactor system, said method
comprising selecting a plant having a high efficiency carbon
assimilation mechanism, rapid growth rate and/or high biomass
production and/or reserves of metabolites or having a capacity to
generate such reserves and/or which possess metabolic and/or
biosynthetic pathways useful in the manufacture of a product of
interest or a precursor form thereof; genetically modifying cells
of the plant to enable access to said metabolites and/or metabolic
or biosynthetic pathways; and then regenerating a genetically
modified plant from said cells.
[0020] The present invention is particularly directed to C4
grasses, however, other non-grass C4 plants such as woody or
herbaceous plants which utilise the C4 pathway are also
contemplated by, and are within the scope of, the present
invention.
[0021] In a preferred embodiment of the present invention, the
subject plant is a C4 grass. In an even more preferred embodiment
the plant is sugarcane. As used herein, the term sugarcane is to be
understood to include, inter alia, plants of the Saccharum genus,
incl. S. robustum, S. offinarum, and S. spontaneum and hybrid
Saccharum sp., incl. modern sugarcane cultivars.
[0022] The preferred compounds to be produced by the plant
bioreactor include: vanillin, sorbitol, polyhydroxyalkanoates (PHA)
such as poly-(D-3-hydroxybutyrate) (PHB), indigo, fructan, lactic
acid, adipic acid, 1,3-propanediol, 2-phenylethanol and pHBA.
However, the present invention also extends to the use of C4
grasses as bioreactors to generate a compounds such as
therapeutics, nutrapharmaceuticals, diagnostic agents including,
for example, single chain antibodies, industrial enzymes and the
like.
[0023] The present invention contemplates, therefore, a method for
producing a product of interest including a product or intermediate
of a biosynthetic or metabolic pathway in a C4 grass, said method
comprising expressing one or more genetic sequences which encode
one or more enzymes or proteins required for the production of the
product or intermediate or a homolog or precursor thereof or which
induces gene silencing of genetic material which encodes an enzyme
or protein in a biosynthetic or metabolic pathway in cell of a C4
grass plant such that the product or intermediate accumulates in
the cytosol, storage vacuole, plastid or non-plastid organelles of
the cell, or accumulates in the juice or vascular fluid of the
plant.
[0024] The present invention therefore provides for the production
of a product in a C4 grass wherein product accumulation is at least
in part predicated on the direct activation or inhibition
(including down-regulation) of an enzyme in a biosynthetic or
metabolic pathway by the administration to the plant of an enzyme
inhibitor or activator. Reference to an "enzyme inhibitor or
activator" includes genetic materials which, for example, induce
(post-transcriptional or transcriptional gene silencing of a
structural gene or positive or negative regulator gene.
[0025] In one preferred embodiment, increased accumulation of a
product or intermediate from a biosynthetic or metabolic pathway is
a result of inhibition of one or more biosynthetic or metabolic
enzymes and optionally re-directing metabolites down another
biosynthetic or metabolic pathway.
[0026] In another preferred embodiment, the present invention
contemplates the use of sugarcane as a bioreactor. Therefore,
alteration to the gene expression profile of sugarcane to effect
the production of an endogenous metabolite at an increased level,
or to produce any heterologous metabolite is within the scope of
the present invention. Accordingly, induction or supression of any
biosythetic genes in sugarcane, such as described herein, is to be
considered within the scope of the present invention.
[0027] Typically, the production of one or more metabolites or
heterologous proteins, polypeptides or peptides in a plant is
achieved by expression of a nucleic acid molecule encoding the
metabolite or protein, polypeptide or peptide of interest. Any
nucleic acid which encodes a protein, polypeptide or peptide of
interest is contemplated by the present invention. However,
preferred nucleic acids include those encoding: [0028] (i) vanillin
biosynthetic enzymes, including 3-dehydroshikimate dehydratase,
catechol-o-methyltransferase, aryl aldehyde dehdrogenase,
feruloyl-CoA synthetase, enoyl-CoA hydratase/aldolase; [0029] (ii)
sorbitol biosynthetic enzymes, including glucose/fructose
oxidoreductase; [0030] (iii) PHA biosynthetic enzymes, including
3-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, enoyl
hydratase, 3-hydroxyacyl-acyl carrier protein:CoA tranferase;
[0031] (iv) indigo biosynthetic enzymes, including tryptophanase,
L-tryptophan indole lyase, napthalene dioxygenase, R. eutrophica
bec gene product; [0032] (v) fructan biosynthetic enzymes,
including fructosyltransferases and levansucrases; [0033] (vi)
lactic acid biosynthetic enzymes, including lactate dehydrogenase;
[0034] (vii) adipic acid biosynthetic enzymes, including
3-dehydroshikimate dehydratase, protocatechuate decarboxylase and
catechol 1,2-dioxygenase; [0035] (viii) petroselinic acid
biosynthetic enzymes, including 3-ketoacyl-ACP synthase; [0036]
(ix) 1,3-propanediol biosynthetic enzymes including glycerol
dehydratase, 1,3-propanediol oxidoreductase, glycerol-3-phosphate
dehydrogenase, and glycerol-3-phosphatase; and/or [0037] (x)
2-phenylethanol biosynthetic enzymes including aromatic-L-amino
acid decarboxylase, 2-phenylethylamine oxidase and aryl alcohol
dehydrogenase. [0038] (xi) pHBA biosynthetic enzymes including
4-hydroxycinnamoyl-CoA hydratas/lyase (HCHL) and chorismate
pyruvate lyase (CPL).
[0039] Any of a number of products may be produced according to the
present invention. Examples of compounds that may be produced via
metabolic engineering of a subject plant include: vanillin
(4-hydroxy-3-methoxybenzaldehyde); sorbitol; PHAs; indigo; fructan;
lactic acid (2-hydroxypropanoic Acid); adipic acid; 1,3
propanediol, 2-phenylethanol and pHBA. These compounds, however,
are only examplary, and the present invention is predicated on the
use of C4 plants as bioreactors for any compound that can be
synthesised in the plant. Accordingly, the present invention is not
limited to any one product or method for producing the product.
[0040] In a particularly preferred embodiment, the present
invention contemplates a method for accumulating polymers
comprising one or more species of hydroxyalkanoic acid monomer in a
C4 grass, said method comprising expressing one or more genetic
sequences which encode enzymes required for the production of the
polymers or a homolog or precursor thereof in a cell of a C4 grass
such that PHA polymers accumulate in the cytosol, storage vacuole
or plastid or non-plastid organelle of said cell.
[0041] The present invention further contemplates a method for
generating a plant which produces PHAs, said method comprising
introducing into cells of said plant a genetic sequence
comprising:-- [0042] (i) a nucleotide sequence encoding a phaA or
homolog thereof; [0043] (ii) a nucleotide sequence encoding phaB or
homolog thereof; [0044] (iii) a nucleotide sequence encoding phaC
or homolog thereof; [0045] (iv) a nucleotide sequence encoding
phaC1 or homolog thereof; [0046] (v) a nucleotide sequence encoding
phaG or homolog thereof; [0047] (vi) a nucleotide sequence encoding
phaJ or homolog thereof [0048] (vii) SEQ ID NO:1 or SEQ ID NO:3 or
SEQ ID NO:10 or SEQ ID NO:12 or a nucleotide sequence having at
least 60% identity thereto after optimal alignment, or capable of
hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:10 or SEQ ID
NO:12 or a complementary form thereof under low stringency
conditions; [0049] (viii) SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID
NO:13 or SEQ ID NO:15 or a nucleotide sequence having at least 60%
identity thereto after optimal alignment, or capable of hybridizing
to SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID NO:15 or a
complementary form thereof under low stringency conditions; [0050]
(ix) SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18 or
a nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to SEQ ID NO:7 or SEQ
ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18 or a complementary form
thereof under low stringency conditions; [0051] (x) SEQ ID NO:19 or
SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:25 or SEQ
ID NO:27 or a nucleotide sequence having at least 60% identity
thereto after optimal alignment, or capable of hybridizing to SEQ
ID NO:19 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID
NO:25 or SEQ ID NO:27 or a complementary form thereof under low
stringency conditions; [0052] (xi) SEQ ID NO:28 or SEQ ID NO:30 or
a nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to SEQ ID NO:28 or SEQ
ID NO:30 or a complementary form thereof under low stringency
conditions; [0053] (xii) SEQ ID NO:31 or SEQ ID NO:33 or a
nucleotide sequence having at least 60% similarity thereto or
capable of hybridizing to SEQ ID NO:31 or SEQ ID NO:33 or a
complementary form thereof under low stringency conditions; and
then regenerating a plant from said cells.
[0054] A convenient C4 grass for use in the present invention is
sugarcane. Sugarcane has certain advantages, which make it a useful
crop for use as a bioreactor including, inter alia, its efficient
carbon fixation, high biomass accumulation, rapid growth and
natural ability to accumulate large quantities of sucrose.
Moreover, it achieves this very efficiently by collecting solar
radiation and converting it into a carbon sink (i.e. sucrose).
Sugarcane is also a hardy crop, is relatively easy to grow and
provides a large biomass capability. A micropropagation system is
already available and an industry infrastructure in existence.
[0055] In another embodiment, the present invention provides a
method for generating a plant which produces vanillin or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence comprising at least one of the
following:-- [0056] (i) a nucleotide sequence encoding a
3-dehydroshikimate dehydratase; [0057] (ii) a nucleotide sequence
encoding catechol-o-methyltransferase; [0058] (iii) a nucleotide
sequence encoding aryl aldehyde dehydrogenase; [0059] (iv) a
nucleotide sequence encoding feruloyl-CoA synthetase; [0060] (v) a
nucleotide sequence encoding enoyl-CoA hydratase; [0061] (vi) a
nucleotide sequence encoding enoyl-CoA aldolase; and/or [0062]
(vii) a nucleotide sequence encoding a homolog of any one of (i)
through (vi) and then regenerating a plant from said cells.
[0063] Another aspect of the present invention contemplates a
method for producing sorbitol in a C4 grass, said method comprising
expressing one or more genetic sequences encoding a
glucose-fructose oxidoreductase, in cells of a C4 grass such that
sorbitol accumulates anywhere in the cell or extracellular matrix
of the plant.
[0064] In another embodiment, the present invention is directed to
a method for generating a plant which produces indigo or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence comprising at least one of the
following:-- [0065] (i) a nucleotide sequence encoding genetic
sequences encoding tryptophanase; [0066] (ii) a nucleotide sequence
encoding L-tryptophan indole lyase; [0067] (iii) a nucleotide
sequence encoding napthalene dioxygenase; [0068] (iv) a nucleotide
sequence comprising the R. eutropha bec gene; [0069] (v) the
nucleotide sequence set forth in Genbank accession number D14279,
or a nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to Genbank D14279
under low stringency conditions. [0070] (vi) the nucleotide
sequence set forth in Genbank accession number M83949, or a
nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to Genbank M83949
under low stringency conditions. [0071] (vii) the nucleotide
sequence set forth in Genbank accession number AF306552, or a
nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to Genbank AF306552
under low stringency conditions. and then regenerating a plant from
said cells.
[0072] In another embodiment, the present invention relates to a
method for generating a C4 grass plant which produces a fructan or
a precursor thereof, said method comprising introducing into cells
of said plant a genetic sequence comprising at least one of the
following:-- [0073] (i) a nucleotide sequence encoding a
fructosyltransferase [0074] (ii) a nucleotide sequence encoding a
levansucrase; [0075] (iii) the nucleotide sequence set forth in
Genbank accession number AY150365, or a nucleotide sequence having
at least 60% identity thereto after optimal alignment, or capable
of hybridizing to Genbank AY150365 under low stringency conditions.
and then regenerating a plant from said cells.
[0076] In another embodiment, the present invention contemplates a
method for generating a plant which produces lactic acid or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence encoding lactate dehydrogenase and
then regenerating a plant from said cells.
[0077] In another embodiment, the present invention contemplates a
method for generating a plant which produces adipic acid or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence comprising at least one of the
following:-- [0078] (i) a nucleotide sequence encoding a
3-dehydroshikimate dehydratase and/or; [0079] (ii) a nucleotide
sequence encoding protochatechuate decarboxylase; [0080] (iii) a
nucleotide sequence encoding catechol 1,2-dioxygenase; [0081] (iv)
a nucleotide sequence encoding 3-ketoacyl-ACP synthase; and/or
[0082] (v) a nucleotide sequence encoding a homolog of any one of
(i) though (iv) and then regenerating a plant from said cells.
[0083] In another embodiment, the present invention contemplates a
method for generating a plant which produces 1,3-propanediol or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence comprising at least one of the
following:-- [0084] (i) a nucleotide sequence encoding a glycerol
dehydratase and/or; [0085] (ii) a nucleotide sequence comprising
the dhaB gene from Klebsiella pneumoniae, or a homolg thereof;
[0086] (iii) a nucleotide sequence encoding 1,3-propanediol
oxidoreductase; [0087] (iv) a nucleotide sequence comprising the
dhaT gene from Klebsiella pneumoniae or homolg thereof; [0088] (v)
a nucleotide sequence encoding glycerol-3-phosphate dehydrogenase;
[0089] (vi) a nucleotide sequence encoding glycerol-3-phosphatase;
and/or [0090] (vi) a nucleotide sequence encoding a homolog of any
one of (i) though (vi) and then regenerating a plant from said
cells.
[0091] In another embodiment, the present invention contemplates a
method for generating a plant which produces 2-phenylethanol or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence comprising at least one of the
following:-- [0092] (i) a nucleotide sequence encoding a
aromatic-L-amino acid decarboxylase; [0093] (ii) a nucleotide
sequence encoding 2-phenylethylamine oxidase; [0094] (iii) a
nucleotide sequence encoding aryl-alcohol dehydrogenase; and/or
[0095] (iv) a nucleotide sequence encoding a homolog of any one of
(i) though (iii) and then regenerating a plant from said cells.
[0096] In another aspect, the present invention contemplates a
method for generating a plant which produces pHBA or a precursor
thereof, said method comprising introducing into cells of said
plant a genetic sequence comprising at least one of the
following:-- [0097] (i) a nucleotide sequence encoding
hydroxycinnamoyl-CoA hydratase/lyase; [0098] (ii) a nucleotide
sequence encoding chorismate pyruvate lyase; [0099] (iii) a
nucleotide sequence comprising the ubiC gene from E. coli, or a
homolg thereof; and/or [0100] (iv) a nucleotide sequence comprising
the HCHL gene from Pseudomonas fluorescens or homolg thereof; and
then regenerating a plant from said cells.
[0101] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, sugarcane.
[0102] A further aspect of the present invention provides a
transfected or transformed cell, tissue, or organ from a C4 grass,
which comprises a nucleotide sequence encoding one or more enzymes
required for the production of a useful product as well as severed
or cut parts of a genetically modified plant including stem,
flower, seed or other reproductive parts.
[0103] Accordingly, another aspect of the present invention
provides a genetically modified C4 grass having cells carrying one
or more genetic sequences such that one of the following products
in the cytosol, storage vacuole, non-plastid organelle or
extracellular matrix of said cells: [0104] (i)
polyhydroxy-alkanoate polymers [0105] (ii) vanillin [0106] (iii)
sorbitol [0107] (iv) indigo [0108] (v) fructans [0109] (vi) lactic
acid [0110] (vii) adipic acid [0111] (viii) 1,3-propanediol [0112]
(ix) 2-phenylethanol [0113] (x) pHBA
[0114] The present invention extends to parts of plants tissue
including leaves, stems, vascular bundles, bark, reproductive
material, roots and any extracted liquid ("juice") from said
plant.
[0115] In order to direct product accumulation to a desired
sub-cellular location, particular specific "target sequences" may
be incorporated into the genetic constructs described above.
[0116] A target sequence includes a signal sequence such as a
signal sequence to direct the protein to a plastid, vacuole,
mitochondrion, peroxisome or ontologically related organelles, or
other appropriate organ or tissue.
[0117] The plants of the present invention may also be further
"tagged" with a reporter that identifies the plant as a plant
bioreactor. Any number of physiological or genetic tags would be
suitable, and readily identified by one of skill in the art.
[0118] Accordingly, the present invention contemplates a plant
suitable for use as a bioreactor that has been tagged with a
genetic sequence which encodes or comprises a genotypic or
phenotypic feature that allows differentiation of the plant
bioreactor from a wild-type plant, or which identifies the plant as
a propietary plant.
[0119] The plant-based bioreactor system of the present invention
is useful in enabling the production of molecules such as PHAs,
pHBA, vanillin, sorbitol, indigo, fructans, lactic acid, adipic
acid, 1,3-propanediol, 2-phenylethanol, inter alia, by a number of
different parties such as different commercial entities. The
present invention extends, therefore, to a data processing system
to monitor the use of the plants and/or the production of target
molecules.
[0120] Accordingly, another aspect of the present invention
contemplates a method for generating a target molecule in a
sucrose-accumulating plant, said method comprising:-- [0121] (i)
providing a plant or cells of a plant to a party; and [0122] (ii)
permitting the party to generate and harvest molecules from said
plant or cells of said plant receiving and processing data from
said party.
[0123] The data received from the party includes, for example,
numbers of plants grown and/or harvested, the types of genetic
constructs introduced into the cells and/or income received from
sale of the products.
BRIEF DESCRIPTION OF THE FIGURES
[0124] FIG. 1 is a schematic representation showing the four
different biosynthetic strategies employed by bacteria that use
PHAs as an energy source.
[0125] FIG. 2 is a diagrammatic representation of the biosynthetic
pathway of PHB in the bacterium R. eutropha. This pathway comprises
three steps catalyzed, respectively, by three enzymes: first, two
molecules of acetyl-CoA are condensed to acetoacetyl-CoA by
3-ketothiolase (encoded by phaA); secondly, acetoacetyl-CoA is
reduced to D-3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase
(phaB); and thirdly, D-3-hydroxybutyryl-CoA is polymerized to PHB
by PHB synthase (phaC).
[0126] FIG. 3 is a diagrammatic representation of the flow of
carbon in sub-cellular compartments within a sugarcane cell.
[0127] FIG. 4 shows a graphical representation of the detection of
PHB in chloroplasts of transgenic sugarcane. A-C: Detection of PHB
by HPLC. A: WT sugarcane (s/c) -ve control; B: sample in A spiked
with PHB; C: plastid-targeted, PHB +ve s/c line. Arrows: elution
point of crotonic acid (PHB breakdown product). Insert in C shows
that the peak at 30 min in C has the same spectrum as crotonic
acid. D-F: Detection of PHB granules by transmission electron
microscopy. D: Chloroplast (c/p) of mesophyll cell of PHB +ve
Arabidopsis control; E: c/p of mesophyll cell of PHB +ve s/c line;
F: c/p of bundle-sheath cell of same line in E. Scale bars=200
nm.
[0128] FIG. 5 illustrates the agronomic performance of
PHB-producing sugarcane lines. Four transgenic sugarcane lines
expressing the PHB biosynthesis genes of R. eutropha (filled bars)
were grown for 3 months in a randomised glasshouse plot, together
with GFP expressing (open bars) and tissue-culture-regenerated WT
(hatched bars) plants as controls. PHB content in lamina from the
tips of mature leaves was quantified by HPLC analysis. Data are the
mean.+-.SE (n=3). DW=dry-weight.
[0129] FIG. 6 shows the affect of PHB production on sugarcane sugar
accumulation. Mature-(A-D) and intermediate-aged (E-H) stem
internodes from the PHB producing (solid bars), GFP expressing
(open bars) and WT (hatched bars) sugarcane plants in FIG. 5 were
assayed for sucrose, glucose and fructose concentrations. Data are
the mean.+-.SE (n=3). DW=dry-weight.
[0130] FIG. 7 is a graphical representation showing the
distribution of PHB throughout a PHB-producing sugarcane line. The
distribution of PHB throughout transgenic sugarcane line PHB3 in
FIG. 5 was determined by HPLC analysis. Samples were taken from:
lamina of the tip, midpoint and base of young, intermediate and
mature leaves; rind+pith of young, intermediate and mature stem
internodes; and roots. Data are the mean % of leaf DW as PHB.+-.SE
(n=3). ND=not detected.
[0131] FIG. 8 is a graphical representation showing the indigo
biosynthetic pathway.
[0132] FIG. 9 is a graphical representation showing adipic acid
biosynthesis from glucose via a cis, cis-muconic acid intermediate.
d=3-dehydroshikimate dehydratase, e=protochatechuate decarboxylase,
f=catechol-1,2-dioxygenase.
[0133] FIG. 10 is a graphical representation showing adipic acid
biosynthesis by petroselinic acid ozonolysis.
[0134] FIG. 11 is a graphical representation showing
2-phenylethanol biosynthesis.
[0135] FIG. 12 is a graphical representation showing the basic
steps of C4 carbon assimilation.
[0136] FIG. 13 is a graphical representation depicting the
detection of pHBA and vanillate by HPLC in acid hydrolysed leaf
samples taken from sugarcane leaves expressing the HCHL transgene.
A: Indicated peaks show the presence of pHBA and vanillate in the
leaf extract. Insets show the characteristic spectrum profiles for
the respective compounds. B: Peaks produced by pHBA and vanillate
synthetic standards.
[0137] FIG. 14 is a graphical representation of p-hydroxybenzoic
acid (pHBA) synthesis in planta can be accomplished by the
introduction of E. coli chorismate pyruvate-lyase (CPL) or P.
fluorescens 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL). CPL
converts plastidal chorismate into pHBA whilst HCHL converts
cytosolic 4-coumaroyl-CoA into 4-hydroxybenzaldehyde via a
a-hydroxy thioester intermediate which is subsequently oxidized to
pHBA by endogenous NAD+-linked dehydrogenases. In both instances,
the majority of the resultant free acid is glucosylated and
transported into vacuoles. Abbreviations: E4P,
erythrose-4-phosphate; PEP, phosphoenolpyruvate; UDP-GT,
UDP-glucosyltransferase.
[0138] FIG. 15 is a graphical representation showing the
distribution pattern of p-hydroxybenozic acid (pHBA) in UH1 at 20
weeks. (a) Leaf and internode pHBA levels are compared. There is
generally more pHBA in the leaf than the stalk and the content in
older tissue is generally higher than younger tissue. (b) pHBA
levels at specific locations in the tissue are compared. The
largest quantities of pHBA are found in the leaf lamina and the
rind tissue of the stem. (LL=leaf lamina; LM=leaf midrib; R=rind;
P=pith; VB=vascular bundles of stem tissue).
[0139] FIG. 16 is a photographic representation showing a
comparison of the growth phenotype between the highest pHBA
producer, UH98 and the control line TC1 reveals no obvious
differences. (a) Plants of approximately equivalent age were
compared. TC1, (left), UH98, (right). Inset: A close-up view of the
under surface of a leaf is shown for TC1 (b) and UH98 (c).
[0140] A summary of sequence identifiers used throughout the
subject specification is provided below in Table 1:
TABLE-US-00001 TABLE 1 SUMMARY OF SEQUENCE IDENTIFIERS SEQUENCE ID
NO: DESCRIPTION 1 Nucleotide sequence (phaA) encoding PhaA without
signal sequence (hence, the PhaA remains in the cytosol) 2 Amino
acid sequence of PhaA without signal sequence 3 Nucleotide sequence
(phaA) encoding PhaA without signal sequence (hence, the PhaA
remains in the cytosol) modified at 5' and 3' ends for insertion
into a vector 4 Nucleotide sequence (phaB) encoding PhaB without
signal sequence (hence, the PhaB remains in the cytosol) 5 Amino
acid sequence of PhaB without signal sequence 6 Nucleotide sequence
(phaB) encoding PhaB without signal sequence (hence, the PhaB
remains in the cytosol) modified at 5' and 3' ends for insertion
into a vector 7 Nucleotide sequence (phaC) encoding PhaC without
signal sequence (hence, the PhaC remains in the cytosol) 8 Amino
acid sequence of PhaC without signal sequence 9 Nucleotide sequence
(phaC) encoding PhaC without signal sequence (hence, the PhaC
remains in the cytosol) modified at 5' and 3' ends for insertion
into a vector 10 Nucleotide sequence (phaA) encoding PhaA targeted
to plastid 11 Amino acid sequence of PhaA with signal sequence to
target to the plastid 12 Nucleotide sequence (phaA) encoding PhaA
targeted to plastid modified at 5' and 3' ends for insertion into a
vector 13 Nucleotide sequence (phaB) encoding PhaB targeted to
plastid 14 Amino acid sequence of PhaB with signal sequence to
target to the plastid 15 Nucleotide sequence (phaB) encoding PhaB
targeted to plastid modified at 5' and 3' ends for insertion into a
vector 16 Nucleotide sequence (phaC) encoding PhaC targeted to
plastid 17 Amino acid of PhaC with signal sequence to target to the
plastid 18 Nucleotide sequence (phaC) encoding PhaC targeted to
modified at 5' and 3' ends for insertion into a vector 19
Nucleotide sequence (phaC1) encoding PhaC1 without signal sequence
(hence, the PhaC1 remains in the cytosol) 20 Amino acid sequence of
PhaC1 without signal sequence 21 Nucleotide sequence (phaC1)
encoding PhaC1 without signal sequence modified at 5' and 3' ends
for insertion into a vector 22 Nucleotide sequence (phaC1) encoding
PhaC1 targeted to the peroxisome 23 Amino acid sequence of PhaC1
with signal sequence to target to the peroxisome 24 Nucleotide
sequence (phaC1) encoding PhaC1 targeted to the peroxisome modified
to 5' and 3' ends for insertion into a vector 25 Nucleotide
sequence of (phaC1) encoding PhaC1 targeted to the plastid 26 Amino
acid sequence of PhaC1 with signal sequence to target to the
plastid 27 Nucleotide sequence (phaC1)) encoding PhaC1 targeted to
the plastid modified at 5' and 3' ends for insertion into a vector
28 Nucleotide sequence (phaG) encoding PhaG targeted to the plastid
29 Amino acid sequence of PhaG with signal sequence to target to
the plastid 30 Nucleotide sequence (phaG) encoding PhaG targeted to
the plastid modified to 5' and 3' ends for insertion into a vector
31 Nucleotide sequence (phaJ) encoding PhaJ targeted to the
peroxisome 32 Amino acid sequence of PhaJ with signal sequence to
target to the peroxisome 33 Nucleotide sequence (phaJ) encoding
PhaJ targeted to the peroxisome modified to 5' and 3' ends for
insertion into a vector 34 TphaF 35 PhaF 36 PhbF 37 PhcF 38 PhaR 39
PhbR 40 PhcR 41 PhaC1Cf 42 PhaC1Cr 43 PhaC1PF 44 PhaJF 45 PhaJR 46
PhaGF 47 PhaGR 48 SSP-F 49 SSP-R 50 primer 3 51 primer 4 52 primer
5 53 primer 6
[0141] A list of abbreviations used herein is provided in table
2.
TABLE-US-00002 TABLE 2 Abbreviations ABBREVIATION Description
1,3-PD 1,3-Propanediol 2-PE 2-Phenylethanol CAM Crassulacean Acid
Metabolism DW Dry weight GFOR Glucose-Fructose Oxidoreductase GFP
Green Fluorescent Protein PEP Phosphoenolpyruvate PEPcase
Phosphoenolpyruvate carboxylase PET Polyethylene terephthalate sPET
Isosorbide PET PHA Polyhydroxyalkanoate PHB Polyhydroxybutyrate
pHBA poly hydroxybenzaldehyde PHV
Polyhydroxyvalerate/Poly-3-hydroxypentanoate Rubisco Ribulose
bisphosphate carboxylase/oxygenase WT Wild type
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0142] The present invention provides a plant-derived bioreactor
system, which is capable of withstanding the extra metabolic load
placed on individual plants without resulting in deleterious growth
effects. Hence, until the advent of the present invention, plants
have been engineered to make small quantities of a desired product,
their capacity to do so being limited, and their ability to go on
doing so prevented, by these deleterious growth effects. In
accordance with the present invention, certain plants were selected
on the basis of the presence of highly efficient photosynthetic
mechanisms for the assimilation of carbon, particular metabolic
reserves, and/or useful metabolic and/or biosynthetic pathways.
Grasses, and particularly C4 grasses, were identified as having
particularly efficient mechanisms for the assimilation of carbon, a
high growth rate and high accumulation of biomass and hence are
useful as bioreactors for the production of a wide range of
products. Furthermore, the C4 grass, sugarcane, is particularly
useful, as this plant stores sugars in dimeric and/or polymeric
forms that may be utilized when needed, for example, to supply
energy during times of significant environmental stress. This store
of carbohydrate would provide a ready supply of substrate for many
metabolic pathways, and utilization of this store would not stress
the producing plant. Therefore, the present invention is
predicated, in part, on the identification of a subset of plants,
namely the C4 grasses, and particularly sugarcane, as useful
bioreactors on the basis of their high carbon assimilation rate,
rapid growth, high biomass production and large store of
carbohydrate.
[0143] Accordingly, one aspect of the present invention provides a
method for generating a plant-based bioreactor system, said method
comprising selecting a plant having a high efficiency carbon
assimilation mechanism, rapid growth rate and/or high biomass
production and/or reserves of metabolites or having a capacity to
generate such reserves and/or which possess metabolic and/or
biosynthetic pathways useful in the manufacture of a product of
interest or a precursor form thereof; genetically modifying cells
of the plant to enable access to said metabolites and/or metabolic
or biosynthetic pathways; and then regenerating a genetically
modified plant from said cells.
[0144] The terms "grass", "grasses" and the like are to be
understood as reference to any member of the Gramineae plant family
whether currently known or not. This family currently encompasses
approximately 660 plant genera including 10,000 plant species. It
will be readily apparent to the skilled artisan when examining a
given plant, either known or novel, whether the plant is a
grass.
[0145] Preferably, the grass is a C4 grass.
[0146] To minimise losses of carbon and nitrogen resulting from
photorespiration, some plants such as corn and sugarcane that grow
in hot climates have a different system for fixing CO.sub.2, called
C4 photosynthesis, than plants that grow in more temperate climates
(which have C3 photosynthesis). The leaf anatomy of plants such as
corn and sugarcane is different from that of temperate plants. The
vascular bundles of these leaves are surrounded by a wreath of
thick-walled parenchyma cells called bundle sheath cells, where
most of the carbon-fixation takes place.
[0147] During C4 photosynthesis, CO.sub.2 in the mesophyll cells is
condensed with a 3C compound called phosphoenolpyruvic acid (PEP),
by the action of the enzyme PEP carboxylase. This produces the 4C
compound oxaloacetic acid which is then converted to malic or
aspartic acid. The malic or aspartic acid is then moved through
plasmodesmata (at the expense of ATP) into the bundle sheath
cells.
[0148] In the bundle sheath cells, the 4C compounds are
decarboxylated to release CO.sub.2 and PEP. The CO.sub.2 collected
in the many mesophyll cells is concentrated into a few bundle
sheath cells. Therefore, the plants can maintain a higher
concentration of CO.sub.2 in the bundle sheath cells (where the
Calvin-Benson cycle of photosythesis occurs) than it can elsewhere
in the leaf. This higher concentration of CO.sub.2 minimises
photorespiration.
[0149] The C4 pathway is more expensive energetically than C3
photosynthesis, but this is offset by the resulting decrease in
photorespiration (where under certain conditions, plants may lose
30% of fixed carbon). For this reason, C4 plants are well adapted
to environments that promote high levels of photorespiration (viz.
subtropical and tropical climes).
[0150] The fundamentals of C4 photosynthesis are shown
schematically in Figure *. The photosynthesis processes of C4
plants are divided between mesophyll and bundle sheath cells. Two
steps of C4 photosynthesis which occur in the mesophyll cells are
the light-dependent reactions and a preliminary fixation of
CO.sub.2 into malate or aspartate (a 4C compound). This C4 compound
is transported to the bundle sheath cells, and is decarboxylated to
form CO.sub.2 and PEP. The released CO.sub.2 is re-fixed by Rubisco
and the Calvin-Benson cycle. The PEP is then recycled back to the
mesophyll cells, and the photosynthates are distributed throughout
the plant.
[0151] One defining aspect of a C4 plant is "Kranz anatomy" (German
for "wreath"). This term refers to the characteristic one, or two
concentric, layer(s) (wreath[s]) of bundle sheath cells, around the
vascular bundles of the leaves. The mesophyll cells surrounding the
bundle sheath cells fix CO.sub.2 via PEP carboxylase to form 4C
organic acids. These are transported to the bundle sheath cells and
are decarboxylated to regenerate CO.sub.2, which is then refixed by
the typical C3 photosynthetic pathway found in non-C4 plants. The
system thus acts as a CO.sub.2 pump, increasing the CO.sub.2
concentration in the bundle sheath to a level where
photorespiration is minimised.
[0152] Accordingly, for the purposes of the present invention C4
plants are to be understood as plants which exhibit at least one of
the following characteristics in at least some part of the plant:
[0153] (i) Kranz anatomy; [0154] (ii) fixation of CO.sub.2 into a 4
carbon compound; and/or [0155] (iii) decarboxylation of a 4 carbon
compound.
[0156] For the purposes of the present invention, a given plant
need not exhibit all of the above characteristics to be considered
a C4 plant. For example, Borszczowia aralocaspica (Chenopodiaceae)
has the photosynthetic features of C4 plants, yet lacks Kranz
anatomy. This species accomplishes C4 photosynthesis through
spatial compartmentation of photosynthetic enzymes, and by
separation of two types of chloroplasts and other organelles in
distinct positions within the chlorenchyma cell cytoplasm.
Accordingly, insofar as the present invention relates to C4 plants,
this is to be understood as those which exhibit 1, 2 or all 3 of
the above characteristics.
[0157] It is also to be understood that the subject plant need not
exhibit one or all of these criteria at all times. Some plant
species, such as the amphibious leafless sedge, Eleocharis
vivipara, can exhibit C3 and C4 characteristics such as those shown
above depending on whether it is grown in a terrestrial or aquatic
environment. In addition, the Cassava plant has photosynthetic
mechanisms which are typical of a C3 plant, yet some studies have
shown that both C3 and C4 enzymatic systems function in Cassava.
The dominant photosynthetic pathway varies between C3 and C4
depending on temperature: at lower temperatures, photosynthesis
follows a C3 path, and at higher temperatures, a C4 path.
[0158] Therefore, for the purposes of the present invention, C4
plants are to be understood as those plants capable of exhibiting
one or more of the above characteristics under any given
environmental condition. The present is not limited by the method
of photosynthesis used at a given time by a given plant, the plant
need only be capable of expressing at least one of the above
characteristics associated with C4 photosynthesis.
[0159] In addition, plants utilising the Crassulacean Acid
Metabolism (CAM) pathway are also to be considered within the
definition of a C4 plant for the purposes of the present
invention.
[0160] The term "Crassulacean" refers to the Stonecrop family
(Crassulaceae) and related succulents in which this process is
common. To date, plants in more than 18 different families
including Cactaceae (Cactus family) and Bromeliaceae (Pineapple
family) have been shown to carry out CAM metabolism. The term
"Acid" is derived from the observation that these plants accumulate
large amounts of C4 organic acids in the dark.
[0161] Plants with CAM metabolism are typically adapted to dry,
hot, high-light environments. CAM is largely a mechanism to
conserve water. Plants in dry environments utilise CAM as they
cannot afford to lose water by opening their stomata during the
day. CAM plants circumvent water loss during the day by opening up
the stomates at night to obtain carbon dioxide.
[0162] Carbon dioxide is accumulated in CAM plants using PEP
carboxylase, and the fixed carbon is stored as 4-carbon compounds
such as malate, as in C4 plants.
[0163] The CO.sub.2 obtained during the night is stored as a C4
acid until ATP and NADPH are available the following day as a
result of the light reactions of photosynthesis. The C4 acid is
then decarboxylated and the CO.sub.2 fixed by the Calvin-Benson
cycle. Thus, in CAM plants there is a temporal separation of
initial carbon fixation and the Calvin-Benson cycle, whereas in
other C4 plants there is a spatial separation.
[0164] In summary the sequence of events in CAM plants is:
[0165] Night.fwdarw.stomates.fwdarw.open nocturnal transpiration
(lower than diurnal) and carbon fixation by PEPcase.fwdarw.OAA
produced.fwdarw.reduced with NADPH to malate.fwdarw.shuttled into
vacuole as malic acid.fwdarw.malic acid content of vacuole
increases.fwdarw.starch depleted to provide PEP for
carboxylation.fwdarw.day.fwdarw.stomates close.fwdarw.transpiration
decreased.fwdarw.malic acid content decreases.fwdarw.resulting
malate decarboxylated to provide carbon dioxide for Calvin
cycle.fwdarw.starch content increases.
[0166] Accordingly, as CAM plants exhibit fixation of CO.sub.2 into
a 4C compound, and decarboxylation of a 4C compound, they are to be
understood as within the definition of C4 plants for the purposes
of the present invention.
[0167] The present invention is particularly directed to C4
grasses; however, other non-grass C4 plants such as woody or
herbaceous plants which utilise the C4 pathway are also
contemplated by, and are within the scope of, the present
invention.
[0168] In a preferred embodiment of the present invention the
subject plant is a grass, more preferrably a C4 grass. In a
particularly preferred embodiment the C4 grass is a member of the
Saccharum genus, and particularly the Saccharum hybrid,
sugarcane.
[0169] Commercially grown sugarcane varieties are mainly
interspecific hybrids and are vegetatively propagated. There are
about six different species contributing to the gene pool:
Saccharum officinarum, Saccharum robustum, Saccharum barberi,
Saccharum spontaneum, Saccharum sinense and Erianthus sp. Hence,
the scope of present invention is not to be limited to any one
variety but should be regarded as extending to and encompassing
other species of Saccharum.
[0170] The preferred compounds to be produced by the plant
bioreactor include: vanillin, sorbitol, PHAs, indigo, fructan,
lactic acid, adipic acid, 1,3-propanediol and 2-phenylethanol.
However, the present invention extends to the use of C4 grasses as
bioreactors to generate a compounds such as therapeutics,
nutrapharmaceuticals, diagnostic agents including single chain
antibodies, industrial enzymes and the like. However, the present
invention is in no way limited by the exemplified compounds and
methods.
[0171] The present invention contemplates, therefore, a method for
producing a product of interest including a product or intermediate
of a biosynthetic or metabolic pathway in a C4 grass, said method
comprising expressing one or more genetic sequences which encode
one or more enzymes or proteins required for the production of the
product or intermediate or a homolog or precursor thereof or which
induces gene silencing of genetic material which encodes an enzyme
or protein in a biosynthetic or metabolic pathway in cell of a C4
grass plant such that the product or intermediate accumulates in
the cytosol, storage vacuole, plastid or non-plastid organelles of
the cell, or accumulates in the juice or vascular fluid of the
plant.
[0172] For the purposes of the present invention, the application
of a plant as a bioreactor, is to be also understood as the
alteration of existing plant metabolism or the introduction of new
plant metabolism to generate a non-endogenous plant product or an
endogenous plant product at non-native levels.
[0173] In the case of the metabolic engineering of native plant
biochemical pathways, this may be achieved via a number of means.
Alterations to the metabolic activity of an organism can be made at
the gene, gene expression and protein levels.
[0174] Metabolic engineering may be affected at the protein level
in an organism by the administration of particular enzyme
activators or inhibitors. For example, the activity of particular
biosynthetic enzymes may be regulated by the administration of
particular enzyme inhibitors to the plant. These inhibitors may
directly effect the accumulation of a product by reducing the
activity of a particular biosynthetic enzyme. In addition indirect
effects such as the redirection of metabolic flux into other
pathways may be a product of a particular enzyme inhibition. For
example, the blocking of a particular enzyme may lead to the
buildup of an intermediate which may then be directed into a
different metabolic pathway, leading to the increased accumulation
of the product of the second pathway.
[0175] The present invention therefore contemplates the production
of a product in a C4 grass wherein product accumulation is at least
in part predicated on the direct activation or inhibition of an
enzyme in a biosynthetic pathway by the administration to the plant
of an enzyme inhibitor or activator. An enzyme "inhibitor" or
"activator" also includes genetic inhibitors or activators; ie.
nucleic acid molecules or RNAi-type molecules which induce gene
silencing or a structural gene or a regulatory gene which
positively or negatively regulates structural gene expression.
[0176] In a preferred embodiment, increased accumulation of a
product or intermediate from a biosynthetic or metabolic pathway is
a result of inhibition of one or more biosynthetic enzymes in the
pathway or re-direction of metabolite flow down another
pathway.
[0177] Particular biosynthetic or metabolic pathways may be induced
in plants, parts of plants and/or plant cells in culture by the
addition of elicitors, or by changes in environmental conditions.
Gene expression in plants and other organisms is mediated by a
number of physical, chemical and biotic factors. Physical factors
such as light intensity and photoperiod have been implicated in the
expression of many plant genes including genes involved in
morphogenesis and plant secondary metabolism. For example, the
anthocyanin biosynthetic genes, PAL and CHS are induced by
increased light intensity and increased photoperiod. In a similar
way, temperature and osmotic stresses have also been shown to alter
gene expression in a broad range of biological systems. For
example, the KIN1, COR15a, and LTI78 genes in Arabidopsis thaliana
are sensitive to induction by low temperatures (Knight et al.,
Plant Cell 11(5): 875-886, 1999), and a range of heat shock
proteins and glycolytic enzymes are induced in the bacterium
Lactobacillus rhamnosus in response to heat and osmotic stress
(Prasad et al., Appl. Environ. Microbiol. 69(2): 917-925, 2003).
Many of these gene expression changes are a result of stress to the
cells. In addition, other physical factors such as wounding and
drought have also been associated with altered gene expression in
plants. For example in the fig tree Ficus carica, drought stress
induced genes encoding a peroxidase, a chitinase and a trypsin
inhibitor (Kim et al., Plant Cell Physiol. 44(4): 412-414).
[0178] Chemical inducers of gene expression have been identified
for many biological systems and specific gene promoters. Examples
of these include the induction of chalcone synthase, a
phenylpropanoid pathway enzyme, by chemical elictors such as
jasmonate. Also, bacterially synthesized lipochitooligosaccharides
(Nod factors) induce the early nodulin (ENOD) genes in leguminous
plants (Fang and Hirsch, Plant Physiol. 115:53-68, 1998). Several
compounds have also been demonstrated to induce gene expression
associated with plant defence, such as silicon dioxide, phosphate
salts, and polyunsaturated fatty acids (Sticher et al., Annu. Rev.
Phytopathol. 35: 235-270, 1997).
[0179] A number of biological agents have also been demonstrated to
alter gene expression in other organisms. For example, many
phytopathogenic fungi and bacteria induce the expression of a
number of defence-related genes, such as the PR proteins,
.beta.-glucanases, terpenoid biosynthetic enzymes and genes in the
`salicylic acid` defence pathway. Non-pathogenic, microbial
colonists of plant induce yet another different set of genes in the
plant host (Han et al., Phytopathology 90: 327-332, 2000).
[0180] Alterations or changes to cultural practices, culture
conditions and growth conditions, including photoperiod and/or
temperature, are considered to be conditions which are not standard
conditions for the growth of the plant or cell. For example, growth
of a plant or plant cell culture under a 24 hour light, would be
considered an altered photoperiod for the purposes of the present
invention. Second, growth of a plant at a temperature substantially
above or below the optimum growth temperature of said plant, plant
cell culture or bacterial culture would be considered an altered
temperature for the purposes of the present invention. The
preceeding examples in no way limit the invention and it will be
clear to those of skill in the art what constitutes altered,
changed or abnormal conditions pertaining to a given cell, cell
culture, plant or organism.
[0181] Methods of altering gene expression in sugarcane
contemplated by the present invention are to be understood as
physical processes or conditions, chemical compounds and
biological, including genetic agents. Non-limiting examples of
physical agents include alterations to culture and/or growth
conditions of the cell or organism, light intensity and/or
photoperiod, temperature, growing season, and or physical wounding.
Examples of chemical agents that may alter gene expression in
plants include phytohormones such as auxins, cytokinins and
gibberellins; signalling molecules such as flavanoids, saccharides,
sterols and peptides; herbicides and antibiotics. Examples of
biological agents contemplated by the present invention include
microorganisms, such as bacteria and fungi; viruses; transposons
and plasmids as well as RNAi-inducing genetic molecules including a
hairpin loop or other means to induce gene silencing (eg.
post-transcriptional gene silencing). The preceeding examples are
only illustrative in nature and in no way limit the invention to
the said agents.
[0182] Accordingly, the present invention contemplates the use of
sugarcane as a bioreactor. Therefore, alteration to the gene
expression profile of sugarcane to effect the production of an
endogenous metabolite at an increased level, or to produce any
heterologous metabolite is within the scope of the present
invention. Accordingly, induction or supression of any biosythetic
or metabolic genes in sugarcane, such as those exemplified herein,
is to be considered within the scope of the present invention.
Reference to a biosynthetic or metabolic gene also includes a
regulatory gene.
[0183] The application of a plant as a bioreactor may be affected
by the introduction of a new biosynthetic or metabolic pathway into
the plant, or the redirection of metabolic flux down a particular
pathway in a plant. For the purposes of the present invention,
"introduction of a new biosynthetic or metabolic pathway"
encompasses where the introduced pathway is a single protein or
enzyme, which may in itself be the end-product. For example, the
introduction of a nucleic acid molecule, whether or not it encodes
a protein of interest, would fall within the scope of the subject
invention. In this regard, a "protein" includes a protein,
polypeptide or peptide as well as a glycoprotein, phosphoprotein or
phospholipoprotein. Alternatively, the invention also contemplates
the introduction of one or more enzymes or proteins, wherein the
introduced enzyme or protein catalyses one or more reactions in the
synthesis of the product of interest. For example, inplanta
synthesis of vanillin could be introduced to sugarcane by the
introduction of the enzymes feruloyl-CoA synthetase and enoyl-CoA
hydratase.
[0184] Typically, the production of one or more metabolites or
heterologous proteins, polypeptides or peptides in a plant is
achieved by expression of a nucleic acid molecule encoding the
protein, polypeptide or peptide of interest. Any nucleic acid which
encodes a protein, polypeptide or peptide of interest is
contemplated by the present invention. However, preferred nucleic
acids include those encoding: [0185] (i) vanillin biosynthetic
enzymes, including 3-dehydroshikimate dehydratase,
catechol-o-methyltransferase, aryl aldehyde dehdrogenase,
feruloyl-CoA synthetase, enoyl-CoA hydratase/aldolase; [0186] (ii)
sorbitol biosynthetic enzymes, including glucose/fructose
oxidoreductase; [0187] (iii) PHA biosynthetic enzymes, including
3-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, enoyl
hydratase, 3-hydroxyacyl-acyl carrier protein:CoA tranferase;
[0188] (iv) indigo biosynthetic enzymes, including tryptophanase,
L-tryptophan indole lyase, napthalene dioxygenase, R. eutrophica
bec gene product; [0189] (v) fructan biosynthetic enzymes,
including fructosyltransferases and levansucrases; [0190] (vi)
lactic acid biosynthetic enzymes, including lactate dehydrogenase;
[0191] (vii) adipic acid biosynthetic enzymes, including
3-dehydroshikimate dehydratase, protocatechuate decarboxylase and
catechol 1,2-dioxygenase; [0192] (viii) petroselinic acid
biosynthetic enzymes, including 3-ketoacyl-ACP synthase; [0193]
(ix) 1,3-propanediol biosynthetic enzymes including glycerol
dehydratase, 1,3-propanediol oxidoreductase, glycerol-3-phosphate
dehydrogenase, and glycerol-3-phosphatase; and/or [0194] (x)
2-phenylethanol biosynthetic enzymes including aromatic-L-amino
acid decarboxylase, 2-phenylethylamine oxidase and aryl alcohol
dehydrogenase. [0195] (xi) pHBA biosynthetic enzymes including
4-hydroxycinnamoyl hydratase/lyase and chorismate pyruvate
lyase.
[0196] Nucleic acids encoding a particular protein or enzyme may be
chemically synthesised or isolated from another organism. In a
preferred embodiment of the present invention, the gene encoding a
protein or enzyme of interest is isolated from bacteria, fungi,
animals, plants, protists or archaea. Bacteria provide a convenient
source of genes encoding useful enzymes and proteins, although the
present invention should not be limited by the source of the gene
encoding the protein or enzyme of interest. Particularly useful
microorganisms for the isolation of useful genes in the context of
the present invention include: R. eutropha, Aeromonas spp.,
Pseudomonas aeruginosa, Rhodococcus ruber, Nocardia corallina,
Zymomonas mobilis, Enterobacter aerogenes, Pseudomonas putida,
Bacillus subtilis, Klebsiella pneumoniae, Acinetobacter
calcoaceticus the actinomycetes (particularly Streptomyces spp.),
Escherichia coli and yeast such as Saccharomyces cereviseae.
However, it should be noted that these microorganisms represent
only examples of the possible source of a gene encoding a protein
or enzyme of interest, and the present invention is in no way
limited by the source of the nucleic acid encoding the protein or
enzyme of interest. Furthermore, as indicated above, the nucleic
acid molecule may not necessarily encode an enzyme or protein but
may encode a sense RNA or an antisense RNA, for use in gene
silencing for example.
[0197] In order to maximise transcription, and/or transcript
stability and/or translation and/or post-translational stability of
the gene product of a heterologous gene in a plant, particularly a
gene from a bacterium, it may be necessary to alter the sequence of
the gene. For example, to maximise translation of the gene
transcript, it may be necessary to alter the coding sequence of the
gene to reflect the preferred codon usage of the host plant.
Similarly, to maximise translation of the gene transcript it may be
necessary to alter the sequence context of the gene's translation
initiation site to reflect the preferred sequence context
recognised by the host's translational machinery. Similarly, it may
be necessary to add 5' and/or 3' non-translated regions to the
coding sequence of the gene to maximise transcript stability within
the host cell. Similarly, it may be necessary to alter the coding
sequence of the gene to remove cryptic protease-recognition sites.
Therefore, the present invention encompasses genes isolated from a
bacterial, fungal, animal, protist or archaeal source, which have
undergone modification to maximise transcription, and/or transcript
stability, and/or translation, and or post-translational stability
in a plant host. Other methods for the alteration of the sequence
of a gene will be readily ascertained by those of skill in the art
and need not be elaborated further here.
[0198] In a preferred embodiment of the present invention, a number
of products may be produced according to any of a number of methods
including those herein described. Examples of compounds that may be
produced via metabolic engineering of a subject plant include:
vanillin (4-hydroxy-3-methoxybenzaldehyde); sorbitol; PHAs; indigo;
fructan; lactic acid (2-hydroxypropanoic Acid); adipic acid; 1,3
propanediol and 2-phenylethanol. These compounds, however, are only
examplary, and the present invention is predicated on the use of C4
plants as bioreactors for any compound that can be synthesised in
the plant. Accordingly, the present invention is not limited to any
one product or method for producing the product.
[0199] In a preferred embodiment of the present invention, the
plant selected is a C4 grass and the product of interest is a
mixture of different chain length polymers of hydroxyalkanoic acid
monomers. The present invention extends, however, to the use of C4
grasses as bioreactors to generate a range of compounds such as
therapeutics, nutrapharmaceuticals and diagnostic agents such as
single chain antibodies.
[0200] Accordingly, the present invention contemplates a method for
accumulating polymers comprising one or more species of
hydroxyalkanoic acid monomer in a C4 grass, said method comprising
expressing one or more genetic sequences which encode enzymes
required for the production of the polymers or a homolog or
precursor thereof in a cell of a C4 grass such that PHA polymers
accumulate in the cytosol, storage vacuole, peroxisome and
ontologically related organelles, or plastid or non-plastid
organelles of said cell.
[0201] Polyhydroxyalkanoates (PHAs) are polyesters of one or more
species of hydroxyalkanoic acid monomers. PHAs, which are bacterial
carbon-storage polymers analogous to starch in plants and glycogen
in animals, are a diverse class of compounds, with over 100
different hydroxyalkanoic acid sidechains identified to date. For
example, PHB is a polymer of 3-hydroxybutyrate, and PHV is a
polymer of hydroxyvalerate. PHAs can also be co-polymers, for
example, poly-(3-hydroxybutyrate-co-3-hydroxyvalerate). As defined
herein, therefore, "polymers comprising one or more species of
hydroxyalkanoic acid monomer" and "PHAs" are synonymous and
encompass any such carbon-storage polymer.
[0202] PHAs are polymers that share many properties with
petrochemically derived, synthetic polymers. The main advantages of
PHAs over synthetic polymers are that they are readily
biodegradable and are made from renewable resources such as sugars
and fatty acids.
[0203] While different bacterial species have developed different
mechanisms for PHA biosynthesis, 3-hydroxyacylCoA is the precursor
for them all. FIG. 1 outlines four different strategies utilised by
a wide range of bacteria including, for example, R. eutropha,
Aeromonas spp., Pseudomonas aeruginosa, Rhodococcus ruber and
Nocardia corallina, to synthesize their requirements of polymers of
3-hydroxy acids as an energy source. Precursors may be generated by
sugar glycolysis (strategy I), from metabolic intermediates of the
.beta.-oxidative (strategy II) and biosynthetic fatty acid
(strategy III) pathways and from metabolites in other pathways such
as the methylmalonyl-CoA pathway (strategy IV).
[0204] In the bacterium R. eutropha, for example, strategy I is
used and the PHB biosynthetic pathway consists of three steps
catalyzed by three enzymes, respectively: first, two molecules of
acetyl CoA are condensed to acetoacetyl-CoA by 3-ketothiolase
(encoded by phaA); secondly, aceotacetyl-CoA is reduced to
D-3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase (encoded by
phaB); and thirdly, D-3-hydroxybutyryl-CoA is polymerized into PHB
by PHB synthase (encoded by phaC). The genes are clustered in a
single operon in the order pha-CAB A diagrammatic representation of
this particular biosynthetic pathway is provided in FIG. 2.
[0205] Aeromonas spp. are examples of organisms that employ
strategy II. They express a (R)-specific enoyl hydratase (PhaJ).
This enzyme catalyzes the formation of 3-hydroxyacyl-CoA from
enoyl-CoA intermediates in .beta.-oxidation of fatty acids, thereby
generating substrates for the polymerase. Alternatively, organisms
like P. aeruginosa produce PHAs from intermediates in the de novo
fatty acid biosynthetic pathway (strategy III). A
3-hydroxyacyl-acyl carrier protein:CoA transferase enzyme
designated PhaG converts 3-hydroxyacyl-acyl carrier protein to its
CoA analog. The hydroxyacyl-CoA is then incorporated into the
nascent polymer. Finally, organisms such as R. ruber and N.
corallina are able to generate precursors for PHA production from
the methylmalonly-CoA pathway (strategy IV).
[0206] Many other micro-organisms have developed alternative
biosynthetic pathways for the manufacture of PHAs, including PHB,
to meet their energy-requiring needs. Biosynthetic genes from
almost 40 different organisms have been cloned. Only limited
homologies are exhibited at both the nucleotide and amino acid
levels, which is not surprising considering the large number of
PHAs naturally produced by bacteria. The structural organisation of
loci encoding PHA genes is equally diverse.
[0207] Any one or more or a combination of these systems may be
adapted to provide suitable genetic sequences for use in accordance
with the present invention. Consequently, genetic sequences which
"encode enzymes required for the production of polymers" of
hydroxyalkanoic acids as used herein in the context of the present
invention may comprise a combination of one or more of any sequence
wherein the enzyme or enzymes thereby encoded usually operate in
vivo singly or together to effect the biosynthesis of PHAs,
including PHB.
[0208] Preferred suitable genetic sequences comprise a combination
of one or more of phaA, phaB, phaC, phaC1, phaG, phaJ. These genes
encode enzyme products referred to herein as PhaA, PhaB and PhaC,
PhaC1, PhaG and PhaJ.
[0209] In one preferred embodiment, the C4 grass is engineered to
express one or more of phaA, phaB and phaC such that it does not
accumulate the PHAs in the plastid. In another preferred
embodiment, the plant is engineered to express, in addition, one or
more of phaC1, phaG and phaJ, such that it accumulates the PHAs in
the plastid.
[0210] The nucleotide sequences encoding phaA, phaB and phaC may
come from any suitable source but the genes from R. eutrophia are
particularly useful in the practice of the present invention. The
nucleotide sequences for these genes are given in SEQ ID NO:1
(phaA), SEQ ID NO:4 (phaB) and SEQ ID NO:7 (phaC) where the
nucleotide sequence does not include a signal sequence and, hence,
the products, i.e. PhaA, PhaB and PhaC, respectively, are located
in the cytosol.
[0211] Nucleotide sequences of phaA, phaB and phaC with a signal
sequence to direct the products to the plastid are shown in SEQ ID
NO:10, SEQ ID NO:13 and SEQ ID NO:16, respectively.
[0212] The nucleotide sequences encoding phaC1, phaG and phaJ may
likewise come from any suitable source. In the case of phaC1, P.
aeruginosa provides a suitable source. Nucleotide sequences
encoding phaG and phaJ may be derived from, for example,
Pseudomonas putida and Aeromonas caviae, respectively. The
nucleotide sequence of phaC1 is given in SEQ ID NO:19, where the
nucleotide sequence does not include a signal sequence and, hence,
the product, i.e. PhaC1 is located in the cytosol.
[0213] Nucleotide sequences of phaC1, phaG and phaJ with a signal
sequence to direct the products to the peroxisome and plastid
(phaC1), and to the plastid (phaG) and peroxisome (phaJ),
respectively, are shown in SEQ ID NO:22 and SEQ ID NO:25 and SEQ ID
NO:28 and SEQ ID NO:31, respectively.
[0214] Clearly, the genetic sequences may be modified to insert any
leader or tail sequence to direct the enzyme to an appropriate
location in the cell.
[0215] Another aspect of the present invention contemplates a
method for producing PHAs in a C4 grass, said method comprising
expressing one or more genetic sequences comprising phaA, phaB,
phaC, phaC1, phaG and/or phaJ or a derivative or homolog of any one
of these in cells of a C4 grass such that polymers of a PHA
accumulate in the cytosol, storage vacuole, plastid or non-plastid
organelle of said cell.
[0216] Where accumulation is in the cytosol, the PHA is preferably
a PHB.
[0217] A homolog of a phaA, phaB, phaC, phaC1, phaG and phaJ
includes nucleotide sequences having at least about 60% identity to
one of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:19, SEQ ID
NO:28 or SEQ ID NO:31 (or one of SEQ ID NO:10, SEQ ID NO:13, SEQ ID
NO:16, SEQ ID NO:22 or SEQ ID NO:25) after optimal alignment or
nucleotide sequences capable of hybridizing to SEQ ID NO:1, SEQ ID
NO:4, SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:31, SEQ ID
NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO: 22 or SEQ ID NO:25 or
their complementary forms under low stringency conditions.
[0218] Alternatively, or in addition, a homolog at the amino acid
level includes an enzyme having an amino acid sequence with at
least about 60% identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8,
SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID
NO:23, SEQ ID NO:26, SEQ ID NO:29 or SEQ ID NO:32.
[0219] Preferably, percentage similarities include at least about
70%, at least about 80%, at least about 90% and at least about 95%
or above at the nucleotide and amino acid sequence levels such as
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
[0220] Accordingly, reference herein to phaA, phaB, phaC phaC1,
phaG, phaJ or PhaA, PhaB, PhaC, PhaC1, PhaG and PhaJ includes all
homologs thereof.
[0221] In another embodiment, the present invention contemplates a
method for generating a plant which produces PHAs, said method
comprising introducing into cells of said plant a genetic sequence
comprising:-- [0222] (i) a nucleotide sequence encoding a phaA or
homolog thereof; [0223] (ii) a nucleotide sequence encoding phaB or
homolog thereof; [0224] (iii) a nucleotide sequence encoding phaC
or homolog thereof; [0225] (iv) a nucleotide sequence encoding
phaC1 or homolog thereof; [0226] (v) a nucleotide sequence encoding
phaG or homolog thereof; [0227] (vi) a nucleotide sequence encoding
phaJ or homolog thereof [0228] (vii) SEQ ID NO:1 or SEQ ID NO:3 or
SEQ ID NO:10 or SEQ ID NO:12 or a nucleotide sequence having at
least 60% identity thereto after optimal alignment, or capable of
hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:10 or SEQ ID
NO:12 or a complementary form thereof under low stringency
conditions; [0229] (viii) SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID
NO:13 or SEQ ID NO:15 or a nucleotide sequence having at least 60%
identity thereto after optimal alignment, or capable of hybridizing
to SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID NO:15 or a
complementary form thereof under low stringency conditions; [0230]
(ix) SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18 or
a nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to SEQ ID NO:7 or SEQ
ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18 or a complementary form
thereof under low stringency conditions; [0231] (x) SEQ ID NO:19 or
SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:25 or SEQ
ID NO:27 or a nucleotide sequence having at least 60% identity
thereto after optimal alignment, or capable of hybridizing to SEQ
ID NO:19 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID
NO:25 or SEQ ID NO:27 or a complementary form thereof under low
stringency conditions; [0232] (xi) SEQ ID NO:28 or SEQ ID NO:30 or
a nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to SEQ ID NO:28 or SEQ
ID NO:30 or a complementary form thereof under low stringency
conditions; [0233] (xii) SEQ ID NO:31 or SEQ ID NO:33 or a
nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to SEQ ID NO:31 or SEQ
ID NO:33 or a complementary form thereof under low stringency
conditions; and then regenerating a plant from said cells.
[0234] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, a sucrose-storing monocotyledonous plant.
This aspect of the present invention includes progeny of the first
generation plants.
[0235] A convenient C4 grass for use in the present invention is
sugarcane. Sugarcane has certain advantages which make it a useful
crop for use as a bioreactor including, inter alia, its efficient
carbon fixation, high biomass accumulation, rapid growth in
subtropical and tropical climates, natural ability to accumulate
large quantities of sucrose, hardiness, and ease of growth. In
addition, a micropropagation system is already available and an
extensive industry infrastructure exists.
[0236] In order that PHAs may be produced in cells of a C4 grass,
suitable sequences such as those derived from R. eutropha must be
introduced into and expressed in the cells. That is, the plant
needs to undergo genetic modification so that the metabolites
and/or metabolic and/or biosynthetic pathways can be harnassed for
the production of the PHAs. This may conveniently be achieved
through the use of genetic constructs, engineered to comprise
nucleotide sequences required to effect PHA production.
[0237] In another preferred embodiment of the present invention,
the plant selected is a C4 grass and the product of interest is
pHBA . . . .
[0238] In another preferred embodiment of the present invention,
the plant selected is a C4 grass and the product of interest is
vanillin.
[0239] Vanillin (4-hydroxy-3-methoxybenzaldehyde) may be produced
as a co-product with sucrose in sugarcane. A number of biological
pathways have been discovered for the biosynthesis/biodegradation
of vanillin. At least 2 of these have substrates which are
available in plants, viz: [0240] (i) 3-dehydroshikimic acid is a
compound which is produced as an intermediate in the shikimate
pathway. A pathway has been determined which converts this
substrate via 3-dehydroshikimate dehydratase to protocatechuic acid
then to vanillic acid via catechol-o-methyltransferase and finally
to vanillin via aryl aldehyde dehydrogenase. [0241] (ii) Ferulic
acid is a secondary metabolite of the phenylpropanoid pathway
involved in lignin synthesis. It is converted in planta to
feruloyl-CoA by feruloyl-CoA synthetase which in turn is converted
to vanillin by enoyl-CoA hydratase/aldolase.
[0242] Accordingly, the present invention further contemplates a
method for producing vanillin in a C4 grass, said method comprising
expressing one or more genetic sequences which encode enzymes
required for the production of vanillin, or a homolog or precursor
thereof in a cell of a C4 grass such that the vanillin accumulates
in the cytosol, storage vacuole, plastid or non-plastid organelle,
or is extra-cellularly secreted.
[0243] Either of these pathways, or a combination of these
pathways, may be adapted to provide suitable genetic sequences for
use in the production of vanillin or precursors thereof in
Sugarcane. Consequently, genetic sequences which "encode enzymes
required for the production of vanillin" as used herein in the
context of the present invention may comprise a combination of one
or more of any sequence wherein the enzyme or enzymes thereby
encoded usually operate in vivo singly or together to effect the
biosynthesis of vanillin or a precursor thereof.
[0244] Clearly, the genetic sequences may be modified to insert any
leader or tail sequence to direct the enzyme to an appropriate
location in the cell.
[0245] Another aspect of the present invention contemplates a
method for producing Vanillin in a C4 grass, said method comprising
expressing one or more genetic sequences encoding
3-dehydroshikimate dehydratase, catechol-o-methyltransferase, aryl
aldehyde dehydrogenase, feruloyl-CoA synthetase, enoyl-CoA
hydratase and/or enoyl-CoA aldolase, in cells of a C4 grass such
that vanillin accumulates anywhere in the cell or extra-cellular
matrix of the plant.
[0246] Accordingly, reference herein to 3-dehydroshikimate
dehydratase, catechol-o-methyltransferase, aryl aldehyde
dehydrogenase, feruloyl-CoA synthetase, enoyl-CoA hydratase and/or
enoyl-CoA aldolase, includes all homologs thereof.
[0247] In another embodiment, the present invention provides a
method for generating a plant which produces vanillin or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence comprising at least one of the
following:-- [0248] (i) a nucleotide sequence encoding a
3-dehydroshikimate dehydratase and/or; [0249] (ii) a nucleotide
sequence encoding catechol-o-methyltransferase; [0250] (iii) a
nucleotide sequence encoding aryl aldehyde dehydrogenase; [0251]
(iv) a nucleotide sequence encoding feruloyl-CoA synthetase; [0252]
(v) a nucleotide sequence encoding enoyl-CoA hydratase; [0253] (vi)
a nucleotide sequence encoding enoyl-CoA aldolase; [0254] (vii) a
nucleotide sequence encoding a homolog of any one of (i) though
(vi) and then regenerating a plant from said cells.
[0255] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, sugarcane. Included in this aspect of the
present invention are progeny of the first generation plants.
[0256] In order that Vanillin may be produced in cells of a C4
grass, suitable sequences such as those encoding one or more
Vanillin biosynthetic enzymes must be introduced into and expressed
in the cells. That is, the plant needs to undergo genetic
modification so that the metabolites and/or metabolic and/or
biosynthetic pathways can be harnessed for the production of the
vanillin or a precursor thereof. This may conveniently be achieved
through the use of genetic constructs, engineered to comprise
nucleotide sequences required to effect vanillin production.
[0257] In another preferred embodiment of the present invention,
the plant selected is a C4 grass and the product of interest is
sorbitol.
[0258] Sorbitol is a polyol that is found naturally in many fruits.
To satisfy the high demand for this compound it is synthesized
industrially by hydrogenation of corn-derived glucose in aqueous
solution using nickel-containing catalysts. The majority of the
sorbitol produced is consumed in the manufacture of toothpaste,
confectionary, and ascorbic acid.
[0259] A new market for sorbitol in the polymer sector has been
described by industry. Isosorbide or 1,4-3,6-dianhydrosorbitol is
produced by the acid catalyzed dehydration of sorbitol. Recent
patents have demonstrated that copolymers of isosorbide and
polyethylene terephthalate (PET) exhibit superior strength and
rigidity compared to PET alone. 4.4 billion lb of PET is currently
used in food and beverage containers (Source: US Dept. Energy,
2001). Replacing PET with the isosorbide-PET copolymer (sPET) would
reduce the overall consumption of petroleum-derived PET because
less sPET is needed to achieve the equivalent strength. The
projected sPET production is 1 billion lb per year by 2020,
utilizing 100 million lb of isosorbide (Source: US Dept. Energy,
2001).
[0260] Sorbitol is already produced from a renewable feedstock. The
main incentive to use sugarcane as a sorbitol biofactory is to
capitalize upon a potential future demand for this product by the
plastics industry.
[0261] Sorbitol can also be converted to other useful chemicals.
Propylene glycol, ethylene glycol, and glycerol can be derived from
catalytic hydrogenolysis of sorbitol. These chemical feedstocks are
currently derived from petrochemicals.
[0262] The biosynthesis of sorbitol produces the coproduct
gluconolactone. The enzyme glucono-.delta.-lactonase can convert
the gluconolactone into gluconic acid. Gluconic acid is used as a
food acidulant, antioxidant, and a clarifier in wines and
softdrinks.
[0263] Zymomonas mobilis is able to produce sorbitol from sucrose
or a mixture of glucose and fructose in a one-step reaction
catalysed by the glucose-fructose oxidoreductase GFOR (Genbank
accession no. Z80356, M97379). The glucose is oxidized to
gluconolactone while the fructose is reduced to sorbitol.
glucose+fructose.fwdarw.sorbitol+gluconolactone
[0264] Without limiting the present invention to any one method or
mode of action, sorbitol production in sugarcane could be achieved
by using GFOR. This involves constructing an expression cassette by
fusing GFOR to the maize polyubiquitin promoter and nopaline
synthase terminator and introducing the cassette into sugarcane
callus by biolistic transformaton. The Z. mobilis GFOR is not
membrane-bound and resides in the periplasm and should work equally
well as a cytosolic enzyme in sugarcane.
[0265] Sorbitol production is unlikely to be toxic in sugarcane
since sorbitol is found in numerous fruits (apples, pears, plums,
berries, cherries). Sorbitol functions physiologically to regulate
osmotic stress hence extremely high levels may be detrimental and
vacuolar storage may circumvent this problem.
[0266] Accordingly, the present invention further contemplates a
method for producing sorbitol in a C4 grass, said method comprising
expressing one or more genetic sequences which encode enzymes
required for the production of sorbitol, or a homolog or precursor
thereof in a cell of a C4 grass such that the sorbitol accumulates
in the cytosol, storage vacuole, plastid or non-plastid organelle,
or is secreted extra-cellularly.
[0267] In addition to the glucose-fructose oxidoreductase pathway,
other nucleotide sequences may encode other enzymes suitable for
use in the production of sorbitol or precursors thereof in
Sugarcane. Consequently, genetic sequences which "encode enzymes
required for the production of sorbitol" as used herein in the
context of the present invention may comprise a combination of one
or more of any sequence wherein the enzyme or enzymes thereby
encoded usually operate in vivo singly or together to effect the
biosynthesis of sorbitol or a precursor thereof.
[0268] Clearly, the genetic sequences may be modified to insert any
leader or tail sequence to direct the enzyme to an appropriate
location in the cell.
[0269] Another aspect of the present invention contemplates a
method for producing sorbitol in a C4 grass, said method comprising
expressing one or more genetic sequences encoding a
glucose-fructose oxidoreductase or homolog thereof, in cells of a
C4 grass such that sorbitol accumulates anywhere in the cell or
extra-cellular matrix of the plant.
[0270] In a preferred embodiment, the glucose-fructose
oxidoreductase is that encoded by the nucleic acid sequence set
forth in Genbank accession number Z80356 or M97379, or a nucleotide
sequence having at least 60% identity thereto after optimal
alignment, or capable of hybridizing to these sequences under low
stringency conditions.
[0271] Accordingly, reference herein to glucose-fructose
oxidoreductase, includes all homologs thereof.
[0272] In another embodiment, the present invention contemplates a
method for generating a plant which produces sorbitol or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence comprising a nucleotide sequence
encoding a glucose-fructose oxidoreductase or homolog thereof and
then regenerating a plant from said cells.
[0273] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, sugarcane.
[0274] In order that sorbitol may be produced in cells of a C4
grass, suitable sequences such as those encoding one or more
sorbitol biosynthetic enzymes must be introduced into and expressed
in the cells. That is, the plant needs to undergo genetic
modification so that the metabolites and/or metabolic and/or
biosynthetic pathways can be harnessed for the production of the
sorbitol or a precursor thereof. This may conveniently be achieved
through the use of genetic constructs, engineered to comprise
nucleotide sequences required to effect sorbitol production.
[0275] Clearly, the genetic sequences may be modified to insert any
leader, tail or signal sequences to direct the enzyme to an
appropriate location in the cell.
[0276] In another preferred embodiment of the present invention,
the plant selected is a C4 grass and the product of interest is
indigo.
[0277] Until the end of the 19th century, the sole source of indigo
was from plants, woad (Isatis tinctoria) and Dyer's Knotweed
(Polygonum tinctorum) in temperate climates and Indigofera species
in the tropics. Woad was widely grown in Europe, making some
regions, especially Toulouse (France) and Erfurt (Germany), very
wealthy until the end of the 16th century.
[0278] Plant-based indigo was almost entirely replaced in the 20th
century by synthetic indigo. Today indigo is still regarded as a
high value specialty chemical used mainly as a dye in the textile
industry. It is produced synthetically from naphthalene by the
Heumann synthesis reaction.
[0279] The chief incentive to use sugarcane as an indigo biofactory
is to provide a manufacturing route that will produce relatively
inexpensive indigo from a renewable feedstock.
[0280] Indigo production by microbial fermentation has been
demonstrated by expressing the genes that mediate indigo formation
in E. coli. The pigment is derived by converting endogenous
tryptophan to indole using the Enterobacter aerogenes tryptophanase
or L-tryptophan indole lyase EC 4.1.99.1 (Genbank accession no.
D14297). Subsequently the indole is converted to indigo via two
possible reactions.
[0281] Route A: Pseudomonas putida napthalene dioxygenase (Genbank
accession no. M83949)
[0282] Route B: Ralstonia eutropha bec gene (Genbank accession no.
AF306552)
[0283] These pathways are graphically depicted in FIG. 8.
[0284] Without limiting the present invention to any one method or
mode of action, indigo production in sugarcane involves
constructing an expression cassette by fusing the aforementioned
genes to the maize polyubiquitin promoter and nopaline synthase
terminator and introducing the cassette into sugarcane callus by
biolistic transformaton. Tryptophan is a product of the plant
shikimate pathway, which is responsible for synthesizing lignin
precursors. The cloned genes may be plastid-targeted since the
shikimate pathway reactions reside in this compartment. The
available metabolic flux in this pathway is expected to be
significant.
[0285] Aeration of the sugarcane juice will lead to spontaneous
oxidation of indoxyl to an insoluble indigo precipitate. The solid
precipitate should be easy to separate from the solution by
filtration or centrifugation.
[0286] Another aspect of the present invention contemplates a
method for producing indigo in a C4 grass, said method comprising
expressing one or more genetic sequences encoding tryptophanase,
L-tryptophan indole lyase, napthalene dioxygenase, and/or the
Ralstonia eutropha bec gene, or homolgs thereof, in cells of a C4
grass such that indigo accumulates anywhere in the cell or
extracellular matrix of the plant.
[0287] In a preferred embodiment, indigo accumulates in the plastid
of the plant cell.
[0288] Accordingly, reference herein to genetic sequences encoding
tryptophanase, L-tryptophan indole lyase, napthalene dioxygenase,
and/or the Ralstonia eutropha bec gene includes all homologs
thereof.
[0289] In another embodiment, the present invention contemplates a
method for generating a plant which produces indigo or a precursor
thereof, said method comprising introducing into cells of said
plant a genetic sequence comprising at least one of the
following:-- [0290] (i) a nucleotide sequence encoding genetic
sequences encoding tryptophanase; [0291] (ii) a nucleotide sequence
encoding L-tryptophan indole lyase; [0292] (iii) a nucleotide
sequence encoding napthalene dioxygenase; [0293] (iv) a nucleotide
sequence comprising the Ralstonia eutropha bec gene; [0294] (v) the
nucleotide sequence set forth in Genbank accession number D14279,
or a nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to Genbank D14279
under low stringency conditions. [0295] (vi) the nucleotide
sequence set forth in Genbank accession number M83949, or a
nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to Genbank M83949
under low stringency conditions. [0296] (vii) the nucleotide
sequence set forth in Genbank accession number AF306552, or a
nucleotide sequence having at least 60% identity thereto after
optimal alignment, or capable of hybridizing to Genbank AF306552
under low stringency conditions. and then regenerating a plant from
said cells.
[0297] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, sugarcane.
[0298] In order that indigo may be produced in cells of a C4 grass,
suitable sequences such as those encoding one or more indigo
biosynthetic enzymes must be introduced into and expressed in the
cells. That is, the plant needs to undergo genetic modification so
that the metabolites and/or metabolic and/or biosynthetic pathways
can be harnessed for the production of the indigo or a precursor
thereof. This may conveniently be achieved through the use of
genetic constructs, engineered to comprise nucleotide sequences
required to effect indigo production.
[0299] Clearly, the genetic sequences may be modified to insert any
leader or tail sequence to direct the enzyme to an appropriate
location in the cell. In a preferred embodiment the leader, tail or
signal sequence directs the indigo biosynthetic enzyme to be
localized in the plastid.
[0300] In another preferred embodiment of the present invention,
the plant selected is a C4 grass and the product of interest is a
mixture of different chain length polymers of fructose monomers,
such as fructans.
[0301] Fructan, or levan as it is often called, is a fructose
homopolysaccharide that is linked to a terminal glucose residue.
Fructans are a storage carbohydrate in some plants such as
Jerusalem artichoke and chicory. Certain bacilli can also
synthesize fructans.
[0302] Despite a plethora of potential applications, this polymer
is not yet widely used. Some of the possible uses cited by the
literature are: [0303] (i) Low calorie sweetener. Fructans possess
a sweet taste but cannot be degraded in humans. [0304] (ii) Dietary
fibre [0305] (iii) Bulking agent [0306] (iv) Raw material for
biodegradable plastics, detergents, and adhesives
[0307] Fructans may also be an inexpensive source of fructose in
the future. The food industry is rapidly adopting fructose as the
preferred sweetener over sucrose. Fructose may be up to 1.8 times
sweeter than sucrose. Consequently, less fructose is needed to
derive the same effect. Fructose syrup is presently obtained by
hydrolysis of starch to glucose followed by enzymatic isomerization
of glucose to fructose. The resultant solution is an equilibrium
mixture of glucose/fructose that must be further purified by ion
chromatography to obtain near pure fructose. This final step
purportedly adds significantly to the cost of fructose manufacture.
It would be possible to avoid this step if the starting material
contained only fructose. Simple hydrolysis of fructans will yield
pure fructose at a reduced cost compared with using starch as the
raw material.
[0308] Incentives to use sugarcane as fructan biofactory include:
[0309] (i) To create a market for this product. A demand for
fructans would develop if sufficient amounts were made available.
The disadvantage of the existing fructan flora is the low
harvestable weight per plant. [0310] (ii) To provide an alternative
and inexpensive route for fructose production. [0311] (iii) In
subtropical and tropical climates sugarcane exhibits fast growth
and very high biomass yields. The high rate of CO.sub.2 fixation
due to C4 photosynthesis should facilitate a rapid accumulation of
fructans. [0312] (iv) Vegetative propagation ensures a stable
germplasm and hence predictable product yields.
[0313] Naturally occurring fructans may contain 10 to 100,000
fructose residues. Bacteria produce the larger fructans whilst
those occurring in plants are smaller. The larger polymers are
desirable because they are less soluble in water and consequently
easier to extract. Larger fructans will not affect the osmotic
pressure in the cell to the same degree as smaller molecules.
Therefore it is possible to store greater quantities of fructan
before the cell is affected.
[0314] Numerous bacterial fructosyltransferases or levansucrases
have been characterized such as Genbank accession no. AY150365,
from Bacillus subtilis. These enzymes catalyze the transfer of the
D-fructosyl residue from sucrose to the .beta.-2,6-linked residues
of fructan.
Sucrose.fwdarw.fructan+glucose
[0315] Without limiting the present invention to any one method or
mode of action, fructan production in sugarcane would be achieved
by constructing an expression cassette containing levansucrase, the
maize polyubiquitin promoter and nopaline synthase terminator and
introducing the cassette into sugarcane callus by biolistic
transformaton.
[0316] Another aspect of the present invention contemplates a
method for producing fructans in a C4 grass, said method comprising
expressing one or more genetic sequences encoding a bacterial
fructosyltransferase or levansucrase in cells of a C4 grass such
that a fructan accumulates anywhere in the cell or extra-cellular
matrix of the plant.
[0317] In a preferred embodiment, fructan accumulates in the
apoplast or vacuole of the plant cell.
[0318] Accordingly, reference herein to genetic sequences encoding
fructosyltransferases and levansucreases includes all homologs
thereof.
[0319] In another embodiment, the present invention relates to a
method for generating a C4 grass plant which produces a fructan or
a precursor thereof, said method comprising introducing into cells
of said plant a genetic sequence comprising at least one of the
following:-- [0320] (i) a nucleotide sequence encoding a
fructosyltransferase or homolog thereof; [0321] (ii) a nucleotide
sequence encoding a levansucrase or homolg thereof; [0322] (iii)
the nucleotide sequence set forth in Genbank accession number
AY150365, or a nucleotide sequence having at least 60% identity
thereto after optimal alignment, or capable of hybridizing to
Genbank AY150365 under low stringency conditions. and then
regenerating a plant from said cells.
[0323] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, sugarcane.
[0324] In order that a fructan may be produced in cells of a C4
grass, suitable sequences such as those encoding one or more indigo
biosynthetic enzymes must be introduced into and expressed in the
cells. That is, the plant needs to undergo genetic modification so
that the metabolites and/or metabolic and/or biosynthetic pathways
can be harnessed for the production of the indigo or a precursor
thereof. This may conveniently be achieved through the use of
genetic constructs, engineered to comprise nucleotide sequences
required to effect fructan production.
[0325] Clearly, the genetic sequences may be modified to insert any
leader, tail or signal sequence to direct the enzyme to an
appropriate location in the cell. In a preferred embodiment, to
maximise fructan production in sugarcane, levansucrase is directed
to the apoplast or vacuole to maximize access to substrate for
conversion.
[0326] In another preferred embodiment of the present invention,
the plant selected is a C4 grass and the product of interest is
lactic acid (2-Hydroxypropanoic acid).
[0327] The world market for solvent replacement, biodegradable
plastics and oxygenated chemicals derived from lactic acid exceeds
US$ 10 billion (Argonne National Laboratory, US DOE).
[0328] Without limiting the present invention to any one method or
mode of action, lactic acid production in sugarcane involves the
following general steps: [0329] (i) Obtain or clone lactate
dehydrogenase (LDH) or a homolog thereof: [0330] (ii) Express gene
in sugarcane (cytosol, therefore no targeting is required) [0331]
(iii) Regenerate plants and evaluate for lactate (or derivative)
production
[0332] Traditionally, lactic acid purification has been a complex
chemical process. However, recent advances have simplified this
process and made it significantly cheaper. It is anticipated that
lactic acid can be removed from the post-crushing millstream
without great difficulty or extensive modification of existing
structures.
[0333] Accordingly, the present invention further contemplates a
method for producing lactic acid in a C4 grass, said method
comprising expressing one or more genetic sequences which encode
enzymes required for the production of lactic acid, or a homolog or
precursor thereof in a cell of a C4 grass such that the lactic acid
accumulates in the cytosol, storage vacuole, plastid or non-plastid
organelle, or is secreted extra-cellularly.
[0334] Genetic sequences which "encode enzymes required for the
production of lactic acid" as used herein in the context of the
present invention may comprise a combination of one or more of any
sequence wherein the enzyme or enzymes thereby encoded usually
operate in vivo singly or together to effect the biosynthesis of
lactic acid or a precursor thereof.
[0335] Clearly, the genetic sequences may be modified to insert any
leader or tail sequence to direct the enzyme to an appropriate
location in the cell.
[0336] In a preferred embodiment the lactate dehydrogenase nucleic
acid sequence is expressed without a signal sequence such that the
enzyme is active in the cytosol.
[0337] Another aspect of the present invention contemplates a
method for producing lactic acid in a C4 grass, said method
comprising expressing one or more genetic sequences encoding
lactate dehydrogenase or a homolog thereof in cells of a C4 grass
such that lactic acid accumulates anywhere in the cell or
extracellular matrix of the plant.
[0338] Accordingly, reference herein to lactate dehydrogenase,
includes all homologs thereof.
[0339] In another embodiment, the present invention contemplates a
method for generating a plant which produces lactic acid or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence encoding lactate dehydrogenase or
homolog thereof, and then regenerating a plant from said cells.
[0340] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, sugarcane.
[0341] In order that lactic acid may be produced in cells of a C4
grass, suitable sequences such as those encoding one or more lactic
acid biosynthetic enzymes must be introduced into and expressed in
the cells. That is, the plant needs to undergo genetic modification
so that the metabolites and/or metabolic and/or biosynthetic
pathways can be harnessed for the production of the lactic acid or
a precursor thereof. This may conveniently be achieved through the
use of genetic constructs, engineered to comprise nucleotide
sequences required to effect lactic acid production.
[0342] In another preferred embodiment of the present invention,
the plant selected is a C4 grass and the product of interest is
adipic acid.
[0343] Adipic acid is classified as a bulk chemical and is among
the top fifty chemicals produced in the US. It is principally used
in the production of nylon 66, polyeurethane resins and
plasticizers. Nearly 90% is used to produce nylon-6,6, a synthetic
polymer developed by DuPont in the 1930's. This polyamide is formed
by the condensation of adipic acid and 1,6-diaminohexane.
[0344] Adipic acid is presently produced industrially by
benzene-based synthetic chemistry. Catalytic hydrogenation of
benzene followed by air oxidation yields a ketone/alcohol mixture
(cyclohexanone/cyclohexanol) that is further oxidized with nitric
acid to produce adipic acid.
[0345] Incentives to use sugarcane as an adipic acid biofactory
include: [0346] (i) Provide a renewable feedstock for adipic acid
manufacture. [0347] (ii) Eliminate the production of the toxic
nitrous oxide byproduct that accompanies traditional adipic acid
synthesis. [0348] (iii) Capitalize upon the high demand for this
product. [0349] (iv) Access to low cost molasses to produce
products by fermentation. [0350] (v) Access to low operating and
infrastructure costs through co-location of an extraction facility
(or fermentation facility) with a sugar mill. [0351] (vi) In
subtropical and tropical climated sugarcane exhibits fast growth
and very high biomass yields. This is a prerequisite for economical
bulk chemical production. [0352] (vii) Vegetative propagation
ensures a stable germplasm and hence predictable product
yields.
[0353] Without limiting the present invention to any one method or
mode of action, adipic acid may be produced in sugarcane by one of
two approaches.
I. Synthesis from Cis, Cis-Muconic Acid
[0354] Niu et al. (Biotechnol. Prog., 18: 201-211, 2002) describe a
microbiological route for the production of adipic acid using E.
coli. Three genes were introduced into E. coli to produce cis,
cis-muconic acid that was subsequently purified from the
fermentation broth and converted to adipic acid by catalytic
hydrogenation (step g, 10% Pt/C, H.sub.2, 3400 kPa, 25.degree. C.).
This final step has a 97% conversion efficiency.
[0355] The synthesis of cis, cis-muconic acid in sugarcane involves
making use of the shikimate pathway. In order to use the shikimate
pathway to produce cis, cis-muconic acid the following biosynthetic
enzymes, or homologs thereof are introduced into sugarcane:
[0356] Klebsiella pneumoniae 3-dehydroshikimate dehydratase
(aroZ)-enzyme d
3-dehydroshikimate.fwdarw.protocatechuate
[0357] Klebsiella pneumoniae protocatechuate decarboxylase
(aroY)-enzyme e
Protocatechuate.fwdarw.catechol
[0358] Acinetobacter calcoaceticus catechol 1,2-dioxygenase
(catA)-enzyme f
Catechol+O.sub.2.fwdarw.cis, cis-muconic acid
[0359] Introduction of these genes into sugarcane involves
constructing an expression cassette by fusing the genes described
above to the maize polyubiquitin promoter and nopaline synthase
terminator and introducing the cassette into sugarcane callus by
biolistic transformation. Catechol is probably produced in most
plants, and therefore, it may be unnecessary to clone additional
copies of 3-dehydroshikimate dehydratase or protocatechuate
decarboxylase. Preferrably, the cloned gene(s) are plastid-targeted
since the shikimate pathway reactions reside in this
compartment.
[0360] The merits of using plant secondary metabolism to synthesize
interesting products have often been promoted in the literature
(Verpoorte and Memelink, Curr. Opin. Biotech. 13: 181-187, 2002).
The shikimate pathway executes a central role in plant secondary
metabolism. This is one of the most active pathways in plants in
terms of carbon flux owing to the fact that it is the source of
lignin precursors. This makes it an attractive candidate for
metabolic engineering.
II. Synthesis from Petroselinic Acid
[0361] Bio-based adipic acid can be obtained through ozonolysis
(O.sub.3) of petroselinic acid (18:1 .DELTA..sup.6 cis), as
depicted in FIG. 10. The coproduct lauric acid is also a potential
source of feedstock for detergent manufacture.
[0362] The seed oil of the coriander spice plant contains 80-90%
petroselinic acid. A 36 kDa putative acyl-ACP desaturase (Genbank
accession no. M93115) has been identified from coriander seed
extracts and the corresponding cDNA was able to confer the ability
to produce petroselinic acid in tobacco callus (Cahoon et al. Proc.
Natl. Acad. Sci. USA 89: 11184-11188, 1992).
[0363] The metabolic pathway for producing petroselinic acid is
unclear, however, evidence suggests that it is formed by the
desaturation of palmitoyl-ACP by the 36 kDa desaturase followed by
elongation to form petroselinic acid (Cahoon and Ohlrogge, Plant
Physiol., 104: 827-837, 1994).
16:0-ACP.fwdarw.16:1.DELTA..sup.4-ACP.fwdarw.18:1.DELTA..sup.6-ACP
[0364] Recent studies have identified a 3-ketoacyl-ACP synthase
(Genbank accession no. AF263992) associated with the two-carbon
elongation of 16:1 .DELTA..sup.4-ACP (Mekhedov et al., Plant Mol.
Biol. 47: 507-518, 2001).
[0365] Cis, cis-muconic acid in sugarcane juice would be converted
to adipic acid by catalytic hydrogenation. The adipic acid in the
resultant solution can be recovered by solvent extraction. The
solution is contacted with chloroform or methylene chloride and the
adipic acid recovered in the aqueous fraction. The aqueous fraction
would then be evaporated to yield crystalline adipic acid.
[0366] Accordingly, the present invention further contemplates a
method for producing adipic acid, or a precursor thereof such as
cis, cis-muconic acid, in a C4 grass, said method comprising
expressing one or more genetic sequences which encode enzymes
required for the production of adipic acid and/or cis, cis-muconic
acid, or a homolog or precursor thereof in a cell of a C4 grass
such that adipic acid and/or cis, cis-muconic acid accumulates in
the cytosol, storage vacuole, plastid or non-plastid organelle, or
is secreted extra-cellularly.
[0367] Either of the hereinbefore described pathways for the
production of cis, cis-muconic acid, or a combination of these
pathways, may be adapted to provide suitable genetic sequences for
use in the production of vanillin or precursors thereof in
Sugarcane. Consequently, genetic sequences which "encode enzymes
required for the production of cis, cis-muconic acid" as used
herein in the context of the present invention may comprise a
combination of one or more of any sequence wherein the enzyme or
enzymes thereby encoded usually operate in vivo singly or together
to effect the biosynthesis of cis, cis-muconic acid or a precursor
thereof.
[0368] Clearly, the genetic sequences may be modified to insert any
leader, tail or signal sequence to direct the enzyme to an
appropriate location in the cell. Preferrably the leader tail or
signal sequences lead to the co-localization of the adipic acid
biosynthetic enzymes with the endogenous skimimate pathway enzymes
in the plant. More preferably, said enzymes are localized in the
plastid.
[0369] Another aspect of the present invention contemplates a
method for producing cis, cis-muconic acid or adipic acid in a C4
grass, said method comprising expressing one or more genetic
sequences encoding 3-dehydroshikimate dehydratase, protochatechuate
decarboxylase, catechol 1,2-dioxygenase and/or 3-ketoacyl-ACP
synthase in cells of a C4 grass such that adipic acid or an adipic
acid precursor accumulates anywhere in the cell or extracellular
matrix of the plant.
[0370] Accordingly, reference herein to 3-dehydroshikimate
dehydratase, protochatechuate decarboxylase, catechol
1,2-dioxygenase and/or 3-ketoacyl-ACP synthase, includes all
homologs thereof.
[0371] In another embodiment, the present invention contemplates a
method for generating a plant which produces adipic acid or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence comprising at least one of the
following:-- [0372] (i) a nucleotide sequence encoding a
3-dehydroshikimate dehydratase and/or; [0373] (ii) a nucleotide
sequence encoding protochatechuate decarboxylase; [0374] (iii) a
nucleotide sequence encoding catechol 1,2-dioxygenase; [0375] (iv)
a nucleotide sequence encoding 3-ketoacyl-ACP synthase; and/or
[0376] (v) a nucleotide sequence encoding a homolog of any one of
(i) through (iv). and then regenerating a plant from said
cells.
[0377] In a preferred embodiments: [0378] (i) the nucleotide
sequence encoding a 3-dehydroshikimate dehydatase is the aroZ gene
from Klebsiella pneumoniae, or a homolog thereof; [0379] (ii) the
nucleotide sequence encoding a protochatechuate decarboxylase is
the aroY gene from Klebsiella pneumoniae, or a homolog thereof;
[0380] (iii) the nucleotide sequence encoding a 1,2-dioxygenase is
the catA gene from Acinetobacter calcoaceticus, or a homolog
thereof; [0381] (iv) the nucleotide sequence encoding a
3-ketoacyl-ACP synthase is the nucleotide sequence set forth in
Genbank Accession number AF263992, or a nucleotide sequence having
at least 60% identity thereto after optimal alignment, or capable
of hybridizing to Genbank AF263992 under low stringency
conditions.
[0382] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, sugarcane.
[0383] In order that adipic acid or a precursor thereof may be
produced in cells of a C4 grass, suitable sequences such as those
encoding one or more adipic acid biosynthetic enzymes must be
introduced into and expressed in the cells. That is, the plant
needs to undergo genetic modification so that the metabolites
and/or metabolic and/or biosynthetic pathways can be harnessed for
the production of adipic acid or a precursor thereof. This may
conveniently be achieved through the use of genetic constructs,
engineered to comprise nucleotide sequences required to effect the
production of adipic acid and/or precursors thereof.
[0384] In another preferred embodiment of the present invention,
the plant selected is a C4 grass and the product of interest is
1,3-propanediol (1,3-PD).
[0385] 1,3-PD is a bifunctional alcohol that can be used as a
monomer in numerous polycondensation reactions to produce
polyesters, polyurethanes, and polyethers. The high cost of
chemical synthesis, reportedly US$30/kg (Biebl et al., Appl.
Microbiol. Biotechnol., 52: 289-297, 1999) has restricted its use
in the past to specialty markets such as dioxane production and the
solvent market.
[0386] 1,3-PD is synthesized using a process in which ethylene
oxide is reacted with carbon dioxide and hydrogen. An alternative
method, the Degussa process, is based upon hydrolysis of acrolein
followed by catalytic hydrogenation. Both routes involve the use of
petrochemical feedstock.
[0387] 1,3-PD is a natural product of glycerol fermentation in a
few enterobacteria and clostridia.
[0388] Incentives to use sugarcane as 1,3-PD biofactory include:
[0389] (i) Provide a renewable feedstock for 1,3-PD manufacture.
[0390] (ii) Provide a high volume, low cost source of 1,3-PD to
facilitate expansion of the market. [0391] (iii) Capitalize upon
the high demand for this product. [0392] (iv) Access to low cost
molasses to produce products by fermentation. [0393] (v) Access to
low operating and infrastructure costs through co-location of an
extraction facility (or fermentation facility) with a sugar mill.
[0394] (vi) In subtropical and tropical climated sugarcane exhibits
fast growth and very high biomass yields. This is a prerequisite
for economical bulk chemical production. [0395] (vii) Vegetative
propagation ensures a stable germplasm and hence predictable
product yields.
[0396] The metabolic reactions that convert glycerol to 1,3-PD have
been established from Klebsiella pneumoniae:
[0397] Klebsiella pneumoniae glycerol dehydratase (dhaB)
glycerol.fwdarw.3-hydroxypropionaldehyde+H.sub.2O
[0398] Klebsiella pneumoniae 1,3-propanediol oxidoreductase
(dhaT)
3-hydroxypropionaldehyde+NADH.fwdarw.1,3-propanediol+NAD
[0399] Sugarcane does not naturally produce glycerol therefore the
reactions that convert triose phosphates to glycerol must also be
engineered into sugarcane.
[0400] Saccharomyces cerevisiae glycerol-3-phosphate
dehydrogenase
dihydroxyacetone phosphate+NADH.fwdarw.glycerol-3-phosphate+NAD
[0401] Saccharomyces cerevisiae glycerol-3-phosphatase
glycerol-3-phosphate+ADP.fwdarw.glycerol+ATP
[0402] Without limiting the present invention to any one method or
mode of action, all four new genes are cloned into sugarcane to
convert it into a 1,3-PD biofactory. These genes are assembled into
an expression cassette containing the maize polyubiquitin promoter
and nopaline synthase terminator. The cassette is introduced into
sugarcane callus by biolistic transformation and expression will be
targeted to the cytosol. The accumulation of 1,3-PD in plant tissue
will be assayed from plant extracts by conventional HPLC.
[0403] 1,3-PD can be recovered from sugarcane juice by extraction
with cyclohexane followed by vaporization of the residual solvent.
Alternatively, distillation may be employed. Use of cyclohexane is
environmentally unsound and distillation is energy intensive.
Consequently, a method has been patented that describes the use of
ion exclusion resins to recover 1,3-PD (WO0173097 Method of
recovering 1,3-propanediol from fermentation broth, Archer Daniels
Midland Co., 2001).
[0404] Accordingly, the present invention further contemplates a
method for producing 1,3-propanediol in a C4 grass, said method
comprising expressing one or more genetic sequences which encode
enzymes required for the production of 1,3-propanediol, or a
homolog or precursor thereof in a cell of a C4 grass such that the
1,3-propanediol accumulates in the cytosol, storage vacuole,
plastid or non-plastid organelle, or is secreted
extra-cellularly.
[0405] Any of the disclosed biosynthetic steps, or a combination of
these, may be adapted to provide suitable genetic sequences for use
in the production of 1,3-propanediol, or precursors thereof, in
sugarcane. Consequently, genetic sequences which "encode enzymes
required for the production of 1,3-propanediol" as used herein in
the context of the present invention may comprise a combination of
one or more of any sequence wherein the enzyme or enzymes thereby
encoded usually operate in vivo singly or together to effect the
biosynthesis of 1,3-propanediol or a precursor thereof.
[0406] Clearly, the genetic sequences may be modified to insert any
leader or tail sequence to direct the enzyme to an appropriate
location in the cell.
[0407] The present invention contemplates a method for producing
1,3-propanediol in a C4 grass, the method comprising expressing one
or more genetic sequences encoding glycerol dehydratase,
1,3-propanediol oxidoreductase, glycerol-3-phosphate dehydrogenase
and glycerol-3-phosphatase in cells of a C4 grass such that
1,3-propanediol accumulates anywhere in the cell or extra-cellular
matrix of the plant.
[0408] Accordingly, reference herein to glycerol dehydratase,
1,3-propanediol oxidoreductase, glycerol-3-phosphate dehydrogenase
and glycerol-3-phosphatase includes all homologs thereof.
[0409] In another aspect, the present invention contemplates a
method for generating a plant which produces 1,3-propanediol or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence comprising at least one of the
following:-- [0410] (i) a nucleotide sequence encoding a glycerol
dehydratase and/or; [0411] (ii) a nucleotide sequence comprising
the dhaB gene from Klebsiella pneumoniae, or a homolg thereof;
[0412] (iii) a nucleotide sequence encoding 1,3-propanediol
oxidoreductase; [0413] (iv) a nucleotide sequence comprising the
dhaT gene from Klebsiella pneumoniae or homolg thereof [0414] (v) a
nucleotide sequence encoding glycerol-3-phosphate dehydrogenase;
[0415] (vi) a nucleotide sequence encoding glycerol-3-phosphatase;
and/or [0416] (vii) a nucleotide sequence encoding a homolog of any
one of (i) through (iv) and then regenerating a plant from said
cells.
[0417] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, sugarcane.
[0418] In order that 1,3-propanediol may be produced in cells of a
C4 grass, suitable sequences such as those encoding one or more
1,3-propanediol biosynthetic enzymes must be introduced into and
expressed in the cells. That is, the plant needs to undergo genetic
modification so that the metabolites and/or metabolic and/or
biosynthetic pathways can be harnessed for the production of the
1,3-propanediol or a precursor thereof. This may conveniently be
achieved through the use of genetic constructs, engineered to
comprise nucleotide sequences required to effect 1,3-propanediol
production.
[0419] Clearly, the genetic sequences may be modified to insert any
leader, tail or signal sequence to direct the enzyme to an
appropriate location in the cell.
[0420] In another preferred embodiment of the present invention,
the plant selected is a C4 grass and the product of interest is
2-phenylethanol (2-PE).
[0421] 2-phenylethanol (2-PE) is an important flavour and fragrance
compound with a rose-like odour. Most of the world's annual
production of several thousand tons is synthesised by chemical
means but, due to increasing demand for natural flavours,
alternative production methods are being sought (Etschmann et al.
Appl Micribiol Biotechnol 59:1-8, 2002)
[0422] A biological pathway for the biosynthesis of 2-PE is
presented in FIG. 11.
[0423] Sugarcane has a productive phenylpropanoid pathway and
should adapt readily to increased demands placed on it for
synthesis of 2-PE.
[0424] Accordingly, in another aspect, the present invention
further contemplates a method for producing 2-phenylethanol in a C4
grass, said method comprising expressing one or more genetic
sequences which encode enzymes required for the production of
2-phenylethanol, or a homolog or precursor thereof in a cell of a
C4 grass such that the 2-phenylethanol accumulates in the cytosol,
storage vacuole, plastid or non-plastid organelle, or is secreted
extra-cellularly.
[0425] Any of the disclosed biosynthetic steps, or a combination of
these, may be adapted to provide suitable genetic sequences for use
in the production of 2-phenylethanol, or precursors thereof, a C4
grass such as in sugarcane. Consequently, genetic sequences which
"encode enzymes required for the production of 2-phenylethanol" as
used herein in the context of the present invention may comprise a
combination of one or more of any sequence wherein the enzyme or
enzymes thereby encoded usually operate in vivo singly or together
to effect the biosynthesis of 2-phenylethanol or a precursor
thereof.
[0426] Another aspect of the present invention contemplates a
method for producing 2-phenylethanol in a C4 grass, said method
comprising expressing one or more genetic sequences encoding
aromatic-L-amino acid decarboxylase, 2-phenylethylamine oxidase and
aryl-alcohol dehydrogenase in cells of a C4 grass such that
2-phenylethanol accumulates anywhere in the cell or extracellular
matrix of the plant.
[0427] Accordingly, reference herein to aromatic-L-amino acid
decarboxylase, 2-phenylethylamine oxidase and aryl-alcohol
dehydrogenase includes all homologs thereof.
[0428] In another embodiment, the present invention contemplates a
method for generating a plant which produces 2-phenylethanol or a
precursor thereof, said method comprising introducing into cells of
said plant a genetic sequence comprising at least one of the
following:-- [0429] (i) a nucleotide sequence encoding a
aromatic-L-amino acid decarboxylase and/or; [0430] (ii) a
nucleotide sequence encoding 2-phenylethylamine oxidase; [0431]
(iii) a nucleotide sequence encoding aryl-alcohol dehydrogenase;
and/or [0432] (iv) a nucleotide sequence encoding a homolog of any
one of (i) through (iii) and then regenerating a plant from said
cells.
[0433] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, sugarcane.
[0434] In order that 2-phenylethanol may be produced in cells of a
C4 grass, suitable sequences such as those encoding one or more
2-phenylethanol biosynthetic enzymes must be introduced into and
expressed in the cells. That is, the plant needs to undergo genetic
modification so that the metabolites and/or metabolic and/or
biosynthetic pathways can be harnessed for the production of the
2-phenylethanol or a precursor thereof. This may conveniently be
achieved through the use of genetic constructs, engineered to
comprise nucleotide sequences required to effect 2-phenylethanol
production.
[0435] Clearly, the genetic sequences may be modified to insert any
leader, tail or signal sequence to direct the enzyme to an
appropriate location in the cell.
[0436] In another preferred embodiment of the present invention,
the plant selected is a C4 grass and the product of interest is
pHBA.
[0437] A schematic depicting the pHBA biosynthetic pathway is shown
in FIG. 14.
[0438] In order to effect pHBA production in sugarcane, a
chloroplast-targeted version of E. coli situated between the maize
ubi-1 promoter and nos terminator of the expression construct
pU3z-mcs-nos, was co-bombarded with a plasmid containing a
selectable marker (pUKN) into embryogenic sugarcane callus to yield
the UC series of transgenic lines. The UH series of plants was
generated in the same manner using an analogous expression
construct that contained the ORF of the P. fluorescens HCHL gene.
The regenerated plants were grown in a greenhouse for four weeks
and were then analyzed for pHBA accumulation in leaf tissue using
HPLC.
[0439] pHBA accumulated in the transformed plants as two glucose
conjugates, ie, a phenolic glucoside and a glucose ester. Both
compounds contained a single glucose molecule that was attached by
a 1-O-- -D linkage to the hydroxyl or carboxyl group of pHBA. The
predominant product in all of the plants examined was the phenolic
glucoside, which accounted for at least 90% of the pHBA.
[0440] The mean value for the population was 0.41%.+-.0.04% of dry
weight (DW), which is almost 30-fold higher than the mean value for
the non-transgenic control plants 0.014%.+-.0.01% DW. More
important, the pHBA glucoside content of the best plant was 1.5%
DW, which is equivalent to 0.69% DW free pHBA after correcting for
the attached glucose molecule. This value is three times higher
than the highest value obtained with transgenic tobacco plants
expressing a different chloroplast-targeted version of CPL. The
HCHL-expressing sugarcane plants accumulated even higher levels of
pHBA. The mean value for total pHBA glucose conjugates in the UH
lines was 0.70%.+-.0.07% DW, and the highest level observed at this
stage of development was 2.6% DW.
[0441] Based on the results obtained with the 4-week-old plants, a
subset of the primary transformants was selected for further
evaluation, and leaf levels of pHBA were determined after 16 weeks
additional growth. Included in this analysis were the two
CPL-expressing plants that previously exhibited the highest levels
of product accumulation (UC63 and UC65) and five HCHL-expressing
plants. The methanol-extracted samples were subjected to acid
hydrolysis, which quantitatively hydrolyzes both pHBA glucose
conjugates, and free pHBA was determined by HPLC.
[0442] It was anticipated that pHBA production would continue
throughout development and that the 20-week-old plants would have
higher levels of pHBA glucosides than the 4-week-old plants.
However, the increase in pHBA content with age was not very
dramatic nor was it universally observed when product accumulation
was expressed on a dry weight basis (FIG. 3A). Part of the
explanation for this is the lower water content of the older plant
leaf tissue.
[0443] For example, the average dry weight to wet weight ratio for
the 20-week-old plants was 0.23, while the corresponding value for
the 4-week-old plants was 0.15. When this phenomenon is taken into
account and product accumulation is expressed on a fresh weight
basis it becomes far more apparent that pHBA levels did increase as
the plants continued to grow (FIG. 3B), except for the two
CPL-expressing plants.
[0444] The 20-week-old primary transformants were large enough to
screen for stalk levels of pHBA without damaging the plants. At
this stage of development, the oldest stem tissue is semi-mature
and new tillers emerge. Since the stalk is the only part of the
sugarcane plant that is normally harvested in the existing sugar
mill infrastructure, pHBA accumulation in this tissue is the most
important gauge for technical success. Leaf and stem samples were
taken from 20-week-old plants, and total pHBA was determined by
HPLC after methanol extraction and acid hydrolysis. The third
internode from the bottom of the plant was the source of stem
tissue for this analysis, and the leaf samples were obtained from
the third fully unfurled leaf from the top of the plant. Generally
speaking, leaf levels of pHBA were considerably higher than stalk
levels.
[0445] However, the difference was much more pronounced for the
CPL-expressing plants. For example, the average stalk to leaf ratio
of pHBA for the five UH lines that were examined was
0.324.+-.0.031, and the highest stalk level of pHBA was 0.24% DW,
which is equivalent to 0.52% pHBA glucose conjugates. In marked
contrast, the corresponding ratios for UC63 and UC65 were 0.135 and
0.133, respectively, and product accumulation in the stalk of the
best plant (UC63) was only 0.06% DW. Since there are no reported
values in the literature for pHBA levels in stem tissue for
transgenic plants expressing CPL or HCHL, it will be very
interesting to see if these observations will extend to other plant
systems. Nevertheless, taken together the above results suggest
that HCHL is a better catalyst for pHBA production in sugarcane
than CPL, and subsequent studies focused on the UH series of
plants.
[0446] To gain a better understanding of pHBA accumulation in
different parts the plant, leaf and stem segments were sampled from
the primary shoot of 20-week-old UH1. The first leaf at the top
with a fully visible dewlap was designated "leaf 1" and consecutive
leaves down the stalk were numbered in ascending order. The stem
segments were numbered similarly with "internode 1" corresponding
to the internode immediately above the point of attachment of leaf
1. Note that the values shown refer to total pHBA after acid
hydrolysis. Except for the youngest leaf examined, product
accumulation in leaves was relatively uniform along the length of
the plant achieving a maximum value of .about.1.0% DW. Product
accumulation also varied along the length of the leaf, with the tip
of the leaf having about twice as much pHBA as the base of leaf A
similar trend was observed in the stalk, but there was a much
larger discrepancy between young stem tissue and old stem tissue.
In agreement with the results described above, pHBA levels in
mature stem tissue were about 3-fold lower than mature leaf tissue.
These results add additional support to the notion that pHBA
accumulation in HCHL-expressing sugarcane plants increases as a
function of time.
[0447] Additional insight on pHBA distribution was obtained from
dissection experiments. Three different compartments of the stalk
were examined: rind, pith, and vascular bundles. The most pHBA was
found in the rind (1% DW), while the pith and vascular bundles had
3- to 4-fold lower levels. Indeed, pHBA levels in the rind were
very similar to values obtained from the leaf midrib and leaf
lamina
[0448] Of all of the HCHL-expressing primary transformants
monitored, UH98 consistently had the highest levels of pHBA in both
leaf and stem tissue. When this plant was 20 weeks old pHBA
accumulation in leaf tissue was 2.8% DW (leaf lamina, 3.35% DW;
leaf midrib, 1.61% DW). The corresponding value for mature stem
tissue was 0.67% DW (rind, 0.96% DW; pith, 0.65% DW). Despite these
very high levels of pHBA glucose conjugates, UH98 was
morphologically indistinguishable from the non-transformed control
line TC1 (FIG. 5).
[0449] The present invention contemplates a method for producing
pHBA in a C4 grass, the method comprising expressing one or more
genetic sequences encoding one or more pHBA biosynthetic enzymes in
cells of a C4 grass such that pHBA accumulates anywhere in the cell
or extra-cellular matrix of the plant.
[0450] Accordingly, reference herein to hydroxycinnamoyl-CoA
hydratase/lyase or chorismate pyruvate lyase includes all homologs
thereof.
[0451] In a preferred embodiment, the present invention
contemplates a method for generating a plant which produces pHBA or
a precursor thereof, said method comprising introducing into cells
of said plant a genetic sequence comprising at least one of the
following:-- [0452] (i) a nucleotide sequence encoding
hydroxycinnamoyl-CoA hydratase/lyase; [0453] (ii) a nucleotide
sequence encoding chorismate pyruvate lyase; [0454] (iii) a
nucleotide sequence comprising the ubiC gene from E. coli, or a
homolg thereof; and/or [0455] (iv) a nucleotide sequence comprising
the HCHL gene from Pseudomonas fluorescens or homolg thereof; and
then regenerating a plant from said cells.
[0456] Preferably, the plant is a C4 grass and, in a particularly
preferred embodiment, sugarcane.
[0457] In order that pHBA may be produced in cells of a C4 grass,
suitable sequences such as those encoding one or more pHBA
biosynthetic enzymes must be introduced into and expressed in the
cells. That is, the plant needs to undergo genetic modification so
that the metabolites and/or metabolic and/or biosynthetic pathways
can be harnessed for the production of the pHBA or a precursor
thereof. This may conveniently be achieved through the use of
genetic constructs, engineered to comprise nucleotide sequences
required to effect pHBA production.
[0458] Clearly, the genetic sequences may be modified to insert any
leader, tail or signal sequence to direct the enzyme to an
appropriate location in the cell. In a preferred embodiment, the
pHBA biosynthetic enzymes are targetted to the plastid.
[0459] To effect expression of the nucleotide sequence of the
present invention, it may conveniently be incorporated into a
chimeric genetic construct comprising inter alia one or more of the
following: a promoter sequence, a 5' non-coding region, a
cis-regulatory region such as a functional binding site for
transcriptional regulatory protein or translational regulatory
protein, an upstream activator sequence, an enhancer element, a
silencer element, a TATA box motif, a CCAAT box motif, an upstream
open reading frame, transcriptional start site, translational start
site, and/or nucleotide sequence which encodes a leader sequence,
and a 3' non-translated region. Preferable the chimeric genetic
construct is designed for transformation of plants as hereinafter
described.
[0460] The term "5' non-coding region" is used herein in its
broadest context to include all nucleotide sequences which are
derived from the upstream region of an expressible gene, other than
those sequences which encode amino acid residues which comprise the
polypeptide product of said gene, wherein 5' non-coding region
confers or activates or otherwise facilitates, at least in part,
expression of the gene.
[0461] The term "gene" is used in its broadest context to include
both a genomic DNA region corresponding to the gene as well as a
cDNA sequence corresponding to exons or a recombinant molecule
engineered to encode a functional form of a product.
[0462] As used herein, the term "cis-acting sequence" or
"cis-regulatory region" or similar term shall be taken to mean any
sequence of nucleotides which is derived from an expressible
genetic sequence wherein the expression of the first genetic
sequence is regulated, at least in part, by said sequence of
nucleotides. Those skilled in the art will be aware that a
cis-regulatory region may be capable of activating, silencing,
enhancing, repressing or otherwise altering the level of expression
and/or cell-type-specificity and/or developmental specificity of
any structural gene sequence.
[0463] Reference herein to a "promoter" is to be taken in its
broadest context and includes the transcriptional regulatory
sequences of a classical genomic gene, including the TATA box which
is required for accurate transcription initiation, with or without
a CCAAT box sequence and additional regulatory elements (i.e.
upstream activating sequences, enhancers and silencers) which alter
gene expression in response to developmental and/or environmental
stimuli, or in a tissue-specific or cell-type-specific manner. A
promoter is usually, but not necessarily, positioned upstream or
5', of a structural gene, the expression of which it regulates.
Furthermore, the regulatory elements comprising a promoter are
usually positioned within 2 kilobase pairs (kb) of the start site
of transcription of the gene.
[0464] In the present context, the term "promoter" is also used to
describe a synthetic or fusion molecule, or derivative which
confers, activates or enhances expression of a structural gene or
other nucleic acid molecule, in a plant cell. Preferred promoters
according to the invention may contain additional copies of one or
more specific regulatory elements to further enhance expression in
a cell, and/or to alter the timing of expression of a gene to which
it is operably connected.
[0465] The term "operably connected" or "operably linked" in the
present context means placing a gene under the regulatory control
of a promoter, which then controls the transcription and optionally
translation of the gene. In the construction of heterologous
promoter/structural gene combinations, it is generally preferred to
position the genetic sequence or promoter at a distance from the
gene transcription start site that is approximately the same as the
distance between that genetic sequence or promoter and the gene it
controls in its natural setting, i.e. the gene from which the
genetic sequence or promoter is derived. As is known in the art,
some variation in this distance can be accommodated without loss of
function. Similarly, the preferred positioning of a regulatory
sequence element with respect to a heterologous gene to be placed
under its control is defined by the positioning of the element in
its natural setting, i.e. the genes from which it is derived.
[0466] Promoter sequences contemplated by the present invention may
be native to the host plant to be transformed or may be derived
from an alternative source, where the region is functional in the
host plant. Other sources include the Agrobacterium T-DNA genes,
such as the promoters for the biosynthesis of nopaline, octapine,
mannopine, or other opine promoters; promoters from plants, such as
the ubiquitin promoter; tissue specific promoters (see, e.g. U.S.
Pat. No. 5,459,252; International Patent Publication No. WO
91/13992); promoters from viruses (including host specific
viruses), or partially or wholly synthetic promoters. Numerous
promoters that are functional in mono- and dicotyledonous plants
are well known in the art (see, for example, Greve, J. Mol. Appl.
Genet. 1: 499-511, 1983; Salomon et al., EMBO J. 3: 141-146, 1984;
Garfinkel et al., Cell 27: 143-153, 1983; Barker et al., Plant Mol.
Biol. 2: 235-350, 1983); including various promoters isolated from
plants (such as the Ubi promoter from the maize ubi-1 gene, e.g.
U.S. Pat. No. 4,962,028) and viruses (such as the cauliflower
mosaic virus promoter, CaMV 35S).
[0467] In the context of the present invention, a particularly
useful tissue-specific promoter is one which drives expression
specifically in the stems of sugarcane plants. Such a stem-specific
promoter is, for example, that described in International Patent
Publication No. WO 01/18211.
[0468] The promoter sequences may include regions which regulate
transcription, where the regulation involves, for example, chemical
or physical repression or induction (e.g. regulation based on
metabolites, light, or other physicochemical factors; see, e.g.
International Patent Publication No. WO 93/06710 disclosing a
nematode responsive promoter) or regulation based on cell
differentiation (such as associated with leaves, roots, seed, or
the like in plants; see, e.g. U.S. Pat. No. 5,459,252 disclosing a
root-specific promoter). Thus, the promoter region, or the
regulatory portion of such region, is obtained from an appropriate
gene that is so regulated. For example, the ribulose
1,5-bisphosphate carboxylase gene is light-induced and may be used
for transcriptional initiation. Other genes are known which are
induced by stress, temperature, wounding, pathogen effects,
etc.
[0469] The chimeric genetic construct of the present invention may
also comprise a 3' non-translated sequence. A 3' non-translated
sequence refers to that portion of a gene comprising a DNA segment
that contains a polyadenylation signal and any other regulatory
signals capable of effecting mRNA processing or gene expression.
The polyadenylation signal is characterized by effecting the
addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. Polyadenylation signals are commonly recognized by the
presence of homology to the canonical form 5' AATAAA-3' although
variations are not uncommon
[0470] The 3' non-translated regulatory DNA sequence preferably
includes from about 50 to 1,000 nucleotide base pairs and may
contain plant transcriptional and translational termination
sequences in addition to a polyadenylation signal and any other
regulatory signals capable of effecting mRNA processing or gene
expression. Examples of suitable 3' non-translated sequences are
the 3' transcribed non-translated regions containing a
polyadenylation signal from the nopaline synthase (nos) gene of
Agrobacterium tumefaciens (Bevan et al., Nucl. Acid. Res. 11: 369,
1983) and the terminator for the T7 transcript from the octopine
synthase gene of Agrobacterium tumefaciens. Alternatively, suitable
3' non-translated sequences may be derived from plant genes such as
the 3' end of the protease inhibitor I or II genes from potato or
tomato, the soybean storage protein genes and the pea E9 small
sub-unit of the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO)
gene, although other 3' elements known to those of skill in the art
can also be employed. Alternatively, 3' non-translated regulatory
sequences can be obtained de novo as, for example, described by An
(Methods of Enzymology 153: 292, 1987), which is incorporated
herein by reference.
[0471] A genetic construct can also be introduced into a vector,
such as a plasmid. Plasmid vectors include additional DNA sequences
that provide for easy selection, amplification, and transformation
of the expression cassette in prokaryotic and eukaryotic cells,
e.g. pUC-derived vectors, pSK-derived vectors, pGEM-derived
vectors, pSP-derived vectors, or pBS-derived vectors. Additional
DNA sequences include origins of replication to provide for
autonomous replication of the vector, selectable marker genes,
preferably encoding, for example, antibiotic or herbicide
resistance or green fluorescent protein or other visible markers,
unique multiple cloning sites providing for multiple sites to
insert DNA sequences or genes encoded in the chimeric genetic
construct, and sequences that enhance transformation of prokaryotic
and eukaryotic cells.
[0472] The vector preferably contains an element(s) that permits
either stable integration of the vector or a chimeric genetic
construct contained therein into the host cell genome, or
autonomous replication of the vector in the cell independent of the
genome of the cell. The vector, or a construct contained therein,
may be integrated into the host cell genome when introduced into a
host cell. For integration, the vector may rely on a foreign or
endogenous DNA sequence present therein or any other element of the
vector for stable integration of the vector into the genome by
homologous recombination. Alternatively, the vector may contain
additional nucleic acid sequences for directing integration by
homologous recombination into the genome of the host cell. The
additional nucleic acid sequences enable the vector or a construct
contained therein to be integrated into the host cell genome at a
precise location in the chromosome. To increase the likelihood of
integration at a precise location, the integrational elements
should preferably contain a sufficient number of nucleic acids,
such as 100 to 1,500 base pairs, preferably 400 to 1,500 base
pairs, and most preferably 800 to 1,500 base pairs, which are
highly homologous with the corresponding target sequence to enhance
the probability of homologous recombination. The integrational
elements may be any sequence that is homologous with the target
sequence in the genome of the host cell. Furthermore, the
integrational elements may be non-encoding or encoding nucleic acid
sequences.
[0473] For cloning and sub-cloning purposes, the vector may further
comprise an origin of replication enabling the vector to replicate
autonomously in a host cell such as a bacterial cell. Examples of
bacterial origins of replication are the origins of replication of
plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting
replication in E. coli, and pUB110, pE194, pTA1060, and pAM.beta.1
permitting replication in Bacillus. The origin of replication may
be one having a mutation to make its function temperature-sensitive
in a Bacillus cell (see, e.g. Ehrlich, Proc. Natl. Acad. Sci. USA
75: 1433, 1978).
[0474] To facilitate identification of transformed cells, the
vector desirably comprises a further genetic construct comprising a
selectable or screenable marker gene. The actual choice of a marker
is not crucial as long as it is functional (i.e. selective) in
combination with the plant cells of choice. The marker gene and the
nucleotide sequence of interest do not have to be linked, since
co-transformation of unlinked genes as, for example, described in
U.S. Pat. No. 4,399,216 is also an efficient process in plant
transformation.
[0475] Included within the terms selectable or screenable marker
genes are genes that encode a "secretable marker" whose secretion
can be detected as a means of identifying or selecting for
transformed cells. Examples include markers that encode a
secretable antigen that can be identified by antibody interaction,
or secretable enzymes that can be detected by their catalytic
activity. Secretable proteins include, but are not restricted to,
proteins that are inserted or trapped in the cell wall (e.g.
proteins that include a leader sequence such as that found in the
expression unit of extensin or tobacco PR-S); small, diffusible
proteins detectable, for example, by ELISA; and small active
enzymes detectable in extracellular solution such as, for example,
.alpha.-amylase, .beta.-lactamase, phosphinothricin
acetyltransferase).
[0476] Examples of bacterial selectable markers are the dal genes
from Bacillus subtilis or Bacillus licheniformis, or markers that
confer antibiotic resistance such as ampicillin, kanamycin,
erythromycin, chloramphenicol or tetracycline resistance. Exemplary
selectable markers for selection of plant transformants include,
but are not limited to, a hyg gene which encodes hygromycin B
resistance; a neomycin phosphotransferase (npt) gene conferring
resistance to kanamycin, paromomycin, G418 and the like as, for
example, described by Potrykus et al. (Mol. Gene. Genet. 199: 183,
1985); a glutathione-S-transferase gene from rat liver conferring
resistance to glutathione derived herbicides as, for example,
described in EP-A 256 223; a glutamine synthetase gene conferring,
upon overexpression, resistance to glutamine synthetase inhibitors
such as phosphinothricin as, for example, described International
Patent Publication No. WO 87/05327, an acetyl transferase gene from
Streptomyces viridochromogenes conferring resistance to the
selective agent phosphinothricin as, for example, described in
European Patent Application No. EP-A 275 957, a gene encoding a
5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance
to N-phosphonomethylglycine as, for example, described by Hinchee
et al. (Biotech 6: 915, 1988), a bar gene conferring resistance
against bialaphos as, for example, described in International
Patent Publication No. WO 91/02071; a nitrilase gene such as bxn
from Klebsiella ozaenae which confers resistance to bromoxynil; a
dihydrofolate reductase (DHFR) gene conferring resistance to
methotrexate (Thillet et al., J. Biol. Chem. 263: 12500, 1988); a
mutant acetolactate synthase gene (ALS), which confers resistance
to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals
(European Patent Application No. EP-A-154 204) or a mutated
anthranilate synthase gene that confers resistance to 5-methyl
tryptophan.
[0477] Preferred screenable markers include, but are not limited
to, a uidA gene encoding a .beta.-glucuronidase (GUS) enzyme for
which various chromogenic substrates are known; a
.beta.-galactosidase gene encoding an enzyme for which chromogenic
substrates are known; an aequorin gene (Prasher et al., Biochem.
Biophys. Res. Comm. 126: 1259, 1985), which may be employed in
calcium-sensitive bioluminescence detection; a green fluorescent
protein gene (Niedz et al., Plant Cell Reports 14: 403, 1995); a
luciferase (luc) gene (Ow et al., Science 234: 856, 1986), which
allows for bioluminescence detection; a .beta.-lactamase gene
(Sutcliffe, Proc. Natl. Acad. Sci. USA 75: 3737, 1978), which
encodes an enzyme for which various chromogenic substrates are
known (e.g. PADAC, a chromogenic cephalosporin); an R-locus gene,
encoding a product that regulates the production of anthocyanin
pigments (red colour) in plant tissues (Dellaporta et al., in
Chromosome Structure and Function pp. 263-282, 1988); an
.alpha.-amylase gene (Ikuta et al., Biotech 8: 241, 1990); a
tyrosinase gene (Katz et al, J. Gen. Microbiol. 129: 2703, 1983)
which encodes an enzyme capable of oxidizing tyrosine to dopa and
dopaquinone which in turn condenses to form the easily detectable
compound melanin; or a xylE gene (Zukowsky et al., Proc. Natl.
Acad. Sci. USA 80: 1101, 1983), which encodes a catechol
dioxygenase that can convert chromogenic catechols.
[0478] A further aspect of the present invention provides a
transfected or transformed cell, tissue, or organ from a C4 grass,
which comprises a nucleotide sequence encoding one or more enzymes
required for the production of a useful product.
[0479] The vectors and chimeric genetic construct(s) of the present
invention may be introduced into a cell by various techniques known
to those skilled in the art. The technique used may vary depending
on the known successful techniques for that particular
organism.
[0480] Techniques for introducing vectors, chimeric genetic
constructs and the like into cells include, but are not limited to,
transformation using CaCl.sub.2 and variations thereof, direct DNA
uptake into protoplasts, PEG-mediated uptake to protoplasts,
microparticle bombardment, electroporation, microinjection of DNA,
microparticle bombardment of tissue explants or cells,
vacuum-infiltration of tissue with nucleic acid, and T-DNA-mediated
transfer from Agrobacterium to the plant tissue.
[0481] For microparticle bombardment of cells, a microparticle is
propelled into a cell to produce a transformed cell. Any suitable
ballistic cell transformation methodology and apparatus can be used
in performing the present invention. Exemplary apparatus and
procedures are disclosed by Stomp et al. (U.S. Pat. No. 5,122,466)
and Sanford and Wolf (U.S. Pat. No. 4,945,050). When using
ballistic transformation procedures, the genetic construct may
incorporate a plasmid capable of replicating in the cell to be
transformed.
[0482] Examples of microparticles suitable for use in such systems
include 0.1 to 10 .mu.m and more particularly 0.5 to 5 .mu.m
tungsten particles or gold spheres. The DNA construct may be
deposited on the microparticle by any suitable technique, such as
by precipitation.
[0483] Plant tissue capable of subsequent clonal propagation,
whether by organogenesis or embryogenesis, may be transformed with
a chimeric genetic construct of the present invention and a whole
plant generated therefrom. The particular tissue chosen will vary
depending on the clonal propagation systems available for, and best
suited to, the particular species being transformed. Exemplary
tissue targets include leaf disks, embryos, cotyledons, hypocotyls,
megagametophytes, callus tissue, existing meristematic tissue (e.g.
apical meristem, axillary buds, and root meristems), and induced
meristem tissue (e.g. cotyledon meristem and hypocotyl
meristem).
[0484] The regenerated transformed plants may be propagated by a
variety of means, such as by clonal propagation or classical
breeding techniques. For example, a first generation (or T1)
transformed plant may be selfed to give a homozygous second
generation (or T2) transformant, and the T2 plants further
propagated through classical breeding techniques.
[0485] Even more particularly, the present invention provides a
plant cell or multicellular plant or progeny thereof wherein said
cell, plant, progeny or part thereof exhibits an activity to
manufacture PHAs.
[0486] The term "genetically modified" is used in its broadest
sense and includes introducing gene(s) into cells, mutating gene(s)
in cells and altering or modulating the regulation of gene(s) in
cells. In the context of the present invention, a transgenic cell
or plant line may also be considered as a mutant cell or plant line
when compared with its non-transgenic counterpart. In essence, a
selected plant is first genetically modified to introduce a genetic
sequence encoding a desired product or intermediate.
[0487] Where genetic sequences for more than one gene are to be
used in the performance of the present invention, they may be
introduced simultaneously or sequentially, separately or together,
into the target cells that are to be transformed. For example, a
singe genetic construct may comprise all the required genetic
sequences for the practice of the subject invention, and this
single construct may be introduced into the cells via any number of
different means, as discussed below. Moreover, each genetic
sequence may be operable linked to and under the control of its own
promoter, or may be comprised within a single polycistronic unit.
Alternatively, separate genetic constructs may be utilized, each
comprising one of the needed genetic sequences. In this event, more
than one construct may be introduced into the target cells
simultaneously or sequentially. Here and elsewhere throughout the
subject specification, the terms "target cells" and "cells to be
transformed" should be regarded as being synonymous and refer to
cells of a C4 grass that are to be used in accordance with the
present invention as a bioreactor.
[0488] The one or more genetic sequences, introduced into a C4
grass plant cell, need to be expressed in order to enable the
manufacture and accumulation of a product. The term "expression" is
to be construed in its broadest sense and includes and encompasses
transcription and translation of a genetic sequence to a
translation product.
[0489] Some plant cells may already comprise a homolog of one or
more of the genetic sequences encoding enzymes needed for the
production of a given product. In instances where a target plant
cell, such as a sugarcane cell, already comprises one or more
suitable genetic sequences, capable of directing sufficiently high
expression, only those enzymes missing in a given pathway need be
provided through via a genetic construct as described above.
[0490] The present invention extends to homologs and derivatives of
any suitable sequences, whether found naturally in a target cell or
provided exogenously having been derived from another plant,
animal, protist, fungal, archeal or bacterial source, inter alia.
The derivatives may be at the protein or nucleic acid level.
[0491] By "derivative" in relation to a polypeptide is meant a
polypeptide that has been derived from the basic sequence by
modification, for example, by conjugation or complexing with other
chemical moieties or by post-translational modification techniques
as would be understood in the art. The term "derivative" also
includes within its scope alterations that have been made to a
parent sequence including additions, or deletions that provide for
functionally-equivalent molecules. Accordingly, the term
"derivative" encompasses molecules that affect a plant's phenotype
in the same way as does the parent an amino acid sequence from
which it was generated. Also encompassed are polypeptides in which
one or more amino acids have been replaced by different amino
acids. It is well understood in the art that some amino acids may
be changed to others with broadly similar properties without
changing the nature of the activity of the polypeptide
(conservative substitutions) as described hereinafter. These terms
also encompass polypeptides in which one or more amino acids have
been added or deleted, or replaced with different amino acids.
[0492] "Polypeptide", "peptide" and "an amino acid sequence" are
used interchangeably herein to refer to a polymer of amino acid
residues and to variants and synthetic analogues thereof. Thus,
these terms apply to amino acid polymers in which one or more amino
acid residues is a synthetic non-naturally-occurring amino acid,
such as a chemical analogue of a corresponding naturally-occurring
amino acid, as well as to naturally-occurring amino acid
polymers.
[0493] The term "derivative" also encompasses fragments. A
"fragment", as used herein, means a portion or a part of a
full-length parent polypeptide, which retains the activity of the
parent polypeptide. As used herein, the term "biologically-active
fragment" includes deletion mutants and small peptides, for
example, of at least 10, preferably at least 20 and more preferably
at least 30 contiguous amino acids, which comprise the above
activity. Peptides of this type may be obtained through the
application of standard recombinant nucleic acid techniques or
synthesized using conventional liquid or solid phase synthesis
techniques. For example, reference may be made to solution
synthesis or solid phase synthesis as described, for example, in
Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard
which is included in a publication entitled "Synthetic Vaccines"
edited by Nicholson and published by Blackwell Scientific
Publications. Alternatively, peptides can be produced by digestion
of an amino acid sequence of the invention with proteinases such as
endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease. The
digested fragments can be purified by, for example, high
performance liquid chromatographic (HPLC) techniques. Any such
fragment, irrespective of its means of generation, is to be
understood to be encompassed by the term "derivative" as used
herein.
[0494] The terms "variant" and "homolog" refer to nucleotide
sequences displaying substantial sequence identity with a reference
nucleotide sequences or polynucleotides that hybridize with a
reference sequence under stringency conditions that are defined
hereinafter. The terms "nucleotide sequence", "polynucleotide" and
"nucleic acid molecule" may be used herein interchangeably and
encompass polynucleotides in which one or more nucleotides have
been added or deleted, or replaced with different nucleotides. In
this regard, it is well understood in the art that certain
alterations inclusive of mutations, additions, deletions and
substitutions can be made to a reference nucleotide sequence
whereby the altered polynucleotide retains the biological function
or activity of the reference polynucleotide. The term "variant"
also includes naturally-occurring allelic variants.
[0495] The extent of homology may be determined using sequence
comparison programs such as GAP. In this way, sequences of a
similar or substantially different length to those cited herein
might be compared by insertion of gaps into the alignment, such
gaps being determined, for example, by the comparison algorithm
used by GAP, as is further discussed below.
[0496] Homologous sequences will generally hybridize under
particular specified conditions. The term "hybridization" denotes
the pairing of complementary nucleotide sequences to produce a
DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences
are those sequences that are related by the base-pairing rules. In
DNA-DNA hybridization, A pairs with T and C pairs with G. In
DNA-RNA hybridization, U pairs with A and C pairs with G. In this
regard, the terms "match" and "mismatch" as used herein refer to
the hybridization potential of paired nucleotides in complementary
nucleic acid strands. Matched nucleotides hybridize efficiently,
such as the classical A-T and G-C base pair mentioned above.
Mismatches are other combinations of nucleotides that do not
hybridize efficiently.
[0497] The extent of hybridization that may be displayed by
homologous sequences depends on the conditions of, for example,
temperature, ionic strength, presence or absence of certain organic
solvents, under which hybridization and washing procedures are
carried out. The higher the stringency, the higher will be the
degree of complementarity between immobilised target nucleotide
sequences and the labelled probe polynucleotide sequences that
remain hybridized to the target after washing. "High stringency
conditions" refers to temperature and ionic conditions under which
only nucleotide sequences having a high frequency of complementary
bases will hybridize. The stringency required is
nucleotide-sequence dependent, and further depends upon the various
components present during hybridization and subsequent washes, and
the time allowed for these processes. Generally, in order to
maximize the hybridization rate, relatively low-stringency
hybridization conditions are selected: about 20 to 25.degree. C.
lower than the thermal melting point (T.sub.m). The T.sub.m, is the
temperature at which 50% of specific target sequence hybridizes to
a perfectly complementary probe in solution at a defined ionic
strength and pH. Generally, in order to require at least about 85%
nucleotide complementarity of hybridized sequences, highly
stringent washing conditions are selected to be about 5 to
15.degree. C. lower than the T. In order to require at least about
70% nucleotide complementarity of hybridized sequences,
moderately-stringent washing conditions are selected to be about 15
to 30.degree. C. lower than the T.sub.m. Highly permissive (very
low stringency) washing conditions may be as low as 50.degree. C.
below the T.sub.m, allowing a high level of mis-matching between
hybridized sequences. Those skilled in the art will recognize that
other physical and chemical parameters in the hybridization and
wash stages can also be altered to affect the outcome of a
detectable hybridization signal from a specific level of homology
between target and probe sequences.
[0498] Reference herein to "low stringency conditions" is generally
determined at 42.degree. C. and includes and encompasses from at
least about 0% v/v to at least about 15% v/v formamide, and from at
least about 1 M to at least about 2 M salt for hybridization, and
at least about 1 M to at least about 2 M for washing conditions.
Alternative stringency conditions may be applied where necessary,
such as: medium stringency, which includes and encompasses from at
least about 16% v/v to at least about 30% v/v formamide, and from
at least about 0.5 M to at least about 0.9 M salt for
hybridization, and at least about 0.5 M to at least about 0.9 M
salt for washing conditions, or high stringency, which includes and
encompasses from at least about 31% v/v to at least about 50% v/v
formamide, and from at least about 0.01 M to least about 0.15 M
salt for hybridization, and at least about 0.01 M to at least about
0.15 M salt for washing conditions.
[0499] Terms used to describe sequence relationships between two or
more nucleotide sequences or amino acid sequences include
"reference sequence", "comparison window", "sequence identity",
"percentage of sequence identity" and "substantial identity". A
"reference sequence" is at least 12 but frequently 15 to 18 and
often at least 25 monomer units, inclusive of nucleotides and amino
acid residues, in length. Because two polynucleotides may each
comprise (1) a sequence (i.e. only a portion of the complete
polynucleotide sequence) that is similar between the two
polynucleotides, and (2) a sequence that is divergent between the
two polynucleotides, sequence comparisons between two (or more)
polynucleotides are typically performed by comparing sequences of
the two polynucleotides over a "comparison window" to identify and
compare local regions of sequence similarity. A "comparison window"
refers to a conceptual segment of at least 6 contiguous positions,
usually about 50 to about 100, more usually about 100 to about 150
in which a sequence is compared to a reference sequence of the same
number of contiguous positions after the two sequences are
optimally aligned. The comparison window may comprise additions or
deletions (i.e. gaps) of about 20% or less as compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Optimal alignment of
sequences for aligning a comparison window may be conducted by
computerized implementations of algorithms (GAP, BESTFIT, FASTA,
and TFASTA in the Wisconsin Genetics Software Package Release 7.0,
Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or
by inspection and the best alignment (i.e. resulting in the highest
percentage homology over the comparison window) generated by any of
the various methods selected. Reference also may be made to the
BLAST family of programs as for example disclosed by Altschul et
al., Nucl. Acids Res. 25: 3389, 1997. A detailed discussion of
sequence analysis can be found in Unit 19.3 of Ausubel et al.,
"Current Protocols in Molecular Biology" John Wiley & Sons Inc,
1994-1998, Chapter 15.
[0500] The term "sequence identity" as used herein refers to the
extent that sequences are identical on a nucleotide-by-nucleotide
basis or an amino acid-by-amino acid basis over a window of
comparison. Thus, a "percentage of sequence identity" is calculated
by comparing two optimally aligned sequences over the window of
comparison, determining the number of positions at which the
identical nucleic acid base (e.g. A, T, C, G, I) or the identical
amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile,
Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met)
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison (i.e. the window size), and
multiplying the result by 100 to yield the percentage of sequence
identity. For the purposes of the present invention, "sequence
identity" will be understood to mean the "match percentage"
calculated by the DNASIS computer program (Version 2.5 for windows;
available from Hitachi Software engineering Co., Ltd., South San
Francisco, Calif., USA) using standard defaults as used in the
reference manual accompanying the software.
[0501] The one or more constructs may be introduced into a plant
cell by any number of well-recognized means such as discussed
above.
[0502] Preferably, the genetic constructs of the present invention
are introduced via the use of biolistics.
[0503] Accordingly, another aspect of the present invention
provides a transgenic C4 grass, cells of which have been
transformed with one or more genetic sequences such that one of the
following products in the cytosol, storage vacuole, non-plastid
organelle or extra-cellular matrix of said cells: [0504] (i)
polyhydroxyalkanoates [0505] (ii) vanillin [0506] (iii) sorbitol
[0507] (iv) indigo [0508] (v) fructans [0509] (vi) lactic acid
[0510] (vii) adipic acid [0511] (viii) 1,3-propanediol [0512] (ix)
2-phenylethanol [0513] (x) pHBA
[0514] The present invention extends to parts of plants tissue
including leaves, stems, vascular bundles, bark, reproductive
material, roots and any extracted liquid ("juice") from said
plant.
[0515] While the present invention is exemplified using the
compounds hereinbefore described, it is to be understood that the
invention extends to and encompasses the use of any suitable
genetic sequence capable of effecting the production of any product
in the cells or extracellular matrix of a C4 grass.
[0516] The term "gene" is used in its broadest sense and includes
cDNA corresponding to the exons of a gene. Accordingly, reference
herein to a "gene" is to be taken to include:-- [0517] (i) a
classical genomic gene consisting of transcriptional and/or
translational regulatory sequences and/or a coding region and/or
non-translated sequences (i.e. introns, 5'- and 3'-untranslated
sequences); or [0518] (ii) mRNA or cDNA corresponding to the coding
regions (i.e. exons) and 5'- and 3'-untranslated sequences of the
gene.
[0519] The term "gene" is also used to describe synthetic or fusion
molecules encoding all or part of an expression product. In
particular embodiments, the term "nucleic acid molecule" and "gene"
may be used interchangeably.
[0520] In order to improve the efficiency and accumulation rate of
a product, a suitable genetic sequence or sequences may be more
specifically targeted so as to facilitate generation of expression
products in particular sub-cellular areas or organelles within the
plant. These include, for example, the cytosol, a storage vacuole
or a plastid or non-plastid organelle.
[0521] The usefulness of a given sub-cellular compartment for a
given product depends on the nature and potential toxicity of the
product to the plant. For example, PHA production is dependent on
both the types of polymer produced and the metabolic pathways being
engineered.
[0522] One particularly useful sub-cellular area for the production
of products such as PHB, 1,3-propanediol and sorbitol, is the
cytosol, wherein sucrose is both synthesized via gluconeogenesis
and degraded via glycolysis, leading to the production of pyruvate.
In the cytosol, PHB is the preferred polymer, as moderate amounts
of acetyl-CoA are available for phaA, phaB and phaC. A particularly
useful sub-cellular organelle is the mitochondrion, wherein
acetyl-CoA, which may be produced from pyruvate via mitochondrial
pyruvate dehydrogenase, and/or perhaps from fatty acids via
.beta.-oxidation, is used to fuel the TCA cycle. A second useful
sub-cellular organelle, with moderate to high acetyl-CoA, is the
peroxisome, the site of fatty acid degradation via
.beta.-oxidation. These pathways involve the utilization of
substantial amounts of acetyl-CoA, depleting reserves and rendering
it unavailable for use in manufacture of a product. However, the
pyruvate needed for acetyl-CoA production is generated via
glycolysis, which, in a sink tissue such as sugarcane stems, is
fuelled by sucrose. Hence, the carbon drain that usually results
from effecting the production of a product, such as a PHA, vanillin
and the like, in a plant cell, is able to be overcome by the
sucrose-accumulating plant cell's ability to mobilise its
substantial sucrose stores. The deleterious effects resulting from
product accumulation observed in non-sucrose-accumulating plant
species does not occur in sugarcane, as a concomitant state of
general starvation is precluded by the mobilization of sucrose from
storage vacuoles, which replenishes the reduction of cellular
acetyl-CoA pools caused by the introduced genetic sequences.
[0523] Accumulation of PHAs, and other products (particularly
products such as pHBA, adipic acid and indigo) may also be targeted
to a plastid, such as a chloroplast, where large amounts of
acetyl-CoA are used for fatty acid biosynthesis. For example, for
the production of PHAs other than PHB, plastids and peroxisomes are
the preferred sub-cellular compartment, as PhaG (plastid) and PhaJ
(peroxisome) provide monomers suitable for MCL-PHA polymerases such
as PhaC1 from intermediates in fatty acid biosynthesis and
.beta.-oxidation. In addition, particular biosynthetic pathways
from which a particular product may be derived may exist only in
the plastid. For example, the shikimate pathway is locatized in the
plastid in plants. Products such as indigo and adipic acid may be
derived from intermediates of the shikimate pathway via the
addition of new biosynthetic enzymes. For these new enzymes to
produce the product of interest, they must be localised to the
particular organelles where their substrates would be found.
[0524] In order to direct product accumulation to a desired
sub-cellular location, particular specific "target sequences" may
be incorporated into the genetic constructs described above.
[0525] A target sequence includes a signal sequence such as a
signal sequence to direct the protein to a plastid, vacuole,
mitochondrion or other appropriate organ or tissue.
[0526] Preferably, accumulation of PHA is in the cytosol or
mitochondrion, assisted via mobilization of sucrose reserves
located in the storage vacuoles of sugarcane stem cells.
[0527] Preferably, accumulation of adipic acid and indigo is in the
plastid, wherein the introduced biosynthetic enzymes have access to
intermediates of the shikimate pathway.
[0528] The plants of the present invention may also be further
"tagged" with a reporter that identifies the plant as a plant
bioreactor. Any number of physiological or genetic tags would be
suitable, and readily identified by one of skill in the art.
Examples of physiological "tags" that could be introduced include
marker genes such as the green fluorescent protein gene, the
firefly luciferase gene and the GUS gene.
[0529] Marker genes that alter the physical appearance of the plant
may also be used as identifying tags. Examples include increased or
decreased length of stems and/or alterations to color. Furthermore,
a number of resistance phenotypes may also be used to identify the
plant bioreactors. Genes encoding resistance to pests such as
bacterial, fungal or nematode pests have been identified in the
art, and would be suitable as "tags".
[0530] In addition, a genetic sequence itself may comprise the tag,
referred to herein as "DNA barcoding". Tagging in this manner is
done by introducing a known non-coding polynucleotide sequence into
the plant. The tag may then be amplified from the plant using known
PCR primers. Plants may then be identified as bioreactors according
to the present invention by the presence of a particular size
amplicon after the PCR reaction. Further discrimination, for
example between types of plant bioreactor, may be achieved by
altering the polynucleotide sequence of the DNA barcode in the
region between the PCR primers. In this way, the sequence of the
barcode may be elucidated using automated sequencing techniques to
determine the exact identity of the plant. This technique allows
for a generic test to identify all plant bioreactors, and allows
further discrimination to identify the type of bioreactor based on
the sequence of the DNA barcode.
[0531] The genetic sequences comprising or encoding the "tag" may
be introduced to the plant using the methods hereinbefore
described. The tag may be introduced on the same construct as the
biosynthetic gene, or may be independently introduced. If
introduced independently, the tag may be introduced on a different
construct at the same time as transformation with the biosynthetic
gene, or introduced to the plant before or after the biosynthetic
gene.
[0532] Accordingly, the present invention contemplates a plant
suitable for use as a bioreactor that has been tagged with a
genetic sequence which encodes or comprises a genotypic or
phenotypic feature that allows differentiation of the plant
bioreactor from a wild-type plant.
[0533] Preferably the plant is a C4 grass, and more preferrably,
the plant is sugarcane.
[0534] The plant-based bioreactor system of the present invention
is useful in enabling the production of molecules such as PHAs,
pHBA, vanillin, sorbitol, indigo, fructans, lactic acid, adipic
acid, 1,3-propanediol, 2-phenylethanol, inter alia, by a number of
different parties such as different commercial entities. The
present invention extends, therefore, to a data processing system
to monitor the use of the plants and/or the production of target
molecules.
[0535] Accordingly, another aspect of the present invention
contemplates a method for generating a target molecule in a
sucrose-accumulating plant, said method comprising:-- [0536] (i)
providing a plant or cells of a plant to a party; and [0537] (ii)
permitting the party to generate and harvest molecules from said
plant or cells of said plant receiving and processing data from
said party.
[0538] The data received from the party includes, for example,
numbers of plants grown and/or harvested, the types of genetic
constructs introduced into the cells and/or income received from
sale of the products.
[0539] The present invention is further described by the following
non-limiting Examples.
Example 1
Materials
[0540] Restriction digests, DNA ligations and all other DNA
manipulations were performed as described in Sambrook, et al.,
Molecular Cloning, A Laboratory Manual, 2n.sup.d edition, Cold
Spring Harbor Press, 1989.
Example 2
Cloning of the phaC1 Gene from P. aeruginosa
[0541] The phaC1 gene targeted to plant peroxisomes inserted into
pART27 as an EcoRI/XbaI fragment, was obtained from Y. Poirier
(University of Lausanne, Switzerland). In order to express the gene
in sugarcane, it was excised with the said enzymes, end-filled with
T4 DNA polymerase (Promega) and inserted into the SmaI site of
pUBI-MCS-Nos. To achieve targeting of the phaC1 gene product to
mitochondria and plastids, the gene is modified as described
below.
Example 3
Generation of Genetic Constructs
(a) Constructs Comprising Sequences Encoding PHB-Synthesizing
Enzymes
[0542] Constructs containing the phaA, phaB and phaC genes from
Ralstonia eutropha targeted to plastids and cloned in pUC18 as
XbaI-SacI fragments were obtained from Y. Poirier (University of
Lausanne, Switzerland).
[0543] The phaA, phaB and phaC genes derived from Ralstonia
eutropha were cloned into the vector pU3z. This vector is a
derivative of pGEM3 (Promega) containing the maize polyubiquitin
promoter and nos terminator from A. tumefaciens, and works well as
an expression vector in sugarcane. All genes were
amplified/modified using the polymerase chain reaction (PCR) prior
to insertion into pU3z except phaC1, which was blunt-end cloned
into the same vector. All constructs used for plant transformation
are listed below. Where inserts are modified, they are sequenced in
full to ensure quality.
[0544] PCR modifications were performed as follows. Platinum Pfx
(registered trademark) DNA polymerase, 10.times. buffer and
PCR-enhancer were obtained from Invitrogen. Final concentrations
were one unit of polymerase per reaction, 1.times. buffer and
enhancer, 2 mM Mg.sup.2+, 0.2 mM dNTP, 0.4 .mu.M of each primer.
All primers were purchased from Geneworks, Australia. Reactions
were performed in a MJC PTC-100 thermal cycler. The profile was
initial denaturation at 96.degree. C. for 5 min, followed by 35
cycles of 94.degree. C. for 30 seconds, 42.degree. C. for 30 sec
and 72.degree. C. for 2.5 min. a final extension step of 72.degree.
C. for 10 min preceded a final hold at 4.degree. C. Table 1 lists
primers used in PCR reactions.
TABLE-US-00003 TABLE 1 POSITION, GENE, NAME SEQUENCE TARGET TphaF
N.sub.6ggatccatggcttctatgatatcct 5', phaA-C, plastid [SEQ ID NO:
34] PhaF N.sub.6GGATCCATGACTGACGTTGTCATC 5', phaA, cytosol [SEQ ID
NO: 35] PhbF N.sub.6GGATCCATGACTCAGCGCATTGCG 5', phaB, cytosol [SEQ
ID NO: 36] PhcF N.sub.6GGATCCATGGCGACCGGCAAAGGC 5', phaC, cytosol
[SEQ ID NO: 37] PhaR CTGAGTCATGTCCACTCC 3', phaA, cytosol and [SEQ
ID NO: 38] plastid PhbR CTGCCGACTGGTGGAACC 3', phaB, cytosol and
[SEQ ID NO: 39] plastid PhcR GAAGCGTCATGCCTTGGC 3', phaC, cytosol
and [SEQ ID NO: 40] plastid PhaC1CF N.sub.6GGATCCATGAGCCAGAAGAAC
5', phaC1, cytosol and [SEQ ID NO: 41] mitochondia PhaC1CR
N.sub.6GGTACCTCATCGTTCATGCACG 3', phaC1, cytosol and [SEQ ID NO:
42] plastid PhaC1PF N.sub.6CCCGGGTGAGCCAGAAGAACAATAAC 5', phaC1,
plastid [SEQ ID NO: 43] PhaJF GGATCCATGAGCGCACAATCCCTGG 5', phaJ,
peroxisome [SEQ ID NO: 44] PhaJR AAGCTTTTGAAGGCAGCTTGACCACGGC 3',
phaJ, peroxisome [SEQ ID NO: 45] PhaGF CCCGGGTGAGGCCAGAAATCGCTGTAC
5', phaG, plastid [SEQ ID NO: 46] PhaGR GGTACCTCAGATGGCAAATGCATGC
3', phaG, plastid [SEQ ID NO: 47] SSP-F
NNGAGCTCGATGGGAGGTGCTCGAAGACATATTA 5', stem-specific CC promoter
[SEQ ID NO: 48] SSP-R NNGGATCCTGTACTAGATATGGCAGC 3', stem-specific
[SEQ ID NO: 49] promoter
[0545] Approximately 10 ng of construct was used as template in
each reaction.
[0546] Following PCR, fragments were gel purified and cloned into
the BamHI and SmaI sites of pUSN in the correct orientation between
the maize polyubiquitin promoter and the nos-terminator from A.
tumefaciens. Plasmid constructs were fully sequenced and purified
by anion-exchange chromatography (Qiagen, Australia) prior to
transformation into callus tissue.
(b) Constructs Comprising Marker Sequences
[0547] Two plasmids were obtained from the CSIRO, Brisbane,
Australia. The first comprised the <pUbi-gfp-nos> construct,
which carries the green fluorescent protein (GFP) from Aquorea
victoria under the control of the same promoter as above. The
second plasmid, designated "pEmuKn", harbours an aphA gene
(neomycin phosphotransferase) under the control of the Emu
promoter. These plasmids were used without any further
modification.
Example 4
Sugarcane Transformation
(a) Generation of Embryogenic Callus Tissue
[0548] Embryogenic callus of the sugarcane variety Q117 was
established, as described in Bower et al., Molec. Breeding 2:
239-249, 1996. Briefly, embryogenic callus was established by
excision of inner leaf whorls from cane tops 2-5 cm above the
apical meristem. Disks of approximately 2 mm thickness were placed
on MSC.sub.3 medium that contains 3 .mu.g/ml
2,4-Dichlorophenoxy-acetic acid (2,4-D) and incubated at 28.degree.
C. in the dark for 2-5 months, with fortnightly subculturing. In
order to avoid problems associated with stress arising from the
tissue culture process, such as somaclonal variation, unused callus
tissue was discarded after 6 months in culture.
[0549] Prior to transformation, embryogenic callus was transferred
to osmotic MSC.sub.3, as previously described (Bower et al., 1996,
supra)
(b) Bombardment of Callus Tissue
[0550] DNA was coated onto tungsten particles (Sylvania M-10) and
embryogenic callus bombarded as described by Bower et al. (1996,
supra).
[0551] Callus tissue was co-transformed with up to five individual
constructs, plasmid solutions being mixed to give equimolar
concentrations to facilitate co-integration and expression of genes
required for PHB production. There is a strong correlation between
co-transformation and co-integration of constructs into the genome
of plant hosts. The combinations of constructs introduced into Q117
callus and the target for products of each pha gene are shown in
the following Table 2, wherein "cyt"=cytosol, "pla"=plastid,
"mito"=mitochondrion and "perox"=peroxisome:
TABLE-US-00004 TABLE 2 COMBINATION AND TARGETING OF pha GENE
PRODUCTS phaA (cyt), phaB (cyt), phaC (cyt), Ubi-GFP, Emu-Kn phaA
(pla), phaB (pla), phaC (pla), Ubi-GFP, Emu-Kn phaA (cyt), phaB
(cyt), phaC1 (cyt), Ubi-GFP, Emu-Kn phaC1 (perox), Ub-GFP, Emu-Kn
phaA (cyt), phaB (cyt), phaC (cyt), phaC1 (perox), phaA (pla), phaB
(pla), phaC (pla), phaG (pla), phaC1 (pla), Ubi-GFP, Emu-Kn phaC1
(perox), phaA (pla), phaB (pla), phaC (pla), phaG (pla), phaC1
(pla), Ubi-GFP, Emu-Kn phaA (pla), phaB (pla), phaC (pla), phaG
(pla), phaC1 (pla), Ubi-GFP, Emu-Kn phaG (pla), phaC1 (pla),
Ubi-GFP, Emu-Kn phaA (mito), phaB (mito), phaC (mito), Ubi-GFP,
Emu-Kn
[0552] Microprojectile bombardment was performed as described in
Bower et al. (supra), except that, to improve transformation
efficiencies, the vacuum chamber was evacuated to -100 kPa
atmospheric pressure and particles accelerated by a helium pulse of
3000 kPa for 100 ms.
(c) Selection of Transformed Material
[0553] Following bombardment, callus was allowed to recover for one
hour, before being placed onto MSC.sub.3 medium supplemented with
50 ng/ml Geneticin (registered trademark) (Invitrogen).
[0554] Putatively transformed tissue was screened for the
expression of GFP and resistance to the antibiotic geneticin.
Bombarded callus was examined for the presence of cells expressing
GFP 7 days after transformation and stained in vivo with Nile Red,
a sensitive in vivo stain specific for intracellular lipids, as
previously described (Taguchi et al., FEMS Microbiol. Lett. 198:
65-71, 2001; Greenspan, et al., 1985, supra). For both techniques,
an Olympus SZX 12 stereomicroscope equipped with GFP excitation and
emission filters was used.
Dump
[0555] Antibiotic selection was continued for 3 months in the dark
at 28.degree. C. and continued during plant regeneration. Only
calli expressing both selectable markers were allowed to regenerate
into plantlets.
(d) Regeneration of Transformed Sugarcane Plantlets
[0556] For plant regeneration, callus was transferred to medium
without 2,4-D and incubated at 24-26.degree. C. under illumination.
Plantlets appeared after 2-4 months and were transferred to potting
mix and kept in mini-glasshouse (Yates, Australia) for one week
prior to transfer to glasshouse facilities.
Example 5
Determination of Quantity and Composition of PHA Produced
[0557] If the desired PHA is PHB, quantification in transgenic
sugarcane is conducted by HPLC, as described by Karr et al.,
Applied and Environmental Microbiology 46: 1339-1344, 1983. This
method allows for the analysis of plant extracts with minimal
handling. This technique is illustrated in Figure *.
[0558] For other PHAs, molecular characterization and
quantification of PHA content in transgenic plants is carried out
using gas chromatography analysis.
[0559] For GC analysis, PHA was separated from homogenized leaf
samples by chloroform extraction, followed by methanol extraction,
to remove lipids other than PHA. The polymer was then purified
further by acetone extraction.
[0560] Transesterification of plant extracts was performed as
described in Braunegg et al., Eur. J. Appl. Microbiol. Biotechnol.
6: 29-37, 1978 modified by using boron trifluoride rather than
sulfuric acid as catalyst and decreased incubation time to 1 hr.
Gas chromatography analysis was performed using a Varian 3300
chromatograph, as described by Slater et al., in J. Bacteriol. 180:
667-73, 1998. Purified PHB (Coparsucar, Brazil) and
methyl-3-hydroxybutyrate (Sigma) were used as positive controls
either pure or spiked into negative control plant extracts.
Modifications to this method, namely using Boron trifluoride rather
than sulfuric acid as catalyst and decreasing incubation times to 1
hour, in addition to minor modifications to instrument parameters,
were found to improve peak resolution.
[0561] Millenium software (Waters Corp., Milford, Mass.) is used to
quantify amounts of PHAs produced, by comparison of peak areas from
plant extracts with those from standards of known concentration.
GC-MS is used to determine the composition of PHAs produced in
transgenic sugarcane, as different hydroxy-alkanoates have
different mass-spectrum signatures.
Example 6
Production of PHB in Transgenic Sugarcane Leaves
[0562] In plants accumulating high amounts of PHA, gene copy
number, transcription levels and amount of protein are determined
using standard molecular biology techniques. Gene copy numbers are
determined by Southern blot analysis of genomic DNA from
transformed plants producing PHAs. Transcription levels will be
evaluated by northern blot analysis of RNA from transgenic plants
and gene product levels examined by western blot analysis of
protein extracts using antibodies against the gene products. The
antisera were obtained from Prof. Y. Poirier (University of
Lausanne, Switzerland).
Example 7
Targeting of PHB Production to Non Plastid Sugarcane Organelles
[0563] The constructs pUbi-phaA, pUbi-phaB and pUbi-phaC comprising
the phaA, B and C genes, respectively, transcriptionally fused to
the aforementioned maize polyubiquitin promoter were digested with
BamHI and dephosphorylated with shrimp alkaline phosphatase
(Promega, Maddison, USA). A BglII/BamHI fragment of the plasmid
sB-pma-4-35S-.beta.-del-GFP, containing the leader sequence and
first 12 amino acid residues of the .beta. subunit of the Nicotiana
plumbaginifolia mitochondrial F1-ATPase (Chaumont et al., PMB 24:
631-664, 1994), was ligated with pUbi-phaA, B or C cut with BamHI.
These constructs target PhaA, B or C to the mitochondria with high
efficiency. For targeting of pha gene products to other organelles,
the genes were modified and ligated into plasmids already
containing the required DNA targeting sequences.
[0564] Sugarcane was transformed with these constructs, using the
aforementioned methods.
[0565] Transgenic plants thereby generated are screened for PHB
production, using the aforementioned techniques.
Example 8
Production of PHB in Transgenic Sugarcane Stems
[0566] Plasmids pUbi-phaA, pUbi-phaB, pUbi-phaC, pUi-TP-phaA,
pUbi-TP-phaB and pUbi-TP-phaC were digested with BamHI/EcoRI to
release the pha genes with or without the aforementioned plastid
leader sequence at the 5' end, and with the aforementioned NOS
terminator at the 3' end. These fragments were ligated into the
vector pSSP cut with BamHI/EcoRI. pSSP is a derivative of the
vector p67G-420 (supplied by Prof R Birch, University of
Queensland). p67G-420 houses, immediately upstream of a unique
BamHI site, a stem-specific promoter isolated from sugarcane. To
obtain pSSP, a consensus ribosome binding site was removed from the
3' end of the promoter in the following way. The promoter was PCR
amplified, using the aformentioned technique, with the primers
SPP-F and SPP-R (see Table 1), incorporating BamHI and SacI sites
into the 5' and 3' ends of the promoter, respectively. The PCR
product was digested with BamHI and SacI and then religated into
the backbone of p67G.sub.--420 digested with the same enzymes. The
promoter was fully sequenced to confirm quality. The resulting
constructs, which were fully sequenced to confirm quality, drive
gene expression in the stems and target the gene products to either
the cytosol or the plastids. pSSP was linearized with BamHI and
dephosphorylated with shrimp alkaline phosphatase, and ligated with
the aforementioned BglII/BamHI fragment of the plasmid
sB-pma-4-35S-.beta.-del-GFP, giving the intermediate vector
pSSP-Tm. BamHI/EcoRI fragments of pUbi-phaA, pUbi-phaB and
pUbi-phaC were ligated into pSSP-Tm. The resulting constructs,
which were fully sequenced to confirm quality, drive gene
expression in the stems and target the gene products to the
mitochondria.
[0567] Sugarcane was transformed with these constructs using the
aforementioned methods.
[0568] Transgenic plants thereby generated are screened for PHB
production, using the aforementioned techniques.
Example 9
Detection of PHB in Chloroplasts of Transgenic Sugarcane
[0569] Transgenic plants expressing PHB biosynthetic genes were
produced according to the methods described herein. Accumulation of
PHB in the plastid was assessed using both HPLC and transmission
electron microscopy.
[0570] Figure * shows a graphical representation of the detection
of PHB in chloroplasts of transgenic sugarcane. Panels A-C indicate
detection of PHB using HPLC. Panel A is a wild-type sugarcane
control; Panel B is the plant in A spiked with PHB; Panel C depicts
a transgenic sugarcane line accumulating PHB in the plastids.
Arrows point to the elution point of crotonic acid, which is the
product of acid-hydrolsis of PHB. The insert in Panel C shows that
the peak at 30 min in C has the same spectrum as crotonic acid.
[0571] Panels D-F in FIG. 4 show the detection of PHB granules in
plants by transmission electron microscopy. Panel D shows a
positive control comprising a chloroplast from a mesophyll cell in
a PHB +ve Arabidopsis plant (Bohmert et al. 2000). Panel E is a
electron-micrograph showing PHB granules in a chloroplast of a
mesophyll cell from a PHB-producing sugarcane plant. Panel F shows
PHB granules in a chloroplast of a bundle-sheath cell from the same
plant line in E. Scale bars=200 nm.
Example 10
Agronomic Performance of PHB Producing Sugarcane Lines
[0572] Four transgenic sugarcane lines expressing the PHB
biosynthesis genes of Ralstonia eutropha were grown for 3 months in
a randomised glasshouse plot. Control plants comprised
GFP-expressing and tissue-culture-regenerated wild-type plants. PHB
content was assessed in lamina from the tips of mature leaves and
quantified by HPLC analysis.
[0573] The results are shown in FIG. 5. The production of PHB in
sugarcane at up to 1.6% of leaf dry-weight did not reduce agronomic
performance compared with GFP-expressing and wild-type control
plants. Data are the mean.+-.SE (n=3). DW=dry-weight.
Example 11
Affect of PHB Production on Sugarcane Sugar Accumulation
[0574] The plants assessed in Example 10, ie. PHB producing, GFP
expressing and wild-type sugarcane, were further examined for their
sucrose, glucose and fructose concentrations to determine the
effect of PHB production on sugar content.
[0575] The results are shown in FIG. 6. It was observed that PHB
accumulation of up to 1.6% of leaf dry-weight did not reduce
sucrose, glucose, fructose or total sugar content in PHB producing
(solid bars) plants compared to GFP-expressing (open bars) and
wild-type (hatched bars) controls. Data are the mean.+-.SE (n=3).
DW=dry-weight.
Example 12
Distribution of PHB in PHB Producing Sugarcane
[0576] The distribution of PHB throughout transgenic sugarcane line
PHB3 was determined by HPLC analysis. Samples were taken from:
[0577] (i) lamina of the tip, midpoint and base of young,
intermediate and mature leaves; [0578] (ii) combined rind and pith
of young, intermediate and mature stem internodes; and [0579] (iii)
roots.
[0580] The PHB content data are presented as the mean percentage of
leaf dry-weight.+-.SE (n=3). ND=not detected.
Example 13
Production of Vanillin in Sugarcane
[0581] Vanillin (4-hydroxy-3-methoxybenzaldehyde) would be produced
as a co-product with sucrose. Sucrose yield is expected to decrease
in direct proportion to the amount of vanillin produced.
[0582] Initially, genes for the vanillin biosynthetic pathway from
a known source are cloned. These genes are then expressed in
sugarcane, including any tailoring of the expression pattern as
required. The product is produced as a glucose conjugate, which is
stable.
[0583] A number of biological pathways have been discovered for the
biosynthesis/biodegradation of vanillin. At least 2 of these have
substrates which are available in plants. [0584] 1)
3-Dehydroshikimic acid is produced as an intermediate in the
shikimate pathway. A pathway has been identified which converts
this substrate via 3-Dehydroshikimate dehydratase to protocatechuic
acid then to vanillic acid via Catechol-o-methyltransferase and
finally to vanillin via Aryl aldehyde dehydrogenase. [0585] 2)
Ferulic acid is a secondary metabolite of the phenylpropanoid
pathway involved in lignin synthesis. It is converted in planta to
feruloyl-CoA by feruloyl-CoA synthetase which in turn is converted
to vanillin by enoyl-CoA hydratase/aldolase.
[0586] Glucosylation of the product in vivo is expected to detoxify
the product. Accordingly, inducible expression should not be
required. The maximum level of production is determined by the flux
through the phenylpropanoid pathway. However, Sugarcane has a
productive phenylpropanoid pathway and should adapt readily to
increased demands placed on it for synthesis of vanillin.
Example 14
Production of Sorbitol in Sugarcane
[0587] Zymomonas mobilis is able to produce sorbitol from sucrose
or a mixture of glucose and fructose in a one-step reaction
catalysed by the glucose-fructose oxidoreductase GFOR (Genbank
accession no. Z80356, M97379). The glucose is oxidized to
gluconolactone while the fructose is reduced to sorbitol.
glucose+fructose.fwdarw.sorbitol+gluconolactone
[0588] Sorbitol production in sugarcane could be achieved by using
GFOR. This involves constructing an expression cassette by fusing
GFOR to the maize polyubiquitin promoter and nopaline synthase
terminator and introducing the cassette into sugarcane callus by
biolistic transformaton. The Z. mobilis GFOR is not membrane-bound
and resides in the periplasm and should work equally well as a
cytosolic enzyme in sugarcane.
[0589] Sorbitol production is unlikely to be toxic in sugarcane
since sorbitol is found in numerous fruits (apples, pears, plums,
berries, cherries). Sorbitol functions physiologically to regulate
osmotic stress hence extremely high levels may be detrimental.
Vacuolar storage may circumvent this problem.
[0590] The threshold level at which sorbitol is deleterious to the
host may be determined by growing sugarcane callus on solid medium
containing sorbitol.
[0591] A potential large-scale system for the recovery of sorbitol
from sugarcane involves adding an aqueous organic salt solution,
mixing and then separating a salt water phase from a polyol-rich
phase (see international patent application WO210252).
Example 15
Indigo Production in Sugarcane
[0592] The chief incentive to use sugarcane as an indigo biofactory
is to provide a manufacturing route that will produce relatively
inexpensive indigo from a renewable feedstock.
[0593] Indigo production by microbial fermentation has been
demonstrated by expressing the genes that mediate indigo formation
in E. coli (Drewlo 2001, Berry 2002). The pigment is derived by
converting endogenous tryptophan to indole using the Enterobacter
aerogenes tryptophanase or L-tryptophan indole lyase EC 4.1.99.1
(Genbank accession no. D14297). Subsequently the indole is
converted to indigo via two possible reactions.
[0594] Route A: Pseudomonas putida napthalene dioxygenase (Genbank
accession no. M83949)
[0595] Route B: Ralstonia eutropha bec gene (Genbank accession no.
AF306552)
[0596] Indigo production in sugarcane would involve constructing an
expression cassette by fusing the aforementioned genes to the maize
polyubiquitin promoter and nopaline synthase terminator and
introducing the cassette into sugarcane callus by biolistic
transformaton. Both route A and B should be tested if possible.
Tryptophan is a product of the plant shikimate pathway, which is
responsible for synthesizing lignin precursors. The cloned genes
will need to be plastid-targeted since the shikimate pathway
reactions reside in this compartment. The available metabolic flux
in this pathway is expected to be significant.
Example 16
Production of Fructans in Sugarcane
[0597] Naturally occurring fructans may contain 10 to 100,000
fructose residues. Bacteria produce the larger fructans whilst
those occurring in plants are smaller. The larger polymers are
desirable because they are less soluble in water and consequently
easier to extract. Larger fructans will not affect the osmotic
pressure in the cell to the same degree as smaller molecules.
Therefore it is possible to store greater quantities of fructan
before the cell is affected.
[0598] Numerous bacterial fructosyltransferases or levansucrases
have been characterized (Genbank accession no. AY150365, Bacillus
subtilis). These enzymes catalyze the transfer of the D-fructosyl
residue from sucrose to the .beta.-2,6-linked residues of
fructan.
Sucrose.fwdarw.fructan+glucose
[0599] Fructan production in sugarcane would be achieved by
constructing an expression cassette containing levansucrase, the
maize polyubiquitin promoter and nopaline synthase terminator and
introducing the cassette into sugarcane callus by biolistic
transformaton.
[0600] Levansucrase will probably require apoplastic or vacuolar
targeting to maximize access to substrate for conversion.
[0601] Fructan may then be recovered from sugarcane juice by
ethanol precipitation followed by vacuum-drying.
Example 17
Lactic Acid Production in Sugarcane
[0602] The production of Lactic acid (2-Hydroxypropanoic acid) in
sugarcane proceeds with the following steps: [0603] (i) Obtain or
clone lactate dehydrogenase (LDH) from a number of sources, such a
Lactobacillus spp. bacterium. [0604] (ii) Expression of the gene in
sugarcane, with any necessary changes to the sequence such as codon
preference. It is preferred that the introduced gene is expressed
in the cytosol, therefore no targeting is required. [0605] (iii)
Regenerate plants and evaluate for lactate (or derivative)
production.
[0606] Lactic acid build-up may cause deleterious effect on cells.
There are several ways by which cells can deal with this. One is to
remove the acid either by diffusion or transport. The other is
modification of the offending chemical and export into the vacuole.
Glycosylation is a major signal for this process and lactic acid
possesses two potential glycosylation sites.
[0607] Traditionally, lactic acid purification has been a complex
chemical process. However, recent advances have simplified this
process and made it significantly cheaper. It is anticipated that
lactic acid can be removed from the post-crushing millstream
without great difficulty or extensive modification of existing
structures. It is anticipated that the extraction process will be
product dependent.
Example 18
Adipic Acid Production in Sugarcane
[0608] Adipic acid may be produced in sugarcane by one of two
approaches.
I. Synthesis from Cis, Cis-Muconic Acid
[0609] Adipic acid has been produced in transgenic E. coli using
the metabolic pathway illustrated below. Three genes were
introduced into E. coli to produce cis, cis-muconic acid that was
subsequently purified from the fermentation broth and converted to
adipic acid by catalytic hydrogenation (step g, 10% Pt/C, H.sub.2,
3400 kPa, 25.degree. C.). This final step has a 97% conversion
efficiency.
[0610] The synthesis of cis, cis-muconic acid in sugarcane involves
making use of the shikimate pathway. In order to use the shikimate
pathway to produce cis, cis-muconic acid the following biosynthetic
enzymes, or homologs thereof are introduced into sugarcane:
[0611] Klebsiella pneumoniae 3-dehydroshikimate dehydratase
(aroZ)-enzyme d
3-dehydroshikimate protocatechuate
[0612] Klebsiella pneumoniae protocatechuate decarboxylase
(aroY)-enzyme e
Protocatechuate.fwdarw.catechol
[0613] Acinetobacter calcoaceticus catechol 1,2-dioxygenase
(catA)-enzyme f
Catechol+O.sub.2.fwdarw.cis, cis-muconic acid
[0614] Introduction of these genes into sugarcane involves
constructing an expression cassette by fusing the genes described
above to the maize polyubiquitin promoter and nopaline synthase
terminator and introducing the cassette into sugarcane callus by
biolistic transformation. Catechol is probably produced in most
plants, and therefore, it may be unnecessary to clone additional
copies of 3-dehydroshikimate dehydratase or protocatechuate
decarboxylase. The cloned gene(s) are plastid-targeted since the
shikimate pathway reactions reside in this compartment.
[0615] The shikimate pathway executes a central role in plant
secondary metabolism. This is one of the most active pathways in
plants in terms of carbon flux owing to the fact that it is the
source of lignin precursors. This makes it an attractive candidate
for metabolic engineering.
II. Synthesis from Petroselinic Acid
[0616] Bio-based adipic acid can be obtained through ozonolysis
(O.sub.3) of petroselinic acid (18:1 .DELTA..sup.6 cis), as shown
in FIG. 10. The coproduct lauric acid is also a potential source of
feedstock for detergent manufacture.
[0617] The seed oil of the coriander spice plant contains 80-90%
petroselinic acid. A 36 kDa putative acyl-ACP desaturase (Genbank
accession no. M93115) has been identified from coriander seed
extracts and the corresponding cDNA was able to confer the ability
to produce petroselinic acid in tobacco callus (Cahoon 1992).
Petroselinic acid was quantified from extracted calli by gas
chromatography and GC-MS (to determine double bond position).
Tobacco does not normally produce petroselinic acid hence the
successful expression of the cDNA in tobacco suggested that this
desaturase was sufficient for petroselinic acid formation. This
also infers that it may be feasible in sugarcane.
[0618] The metabolic pathway for producing petroselinic acid is
unclear, however, evidence suggests that it is formed by the
desaturation of palmitoyl-ACP by the 36 kDa desaturase followed by
elongation to form petroselinic acid (Cahoon 1994).
16:0-ACP.fwdarw.16:1.DELTA..sup.4-ACP.fwdarw.18:1.DELTA..sup.6-ACP
[0619] Recent studies have identified a 3-ketoacyl-ACP synthase
(Genbank accession no. AF263992) associated with the two-carbon
elongation of 16:1 .DELTA..sup.4-ACP.
[0620] Cis, cis-muconic acid in sugarcane juice would be converted
to adipic acid by catalytic hydrogenation. The adipic acid in the
resultant solution can be recovered by solvent extraction. The
solution is contacted with chloroform or methylene chloride and the
adipic acid recovered in the aqueous fraction. The aqueous fraction
would then be evaporated to yield crystalline adipic acid.
Example 19
Production of 1,3 propanediol (1,3-PD) in Sugarcane
[0621] 1,3-PD is a natural product of glycerol fermentation in a
few enterobacteria and clostridia. Fermentation-derived 1,3-PD was
not commercially viable for many years due to the high cost of the
glycerol feedstock.
[0622] The metabolic reactions that convert glycerol to 1,3-PD have
been established from Klebsiella pneumoniae.
[0623] Klebsiella pneumoniae glycerol dehydratase (dhaB)
glycerol.fwdarw.3-hydroxypropionaldehyde+H.sub.2O
[0624] Klebsiella pneumoniae 1,3-propanediol oxidoreductase
(dhaT)
3-hydroxypropionaldehyde+NADH.fwdarw.1,3-propanediol+NAD
[0625] Sugarcane does not naturally produce glycerol therefore the
reactions that convert triose phosphates to glycerol must also be
engineered into sugarcane.
[0626] Saccharomyces cerevisiae glycerol-3-phosphate
dehydrogenase
dihydroxyacetone phosphate+NADH.fwdarw.glycerol-3-phosphate+NAD
Saccharomyces cerevisiae glycerol-3-phosphatase
glycerol-3-phosphate+ADP.fwdarw.glycerol+ATP
[0627] Effectively, all four new genes must be cloned into
sugarcane to convert it into a 1,3-PD biofactory. These genes will
be assembled into an expression cassette containing the maize
polyubiquitin promoter and nopaline synthase terminator. The
cassette will be introduced into sugarcane callus by biolistic
transformation and expression will be targeted to the cytosol. The
accumulation of 1,3-PD in plant tissue will be assayed from plant
extracts by conventional HPLC.
[0628] 1,3-PD can be recovered from sugarcane juice by extraction
with cyclohexane followed by vaporization of the residual solvent.
Alternatively, distillation may be employed. Use of cyclohexane is
environmentally unsound and distillation is energy intensive.
Consequently, a method has been patented that describes the use of
ion exclusion resins to recover 1,3-PD (see international patent
application WO 01/73097).
Example 20
Production of 2-phenylethanol (2-PE) in Sugarcane
[0629] The production of 2-PE in sugarcane would be achieved in a
similar way to previous examples. Briefly, cloned genes for the
2-PE biosynthetic pathway, which has previously been determined,
would be obtained. Second, these genes would then be expressed in
sugarcane, tailoring the expression pattern and codon usage if
required. Finally, a stable product, as a glucose conjugate, is
expected.
[0630] A biological pathway for the biosynthesis of 2-PE is
presented in FIG. 11.
[0631] The product is produced naturally in roses and hence should
not be toxic. Glucosylation of the active group is likely to occur
in sugarcane to reduce potential toxicity.
[0632] 2-PE would be recovered by crushing the cane and refining
from juice as for sucrose, and standard production processes for
the synthetic form are well established.
[0633] 2-PE is water-soluble and should be stable for the time
normally taken to process sugarcane for sucrose. If sugarcane
stores 2-PE as a glucose conjugate then alkaline hydrolysis may be
required.
Example 21
Characterization of CPL-Expressing and HCHL-Expressing Sugarcane
Plants
[0634] A chloroplast-targeted version of E. CPL situated between
the maize ubi-1 promoter and nos terminator of the expression
construct pU3z-mcs-nos, was co-bombarded with a plasmid containing
a selectable marker (pUKN) into embryogenic sugarcane callus to
yield the UC series of transgenic lines. The UH series of plants
was generated in the same manner using an analogous expression
construct that contained the ORF of the P. fluorescens HCHL gene.
To serve as controls for the experiments described below, four
non-transgenic lines (TC1-TC4) were also regenerated from the same
callus material omitting the transformation and selection steps.
The regenerated plants were grown in a greenhouse for four weeks
and were then analyzed for pHBA accumulation in leaf tissue using
HPLC. Only plants that had higher levels than the control plants
(46% and 48% of the population for the UC lines and UH lines,
respectively) were included in the analysis shown in FIG. 2.
[0635] Not surprisingly, none of the transgenic plants had
significantly higher levels of "free" pHBA than the control plants.
Similar to the situation reported for tobacco plants expressing CPL
(Siebert et al., Plant Physiol. 112: 811-819, 1996) or HCHL (Mayer
et al., Plant Cell 13:1669-1682, 2001), the only two compounds that
accumulated were pHBA glucose conjugates, a phenolic glucoside and
a glucose ester. Both compounds contained a single glucose molecule
that was attached by a 1-O---D linkage to the hydroxyl or carboxyl
group of pHBA. The predominant product in all of the plants
examined was the phenolic glucoside, which accounted for at least
90% of the pHBA (see below). The mean value for the population was
0.41%.+-.0.04% of dry weight (DW), which is almost 30-fold higher
than the mean value for the non-transgenic control plants
0.014%.+-.0.01% DW. More important, the pHBA glucoside content of
the best plant was 1.5% DW, which is equivalent to 0.69% DW free
pHBA after correcting for the attached glucose molecule. This value
is three times higher than the highest value obtained with
transgenic tobacco plants expressing a different
chloroplast-targeted version of CPL (Siebert et al., supra). The
HCHL-expressing sugarcane plants accumulated even higher levels of
pHBA. The mean value for total pHBA glucose conjugates in the UH
lines was 0.70%.+-.0.07% DW, and the highest level observed at this
stage of development was 2.6% DW, which is very similar to the best
value reported for transgenic tobacco plants expressing the same
enzyme (Mayer et al., supra).
[0636] Based on the results obtained with the 4-week-old plants, a
subset of the primary transformants was selected for further
evaluation, and leaf levels of pHBA were determined after 16 weeks
additional growth (FIG. 15). Included in this analysis were the two
CPL-expressing plants that previously exhibited the highest levels
of product accumulation (UC63 and UC65) and five HCHL-expressing
plants. The methanol-extracted samples were subjected to acid
hydrolysis, which quantitatively hydrolyzes both pHBA glucose
conjugates, and free pHBA was determined by HPLC.
[0637] It was anticipated that pHBA production would continue
throughout development and that the 20-week-old plants would have
higher levels of pHBA glucosides than the 4-week-old plants.
However, the increase in pHBA content with age was not very
dramatic nor was it universally observed when product accumulation
was expressed on a dry weight basis. Part of the explanation for
this is the lower water content of the older plant leaf tissue. For
example, the average dry weight to wet weight ratio for the
20-week-old plants was 0.23, while the corresponding value for the
4-week-old plants was 0.15. When this phenomenon is taken into
account and product accumulation is expressed on a fresh weight
basis it becomes far more apparent that pHBA levels did increase as
the plants continued to grow, except for the two CPL-expressing
plants.
[0638] The 20-week-old primary transformants were large enough to
screen for stalk levels of pHBA without damaging the plants. At
this stage of development, the oldest stem tissue is semi-mature
and new tillers emerge. Since the stalk is the only part of the
sugarcane plant that is normally harvested in the existing sugar
mill infrastructure, pHBA accumulation in this tissue is the most
important gauge for technical success. Leaf and stem samples were
taken from 20-week-old plants, and total pHBA was determined by
HPLC after methanol extraction and acid hydrolysis. The third
internode from the bottom of the plant was the source of stem
tissue for this analysis, and the leaf samples were obtained from
the third fully unfurled leaf from the top of the plant. Generally
speaking, leaf levels of pHBA were considerably higher than stalk
levels.
[0639] However, the difference was much more pronounced for the
CPL-expressing plants. For example, the average stalk to leaf ratio
of pHBA for the five UH lines that were examined was
0.324.+-.0.031, and the highest stalk level of pHBA was 0.24% DW,
which is equivalent to 0.52% pHBA glucose conjugates. In marked
contrast, the corresponding ratios for UC63 and UC65 were 0.135 and
0.133, respectively, and product accumulation in the stalk of the
best plant (UC63) was only 0.06% DW. Since there are no reported
values in the literature for pHBA levels in stem tissue for
transgenic plants expressing CPL or HCHL, it will be very
interesting to see if these observations will extend to other plant
systems. Nevertheless, taken together the above results suggest
that HCHL is a better catalyst for pHBA production in sugarcane
than CPL, and subsequent studies focused on the UH series of
plants.
Example 22
Localization of pHBA in Sugarcane Tissue
[0640] To gain a better understanding of pHBA accumulation in
different parts the plant, leaf and stem segments were sampled from
the primary shoot of 20-week-old UH1. The first leaf at the top
with a fully visible dewlap was designated "leaf 1" and consecutive
leaves down the stalk were numbered in ascending order. The stem
segments were numbered similarly with "internode 1" corresponding
to the internode immediately above the point of attachment of leaf
1. The results from this analysis are summarized in FIG. 15. Note
that the values shown refer to total pHBA after acid hydrolysis.
Except for the youngest leaf examined, product accumulation in
leaves was relatively uniform along the length of the plant
achieving a maximum value of .about.1.0% DW. Product accumulation
also varied along the length of the leaf, with the tip of the leaf
having about twice as much pHBA as the base of leaf (data not
shown). A similar trend was observed in the stalk, but there was a
much larger discrepancy between young stem tissue and old stem
tissue. In agreement with the results described above, pHBA levels
in mature stem tissue were about 3-fold lower than mature leaf
tissue. These results add additional support to the notion that
pHBA accumulation in HCHL-expressing sugarcane plants increases as
a function of time.
[0641] Additional insight on pHBA distribution was obtained from
dissection experiments similar to the one shown in FIG. 15. The
plant that was used for this analysis was 20-week-old UH1. Three
different compartments of the stalk were examined: rind, pith, and
vascular bundles. The most pHBA was found in the rind (1% DW),
while the pith and vascular bundles had 3- to 4-fold lower levels.
Indeed, pHBA levels in the rind were very similar to values
obtained from the leaf midrib and leaf lamina.
[0642] Of all of the HCHL-expressing primary transformants
monitored, UH98 (FIG. 2B) consistently had the highest levels of
pHBA in both leaf and stem tissue. When this plant was 20 weeks old
pHBA accumulation in leaf tissue was 2.8% DW (leaf lamina, 3.35%
DW; leaf midrib, 1.61% DW). The corresponding value for mature stem
tissue was 0.67% DW (rind, 0.96% DW; pith, 0.65% DW). Despite these
very high levels of pHBA glucose conjugates, UH98 was
morphologically indistinguishable from the non-transformed control
line TC1 (FIG. 5).
Example 23
Construction of cTP-CPL
[0643] PCR was used to generate the monocot chloroplast-targeting
sequence that was fused to the N-terminus of E. coli CPL. The
target for amplification was the maize rbcS gene (GenBank accession
number Y00322), which codes for the Rubisco small subunit
precursor. Primer 1 (5'-CTA CTC ATA ACC ATG GCG CCC ACC GTG-3')
hybridized to nucleotides 489-505 and introduced a NcoI site at the
start codon of the transit peptide. Primer 2 (5'-CAT CTT ACT CAT
ATG CCG CAC CTG CAT GCA CCG GAT CCT TCC G-3') hybridized to
nucleotides 616-639 and introduced an NdeI site five amino acid
residues downstream from the chloroplast cleavage site. The PCR
product was cut with NcoI and NdeI and inserted into pET24a-tTP-CPL
(manuscript in preparation), after the latter was cleaved with the
same enzymes. pET24a-tTP-CPL contains the gene for a chimeric
protein that consists of the tomato Rubisco small subunit transit
peptide plus the first four amino acid residues of the `mature`
Rubisco small subunit, fused to the N-terminus of E. coli CPL. The
plasmid DNA was cut with NcoI and NdeI to remove the tomato
chloroplast-targeting sequence, and this was replaced with
PCR-generated maize chloroplast-targeting sequence. The ligation
mixture was introduced into E. coli DH10B, and growth was selected
on LB media containing kanamycin (50 .mu.g mL.sup.-1). A
representative plasmid
[0644] (pET24a-cTP-CPL) was sequenced and no PCR errors were found.
The predicted chloroplast cleavage product of the cTP-CPL fusion
protein is a CPL variant with five extra N-terminal amino acid
residues (i.e. MQVRH-CPL).
Example 24
Generation of CPL and HCHL Expression Constructs Used for Sugarcane
Transformation
[0645] The antibiotic selection plasmid pUKN contains the ubi-1
promoter, the neomycin phosphotransferase gene and the nos
terminator. The plasmid pU3z-mcs-nos was used for cTP-CPL and HCHL
expression in sugarcane. This plasmid is a modification of pAHC20
and contains the maize ubi-1 promoter and nos terminator. Both
genes were inserted in the SpeI and KpnI sites of the multicloning
region that immediately follows the maize ubi-1 intron. The gene
coding for cTP-CPL was amplified from pET24a-cTP-CPL using primers
3 and 4. Primer 3 (5'-CTA CTC ATT TAC TAG TCA CCA TGG CGC CCA CCG
TGA TG-3') (SEQ ID NO: 50) hybridized to the first 18 nucleotides
of the ORF of cTP-CPL and introduced a SpeI site upstream from the
start codon. Primer 3 also contained a consensus monocot ribosomal
binding site, CACC, which is situated between the SpeI site and the
initiator Met codon. Primer 4 (5'-CAT CTT ACT GGT ACC TTT AGT ACA
ACG GTG ACG CC-3') (SEQ ID NO: 51) hybridized to the other end of
the insert and introduced a KpnI site just after the cTP-CPL stop
codon. The PCR product was cut with SpeI and KpnI, and ligated into
similarly digested pU3z-mcs-nos. The ligation reaction mixture was
used to transform E. coli DH10B and growth was selected on LB media
containing ampicillin (100 .mu.g mL.sup.-1). A representative
plasmid (pU3z-mcs-nos-cTP-CPL) was sequenced to confirm the absence
of PCR errors.
[0646] Primers 5 and 6 were used to amplify the Pseudomonas
fluorescens strain AN103 HCHL gene (GenBank accession number
Y13067) from plasmid pFI1039. Primer 5 (5'-CTA CTC ATT TAC TAG TCA
CCA TGA GCA CAT ACG AAG GTC G-3') (SEQ ID NO: 52) hybridized to the
first 20 nucleotides of the ORF for HCHL and introduced a unique
SpeI site upstream from the start codon. Primer 5 also contained a
consensus monocot ribosomal binding site (CACC) that is situated
between the SpeI site and the initiator Met codon. Primer 6 (5'-CAT
CTT ACT GGT ACC TTC AGC GTT TAT ACG CTT GCA-3') (SEQ ID NO: 53)
hybridized to the other end of the insert and introduced a KpnI
site just after the HCHL stop codon. The PCR product was cut with
SpeI and KpnI, and ligated into similarly digested pU3z-mcs-nos.
The ligation reaction mixture was used to transform E. coli DH10B
and growth was selected on LB media containing ampicillin (100
.mu.g mL.sup.-1). A representative plasmid (pU3z-mcs-nos-HCHL) was
sequenced to confirm the absence of PCR errors.
Example 25
Plant Transformation
[0647] Embryogenic callus from sugarcane cultivar Q117 was prepared
essentially as described, and grown in the dark at 27.degree. C. on
MS media supplemented with 3 mg L.sup.-1 of 2,4-dichlorophenoxy
acetic acid (2,4-D). The calli were co-transformed with the
antibiotic selection plasmid pUKN by microprojectile bombardment
Following bombardment and a recovery phase of 2 weeks in the dark,
transformants were placed on MS-2,4D selection media supplemented
with 60 mg L.sup.-1 geneticin. Individual callus clumps were
maintained separately throughout the selection process. After 6
weeks, antibiotic-resistant calli were transferred to MS media
supplemented with geneticin and incubated in the light to initiate
plantlet regeneration. At least four plantlets per callus clump
were transferred to pots in a glasshouse certified for the physical
containment of transgenic plants for further analysis.
Example 26
Measurement of Accumulated Soluble Phenolics by HPLC
[0648] Soluble phenolics were extracted from 100-200 mg of leaf or
stem tissue. The tissue samples was resuspended in 1 mL of 50% v/v
methanol and homogenized in a bead beater (Bio101/Savant, Fastprep
FP120, Holbrook, N.Y.). The sample were then agitated in an orbital
shaker (200 rpm) for 1 hour at 37.degree. C., and clarified by
centrifugation. A 550-.mu.L aliquot of extract was transferred to a
fresh tube and dried under vacuum, and the dry residue was
dissolved in. 200 .mu.L.
[0649] When the goal was to convert pHBA and vanillic acid glucose
conjugates to free pHBA and vanillic acid an acid hydrolysis step
was included. A 200-.mu.L aliquot of the extract was transferred to
a fresh tube and dried under vacuum. After adding 200 .mu.L of 1 N
HCl to the dry residue and vortexing, the sample was incubated for
2 hours at 95.degree. C. The sample was then neutralized by adding
200 .mu.L of 1.2 N NaOH.
[0650] Soluble phenolics were detected by HPLC at 32.degree. C.
using the Novapak C18 column described above. Samples were filtered
through 0.2 .mu.m syringe filters and 20 .mu.L of filtrate was
injected for each analysis. Mobile phases were the same as
previously described. Solvent was pumped at 1 mL min.sup.-1 using
the following gradient conditions: 0 min, 0% B; 80 min, 80% B; 81
min, 100% B; 85 min, 100% B; 86 min, 0% B. Total run time was 90
minutes. An optimized gradient was applied to separate
p-hydroxybenzoic acid and vanillic acid (0 min, 10% B; 20 min, 50%
B; 21 min, 100% B; 24 min, 100% B; 25 min, 10% B; total runtime was
35 minutes). Identified peaks were quantified using authentic
standards (Sigma-Aldrich Co.).
[0651] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to, or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
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Sequence CWU 1
1
5611182DNARastonia EutropiaCDS(1)..(1182) 1atg act gac gtt gtc atc
gta tcc gcc gcc cgc acc gcg gtc ggc aag 48Met Thr Asp Val Val Ile
Val Ser Ala Ala Arg Thr Ala Val Gly Lys1 5 10 15ttt ggc ggc tcg ctg
gcc aag atc ccg gca ccg gaa ctg ggt gcc gtg 96Phe Gly Gly Ser Leu
Ala Lys Ile Pro Ala Pro Glu Leu Gly Ala Val 20 25 30gtc atc aag gcc
gcg ctg gag cgc gcc ggc gtc aag ccg gag cag gtg 144Val Ile Lys Ala
Ala Leu Glu Arg Ala Gly Val Lys Pro Glu Gln Val 35 40 45agc gaa gtc
atc atg ggc cag gtg ctg acc gcc ggt tcg ggc cag aac 192Ser Glu Val
Ile Met Gly Gln Val Leu Thr Ala Gly Ser Gly Gln Asn 50 55 60ccc gca
cgc cag gcc gcg atc aag gcc ggc ctg ccg gcg atg gtg ccg 240Pro Ala
Arg Gln Ala Ala Ile Lys Ala Gly Leu Pro Ala Met Val Pro65 70 75
80gcc atg acc atc aac aag gtg tgc ggc tcg ggc ctg aag gcc gtg atg
288Ala Met Thr Ile Asn Lys Val Cys Gly Ser Gly Leu Lys Ala Val Met
85 90 95ctg gcc gcc aac gcg atc atg gcg ggc gac gcc gag atc gtg gtg
gcc 336Leu Ala Ala Asn Ala Ile Met Ala Gly Asp Ala Glu Ile Val Val
Ala 100 105 110ggc ggc cag gaa aac atg agc gcc gcc ccg cac gtg ctg
ccg ggc tcg 384Gly Gly Gln Glu Asn Met Ser Ala Ala Pro His Val Leu
Pro Gly Ser 115 120 125cgc gat ggt ttc cgc atg ggc gat gcc aag ctg
gtc gac acc atg atc 432Arg Asp Gly Phe Arg Met Gly Asp Ala Lys Leu
Val Asp Thr Met Ile 130 135 140gtc gac ggc ctg tgg gac gtg tac aac
cag tac cac atg ggc atc acc 480Val Asp Gly Leu Trp Asp Val Tyr Asn
Gln Tyr His Met Gly Ile Thr145 150 155 160gcc gag aac gtg gcc aag
gaa tac ggc atc aca cgc gag gcg cag gat 528Ala Glu Asn Val Ala Lys
Glu Tyr Gly Ile Thr Arg Glu Ala Gln Asp 165 170 175gag ttc gcc gtc
ggc tcg cag aac aag gcc gaa gcc gcg cag aag gcc 576Glu Phe Ala Val
Gly Ser Gln Asn Lys Ala Glu Ala Ala Gln Lys Ala 180 185 190ggc aag
ttt gac gaa gag atc gtc ccg gtg ctg atc ccg cag cgc aag 624Gly Lys
Phe Asp Glu Glu Ile Val Pro Val Leu Ile Pro Gln Arg Lys 195 200
205ggc gac ccg gtg gcc ttc aag acc gac gag ttc gtg cgc cag ggc gcc
672Gly Asp Pro Val Ala Phe Lys Thr Asp Glu Phe Val Arg Gln Gly Ala
210 215 220acg ctg gac agc atg tcc ggc ctc aag ccc gcc ttc gac aag
gcc ggc 720Thr Leu Asp Ser Met Ser Gly Leu Lys Pro Ala Phe Asp Lys
Ala Gly225 230 235 240acg gtg acc gcg gcc aac gcc tcg ggc ctg aac
gac ggc gcc gcc gcg 768Thr Val Thr Ala Ala Asn Ala Ser Gly Leu Asn
Asp Gly Ala Ala Ala 245 250 255gtg gtg gtg atg tcg gcg gcc aag gcc
aag gaa ctg ggc ctg acc ccg 816Val Val Val Met Ser Ala Ala Lys Ala
Lys Glu Leu Gly Leu Thr Pro 260 265 270ctg gcc acg atc aag agc tat
gcc aac gcc ggt gtc gat ccc aag gtg 864Leu Ala Thr Ile Lys Ser Tyr
Ala Asn Ala Gly Val Asp Pro Lys Val 275 280 285atg ggc atg ggc ccg
gtg ccg gcc tcc aag cgc gcc ctg tcg cgc gcc 912Met Gly Met Gly Pro
Val Pro Ala Ser Lys Arg Ala Leu Ser Arg Ala 290 295 300gag tgg acc
ccg caa gac ctg gac ctg atg gag atc aac gag gcc ttt 960Glu Trp Thr
Pro Gln Asp Leu Asp Leu Met Glu Ile Asn Glu Ala Phe305 310 315
320gcc gcc cag gcg ctg gcg gtg cac cag cag atg ggc tgg gac acc tcc
1008Ala Ala Gln Ala Leu Ala Val His Gln Gln Met Gly Trp Asp Thr Ser
325 330 335aag gtc aat gtg aac ggc ggc gcc atc gcc atc ggc cac ccg
atc ggc 1056Lys Val Asn Val Asn Gly Gly Ala Ile Ala Ile Gly His Pro
Ile Gly 340 345 350gcg tcg ggc tgc cgt atc ctg gtg acg ctg ctg cac
gag atg aag cgc 1104Ala Ser Gly Cys Arg Ile Leu Val Thr Leu Leu His
Glu Met Lys Arg 355 360 365cgt gac gcg aag aag ggc ctg gcc tcg ctg
tgc atc ggc ggc ggc atg 1152Arg Asp Ala Lys Lys Gly Leu Ala Ser Leu
Cys Ile Gly Gly Gly Met 370 375 380ggc gtg gcg ctg gca gtc gag cgc
aaa taa 1182Gly Val Ala Leu Ala Val Glu Arg Lys385
3902393PRTRastonia Eutropia 2Met Thr Asp Val Val Ile Val Ser Ala
Ala Arg Thr Ala Val Gly Lys1 5 10 15Phe Gly Gly Ser Leu Ala Lys Ile
Pro Ala Pro Glu Leu Gly Ala Val 20 25 30Val Ile Lys Ala Ala Leu Glu
Arg Ala Gly Val Lys Pro Glu Gln Val 35 40 45Ser Glu Val Ile Met Gly
Gln Val Leu Thr Ala Gly Ser Gly Gln Asn 50 55 60Pro Ala Arg Gln Ala
Ala Ile Lys Ala Gly Leu Pro Ala Met Val Pro65 70 75 80Ala Met Thr
Ile Asn Lys Val Cys Gly Ser Gly Leu Lys Ala Val Met 85 90 95Leu Ala
Ala Asn Ala Ile Met Ala Gly Asp Ala Glu Ile Val Val Ala 100 105
110Gly Gly Gln Glu Asn Met Ser Ala Ala Pro His Val Leu Pro Gly Ser
115 120 125Arg Asp Gly Phe Arg Met Gly Asp Ala Lys Leu Val Asp Thr
Met Ile 130 135 140Val Asp Gly Leu Trp Asp Val Tyr Asn Gln Tyr His
Met Gly Ile Thr145 150 155 160Ala Glu Asn Val Ala Lys Glu Tyr Gly
Ile Thr Arg Glu Ala Gln Asp 165 170 175Glu Phe Ala Val Gly Ser Gln
Asn Lys Ala Glu Ala Ala Gln Lys Ala 180 185 190Gly Lys Phe Asp Glu
Glu Ile Val Pro Val Leu Ile Pro Gln Arg Lys 195 200 205Gly Asp Pro
Val Ala Phe Lys Thr Asp Glu Phe Val Arg Gln Gly Ala 210 215 220Thr
Leu Asp Ser Met Ser Gly Leu Lys Pro Ala Phe Asp Lys Ala Gly225 230
235 240Thr Val Thr Ala Ala Asn Ala Ser Gly Leu Asn Asp Gly Ala Ala
Ala 245 250 255Val Val Val Met Ser Ala Ala Lys Ala Lys Glu Leu Gly
Leu Thr Pro 260 265 270Leu Ala Thr Ile Lys Ser Tyr Ala Asn Ala Gly
Val Asp Pro Lys Val 275 280 285Met Gly Met Gly Pro Val Pro Ala Ser
Lys Arg Ala Leu Ser Arg Ala 290 295 300Glu Trp Thr Pro Gln Asp Leu
Asp Leu Met Glu Ile Asn Glu Ala Phe305 310 315 320Ala Ala Gln Ala
Leu Ala Val His Gln Gln Met Gly Trp Asp Thr Ser 325 330 335Lys Val
Asn Val Asn Gly Gly Ala Ile Ala Ile Gly His Pro Ile Gly 340 345
350Ala Ser Gly Cys Arg Ile Leu Val Thr Leu Leu His Glu Met Lys Arg
355 360 365Arg Asp Ala Lys Lys Gly Leu Ala Ser Leu Cys Ile Gly Gly
Gly Met 370 375 380Gly Val Ala Leu Ala Val Glu Arg Lys385
39031280DNARastonia Eutropia 3ggatccatga ctgacgttgt catcgtatcc
gccgcccgca ccgcggtcgg caagtttggc 60ggctcgctgg ccaagatccc ggcaccggaa
ctgggtgccg tggtcatcaa ggccgcgctg 120gagcgcgccg gcgtcaagcc
ggagcaggtg agcgaagtca tcatgggcca ggtgctgacc 180gccggttcgg
gccagaaccc cgcacgccag gccgcgatca aggccggcct gccggcgatg
240gtgccggcca tgaccatcaa caaggtgtgc ggctcgggcc tgaaggccgt
gatgctggcc 300gccaacgcga tcatggcggg cgacgccgag atcgtggtgg
ccggcggcca ggaaaacatg 360agcgccgccc cgcacgtgct gccgggctcg
cgcgatggtt tccgcatggg cgatgccaag 420ctggtcgaca ccatgatcgt
cgacggcctg tgggacgtgt acaaccagta ccacatgggc 480atcaccgccg
agaacgtggc caaggaatac ggcatcacac gcgaggcgca ggatgagttc
540gccgtcggct cgcagaacaa ggccgaagcc gcgcagaagg ccggcaagtt
tgacgaagag 600atcgtcccgg tgctgatccc gcagcgcaag ggcgacccgg
tggccttcaa gaccgacgag 660ttcgtgcgcc agggcgccac gctggacagc
atgtccggcc tcaagcccgc cttcgacaag 720gccggcacgg tgaccgcggc
caacgcctcg ggcctgaacg acggcgccgc cgcggtggtg 780gtgatgtcgg
cggccaaggc caaggaactg ggcctgaccc cgctggccac gatcaagagc
840tatgccaacg ccggtgtcga tcccaaggtg atgggcatgg gcccggtgcc
ggcctccaag 900cgcgccctgt cgcgcgccga gtggaccccg caagacctgg
acctgatgga gatcaacgag 960gcctttgccg cccaggcgct ggcggtgcac
cagcagatgg gctgggacac ctccaaggtc 1020aatgtgaacg gcggcgccat
cgccatcggc cacccgatcg gcgcgtcggg ctgccgtatc 1080ctggtgacgc
tgctgcacga gatgaagcgc cgtgacgcga agaagggcct ggcctcgctg
1140tgcatcggcg gcggcatggg cgtggcgctg gcagtcgagc gcaaataagg
aaggggtttt 1200ccggggccgc gcgcggttgg cgcggacccg gcgacgataa
cgaagccaat caaggagtgg 1260acatgactca ggggggtacc 12804738DNARastonia
EutropiaCDS(1)..(738) 4atg act cag cgc att gcg tat gtg acc ggc ggc
atg ggt ggt atc gga 48Met Thr Gln Arg Ile Ala Tyr Val Thr Gly Gly
Met Gly Gly Ile Gly1 5 10 15acc gcc att tgc cag cgg ctg gcc aag gat
ggc ttt cgt gtg gtg gcc 96Thr Ala Ile Cys Gln Arg Leu Ala Lys Asp
Gly Phe Arg Val Val Ala 20 25 30ggt tgc ggc ccc aac tcg ccg cgc cgc
gaa aag tgg ctg gag cag cag 144Gly Cys Gly Pro Asn Ser Pro Arg Arg
Glu Lys Trp Leu Glu Gln Gln 35 40 45aag gcc ctg ggc ttc gat ttc att
gcc tcg gaa ggc aat gtg gct gac 192Lys Ala Leu Gly Phe Asp Phe Ile
Ala Ser Glu Gly Asn Val Ala Asp 50 55 60tgg gac tcg acc aag acc gca
ttc gac aag gtc aag tcc gag gtc ggc 240Trp Asp Ser Thr Lys Thr Ala
Phe Asp Lys Val Lys Ser Glu Val Gly65 70 75 80gag gtt gat gtg ctg
atc aac aac gcc ggt atc acc cgc gac gtg gtg 288Glu Val Asp Val Leu
Ile Asn Asn Ala Gly Ile Thr Arg Asp Val Val 85 90 95ttc cgc aag atg
acc cgc gcc gac tgg gat gcg gtg atc gac acc aac 336Phe Arg Lys Met
Thr Arg Ala Asp Trp Asp Ala Val Ile Asp Thr Asn 100 105 110ctg acc
tcg ctg ttc aac gtc acc aag cag gtg atc gac ggc atg gcc 384Leu Thr
Ser Leu Phe Asn Val Thr Lys Gln Val Ile Asp Gly Met Ala 115 120
125gac cgt ggc tgg ggc cgc atc gtc aac atc tcg tcg gtg aac ggg cag
432Asp Arg Gly Trp Gly Arg Ile Val Asn Ile Ser Ser Val Asn Gly Gln
130 135 140aag ggc cag ttc ggc cag acc aac tac tcc acc gcc aag gcc
ggc ctg 480Lys Gly Gln Phe Gly Gln Thr Asn Tyr Ser Thr Ala Lys Ala
Gly Leu145 150 155 160cat ggc ttc acc atg gca ctg gcg cag gaa gtg
gcg acc aag ggc gtg 528His Gly Phe Thr Met Ala Leu Ala Gln Glu Val
Ala Thr Lys Gly Val 165 170 175acc gtc aac acg gtc tct ccg ggc tat
atc gcc acc gac atg gtc aag 576Thr Val Asn Thr Val Ser Pro Gly Tyr
Ile Ala Thr Asp Met Val Lys 180 185 190gcg atc cgc cag gac gtg ctc
gac aag atc gtc gcg acg atc ccg gtc 624Ala Ile Arg Gln Asp Val Leu
Asp Lys Ile Val Ala Thr Ile Pro Val 195 200 205aag cgc ctg ggc ctg
cca gaa gag atc gcc tcg atc tgc gcc tgg ttg 672Lys Arg Leu Gly Leu
Pro Glu Glu Ile Ala Ser Ile Cys Ala Trp Leu 210 215 220tcg tcg gag
gag tcc ggt ttc tcg acc ggc gcc gac ttc tcg ctc aac 720Ser Ser Glu
Glu Ser Gly Phe Ser Thr Gly Ala Asp Phe Ser Leu Asn225 230 235
240ggc ggc ctg cat atg ggc 738Gly Gly Leu His Met Gly
2455246PRTRastonia Eutropia 5Met Thr Gln Arg Ile Ala Tyr Val Thr
Gly Gly Met Gly Gly Ile Gly1 5 10 15Thr Ala Ile Cys Gln Arg Leu Ala
Lys Asp Gly Phe Arg Val Val Ala 20 25 30Gly Cys Gly Pro Asn Ser Pro
Arg Arg Glu Lys Trp Leu Glu Gln Gln 35 40 45Lys Ala Leu Gly Phe Asp
Phe Ile Ala Ser Glu Gly Asn Val Ala Asp 50 55 60Trp Asp Ser Thr Lys
Thr Ala Phe Asp Lys Val Lys Ser Glu Val Gly65 70 75 80Glu Val Asp
Val Leu Ile Asn Asn Ala Gly Ile Thr Arg Asp Val Val 85 90 95Phe Arg
Lys Met Thr Arg Ala Asp Trp Asp Ala Val Ile Asp Thr Asn 100 105
110Leu Thr Ser Leu Phe Asn Val Thr Lys Gln Val Ile Asp Gly Met Ala
115 120 125Asp Arg Gly Trp Gly Arg Ile Val Asn Ile Ser Ser Val Asn
Gly Gln 130 135 140Lys Gly Gln Phe Gly Gln Thr Asn Tyr Ser Thr Ala
Lys Ala Gly Leu145 150 155 160His Gly Phe Thr Met Ala Leu Ala Gln
Glu Val Ala Thr Lys Gly Val 165 170 175Thr Val Asn Thr Val Ser Pro
Gly Tyr Ile Ala Thr Asp Met Val Lys 180 185 190Ala Ile Arg Gln Asp
Val Leu Asp Lys Ile Val Ala Thr Ile Pro Val 195 200 205Lys Arg Leu
Gly Leu Pro Glu Glu Ile Ala Ser Ile Cys Ala Trp Leu 210 215 220Ser
Ser Glu Glu Ser Gly Phe Ser Thr Gly Ala Asp Phe Ser Leu Asn225 230
235 240Gly Gly Leu His Met Gly 2456783DNARastonia Eutropia
6ggatccatga ctcagcgcat tgcgtatgtg accggcggca tgggtggtat cggaaccgcc
60atttgccagc ggctggccaa ggatggcttt cgtgtggtgg ccggttgcgg ccccaactcg
120ccgcgccgcg aaaagtggct ggagcagcag aaggccctgg gcttcgattt
cattgcctcg 180gaaggcaatg tggctgactg ggactcgacc aagaccgcat
tcgacaaggt caagtccgag 240gtcggcgagg ttgatgtgct gatcaacaac
gccggtatca cccgcgacgt ggtgttccgc 300aagatgaccc gcgccgactg
ggatgcggtg atcgacacca acctgacctc gctgttcaac 360gtcaccaagc
aggtgatcga cggcatggcc gaccgtggct ggggccgcat cgtcaacatc
420tcgtcggtga acgggcagaa gggccagttc ggccagacca actactccac
cgccaaggcc 480ggcctgcatg gcttcaccat ggcactggcg caggaagtgg
cgaccaaggg cgtgaccgtc 540aacacggtct ctccgggcta tatcgccacc
gacatggtca aggcgatccg ccaggacgtg 600ctcgacaaga tcgtcgcgac
gatcccggtc aagcgcctgg gcctgccaga agagatcgcc 660tcgatctgcg
cctggttgtc gtcggaggag tccggtttct cgaccggcgc cgacttctcg
720ctcaacggcg gcctgcatat gggctgacct gccggcctgg ttccaccagt
cggcaggggt 780acc 78371767DNARastonia EutropiaCDS(1)..(1767) 7atg
gcg acc ggc aaa ggc gcg gca gct tcc acg cag gaa ggc aag tcc 48Met
Ala Thr Gly Lys Gly Ala Ala Ala Ser Thr Gln Glu Gly Lys Ser1 5 10
15caa cca ttc aag gtc acg ccg ggg cca ttc gat cca gcc aca tgg ctg
96Gln Pro Phe Lys Val Thr Pro Gly Pro Phe Asp Pro Ala Thr Trp Leu
20 25 30gaa tgg tcc cgc cag tgg cag ggc act gaa ggc aac ggc cac gcg
gcc 144Glu Trp Ser Arg Gln Trp Gln Gly Thr Glu Gly Asn Gly His Ala
Ala 35 40 45gcg tcc ggc att ccg ggc ctg gat gcg ctg gca ggc gtc aag
atc gcg 192Ala Ser Gly Ile Pro Gly Leu Asp Ala Leu Ala Gly Val Lys
Ile Ala 50 55 60ccg gcg cag ctg ggt gat atc cag cag cgc tac atg aag
gac ttc tca 240Pro Ala Gln Leu Gly Asp Ile Gln Gln Arg Tyr Met Lys
Asp Phe Ser65 70 75 80gcg ctg tgg cag gcc atg gcc gag ggc aag gcc
gag gcc acc ggt ccg 288Ala Leu Trp Gln Ala Met Ala Glu Gly Lys Ala
Glu Ala Thr Gly Pro 85 90 95ctg cac gac cgg cgc ttc gcc ggc gac gca
tgg cgc acc aac ctc cca 336Leu His Asp Arg Arg Phe Ala Gly Asp Ala
Trp Arg Thr Asn Leu Pro 100 105 110tat cgc ttc gct gcc gcg ttc tac
ctg ctc aat gcg cgc gcc ttg acc 384Tyr Arg Phe Ala Ala Ala Phe Tyr
Leu Leu Asn Ala Arg Ala Leu Thr 115 120 125gag ctg gcc gat gcc gtc
gag gcc gat gcc aag acc cgc cag cgc atc 432Glu Leu Ala Asp Ala Val
Glu Ala Asp Ala Lys Thr Arg Gln Arg Ile 130 135 140cgc ttc gcg atc
tcg caa tgg gtc gat gcg atg tcg ccc gcc aac ttc 480Arg Phe Ala Ile
Ser Gln Trp Val Asp Ala Met Ser Pro Ala Asn Phe145 150 155 160ctt
gcc acc aat ccc gag gcg cag cgc ctg ctg atc gag tcg ggc ggc 528Leu
Ala Thr Asn Pro Glu Ala Gln Arg Leu Leu Ile Glu Ser Gly Gly 165 170
175gaa tcg ctg cgt gcc ggc gtg cgc aac atg atg gaa gac ctg aca cgc
576Glu Ser Leu Arg Ala Gly Val Arg Asn Met Met Glu Asp Leu Thr Arg
180 185 190ggc aag atc tcg cag acc gac gag agc gcg ttt gag gtc ggc
cgc aat 624Gly Lys Ile Ser Gln Thr Asp Glu Ser Ala Phe Glu Val Gly
Arg Asn 195 200 205gtc gcg gtg acc gaa ggc gcc gtg gtc ttc gag aac
gag tac ttc cag 672Val Ala Val Thr Glu Gly Ala Val Val Phe Glu Asn
Glu Tyr Phe Gln 210 215 220ctg ttg cag tac aag ccg ctg acc gac aag
gtg cac gcg cgc ccg ctg 720Leu Leu Gln Tyr Lys Pro Leu Thr Asp Lys
Val His Ala Arg Pro Leu225 230 235 240ctg atg gtg ccg ccg tgc atc
aac aag tac tac atc ctg gac ctg cag 768Leu Met Val Pro Pro Cys Ile
Asn Lys Tyr Tyr Ile Leu Asp Leu Gln 245 250 255ccg gag agc tcg ctg
gtg cgc cat gtg gtg
gag cag gga cat acg gtg 816Pro Glu Ser Ser Leu Val Arg His Val Val
Glu Gln Gly His Thr Val 260 265 270ttt ctg gtg tcg tgg cgc aat ccg
gac gcc agc atg gcc ggc agc acc 864Phe Leu Val Ser Trp Arg Asn Pro
Asp Ala Ser Met Ala Gly Ser Thr 275 280 285tgg gac gac tac atc gag
cac gcg gcc atc cgc gcc atc gaa gtc gcg 912Trp Asp Asp Tyr Ile Glu
His Ala Ala Ile Arg Ala Ile Glu Val Ala 290 295 300cgc gac atc agc
ggc cag gac aag atc aac gtg ctc ggc ttc tgc gtg 960Arg Asp Ile Ser
Gly Gln Asp Lys Ile Asn Val Leu Gly Phe Cys Val305 310 315 320ggc
ggc acc att gtc tcg acc gcg ctg gcg gtg ctg gcc gcg cgc ggc 1008Gly
Gly Thr Ile Val Ser Thr Ala Leu Ala Val Leu Ala Ala Arg Gly 325 330
335gag cac ccg gcc gcc agc gtc acg ctg ctg acc acg ctg ctg gac ttt
1056Glu His Pro Ala Ala Ser Val Thr Leu Leu Thr Thr Leu Leu Asp Phe
340 345 350gcc gac acg ggc atc ctc gac gtc ttt gtc gac gag ggc cat
gtg cag 1104Ala Asp Thr Gly Ile Leu Asp Val Phe Val Asp Glu Gly His
Val Gln 355 360 365ttg cgc gag gcc acg ctg ggc ggc ggc gcc ggc gcg
ccg tgc gcg ctg 1152Leu Arg Glu Ala Thr Leu Gly Gly Gly Ala Gly Ala
Pro Cys Ala Leu 370 375 380ctg cgc ggc ctt gag ctg gcc aat acc ttc
tcg ttc ttg cgc ccg aac 1200Leu Arg Gly Leu Glu Leu Ala Asn Thr Phe
Ser Phe Leu Arg Pro Asn385 390 395 400gac ctg gtg tgg aac tac gtg
gtc gac aac tac ctg aag ggc aac acg 1248Asp Leu Val Trp Asn Tyr Val
Val Asp Asn Tyr Leu Lys Gly Asn Thr 405 410 415ccg gtg ccg ttc gac
ctg ctg ttc tgg aac ggc gac gcc acc aac ctg 1296Pro Val Pro Phe Asp
Leu Leu Phe Trp Asn Gly Asp Ala Thr Asn Leu 420 425 430ccg ggg ccg
tgg tac tgc tgg tac ctg cgc cac acc tac ctg cag aac 1344Pro Gly Pro
Trp Tyr Cys Trp Tyr Leu Arg His Thr Tyr Leu Gln Asn 435 440 445gag
ctc aag gta ccg ggc aag ctg acc gtg tgc ggc gtg ccg gtg gac 1392Glu
Leu Lys Val Pro Gly Lys Leu Thr Val Cys Gly Val Pro Val Asp 450 455
460ctg gcc agc atc gac gtg ccg acc tat atc tac ggc tcg cgc gaa gac
1440Leu Ala Ser Ile Asp Val Pro Thr Tyr Ile Tyr Gly Ser Arg Glu
Asp465 470 475 480cat atc gtg ccg tgg acc gcg gcc tat gcc tcg acc
gcg ctg ctg gcg 1488His Ile Val Pro Trp Thr Ala Ala Tyr Ala Ser Thr
Ala Leu Leu Ala 485 490 495aac aag ctg cgc ttc gtg ctg ggt gcg tcg
ggc cat atc gcc ggt gtg 1536Asn Lys Leu Arg Phe Val Leu Gly Ala Ser
Gly His Ile Ala Gly Val 500 505 510atc aac ccg ccg gcc aag aac aag
cgc agc cac tgg act aac gat gcg 1584Ile Asn Pro Pro Ala Lys Asn Lys
Arg Ser His Trp Thr Asn Asp Ala 515 520 525ctg ccg gag tcg ccg cag
caa tgg ctg gcc ggc gcc atc gag cat cac 1632Leu Pro Glu Ser Pro Gln
Gln Trp Leu Ala Gly Ala Ile Glu His His 530 535 540ggc agc tgg tgg
ccg gac tgg acc gca tgg ctg gcc ggg cag gcc ggc 1680Gly Ser Trp Trp
Pro Asp Trp Thr Ala Trp Leu Ala Gly Gln Ala Gly545 550 555 560gcg
aaa cgc gcc gcg ccc gcc aac tat ggc aat gcg cgc tat cgc gca 1728Ala
Lys Arg Ala Ala Pro Ala Asn Tyr Gly Asn Ala Arg Tyr Arg Ala 565 570
575atc gaa ccc gcg cct ggg cga tac gtc aaa gcc aag gca 1767Ile Glu
Pro Ala Pro Gly Arg Tyr Val Lys Ala Lys Ala 580 5858589PRTRastonia
Eutropia 8Met Ala Thr Gly Lys Gly Ala Ala Ala Ser Thr Gln Glu Gly
Lys Ser1 5 10 15Gln Pro Phe Lys Val Thr Pro Gly Pro Phe Asp Pro Ala
Thr Trp Leu 20 25 30Glu Trp Ser Arg Gln Trp Gln Gly Thr Glu Gly Asn
Gly His Ala Ala 35 40 45Ala Ser Gly Ile Pro Gly Leu Asp Ala Leu Ala
Gly Val Lys Ile Ala 50 55 60Pro Ala Gln Leu Gly Asp Ile Gln Gln Arg
Tyr Met Lys Asp Phe Ser65 70 75 80Ala Leu Trp Gln Ala Met Ala Glu
Gly Lys Ala Glu Ala Thr Gly Pro 85 90 95Leu His Asp Arg Arg Phe Ala
Gly Asp Ala Trp Arg Thr Asn Leu Pro 100 105 110Tyr Arg Phe Ala Ala
Ala Phe Tyr Leu Leu Asn Ala Arg Ala Leu Thr 115 120 125Glu Leu Ala
Asp Ala Val Glu Ala Asp Ala Lys Thr Arg Gln Arg Ile 130 135 140Arg
Phe Ala Ile Ser Gln Trp Val Asp Ala Met Ser Pro Ala Asn Phe145 150
155 160Leu Ala Thr Asn Pro Glu Ala Gln Arg Leu Leu Ile Glu Ser Gly
Gly 165 170 175Glu Ser Leu Arg Ala Gly Val Arg Asn Met Met Glu Asp
Leu Thr Arg 180 185 190Gly Lys Ile Ser Gln Thr Asp Glu Ser Ala Phe
Glu Val Gly Arg Asn 195 200 205Val Ala Val Thr Glu Gly Ala Val Val
Phe Glu Asn Glu Tyr Phe Gln 210 215 220Leu Leu Gln Tyr Lys Pro Leu
Thr Asp Lys Val His Ala Arg Pro Leu225 230 235 240Leu Met Val Pro
Pro Cys Ile Asn Lys Tyr Tyr Ile Leu Asp Leu Gln 245 250 255Pro Glu
Ser Ser Leu Val Arg His Val Val Glu Gln Gly His Thr Val 260 265
270Phe Leu Val Ser Trp Arg Asn Pro Asp Ala Ser Met Ala Gly Ser Thr
275 280 285Trp Asp Asp Tyr Ile Glu His Ala Ala Ile Arg Ala Ile Glu
Val Ala 290 295 300Arg Asp Ile Ser Gly Gln Asp Lys Ile Asn Val Leu
Gly Phe Cys Val305 310 315 320Gly Gly Thr Ile Val Ser Thr Ala Leu
Ala Val Leu Ala Ala Arg Gly 325 330 335Glu His Pro Ala Ala Ser Val
Thr Leu Leu Thr Thr Leu Leu Asp Phe 340 345 350Ala Asp Thr Gly Ile
Leu Asp Val Phe Val Asp Glu Gly His Val Gln 355 360 365Leu Arg Glu
Ala Thr Leu Gly Gly Gly Ala Gly Ala Pro Cys Ala Leu 370 375 380Leu
Arg Gly Leu Glu Leu Ala Asn Thr Phe Ser Phe Leu Arg Pro Asn385 390
395 400Asp Leu Val Trp Asn Tyr Val Val Asp Asn Tyr Leu Lys Gly Asn
Thr 405 410 415Pro Val Pro Phe Asp Leu Leu Phe Trp Asn Gly Asp Ala
Thr Asn Leu 420 425 430Pro Gly Pro Trp Tyr Cys Trp Tyr Leu Arg His
Thr Tyr Leu Gln Asn 435 440 445Glu Leu Lys Val Pro Gly Lys Leu Thr
Val Cys Gly Val Pro Val Asp 450 455 460Leu Ala Ser Ile Asp Val Pro
Thr Tyr Ile Tyr Gly Ser Arg Glu Asp465 470 475 480His Ile Val Pro
Trp Thr Ala Ala Tyr Ala Ser Thr Ala Leu Leu Ala 485 490 495Asn Lys
Leu Arg Phe Val Leu Gly Ala Ser Gly His Ile Ala Gly Val 500 505
510Ile Asn Pro Pro Ala Lys Asn Lys Arg Ser His Trp Thr Asn Asp Ala
515 520 525Leu Pro Glu Ser Pro Gln Gln Trp Leu Ala Gly Ala Ile Glu
His His 530 535 540Gly Ser Trp Trp Pro Asp Trp Thr Ala Trp Leu Ala
Gly Gln Ala Gly545 550 555 560Ala Lys Arg Ala Ala Pro Ala Asn Tyr
Gly Asn Ala Arg Tyr Arg Ala 565 570 575Ile Glu Pro Ala Pro Gly Arg
Tyr Val Lys Ala Lys Ala 580 58591800DNARastonia Eutropia
9ggatccatgg cgaccggcaa aggcgcggca gcttccacgc aggaaggcaa gtcccaacca
60ttcaaggtca cgccggggcc attcgatcca gccacatggc tggaatggtc ccgccagtgg
120cagggcactg aaggcaacgg ccacgcggcc gcgtccggca ttccgggcct
ggatgcgctg 180gcaggcgtca agatcgcgcc ggcgcagctg ggtgatatcc
agcagcgcta catgaaggac 240ttctcagcgc tgtggcaggc catggccgag
ggcaaggccg aggccaccgg tccgctgcac 300gaccggcgct tcgccggcga
cgcatggcgc accaacctcc catatcgctt cgctgccgcg 360ttctacctgc
tcaatgcgcg cgccttgacc gagctggccg atgccgtcga ggccgatgcc
420aagacccgcc agcgcatccg cttcgcgatc tcgcaatggg tcgatgcgat
gtcgcccgcc 480aacttccttg ccaccaatcc cgaggcgcag cgcctgctga
tcgagtcggg cggcgaatcg 540ctgcgtgccg gcgtgcgcaa catgatggaa
gacctgacac gcggcaagat ctcgcagacc 600gacgagagcg cgtttgaggt
cggccgcaat gtcgcggtga ccgaaggcgc cgtggtcttc 660gagaacgagt
acttccagct gttgcagtac aagccgctga ccgacaaggt gcacgcgcgc
720ccgctgctga tggtgccgcc gtgcatcaac aagtactaca tcctggacct
gcagccggag 780agctcgctgg tgcgccatgt ggtggagcag ggacatacgg
tgtttctggt gtcgtggcgc 840aatccggacg ccagcatggc cggcagcacc
tgggacgact acatcgagca cgcggccatc 900cgcgccatcg aagtcgcgcg
cgacatcagc ggccaggaca agatcaacgt gctcggcttc 960tgcgtgggcg
gcaccattgt ctcgaccgcg ctggcggtgc tggccgcgcg cggcgagcac
1020ccggccgcca gcgtcacgct gctgaccacg ctgctggact ttgccgacac
gggcatcctc 1080gacgtctttg tcgacgaggg ccatgtgcag ttgcgcgagg
ccacgctggg cggcggcgcc 1140ggcgcgccgt gcgcgctgct gcgcggcctt
gagctggcca ataccttctc gttcttgcgc 1200ccgaacgacc tggtgtggaa
ctacgtggtc gacaactacc tgaagggcaa cacgccggtg 1260ccgttcgacc
tgctgttctg gaacggcgac gccaccaacc tgccggggcc gtggtactgc
1320tggtacctgc gccacaccta cctgcagaac gagctcaagg taccgggcaa
gctgaccgtg 1380tgcggcgtgc cggtggacct ggccagcatc gacgtgccga
cctatatcta cggctcgcgc 1440gaagaccata tcgtgccgtg gaccgcggcc
tatgcctcga ccgcgctgct ggcgaacaag 1500ctgcgcttcg tgctgggtgc
gtcgggccat atcgccggtg tgatcaaccc gccggccaag 1560aacaagcgca
gccactggac taacgatgcg ctgccggagt cgccgcagca atggctggcc
1620ggcgccatcg agcatcacgg cagctggtgg ccggactgga ccgcatggct
ggccgggcag 1680gccggcgcga aacgcgccgc gcccgccaac tatggcaatg
cgcgctatcg cgcaatcgaa 1740cccgcgcctg ggcgatacgt caaagccaag
gcatgacgct tcaatcgaat tgggggtacc 1800101428DNARastonia
EutropiaCDS(1)..(1428) 10atg gct tct atg ata tcc tct tcc gct gtg
aca aca gtc agc cgt gcc 48Met Ala Ser Met Ile Ser Ser Ser Ala Val
Thr Thr Val Ser Arg Ala1 5 10 15tct agg ggg caa tcc gcc gca atg gct
cca ttc ggc ggc ctc aaa tcc 96Ser Arg Gly Gln Ser Ala Ala Met Ala
Pro Phe Gly Gly Leu Lys Ser 20 25 30atg act gga ttc cca gtg aag aag
gtc aac act gac att act tcc att 144Met Thr Gly Phe Pro Val Lys Lys
Val Asn Thr Asp Ile Thr Ser Ile 35 40 45aca agc aat ggt gga aga gta
aag tgc atg cag gtg tgg cct cca att 192Thr Ser Asn Gly Gly Arg Val
Lys Cys Met Gln Val Trp Pro Pro Ile 50 55 60gga aag aag aag ttt gag
act ctt tcc tat ttg cca cca ttg acc aga 240Gly Lys Lys Lys Phe Glu
Thr Leu Ser Tyr Leu Pro Pro Leu Thr Arg65 70 75 80gat tcc cgg gtg
act gac gtt gtc atc gta tcc gcc gcc cgc acc gcg 288Asp Ser Arg Val
Thr Asp Val Val Ile Val Ser Ala Ala Arg Thr Ala 85 90 95gtc ggc aag
ttt ggc ggc tcg ctg gcc aag atc ccg gca ccg gaa ctg 336Val Gly Lys
Phe Gly Gly Ser Leu Ala Lys Ile Pro Ala Pro Glu Leu 100 105 110ggt
gcc gtg gtc atc aag gcc gcg ctg gag cgc gcc ggc gtc aag ccg 384Gly
Ala Val Val Ile Lys Ala Ala Leu Glu Arg Ala Gly Val Lys Pro 115 120
125gag cag gtg agc gaa gtc atc atg ggc cag gtg ctg acc gcc ggt tcg
432Glu Gln Val Ser Glu Val Ile Met Gly Gln Val Leu Thr Ala Gly Ser
130 135 140ggc cag aac ccc gca cgc cag gcc gcg atc aag gcc ggc ctg
ccg gcg 480Gly Gln Asn Pro Ala Arg Gln Ala Ala Ile Lys Ala Gly Leu
Pro Ala145 150 155 160atg gtg ccg gcc atg acc atc aac aag gtg tgc
ggc tcg ggc ctg aag 528Met Val Pro Ala Met Thr Ile Asn Lys Val Cys
Gly Ser Gly Leu Lys 165 170 175gcc gtg atg ctg gcc gcc aac gcg atc
atg gcg ggc gac gcc gag atc 576Ala Val Met Leu Ala Ala Asn Ala Ile
Met Ala Gly Asp Ala Glu Ile 180 185 190gtg gtg gcc ggc ggc cag gaa
aac atg agc gcc gcc ccg cac gtg ctg 624Val Val Ala Gly Gly Gln Glu
Asn Met Ser Ala Ala Pro His Val Leu 195 200 205ccg ggc tcg cgc gat
ggt ttc cgc atg ggc gat gcc aag ctg gtc gac 672Pro Gly Ser Arg Asp
Gly Phe Arg Met Gly Asp Ala Lys Leu Val Asp 210 215 220acc atg atc
gtc gac ggc ctg tgg gac gtg tac aac cag tac cac atg 720Thr Met Ile
Val Asp Gly Leu Trp Asp Val Tyr Asn Gln Tyr His Met225 230 235
240ggc atc acc gcc gag aac gtg gcc aag gaa tac ggc atc aca cgc gag
768Gly Ile Thr Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr Arg Glu
245 250 255gcg cag gat gag ttc gcc gtc ggc tcg cag aac aag gcc gaa
gcc gcg 816Ala Gln Asp Glu Phe Ala Val Gly Ser Gln Asn Lys Ala Glu
Ala Ala 260 265 270cag aag gcc ggc aag ttt gac gaa gag atc gtc ccg
gtg ctg atc ccg 864Gln Lys Ala Gly Lys Phe Asp Glu Glu Ile Val Pro
Val Leu Ile Pro 275 280 285cag cgc aag ggc gac ccg gtg gcc ttc aag
acc gac gag ttc gtg cgc 912Gln Arg Lys Gly Asp Pro Val Ala Phe Lys
Thr Asp Glu Phe Val Arg 290 295 300cag ggc gcc acg ctg gac agc atg
tcc ggc ctc aag ccc gcc ttc gac 960Gln Gly Ala Thr Leu Asp Ser Met
Ser Gly Leu Lys Pro Ala Phe Asp305 310 315 320aag gcc ggc acg gtg
acc gcg gcc aac gcc tcg ggc ctg aac gac ggc 1008Lys Ala Gly Thr Val
Thr Ala Ala Asn Ala Ser Gly Leu Asn Asp Gly 325 330 335gcc gcc gcg
gtg gtg gtg atg tcg gcg gcc aag gcc aag gaa ctg ggc 1056Ala Ala Ala
Val Val Val Met Ser Ala Ala Lys Ala Lys Glu Leu Gly 340 345 350ctg
acc ccg ctg gcc acg atc aag agc tat gcc aac gcc ggt gtc gat 1104Leu
Thr Pro Leu Ala Thr Ile Lys Ser Tyr Ala Asn Ala Gly Val Asp 355 360
365ccc aag gtg atg ggc atg ggc ccg gtg ccg gcc tcc aag cgc gcc ctg
1152Pro Lys Val Met Gly Met Gly Pro Val Pro Ala Ser Lys Arg Ala Leu
370 375 380tcg cgc gcc gag tgg acc ccg caa gac ctg gac ctg atg gag
atc aac 1200Ser Arg Ala Glu Trp Thr Pro Gln Asp Leu Asp Leu Met Glu
Ile Asn385 390 395 400gag gcc ttt gcc gcc cag gcg ctg gcg gtg cac
cag cag atg ggc tgg 1248Glu Ala Phe Ala Ala Gln Ala Leu Ala Val His
Gln Gln Met Gly Trp 405 410 415gac acc tcc aag gtc aat gtg aac ggc
ggc gcc atc gcc atc ggc cac 1296Asp Thr Ser Lys Val Asn Val Asn Gly
Gly Ala Ile Ala Ile Gly His 420 425 430ccg atc ggc gcg tcg ggc tgc
cgt atc ctg gtg acg ctg ctg cac gag 1344Pro Ile Gly Ala Ser Gly Cys
Arg Ile Leu Val Thr Leu Leu His Glu 435 440 445atg aag cgc cgt gac
gcg aag aag ggc ctg gcc tcg ctg tgc atc ggc 1392Met Lys Arg Arg Asp
Ala Lys Lys Gly Leu Ala Ser Leu Cys Ile Gly 450 455 460ggc ggc atg
ggc gtg gcg ctg gca gtc gag cgc aaa 1428Gly Gly Met Gly Val Ala Leu
Ala Val Glu Arg Lys465 470 47511476PRTRastonia Eutropia 11Met Ala
Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala1 5 10 15Ser
Arg Gly Gln Ser Ala Ala Met Ala Pro Phe Gly Gly Leu Lys Ser 20 25
30Met Thr Gly Phe Pro Val Lys Lys Val Asn Thr Asp Ile Thr Ser Ile
35 40 45Thr Ser Asn Gly Gly Arg Val Lys Cys Met Gln Val Trp Pro Pro
Ile 50 55 60Gly Lys Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu
Thr Arg65 70 75 80Asp Ser Arg Val Thr Asp Val Val Ile Val Ser Ala
Ala Arg Thr Ala 85 90 95Val Gly Lys Phe Gly Gly Ser Leu Ala Lys Ile
Pro Ala Pro Glu Leu 100 105 110Gly Ala Val Val Ile Lys Ala Ala Leu
Glu Arg Ala Gly Val Lys Pro 115 120 125Glu Gln Val Ser Glu Val Ile
Met Gly Gln Val Leu Thr Ala Gly Ser 130 135 140Gly Gln Asn Pro Ala
Arg Gln Ala Ala Ile Lys Ala Gly Leu Pro Ala145 150 155 160Met Val
Pro Ala Met Thr Ile Asn Lys Val Cys Gly Ser Gly Leu Lys 165 170
175Ala Val Met Leu Ala Ala Asn Ala Ile Met Ala Gly Asp Ala Glu Ile
180 185 190Val Val Ala Gly Gly Gln Glu Asn Met Ser Ala Ala Pro His
Val Leu 195 200 205Pro Gly Ser Arg Asp Gly Phe Arg Met Gly Asp Ala
Lys Leu Val Asp 210 215 220Thr Met Ile Val Asp Gly Leu Trp Asp Val
Tyr Asn Gln Tyr His Met225 230 235 240Gly Ile Thr Ala Glu Asn Val
Ala Lys Glu Tyr Gly Ile Thr Arg Glu 245 250 255Ala Gln Asp Glu Phe
Ala Val Gly Ser Gln Asn Lys Ala Glu Ala Ala 260 265 270Gln Lys Ala
Gly Lys Phe Asp Glu Glu Ile Val Pro
Val Leu Ile Pro 275 280 285Gln Arg Lys Gly Asp Pro Val Ala Phe Lys
Thr Asp Glu Phe Val Arg 290 295 300Gln Gly Ala Thr Leu Asp Ser Met
Ser Gly Leu Lys Pro Ala Phe Asp305 310 315 320Lys Ala Gly Thr Val
Thr Ala Ala Asn Ala Ser Gly Leu Asn Asp Gly 325 330 335Ala Ala Ala
Val Val Val Met Ser Ala Ala Lys Ala Lys Glu Leu Gly 340 345 350Leu
Thr Pro Leu Ala Thr Ile Lys Ser Tyr Ala Asn Ala Gly Val Asp 355 360
365Pro Lys Val Met Gly Met Gly Pro Val Pro Ala Ser Lys Arg Ala Leu
370 375 380Ser Arg Ala Glu Trp Thr Pro Gln Asp Leu Asp Leu Met Glu
Ile Asn385 390 395 400Glu Ala Phe Ala Ala Gln Ala Leu Ala Val His
Gln Gln Met Gly Trp 405 410 415Asp Thr Ser Lys Val Asn Val Asn Gly
Gly Ala Ile Ala Ile Gly His 420 425 430Pro Ile Gly Ala Ser Gly Cys
Arg Ile Leu Val Thr Leu Leu His Glu 435 440 445Met Lys Arg Arg Asp
Ala Lys Lys Gly Leu Ala Ser Leu Cys Ile Gly 450 455 460Gly Gly Met
Gly Val Ala Leu Ala Val Glu Arg Lys465 470 475121529DNARastonia
Eutropia 12ggatccccat ggcttctatg atatcctctt ccgctgtgac aacagtcagc
cgtgcctcta 60gggggcaatc cgccgcaatg gctccattcg gcggcctcaa atccatgact
ggattcccag 120tgaagaaggt caacactgac attacttcca ttacaagcaa
tggtggaaga gtaaagtgca 180tgcaggtgtg gcctccaatt ggaaagaaga
agtttgagac tctttcctat ttgccaccat 240tgaccagaga ttcccgggtg
actgacgttg tcatcgtatc cgccgcccgc accgcggtcg 300gcaagtttgg
cggctcgctg gccaagatcc cggcaccgga actgggtgcc gtggtcatca
360aggccgcgct ggagcgcgcc ggcgtcaagc cggagcaggt gagcgaagtc
atcatgggcc 420aggtgctgac cgccggttcg ggccagaacc ccgcacgcca
ggccgcgatc aaggccggcc 480tgccggcgat ggtgccggcc atgaccatca
acaaggtgtg cggctcgggc ctgaaggccg 540tgatgctggc cgccaacgcg
atcatggcgg gcgacgccga gatcgtggtg gccggcggcc 600aggaaaacat
gagcgccgcc ccgcacgtgc tgccgggctc gcgcgatggt ttccgcatgg
660gcgatgccaa gctggtcgac accatgatcg tcgacggcct gtgggacgtg
tacaaccagt 720accacatggg catcaccgcc gagaacgtgg ccaaggaata
cggcatcaca cgcgaggcgc 780aggatgagtt cgccgtcggc tcgcagaaca
aggccgaagc cgcgcagaag gccggcaagt 840ttgacgaaga gatcgtcccg
gtgctgatcc cgcagcgcaa gggcgacccg gtggccttca 900agaccgacga
gttcgtgcgc cagggcgcca cgctggacag catgtccggc ctcaagcccg
960ccttcgacaa ggccggcacg gtgaccgcgg ccaacgcctc gggcctgaac
gacggcgccg 1020ccgcggtggt ggtgatgtcg gcggccaagg ccaaggaact
gggcctgacc ccgctggcca 1080cgatcaagag ctatgccaac gccggtgtcg
atcccaaggt gatgggcatg ggcccggtgc 1140cggcctccaa gcgcgccctg
tcgcgcgccg agtggacccc gcaagacctg gacctgatgg 1200agatcaacga
ggcctttgcc gcccaggcgc tggcggtgca ccagcagatg ggctgggaca
1260cctccaaggt caatgtgaac ggcggcgcca tcgccatcgg ccacccgatc
ggcgcgtcgg 1320gctgccgtat cctggtgacg ctgctgcacg agatgaagcg
ccgtgacgcg aagaagggcc 1380tggcctcgct gtgcatcggc ggcggcatgg
gcgtggcgct ggcagtcgag cgcaaataag 1440gaaggggttt tccggggccg
cgcgcggttg gcgcggaccc ggcgacgata acgaagccaa 1500tcaaggagtg
gacatgactc aggggtacc 152913987DNARastonia EutropiaCDS(1)..(987)
13atg gct tct atg ata tcc tct tcc gct gtg aca aca gtc agc cgt gcc
48Met Ala Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala1
5 10 15tct agg ggg caa tcc gcc gca atg gct cca ttc ggc ggc ctc aaa
tcc 96Ser Arg Gly Gln Ser Ala Ala Met Ala Pro Phe Gly Gly Leu Lys
Ser 20 25 30atg act gga ttc cca gtg aag aag gtc aac act gac att act
tcc att 144Met Thr Gly Phe Pro Val Lys Lys Val Asn Thr Asp Ile Thr
Ser Ile 35 40 45aca agc aat ggt gga aga gta aag tgc atg cag gtg tgg
cct cca att 192Thr Ser Asn Gly Gly Arg Val Lys Cys Met Gln Val Trp
Pro Pro Ile 50 55 60gga aag aag aag ttt gag act ctt tcc tat ttg cca
cca ttg acc aga 240Gly Lys Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro
Pro Leu Thr Arg65 70 75 80gat tcc cgg gtg act cag cgc att gcg tat
gtg acc ggc ggc atg ggt 288Asp Ser Arg Val Thr Gln Arg Ile Ala Tyr
Val Thr Gly Gly Met Gly 85 90 95ggt atc gga acc gcc att tgc cag cgg
ctg gcc aag gat ggc ttt cgt 336Gly Ile Gly Thr Ala Ile Cys Gln Arg
Leu Ala Lys Asp Gly Phe Arg 100 105 110gtg gtg gcc ggt tgc ggc ccc
aac tcg ccg cgc cgc gaa aag tgg ctg 384Val Val Ala Gly Cys Gly Pro
Asn Ser Pro Arg Arg Glu Lys Trp Leu 115 120 125gag cag cag aag gca
ctg ggc ttc gat ttc att gcc tcg gaa ggc aat 432Glu Gln Gln Lys Ala
Leu Gly Phe Asp Phe Ile Ala Ser Glu Gly Asn 130 135 140gtg gct gac
tgg gac tcg acc aag acc gca ttc gac aag gtc aag tcc 480Val Ala Asp
Trp Asp Ser Thr Lys Thr Ala Phe Asp Lys Val Lys Ser145 150 155
160gag gtc ggc gag gtt gat gtg ctg atc aac aac gcc ggt atc acc cgc
528Glu Val Gly Glu Val Asp Val Leu Ile Asn Asn Ala Gly Ile Thr Arg
165 170 175gac gtg gtg ttc cgc aag atg acc cgc gcc gac tgg gat gcg
gtg atc 576Asp Val Val Phe Arg Lys Met Thr Arg Ala Asp Trp Asp Ala
Val Ile 180 185 190gac acc aac ctg acc tcg ctg ttc aac gtc acc aag
cag gtg atc gac 624Asp Thr Asn Leu Thr Ser Leu Phe Asn Val Thr Lys
Gln Val Ile Asp 195 200 205ggc atg gcc gac cgt ggc tgg ggc cgc atc
gtc aac atc tcg tcg gtg 672Gly Met Ala Asp Arg Gly Trp Gly Arg Ile
Val Asn Ile Ser Ser Val 210 215 220aac ggg cag aag ggc cag ttc ggc
cag acc aac tac tcc acc gcc aag 720Asn Gly Gln Lys Gly Gln Phe Gly
Gln Thr Asn Tyr Ser Thr Ala Lys225 230 235 240gcc ggc ctg cat ggc
ttc acc atg gca ctg gcg cag gaa gtg gcg acc 768Ala Gly Leu His Gly
Phe Thr Met Ala Leu Ala Gln Glu Val Ala Thr 245 250 255aag ggc gtg
acc gtc aac acg gtc tct ccg ggc tat atc gcc acc gac 816Lys Gly Val
Thr Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Asp 260 265 270atg
gtc aag gcg atc cgc cag gac gtg ctc gac aag atc gtc gcg acg 864Met
Val Lys Ala Ile Arg Gln Asp Val Leu Asp Lys Ile Val Ala Thr 275 280
285atc ccg gtc aag cgc ctg ggc ctg cca gaa gag atc gcc tcg atc tgc
912Ile Pro Val Lys Arg Leu Gly Leu Pro Glu Glu Ile Ala Ser Ile Cys
290 295 300gcc tgg ttg tcg tcg gag gag tcc ggt ttc tcg acc ggc gcc
gac ttc 960Ala Trp Leu Ser Ser Glu Glu Ser Gly Phe Ser Thr Gly Ala
Asp Phe305 310 315 320tcg ctc aac ggc ggc ctg cat atg ggc 987Ser
Leu Asn Gly Gly Leu His Met Gly 32514329PRTRastonia Eutropia 14Met
Ala Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala1 5 10
15Ser Arg Gly Gln Ser Ala Ala Met Ala Pro Phe Gly Gly Leu Lys Ser
20 25 30Met Thr Gly Phe Pro Val Lys Lys Val Asn Thr Asp Ile Thr Ser
Ile 35 40 45Thr Ser Asn Gly Gly Arg Val Lys Cys Met Gln Val Trp Pro
Pro Ile 50 55 60Gly Lys Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro
Leu Thr Arg65 70 75 80Asp Ser Arg Val Thr Gln Arg Ile Ala Tyr Val
Thr Gly Gly Met Gly 85 90 95Gly Ile Gly Thr Ala Ile Cys Gln Arg Leu
Ala Lys Asp Gly Phe Arg 100 105 110Val Val Ala Gly Cys Gly Pro Asn
Ser Pro Arg Arg Glu Lys Trp Leu 115 120 125Glu Gln Gln Lys Ala Leu
Gly Phe Asp Phe Ile Ala Ser Glu Gly Asn 130 135 140Val Ala Asp Trp
Asp Ser Thr Lys Thr Ala Phe Asp Lys Val Lys Ser145 150 155 160Glu
Val Gly Glu Val Asp Val Leu Ile Asn Asn Ala Gly Ile Thr Arg 165 170
175Asp Val Val Phe Arg Lys Met Thr Arg Ala Asp Trp Asp Ala Val Ile
180 185 190Asp Thr Asn Leu Thr Ser Leu Phe Asn Val Thr Lys Gln Val
Ile Asp 195 200 205Gly Met Ala Asp Arg Gly Trp Gly Arg Ile Val Asn
Ile Ser Ser Val 210 215 220Asn Gly Gln Lys Gly Gln Phe Gly Gln Thr
Asn Tyr Ser Thr Ala Lys225 230 235 240Ala Gly Leu His Gly Phe Thr
Met Ala Leu Ala Gln Glu Val Ala Thr 245 250 255Lys Gly Val Thr Val
Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Asp 260 265 270Met Val Lys
Ala Ile Arg Gln Asp Val Leu Asp Lys Ile Val Ala Thr 275 280 285Ile
Pro Val Lys Arg Leu Gly Leu Pro Glu Glu Ile Ala Ser Ile Cys 290 295
300Ala Trp Leu Ser Ser Glu Glu Ser Gly Phe Ser Thr Gly Ala Asp
Phe305 310 315 320Ser Leu Asn Gly Gly Leu His Met Gly
325151032DNARastonia Eutropia 15ggatccatgg cttctatgat atcctcttcc
gctgtgacaa cagtcagccg tgcctctagg 60gggcaatccg ccgcaatggc tccattcggc
ggcctcaaat ccatgactgg attcccagtg 120aagaaggtca acactgacat
tacttccatt acaagcaatg gtggaagagt aaagtgcatg 180caggtgtggc
ctccaattgg aaagaagaag tttgagactc tttcctattt gccaccattg
240accagagatt cccgggtgac tcagcgcatt gcgtatgtga ccggcggcat
gggtggtatc 300ggaaccgcca tttgccagcg gctggccaag gatggctttc
gtgtggtggc cggttgcggc 360cccaactcgc cgcgccgcga aaagtggctg
gagcagcaga aggcactggg cttcgatttc 420attgcctcgg aaggcaatgt
ggctgactgg gactcgacca agaccgcatt cgacaaggtc 480aagtccgagg
tcggcgaggt tgatgtgctg atcaacaacg ccggtatcac ccgcgacgtg
540gtgttccgca agatgacccg cgccgactgg gatgcggtga tcgacaccaa
cctgacctcg 600ctgttcaacg tcaccaagca ggtgatcgac ggcatggccg
accgtggctg gggccgcatc 660gtcaacatct cgtcggtgaa cgggcagaag
ggccagttcg gccagaccaa ctactccacc 720gccaaggccg gcctgcatgg
cttcaccatg gcactggcgc aggaagtggc gaccaagggc 780gtgaccgtca
acacggtctc tccgggctat atcgccaccg acatggtcaa ggcgatccgc
840caggacgtgc tcgacaagat cgtcgcgacg atcccggtca agcgcctggg
cctgccagaa 900gagatcgcct cgatctgcgc ctggttgtcg tcggaggagt
ccggtttctc gaccggcgcc 960gacttctcgc tcaacggcgg cctgcatatg
ggctgacctg ccggcctggt tccaccagtc 1020ggcaggggta cc
1032162016DNARastonia EutropiaCDS(1)..(2016) 16atg gct tct atg ata
tcc tct tcc gct gtg aca aca gtc agc cgt gcc 48Met Ala Ser Met Ile
Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala1 5 10 15tct agg ggg caa
tcc gcc gca atg gct cca ttc ggc ggc ctc aaa tcc 96Ser Arg Gly Gln
Ser Ala Ala Met Ala Pro Phe Gly Gly Leu Lys Ser 20 25 30atg act gga
ttc cca gtg aag aag gtc aac act gac att act tcc att 144Met Thr Gly
Phe Pro Val Lys Lys Val Asn Thr Asp Ile Thr Ser Ile 35 40 45aca agc
aat ggt gga aga gta aag tgc atg cag gtg tgg cct cca att 192Thr Ser
Asn Gly Gly Arg Val Lys Cys Met Gln Val Trp Pro Pro Ile 50 55 60gga
aag aag aag ttt gag act ctt tcc tat ttg cca cca ttg acc aga 240Gly
Lys Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Thr Arg65 70 75
80gat tcc cgg gtg gcg acc ggc aaa ggc gcg gca gct tcc acg cag gaa
288Asp Ser Arg Val Ala Thr Gly Lys Gly Ala Ala Ala Ser Thr Gln Glu
85 90 95ggc aag tcc caa cca ttc aag gtc acg ccg ggg cca ttc gat cca
gcc 336Gly Lys Ser Gln Pro Phe Lys Val Thr Pro Gly Pro Phe Asp Pro
Ala 100 105 110aca tgg ctg gaa tgg tcc cgc cag tgg cag ggc act gaa
ggc aac ggc 384Thr Trp Leu Glu Trp Ser Arg Gln Trp Gln Gly Thr Glu
Gly Asn Gly 115 120 125cac gcg gcc gcg tcc ggc att ccg ggc ctg gat
gcg ctg gca ggc gtc 432His Ala Ala Ala Ser Gly Ile Pro Gly Leu Asp
Ala Leu Ala Gly Val 130 135 140aag atc gcg ccg gcg cag ctg ggt gat
atc cag cag cgc tac atg aag 480Lys Ile Ala Pro Ala Gln Leu Gly Asp
Ile Gln Gln Arg Tyr Met Lys145 150 155 160gac ttc tca gcg ctg tgg
cag gcc atg gcc gag ggc aag gcc gag gcc 528Asp Phe Ser Ala Leu Trp
Gln Ala Met Ala Glu Gly Lys Ala Glu Ala 165 170 175acc ggt ccg ctg
cac gac cgg cgc ttc gcc ggc gac gca tgg cgc acc 576Thr Gly Pro Leu
His Asp Arg Arg Phe Ala Gly Asp Ala Trp Arg Thr 180 185 190aac ctc
cca tat cgc ttc gct gcc gcg ttc tac ctg ctc aat gcg cgc 624Asn Leu
Pro Tyr Arg Phe Ala Ala Ala Phe Tyr Leu Leu Asn Ala Arg 195 200
205gcc ttg acc gag ctg gcc gat gcc gtc gag gcc gat gcc aag acc cgc
672Ala Leu Thr Glu Leu Ala Asp Ala Val Glu Ala Asp Ala Lys Thr Arg
210 215 220cag cgc atc cgc ttc gcg atc tcg caa tgg gtc gat gcg atg
tcg ccc 720Gln Arg Ile Arg Phe Ala Ile Ser Gln Trp Val Asp Ala Met
Ser Pro225 230 235 240gcc aac ttc ctt gcc acc aat ccc gag gcg cag
cgc ctg ctg atc gag 768Ala Asn Phe Leu Ala Thr Asn Pro Glu Ala Gln
Arg Leu Leu Ile Glu 245 250 255tcg ggc ggc gaa tcg ctg cgt gcc ggc
gtg cgc aac atg atg gaa gac 816Ser Gly Gly Glu Ser Leu Arg Ala Gly
Val Arg Asn Met Met Glu Asp 260 265 270ctg aca cgc ggc aag atc tcg
cag acc gac gag agc gcg ttt gag gtc 864Leu Thr Arg Gly Lys Ile Ser
Gln Thr Asp Glu Ser Ala Phe Glu Val 275 280 285ggc cgc aat gtc gcg
gtg acc gaa ggc gcc gtg gtc ttc gag aac gag 912Gly Arg Asn Val Ala
Val Thr Glu Gly Ala Val Val Phe Glu Asn Glu 290 295 300tac ttc cag
ctg ttg cag tac aag ccg ctg acc gac aag gtg cac gcg 960Tyr Phe Gln
Leu Leu Gln Tyr Lys Pro Leu Thr Asp Lys Val His Ala305 310 315
320cgc ccg ctg ctg atg gtg ccg ccg tgc atc aac aag tac tac atc ctg
1008Arg Pro Leu Leu Met Val Pro Pro Cys Ile Asn Lys Tyr Tyr Ile Leu
325 330 335gac ctg cag ccg gag agc tcg ctg gtg cgc cat gtg gtg gag
cag gga 1056Asp Leu Gln Pro Glu Ser Ser Leu Val Arg His Val Val Glu
Gln Gly 340 345 350cat acg gtg ttt ctg gtg tcg tgg cgc aat ccg gac
gcc agc atg gcc 1104His Thr Val Phe Leu Val Ser Trp Arg Asn Pro Asp
Ala Ser Met Ala 355 360 365ggc agc acc tgg gac gac tac atc gag cac
gcg gcc atc cgc gcc atc 1152Gly Ser Thr Trp Asp Asp Tyr Ile Glu His
Ala Ala Ile Arg Ala Ile 370 375 380gaa gtc gcg cgc gac atc agc ggc
cag gac aag atc aac gtg ctc ggc 1200Glu Val Ala Arg Asp Ile Ser Gly
Gln Asp Lys Ile Asn Val Leu Gly385 390 395 400ttc tgc gtg ggc ggc
acc att gtc tcg acc gcg ctg gcg gtg ctg gcc 1248Phe Cys Val Gly Gly
Thr Ile Val Ser Thr Ala Leu Ala Val Leu Ala 405 410 415gcg cgc ggc
gag cac ccg gcc gcc agc gtc acg ctg ctg acc acg ctg 1296Ala Arg Gly
Glu His Pro Ala Ala Ser Val Thr Leu Leu Thr Thr Leu 420 425 430ctg
gac ttt gcc gac acg ggc atc ctc gac gtc ttt gtc gac gag ggc 1344Leu
Asp Phe Ala Asp Thr Gly Ile Leu Asp Val Phe Val Asp Glu Gly 435 440
445cat gtg cag ttg cgc gag gcc acg ctg ggc ggc ggc gcc ggc gcg ccg
1392His Val Gln Leu Arg Glu Ala Thr Leu Gly Gly Gly Ala Gly Ala Pro
450 455 460tgc gcg ctg ctg cgc ggc ctt gag ctg gcc aat acc ttc tcg
ttc ttg 1440Cys Ala Leu Leu Arg Gly Leu Glu Leu Ala Asn Thr Phe Ser
Phe Leu465 470 475 480cgc ccg aac gac ctg gtg tgg aac tac gtg gtc
gac aac tac ctg aag 1488Arg Pro Asn Asp Leu Val Trp Asn Tyr Val Val
Asp Asn Tyr Leu Lys 485 490 495ggc aac acg ccg gtg ccg ttc gac ctg
ctg ttc tgg aac ggc gac gcc 1536Gly Asn Thr Pro Val Pro Phe Asp Leu
Leu Phe Trp Asn Gly Asp Ala 500 505 510acc aac ctg ccg ggg ccg tgg
tac tgc tgg tac ctg cgc cac acc tac 1584Thr Asn Leu Pro Gly Pro Trp
Tyr Cys Trp Tyr Leu Arg His Thr Tyr 515 520 525ctg cag aac gag ctc
aag gta ccg ggc aag ctg acc gtg tgc ggc gtg 1632Leu Gln Asn Glu Leu
Lys Val Pro Gly Lys Leu Thr Val Cys Gly Val 530 535 540ccg gtg gac
ctg gcc agc atc gac gtg ccg acc tat atc tac ggc tcg 1680Pro Val Asp
Leu Ala Ser Ile Asp Val Pro Thr Tyr Ile Tyr Gly Ser545 550 555
560cgc gaa gac cat atc gtg ccg tgg acc gcg gcc tat gcc tcg acc gcg
1728Arg Glu Asp His Ile Val Pro Trp Thr Ala Ala Tyr Ala Ser Thr Ala
565 570 575ctg ctg gcg aac aag ctg cgc ttc gtg ctg ggt gcg tcg ggc
cat atc 1776Leu Leu Ala Asn Lys Leu Arg Phe Val Leu Gly Ala Ser Gly
His Ile 580 585 590gcc ggt gtg atc aac ccg ccg gcc aag aac aag cgc
agc cac tgg act
1824Ala Gly Val Ile Asn Pro Pro Ala Lys Asn Lys Arg Ser His Trp Thr
595 600 605aac gat gcg ctg ccg gag tcg ccg cag caa tgg ctg gcc ggc
gcc atc 1872Asn Asp Ala Leu Pro Glu Ser Pro Gln Gln Trp Leu Ala Gly
Ala Ile 610 615 620gag cat cac ggc agc tgg tgg ccg gac tgg acc gca
tgg ctg gcc ggg 1920Glu His His Gly Ser Trp Trp Pro Asp Trp Thr Ala
Trp Leu Ala Gly625 630 635 640cag gcc ggc gcg aaa cgc gcc gcg ccc
gcc aac tat ggc aat gcg cgc 1968Gln Ala Gly Ala Lys Arg Ala Ala Pro
Ala Asn Tyr Gly Asn Ala Arg 645 650 655tat cgc gca atc gaa ccc gcg
cct ggg cga tac gtc aaa gcc aag gca 2016Tyr Arg Ala Ile Glu Pro Ala
Pro Gly Arg Tyr Val Lys Ala Lys Ala 660 665 67017672PRTRastonia
Eutropia 17Met Ala Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser
Arg Ala1 5 10 15Ser Arg Gly Gln Ser Ala Ala Met Ala Pro Phe Gly Gly
Leu Lys Ser 20 25 30Met Thr Gly Phe Pro Val Lys Lys Val Asn Thr Asp
Ile Thr Ser Ile 35 40 45Thr Ser Asn Gly Gly Arg Val Lys Cys Met Gln
Val Trp Pro Pro Ile 50 55 60Gly Lys Lys Lys Phe Glu Thr Leu Ser Tyr
Leu Pro Pro Leu Thr Arg65 70 75 80Asp Ser Arg Val Ala Thr Gly Lys
Gly Ala Ala Ala Ser Thr Gln Glu 85 90 95Gly Lys Ser Gln Pro Phe Lys
Val Thr Pro Gly Pro Phe Asp Pro Ala 100 105 110Thr Trp Leu Glu Trp
Ser Arg Gln Trp Gln Gly Thr Glu Gly Asn Gly 115 120 125His Ala Ala
Ala Ser Gly Ile Pro Gly Leu Asp Ala Leu Ala Gly Val 130 135 140Lys
Ile Ala Pro Ala Gln Leu Gly Asp Ile Gln Gln Arg Tyr Met Lys145 150
155 160Asp Phe Ser Ala Leu Trp Gln Ala Met Ala Glu Gly Lys Ala Glu
Ala 165 170 175Thr Gly Pro Leu His Asp Arg Arg Phe Ala Gly Asp Ala
Trp Arg Thr 180 185 190Asn Leu Pro Tyr Arg Phe Ala Ala Ala Phe Tyr
Leu Leu Asn Ala Arg 195 200 205Ala Leu Thr Glu Leu Ala Asp Ala Val
Glu Ala Asp Ala Lys Thr Arg 210 215 220Gln Arg Ile Arg Phe Ala Ile
Ser Gln Trp Val Asp Ala Met Ser Pro225 230 235 240Ala Asn Phe Leu
Ala Thr Asn Pro Glu Ala Gln Arg Leu Leu Ile Glu 245 250 255Ser Gly
Gly Glu Ser Leu Arg Ala Gly Val Arg Asn Met Met Glu Asp 260 265
270Leu Thr Arg Gly Lys Ile Ser Gln Thr Asp Glu Ser Ala Phe Glu Val
275 280 285Gly Arg Asn Val Ala Val Thr Glu Gly Ala Val Val Phe Glu
Asn Glu 290 295 300Tyr Phe Gln Leu Leu Gln Tyr Lys Pro Leu Thr Asp
Lys Val His Ala305 310 315 320Arg Pro Leu Leu Met Val Pro Pro Cys
Ile Asn Lys Tyr Tyr Ile Leu 325 330 335Asp Leu Gln Pro Glu Ser Ser
Leu Val Arg His Val Val Glu Gln Gly 340 345 350His Thr Val Phe Leu
Val Ser Trp Arg Asn Pro Asp Ala Ser Met Ala 355 360 365Gly Ser Thr
Trp Asp Asp Tyr Ile Glu His Ala Ala Ile Arg Ala Ile 370 375 380Glu
Val Ala Arg Asp Ile Ser Gly Gln Asp Lys Ile Asn Val Leu Gly385 390
395 400Phe Cys Val Gly Gly Thr Ile Val Ser Thr Ala Leu Ala Val Leu
Ala 405 410 415Ala Arg Gly Glu His Pro Ala Ala Ser Val Thr Leu Leu
Thr Thr Leu 420 425 430Leu Asp Phe Ala Asp Thr Gly Ile Leu Asp Val
Phe Val Asp Glu Gly 435 440 445His Val Gln Leu Arg Glu Ala Thr Leu
Gly Gly Gly Ala Gly Ala Pro 450 455 460Cys Ala Leu Leu Arg Gly Leu
Glu Leu Ala Asn Thr Phe Ser Phe Leu465 470 475 480Arg Pro Asn Asp
Leu Val Trp Asn Tyr Val Val Asp Asn Tyr Leu Lys 485 490 495Gly Asn
Thr Pro Val Pro Phe Asp Leu Leu Phe Trp Asn Gly Asp Ala 500 505
510Thr Asn Leu Pro Gly Pro Trp Tyr Cys Trp Tyr Leu Arg His Thr Tyr
515 520 525Leu Gln Asn Glu Leu Lys Val Pro Gly Lys Leu Thr Val Cys
Gly Val 530 535 540Pro Val Asp Leu Ala Ser Ile Asp Val Pro Thr Tyr
Ile Tyr Gly Ser545 550 555 560Arg Glu Asp His Ile Val Pro Trp Thr
Ala Ala Tyr Ala Ser Thr Ala 565 570 575Leu Leu Ala Asn Lys Leu Arg
Phe Val Leu Gly Ala Ser Gly His Ile 580 585 590Ala Gly Val Ile Asn
Pro Pro Ala Lys Asn Lys Arg Ser His Trp Thr 595 600 605Asn Asp Ala
Leu Pro Glu Ser Pro Gln Gln Trp Leu Ala Gly Ala Ile 610 615 620Glu
His His Gly Ser Trp Trp Pro Asp Trp Thr Ala Trp Leu Ala Gly625 630
635 640Gln Ala Gly Ala Lys Arg Ala Ala Pro Ala Asn Tyr Gly Asn Ala
Arg 645 650 655Tyr Arg Ala Ile Glu Pro Ala Pro Gly Arg Tyr Val Lys
Ala Lys Ala 660 665 670182049DNARastonia Eutropia 18ggatccatgg
cttctatgat atcctcttcc gctgtgacaa cagtcagccg tgcctctagg 60gggcaatccg
ccgcaatggc tccattcggc ggcctcaaat ccatgactgg attcccagtg
120aagaaggtca acactgacat tacttccatt acaagcaatg gtggaagagt
aaagtgcatg 180caggtgtggc ctccaattgg aaagaagaag tttgagactc
tttcctattt gccaccattg 240accagagatt cccgggtggc gaccggcaaa
ggcgcggcag cttccacgca ggaaggcaag 300tcccaaccat tcaaggtcac
gccggggcca ttcgatccag ccacatggct ggaatggtcc 360cgccagtggc
agggcactga aggcaacggc cacgcggccg cgtccggcat tccgggcctg
420gatgcgctgg caggcgtcaa gatcgcgccg gcgcagctgg gtgatatcca
gcagcgctac 480atgaaggact tctcagcgct gtggcaggcc atggccgagg
gcaaggccga ggccaccggt 540ccgctgcacg accggcgctt cgccggcgac
gcatggcgca ccaacctccc atatcgcttc 600gctgccgcgt tctacctgct
caatgcgcgc gccttgaccg agctggccga tgccgtcgag 660gccgatgcca
agacccgcca gcgcatccgc ttcgcgatct cgcaatgggt cgatgcgatg
720tcgcccgcca acttccttgc caccaatccc gaggcgcagc gcctgctgat
cgagtcgggc 780ggcgaatcgc tgcgtgccgg cgtgcgcaac atgatggaag
acctgacacg cggcaagatc 840tcgcagaccg acgagagcgc gtttgaggtc
ggccgcaatg tcgcggtgac cgaaggcgcc 900gtggtcttcg agaacgagta
cttccagctg ttgcagtaca agccgctgac cgacaaggtg 960cacgcgcgcc
cgctgctgat ggtgccgccg tgcatcaaca agtactacat cctggacctg
1020cagccggaga gctcgctggt gcgccatgtg gtggagcagg gacatacggt
gtttctggtg 1080tcgtggcgca atccggacgc cagcatggcc ggcagcacct
gggacgacta catcgagcac 1140gcggccatcc gcgccatcga agtcgcgcgc
gacatcagcg gccaggacaa gatcaacgtg 1200ctcggcttct gcgtgggcgg
caccattgtc tcgaccgcgc tggcggtgct ggccgcgcgc 1260ggcgagcacc
cggccgccag cgtcacgctg ctgaccacgc tgctggactt tgccgacacg
1320ggcatcctcg acgtctttgt cgacgagggc catgtgcagt tgcgcgaggc
cacgctgggc 1380ggcggcgccg gcgcgccgtg cgcgctgctg cgcggccttg
agctggccaa taccttctcg 1440ttcttgcgcc cgaacgacct ggtgtggaac
tacgtggtcg acaactacct gaagggcaac 1500acgccggtgc cgttcgacct
gctgttctgg aacggcgacg ccaccaacct gccggggccg 1560tggtactgct
ggtacctgcg ccacacctac ctgcagaacg agctcaaggt accgggcaag
1620ctgaccgtgt gcggcgtgcc ggtggacctg gccagcatcg acgtgccgac
ctatatctac 1680ggctcgcgcg aagaccatat cgtgccgtgg accgcggcct
atgcctcgac cgcgctgctg 1740gcgaacaagc tgcgcttcgt gctgggtgcg
tcgggccata tcgccggtgt gatcaacccg 1800ccggccaaga acaagcgcag
ccactggact aacgatgcgc tgccggagtc gccgcagcaa 1860tggctggccg
gcgccatcga gcatcacggc agctggtggc cggactggac cgcatggctg
1920gccgggcagg ccggcgcgaa acgcgccgcg cccgccaact atggcaatgc
gcgctatcgc 1980gcaatcgaac ccgcgcctgg gcgatacgtc aaagccaagg
catgacgctt caatcgaatt 2040gggggtacc 2049191680DNAP.
aeruginosaCDS(1)..(1680) 19atg agt cag aag aac aat aac gag ctt ccc
aag caa gcc gcg gaa aac 48Met Ser Gln Lys Asn Asn Asn Glu Leu Pro
Lys Gln Ala Ala Glu Asn1 5 10 15acg ctg aac ctg aat ccg gtg atc ggc
atc cgg ggc aag gac ctg ctc 96Thr Leu Asn Leu Asn Pro Val Ile Gly
Ile Arg Gly Lys Asp Leu Leu 20 25 30acc tcc gcg cgc atg gtc ctg ctc
cag gcg gtg cgc cag ccg ctg cac 144Thr Ser Ala Arg Met Val Leu Leu
Gln Ala Val Arg Gln Pro Leu His 35 40 45agc gcc agg cac gtg gcg cat
ttc agc ctg gag ctg aag aac gtc ctg 192Ser Ala Arg His Val Ala His
Phe Ser Leu Glu Leu Lys Asn Val Leu 50 55 60ctc ggc cag tcg gag cta
cgc cca ggc gat gac gac cga cgc ttt tcc 240Leu Gly Gln Ser Glu Leu
Arg Pro Gly Asp Asp Asp Arg Arg Phe Ser65 70 75 80gat ccg gcc tgg
agc cag aat cca ctg tac aag cgc tac atg cag acc 288Asp Pro Ala Trp
Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Met Gln Thr 85 90 95tac ctg gcc
tgg cgc aag gag ctg cac agc tgg atc agc cac agc gac 336Tyr Leu Ala
Trp Arg Lys Glu Leu His Ser Trp Ile Ser His Ser Asp 100 105 110ctg
tcg ccg cag gac atc agt cgt ggc cag ttc gtc atc aac ctg ctg 384Leu
Ser Pro Gln Asp Ile Ser Arg Gly Gln Phe Val Ile Asn Leu Leu 115 120
125acc gag gcg atg tcg ccg acc aac agc ctg agc aac ccg gcg gcg gtc
432Thr Glu Ala Met Ser Pro Thr Asn Ser Leu Ser Asn Pro Ala Ala Val
130 135 140aag cgc ttc ttc gag acc ggc ggc aag agc ctg ctg gac ggc
ctc ggc 480Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly
Leu Gly145 150 155 160cac ctg gcc aag gac ctg gtg aac aac ggc ggg
atg ccg agc cag gtg 528His Leu Ala Lys Asp Leu Val Asn Asn Gly Gly
Met Pro Ser Gln Val 165 170 175gac atg gac gcc ttc gag gtg ggc aag
aac ctg gcc acc acc gag ggc 576Asp Met Asp Ala Phe Glu Val Gly Lys
Asn Leu Ala Thr Thr Glu Gly 180 185 190gcc gtg gtg ttc cgc aac gac
gtg ctg gaa ctg atc cag tac cgg ccg 624Ala Val Val Phe Arg Asn Asp
Val Leu Glu Leu Ile Gln Tyr Arg Pro 195 200 205atc acc gag tcg gtg
cac gaa cgc ccg ctg ctg gtg gtg ccg ccg cag 672Ile Thr Glu Ser Val
His Glu Arg Pro Leu Leu Val Val Pro Pro Gln 210 215 220atc aac aag
ttc tac gtc ttc gac ctg tcg ccg gac aag agc ctg gcg 720Ile Asn Lys
Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser Leu Ala225 230 235
240cgc ttc tgc ctg cgc aac ggc gtg cag acc ttc atc gtc agt tgg cgc
768Arg Phe Cys Leu Arg Asn Gly Val Gln Thr Phe Ile Val Ser Trp Arg
245 250 255aac ccg acc aag tcg cag cgc gaa tgg ggc ctg acc acc tat
atc gag 816Asn Pro Thr Lys Ser Gln Arg Glu Trp Gly Leu Thr Thr Tyr
Ile Glu 260 265 270gcg ctc aag gag gcc atc gag gta gtc ctg tcg atc
acc ggc agc aag 864Ala Leu Lys Glu Ala Ile Glu Val Val Leu Ser Ile
Thr Gly Ser Lys 275 280 285gac ctc aac ctc ctc ggc gcc tgc tcc ggc
ggg atc acc acc gcg acc 912Asp Leu Asn Leu Leu Gly Ala Cys Ser Gly
Gly Ile Thr Thr Ala Thr 290 295 300ctg gtc ggc cac tac gtg gcc agc
ggc gag aag aag gtc aac gcc ttc 960Leu Val Gly His Tyr Val Ala Ser
Gly Glu Lys Lys Val Asn Ala Phe305 310 315 320acc caa ctg gtc agc
gtg ctc gac ttc gaa ctg aat acc cag gtc gcg 1008Thr Gln Leu Val Ser
Val Leu Asp Phe Glu Leu Asn Thr Gln Val Ala 325 330 335ctg ttc gcc
gac gag aag act ctg gag gcc gcc aag cgt cgt tcc tac 1056Leu Phe Ala
Asp Glu Lys Thr Leu Glu Ala Ala Lys Arg Arg Ser Tyr 340 345 350cag
tcc ggc gtg ctg gag ggc aag gac atg gcc aag gtg ttc gcc tgg 1104Gln
Ser Gly Val Leu Glu Gly Lys Asp Met Ala Lys Val Phe Ala Trp 355 360
365atg cgc ccc aac gac ctg atc tgg aac tac tgg gtc aac aac tac ctg
1152Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu
370 375 380ctc ggc aac cag ccg ccg gcg ttc gac atc ctc tac tgg aac
aac gac 1200Leu Gly Asn Gln Pro Pro Ala Phe Asp Ile Leu Tyr Trp Asn
Asn Asp385 390 395 400acc acg cgc ctg ccc gcc gcg ctg cac ggc gag
ttc gtc gaa ctg ttc 1248Thr Thr Arg Leu Pro Ala Ala Leu His Gly Glu
Phe Val Glu Leu Phe 405 410 415aag agc aac ccg ctg aac cgc ccc ggc
gcc ctg gag gtc tcc ggc acg 1296Lys Ser Asn Pro Leu Asn Arg Pro Gly
Ala Leu Glu Val Ser Gly Thr 420 425 430ccc atc gac ctg aag cag gtg
act tgc gac ttc tac tgt gtc gcc ggt 1344Pro Ile Asp Leu Lys Gln Val
Thr Cys Asp Phe Tyr Cys Val Ala Gly 435 440 445 ctg aac gac cac atc
acc ccc tgg gag tcg tgc tac aag tcg gcc agg 1392Leu Asn Asp His Ile
Thr Pro Trp Glu Ser Cys Tyr Lys Ser Ala Arg 450 455 460ctg ctg ggt
ggc aag tgc gag ttc atc ctc tcc aac agc ggt cac atc 1440Leu Leu Gly
Gly Lys Cys Glu Phe Ile Leu Ser Asn Ser Gly His Ile465 470 475
480cag agc atc ctc aac cca ccg ggc aac ccc aag gca cgc ttc atg acc
1488Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ala Arg Phe Met Thr
485 490 495aat ccg gaa ctg ccc gcc gag ccc aag gcc tgg ctg gaa cag
gcc ggc 1536Asn Pro Glu Leu Pro Ala Glu Pro Lys Ala Trp Leu Glu Gln
Ala Gly 500 505 510aag cac gcc gac tcg tgg tgg ttg cac tgg cag caa
tgg ctg gcc gaa 1584Lys His Ala Asp Ser Trp Trp Leu His Trp Gln Gln
Trp Leu Ala Glu 515 520 525cgc tcc ggc aag acc cgc aag gcg ccc gcc
agc ctg ggc aac aag acc 1632Arg Ser Gly Lys Thr Arg Lys Ala Pro Ala
Ser Leu Gly Asn Lys Thr 530 535 540tat ccg gcc ggc gaa gcc gcg ccc
gga acc tac gtg cat gaa cga tga 1680Tyr Pro Ala Gly Glu Ala Ala Pro
Gly Thr Tyr Val His Glu Arg545 550 55520559PRTP. aeruginosa 20Met
Ser Gln Lys Asn Asn Asn Glu Leu Pro Lys Gln Ala Ala Glu Asn1 5 10
15Thr Leu Asn Leu Asn Pro Val Ile Gly Ile Arg Gly Lys Asp Leu Leu
20 25 30Thr Ser Ala Arg Met Val Leu Leu Gln Ala Val Arg Gln Pro Leu
His 35 40 45Ser Ala Arg His Val Ala His Phe Ser Leu Glu Leu Lys Asn
Val Leu 50 55 60Leu Gly Gln Ser Glu Leu Arg Pro Gly Asp Asp Asp Arg
Arg Phe Ser65 70 75 80Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Lys
Arg Tyr Met Gln Thr 85 90 95Tyr Leu Ala Trp Arg Lys Glu Leu His Ser
Trp Ile Ser His Ser Asp 100 105 110Leu Ser Pro Gln Asp Ile Ser Arg
Gly Gln Phe Val Ile Asn Leu Leu 115 120 125Thr Glu Ala Met Ser Pro
Thr Asn Ser Leu Ser Asn Pro Ala Ala Val 130 135 140Lys Arg Phe Phe
Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Gly145 150 155 160His
Leu Ala Lys Asp Leu Val Asn Asn Gly Gly Met Pro Ser Gln Val 165 170
175Asp Met Asp Ala Phe Glu Val Gly Lys Asn Leu Ala Thr Thr Glu Gly
180 185 190Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr
Arg Pro 195 200 205Ile Thr Glu Ser Val His Glu Arg Pro Leu Leu Val
Val Pro Pro Gln 210 215 220Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser
Pro Asp Lys Ser Leu Ala225 230 235 240Arg Phe Cys Leu Arg Asn Gly
Val Gln Thr Phe Ile Val Ser Trp Arg 245 250 255Asn Pro Thr Lys Ser
Gln Arg Glu Trp Gly Leu Thr Thr Tyr Ile Glu 260 265 270Ala Leu Lys
Glu Ala Ile Glu Val Val Leu Ser Ile Thr Gly Ser Lys 275 280 285Asp
Leu Asn Leu Leu Gly Ala Cys Ser Gly Gly Ile Thr Thr Ala Thr 290 295
300Leu Val Gly His Tyr Val Ala Ser Gly Glu Lys Lys Val Asn Ala
Phe305 310 315 320Thr Gln Leu Val Ser Val Leu Asp Phe Glu Leu Asn
Thr Gln Val Ala 325 330 335Leu Phe Ala Asp Glu Lys Thr Leu Glu Ala
Ala Lys Arg Arg Ser Tyr 340 345 350Gln Ser Gly Val Leu Glu Gly Lys
Asp Met Ala Lys Val Phe Ala Trp 355 360 365Met Arg Pro Asn Asp Leu
Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu 370 375 380Leu Gly Asn Gln
Pro Pro Ala Phe Asp Ile Leu Tyr Trp Asn Asn Asp385 390 395 400Thr
Thr Arg Leu Pro Ala Ala Leu His Gly Glu Phe Val Glu Leu Phe 405 410
415Lys Ser Asn Pro Leu Asn Arg Pro Gly Ala Leu Glu Val Ser Gly Thr
420
425 430Pro Ile Asp Leu Lys Gln Val Thr Cys Asp Phe Tyr Cys Val Ala
Gly 435 440 445Leu Asn Asp His Ile Thr Pro Trp Glu Ser Cys Tyr Lys
Ser Ala Arg 450 455 460Leu Leu Gly Gly Lys Cys Glu Phe Ile Leu Ser
Asn Ser Gly His Ile465 470 475 480Gln Ser Ile Leu Asn Pro Pro Gly
Asn Pro Lys Ala Arg Phe Met Thr 485 490 495Asn Pro Glu Leu Pro Ala
Glu Pro Lys Ala Trp Leu Glu Gln Ala Gly 500 505 510Lys His Ala Asp
Ser Trp Trp Leu His Trp Gln Gln Trp Leu Ala Glu 515 520 525Arg Ser
Gly Lys Thr Arg Lys Ala Pro Ala Ser Leu Gly Asn Lys Thr 530 535
540Tyr Pro Ala Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg545
550 555211692DNAP. aeruginosa 21ggatccatga gtcagaagaa caataacgag
cttcccaagc aagccgcgga aaacacgctg 60aacctgaatc cggtgatcgg catccggggc
aaggacctgc tcacctccgc gcgcatggtc 120ctgctccagg cggtgcgcca
gccgctgcac agcgccaggc acgtggcgca tttcagcctg 180gagctgaaga
acgtcctgct cggccagtcg gagctacgcc caggcgatga cgaccgacgc
240ttttccgatc cggcctggag ccagaatcca ctgtacaagc gctacatgca
gacctacctg 300gcctggcgca aggagctgca cagctggatc agccacagcg
acctgtcgcc gcaggacatc 360agtcgtggcc agttcgtcat caacctgctg
accgaggcga tgtcgccgac caacagcctg 420agcaacccgg cggcggtcaa
gcgcttcttc gagaccggcg gcaagagcct gctggacggc 480ctcggccacc
tggccaagga cctggtgaac aacggcggga tgccgagcca ggtggacatg
540gacgccttcg aggtgggcaa gaacctggcc accaccgagg gcgccgtggt
gttccgcaac 600gacgtgctgg aactgatcca gtaccggccg atcaccgagt
cggtgcacga acgcccgctg 660ctggtggtgc cgccgcagat caacaagttc
tacgtcttcg acctgtcgcc ggacaagagc 720ctggcgcgct tctgcctgcg
caacggcgtg cagaccttca tcgtcagttg gcgcaacccg 780accaagtcgc
agcgcgaatg gggcctgacc acctatatcg aggcgctcaa ggaggccatc
840gaggtagtcc tgtcgatcac cggcagcaag gacctcaacc tcctcggcgc
ctgctccggc 900gggatcacca ccgcgaccct ggtcggccac tacgtggcca
gcggcgagaa gaaggtcaac 960gccttcaccc aactggtcag cgtgctcgac
ttcgaactga atacccaggt cgcgctgttc 1020gccgacgaga agactctgga
ggccgccaag cgtcgttcct accagtccgg cgtgctggag 1080ggcaaggaca
tggccaaggt gttcgcctgg atgcgcccca acgacctgat ctggaactac
1140tgggtcaaca actacctgct cggcaaccag ccgccggcgt tcgacatcct
ctactggaac 1200aacgacacca cgcgcctgcc cgccgcgctg cacggcgagt
tcgtcgaact gttcaagagc 1260aacccgctga accgccccgg cgccctggag
gtctccggca cgcccatcga cctgaagcag 1320gtgacttgcg acttctactg
tgtcgccggt ctgaacgacc acatcacccc ctgggagtcg 1380tgctacaagt
cggccaggct gctgggtggc aagtgcgagt tcatcctctc caacagcggt
1440cacatccaga gcatcctcaa cccaccgggc aaccccaagg cacgcttcat
gaccaatccg 1500gaactgcccg ccgagcccaa ggcctggctg gaacaggccg
gcaagcacgc cgactcgtgg 1560tggttgcact ggcagcaatg gctggccgaa
cgctccggca agacccgcaa ggcgcccgcc 1620agcctgggca acaagaccta
tccggccggc gaagccgcgc ccggaaccta cgtgcatgaa 1680cgatgaggta cc
1692221794DNAP. aeruginosaCDS(1)..(1794) 22atg agt cag aag aac aat
aac gag ctt ccc aag caa gcc gcg gaa aac 48Met Ser Gln Lys Asn Asn
Asn Glu Leu Pro Lys Gln Ala Ala Glu Asn1 5 10 15acg ctg aac ctg aat
ccg gtg atc ggc atc cgg ggc aag gac ctg ctc 96Thr Leu Asn Leu Asn
Pro Val Ile Gly Ile Arg Gly Lys Asp Leu Leu 20 25 30acc tcc gcg cgc
atg gtc ctg ctc cag gcg gtg cgc cag ccg ctg cac 144Thr Ser Ala Arg
Met Val Leu Leu Gln Ala Val Arg Gln Pro Leu His 35 40 45agc gcc agg
cac gtg gcg cat ttc agc ctg gag ctg aag aac gtc ctg 192Ser Ala Arg
His Val Ala His Phe Ser Leu Glu Leu Lys Asn Val Leu 50 55 60ctc ggc
cag tcg gag cta cgc cca ggc gat gac gac cga cgc ttt tcc 240Leu Gly
Gln Ser Glu Leu Arg Pro Gly Asp Asp Asp Arg Arg Phe Ser65 70 75
80gat ccg gcc tgg agc cag aat cca ctg tac aag cgc tac atg cag acc
288Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Met Gln Thr
85 90 95tac ctg gcc tgg cgc aag gag ctg cac agc tgg atc agc cac agc
gac 336Tyr Leu Ala Trp Arg Lys Glu Leu His Ser Trp Ile Ser His Ser
Asp 100 105 110ctg tcg ccg cag gac atc agt cgt ggc cag ttc gtc atc
aac ctg ctg 384Leu Ser Pro Gln Asp Ile Ser Arg Gly Gln Phe Val Ile
Asn Leu Leu 115 120 125acc gag gcg atg tcg ccg acc aac agc ctg agc
aac ccg gcg gcg gtc 432Thr Glu Ala Met Ser Pro Thr Asn Ser Leu Ser
Asn Pro Ala Ala Val 130 135 140aag cgc ttc ttc gag acc ggc ggc aag
agc ctg ctg gac ggc ctc ggc 480Lys Arg Phe Phe Glu Thr Gly Gly Lys
Ser Leu Leu Asp Gly Leu Gly145 150 155 160cac ctg gcc aag gac ctg
gtg aac aac ggc ggg atg ccg agc cag gtg 528His Leu Ala Lys Asp Leu
Val Asn Asn Gly Gly Met Pro Ser Gln Val 165 170 175gac atg gac gcc
ttc gag gtg ggc aag aac ctg gcc acc acc gag ggc 576Asp Met Asp Ala
Phe Glu Val Gly Lys Asn Leu Ala Thr Thr Glu Gly 180 185 190gcc gtg
gtg ttc cgc aac gac gtg ctg gaa ctg atc cag tac cgg ccg 624Ala Val
Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Arg Pro 195 200
205atc acc gag tcg gtg cac gaa cgc ccg ctg ctg gtg gtg ccg ccg cag
672Ile Thr Glu Ser Val His Glu Arg Pro Leu Leu Val Val Pro Pro Gln
210 215 220atc aac aag ttc tac gtc ttc gac ctg tcg ccg gac aag agc
ctg gcg 720Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser
Leu Ala225 230 235 240cgc ttc tgc ctg cgc aac ggc gtg cag acc ttc
atc gtc agt tgg cgc 768Arg Phe Cys Leu Arg Asn Gly Val Gln Thr Phe
Ile Val Ser Trp Arg 245 250 255aac ccg acc aag tcg cag cgc gaa tgg
ggc ctg acc acc tat atc gag 816Asn Pro Thr Lys Ser Gln Arg Glu Trp
Gly Leu Thr Thr Tyr Ile Glu 260 265 270gcg ctc aag gag gcc atc gag
gta gtc ctg tcg atc acc ggc agc aag 864Ala Leu Lys Glu Ala Ile Glu
Val Val Leu Ser Ile Thr Gly Ser Lys 275 280 285gac ctc aac ctc ctc
ggc gcc tgc tcc ggc ggg atc acc acc gcg acc 912Asp Leu Asn Leu Leu
Gly Ala Cys Ser Gly Gly Ile Thr Thr Ala Thr 290 295 300ctg gtc ggc
cac tac gtg gcc agc ggc gag aag aag gtc aac gcc ttc 960Leu Val Gly
His Tyr Val Ala Ser Gly Glu Lys Lys Val Asn Ala Phe305 310 315
320acc caa ctg gtc agc gtg ctc gac ttc gaa ctg aat acc cag gtc gcg
1008Thr Gln Leu Val Ser Val Leu Asp Phe Glu Leu Asn Thr Gln Val Ala
325 330 335ctg ttc gcc gac gag aag act ctg gag gcc gcc aag cgt cgt
tcc tac 1056Leu Phe Ala Asp Glu Lys Thr Leu Glu Ala Ala Lys Arg Arg
Ser Tyr 340 345 350cag tcc ggc gtg ctg gag ggc aag gac atg gcc aag
gtg ttc gcc tgg 1104Gln Ser Gly Val Leu Glu Gly Lys Asp Met Ala Lys
Val Phe Ala Trp 355 360 365atg cgc ccc aac gac ctg atc tgg aac tac
tgg gtc aac aac tac ctg 1152Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr
Trp Val Asn Asn Tyr Leu 370 375 380ctc ggc aac cag ccg ccg gcg ttc
gac atc ctc tac tgg aac aac gac 1200Leu Gly Asn Gln Pro Pro Ala Phe
Asp Ile Leu Tyr Trp Asn Asn Asp385 390 395 400acc acg cgc ctg ccc
gcc gcg ctg cac ggc gag ttc gtc gaa ctg ttc 1248Thr Thr Arg Leu Pro
Ala Ala Leu His Gly Glu Phe Val Glu Leu Phe 405 410 415aag agc aac
ccg ctg aac cgc ccc ggc gcc ctg gag gtc tcc ggc acg 1296Lys Ser Asn
Pro Leu Asn Arg Pro Gly Ala Leu Glu Val Ser Gly Thr 420 425 430ccc
atc gac ctg aag cag gtg act tgc gac ttc tac tgt gtc gcc ggt 1344Pro
Ile Asp Leu Lys Gln Val Thr Cys Asp Phe Tyr Cys Val Ala Gly 435 440
445ctg aac gac cac atc acc ccc tgg gag tcg tgc tac aag tcg gcc agg
1392Leu Asn Asp His Ile Thr Pro Trp Glu Ser Cys Tyr Lys Ser Ala Arg
450 455 460ctg ctg ggt ggc aag tgc gag ttc atc ctc tcc aac agc ggt
cac atc 1440Leu Leu Gly Gly Lys Cys Glu Phe Ile Leu Ser Asn Ser Gly
His Ile465 470 475 480cag agc atc ctc aac cca ccg ggc aac ccc aag
gca cgc ttc atg acc 1488Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys
Ala Arg Phe Met Thr 485 490 495aat ccg gaa ctg ccc gcc gag ccc aag
gcc tgg ctg gaa cag gcc ggc 1536Asn Pro Glu Leu Pro Ala Glu Pro Lys
Ala Trp Leu Glu Gln Ala Gly 500 505 510aag cac gcc gac tcg tgg tgg
ttg cac tgg cag caa tgg ctg gcc gaa 1584Lys His Ala Asp Ser Trp Trp
Leu His Trp Gln Gln Trp Leu Ala Glu 515 520 525cgc tcc ggc aag acc
cgc aag gcg ccc gcc agc ctg ggc aac aag acc 1632Arg Ser Gly Lys Thr
Arg Lys Ala Pro Ala Ser Leu Gly Asn Lys Thr 530 535 540tat ccg gcc
ggc gaa gcc gcg ccc gga acc tac gtg cat gaa cga tca 1680Tyr Pro Ala
Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg Ser545 550 555
560aaa gct ttg ggc aaa ggt gtt acc gag gaa caa ttc aaa gag acc tgg
1728Lys Ala Leu Gly Lys Gly Val Thr Glu Glu Gln Phe Lys Glu Thr Trp
565 570 575acg agg ccg gga gct gct gga atg ggc gaa ggg act agc ctt
gtg gtg 1776Thr Arg Pro Gly Ala Ala Gly Met Gly Glu Gly Thr Ser Leu
Val Val 580 585 590gcc aag tcc aga atg taa 1794Ala Lys Ser Arg Met
59523597PRTP. aeruginosa 23Met Ser Gln Lys Asn Asn Asn Glu Leu Pro
Lys Gln Ala Ala Glu Asn1 5 10 15Thr Leu Asn Leu Asn Pro Val Ile Gly
Ile Arg Gly Lys Asp Leu Leu 20 25 30Thr Ser Ala Arg Met Val Leu Leu
Gln Ala Val Arg Gln Pro Leu His 35 40 45Ser Ala Arg His Val Ala His
Phe Ser Leu Glu Leu Lys Asn Val Leu 50 55 60Leu Gly Gln Ser Glu Leu
Arg Pro Gly Asp Asp Asp Arg Arg Phe Ser65 70 75 80Asp Pro Ala Trp
Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Met Gln Thr 85 90 95Tyr Leu Ala
Trp Arg Lys Glu Leu His Ser Trp Ile Ser His Ser Asp 100 105 110Leu
Ser Pro Gln Asp Ile Ser Arg Gly Gln Phe Val Ile Asn Leu Leu 115 120
125Thr Glu Ala Met Ser Pro Thr Asn Ser Leu Ser Asn Pro Ala Ala Val
130 135 140Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly
Leu Gly145 150 155 160His Leu Ala Lys Asp Leu Val Asn Asn Gly Gly
Met Pro Ser Gln Val 165 170 175Asp Met Asp Ala Phe Glu Val Gly Lys
Asn Leu Ala Thr Thr Glu Gly 180 185 190Ala Val Val Phe Arg Asn Asp
Val Leu Glu Leu Ile Gln Tyr Arg Pro 195 200 205Ile Thr Glu Ser Val
His Glu Arg Pro Leu Leu Val Val Pro Pro Gln 210 215 220Ile Asn Lys
Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser Leu Ala225 230 235
240Arg Phe Cys Leu Arg Asn Gly Val Gln Thr Phe Ile Val Ser Trp Arg
245 250 255Asn Pro Thr Lys Ser Gln Arg Glu Trp Gly Leu Thr Thr Tyr
Ile Glu 260 265 270Ala Leu Lys Glu Ala Ile Glu Val Val Leu Ser Ile
Thr Gly Ser Lys 275 280 285Asp Leu Asn Leu Leu Gly Ala Cys Ser Gly
Gly Ile Thr Thr Ala Thr 290 295 300Leu Val Gly His Tyr Val Ala Ser
Gly Glu Lys Lys Val Asn Ala Phe305 310 315 320Thr Gln Leu Val Ser
Val Leu Asp Phe Glu Leu Asn Thr Gln Val Ala 325 330 335Leu Phe Ala
Asp Glu Lys Thr Leu Glu Ala Ala Lys Arg Arg Ser Tyr 340 345 350Gln
Ser Gly Val Leu Glu Gly Lys Asp Met Ala Lys Val Phe Ala Trp 355 360
365Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu
370 375 380Leu Gly Asn Gln Pro Pro Ala Phe Asp Ile Leu Tyr Trp Asn
Asn Asp385 390 395 400Thr Thr Arg Leu Pro Ala Ala Leu His Gly Glu
Phe Val Glu Leu Phe 405 410 415Lys Ser Asn Pro Leu Asn Arg Pro Gly
Ala Leu Glu Val Ser Gly Thr 420 425 430Pro Ile Asp Leu Lys Gln Val
Thr Cys Asp Phe Tyr Cys Val Ala Gly 435 440 445Leu Asn Asp His Ile
Thr Pro Trp Glu Ser Cys Tyr Lys Ser Ala Arg 450 455 460Leu Leu Gly
Gly Lys Cys Glu Phe Ile Leu Ser Asn Ser Gly His Ile465 470 475
480Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ala Arg Phe Met Thr
485 490 495Asn Pro Glu Leu Pro Ala Glu Pro Lys Ala Trp Leu Glu Gln
Ala Gly 500 505 510Lys His Ala Asp Ser Trp Trp Leu His Trp Gln Gln
Trp Leu Ala Glu 515 520 525Arg Ser Gly Lys Thr Arg Lys Ala Pro Ala
Ser Leu Gly Asn Lys Thr 530 535 540Tyr Pro Ala Gly Glu Ala Ala Pro
Gly Thr Tyr Val His Glu Arg Ser545 550 555 560Lys Ala Leu Gly Lys
Gly Val Thr Glu Glu Gln Phe Lys Glu Thr Trp 565 570 575Thr Arg Pro
Gly Ala Ala Gly Met Gly Glu Gly Thr Ser Leu Val Val 580 585 590Ala
Lys Ser Arg Met 595241883DNAP. aeruginosa 24ggatccccaa ttcccgatga
gtcagaagaa caataacgag cttcccaagc aagccgcgga 60aaacacgctg aacctgaatc
cggtgatcgg catccggggc aaggacctgc tcacctccgc 120gcgcatggtc
ctgctccagg cggtgcgcca gccgctgcac agcgccaggc acgtggcgca
180tttcagcctg gagctgaaga acgtcctgct cggccagtcg gagctacgcc
caggcgatga 240cgaccgacgc ttttccgatc cggcctggag ccagaatcca
ctgtacaagc gctacatgca 300gacctacctg gcctggcgca aggagctgca
cagctggatc agccacagcg acctgtcgcc 360gcaggacatc agtcgtggcc
agttcgtcat caacctgctg accgaggcga tgtcgccgac 420caacagcctg
agcaacccgg cggcggtcaa gcgcttcttc gagaccggcg gcaagagcct
480gctggacggc ctcggccacc tggccaagga cctggtgaac aacggcggga
tgccgagcca 540ggtggacatg gacgccttcg aggtgggcaa gaacctggcc
accaccgagg gcgccgtggt 600gttccgcaac gacgtgctgg aactgatcca
gtaccggccg atcaccgagt cggtgcacga 660acgcccgctg ctggtggtgc
cgccgcagat caacaagttc tacgtcttcg acctgtcgcc 720ggacaagagc
ctggcgcgct tctgcctgcg caacggcgtg cagaccttca tcgtcagttg
780gcgcaacccg accaagtcgc agcgcgaatg gggcctgacc acctatatcg
aggcgctcaa 840ggaggccatc gaggtagtcc tgtcgatcac cggcagcaag
gacctcaacc tcctcggcgc 900ctgctccggc gggatcacca ccgcgaccct
ggtcggccac tacgtggcca gcggcgagaa 960gaaggtcaac gccttcaccc
aactggtcag cgtgctcgac ttcgaactga atacccaggt 1020cgcgctgttc
gccgacgaga agactctgga ggccgccaag cgtcgttcct accagtccgg
1080cgtgctggag ggcaaggaca tggccaaggt gttcgcctgg atgcgcccca
acgacctgat 1140ctggaactac tgggtcaaca actacctgct cggcaaccag
ccgccggcgt tcgacatcct 1200ctactggaac aacgacacca cgcgcctgcc
cgccgcgctg cacggcgagt tcgtcgaact 1260gttcaagagc aacccgctga
accgccccgg cgccctggag gtctccggca cgcccatcga 1320cctgaagcag
gtgacttgcg acttctactg tgtcgccggt ctgaacgacc acatcacccc
1380ctgggagtcg tgctacaagt cggccaggct gctgggtggc aagtgcgagt
tcatcctctc 1440caacagcggt cacatccaga gcatcctcaa cccaccgggc
aaccccaagg cacgcttcat 1500gaccaatccg gaactgcccg ccgagcccaa
ggcctggctg gaacaggccg gcaagcacgc 1560cgactcgtgg tggttgcact
ggcagcaatg gctggccgaa cgctccggca agacccgcaa 1620ggcgcccgcc
agcctgggca acaagaccta tccggccggc gaagccgcgc ccggaaccta
1680cgtgcatgaa cgatcaaaag ctttgggcaa aggtgttacc gaggaacaat
tcaaagagac 1740ctggacgagg ccgggagctg ctggaatggg cgaagggcga
agggactagc cttgtggtgg 1800ccaagtccag aatgtaagac agacgttcat
tgcggcggag cggccaaggc ggttcggcat 1860cttcgcagaa aaacaactag ggg
1883251929DNAP. aeruginosaCDS(1)..(1929) 25atg gct tct atg ata tcc
tct tcc gct gtg aca aca gtc agc cgt gcc 48Met Ala Ser Met Ile Ser
Ser Ser Ala Val Thr Thr Val Ser Arg Ala1 5 10 15tct agg ggg caa tcc
gcc gca atg gct cca ttc ggc ggc ctc aaa tcc 96Ser Arg Gly Gln Ser
Ala Ala Met Ala Pro Phe Gly Gly Leu Lys Ser 20 25 30atg act gga ttc
cca gtg aag aag gtc aac act gac att act tcc att 144Met Thr Gly Phe
Pro Val Lys Lys Val Asn Thr Asp Ile Thr Ser Ile 35 40 45aca agc aat
ggt gga aga gta aag tgc atg cag gtg tgg cct cca att 192Thr Ser Asn
Gly Gly Arg Val Lys Cys Met Gln Val Trp Pro Pro Ile 50 55 60gga aag
aag aag ttt gag act ctt tcc tat ttg cca cca ttg acc aga 240Gly Lys
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Thr Arg65 70 75
80gat tcc cgg gtg agt cag aag aac aat aac gag ctt ccc aag caa gcc
288Asp Ser Arg Val Ser Gln Lys Asn Asn Asn Glu Leu Pro Lys Gln Ala
85 90 95gcg gaa aac acg ctg aac ctg aat ccg gtg atc ggc atc cgg ggc
aag
336Ala Glu Asn Thr Leu Asn Leu Asn Pro Val Ile Gly Ile Arg Gly Lys
100 105 110gac ctg ctc acc tcc gcg cgc atg gtc ctg ctc cag gcg gtg
cgc cag 384Asp Leu Leu Thr Ser Ala Arg Met Val Leu Leu Gln Ala Val
Arg Gln 115 120 125ccg ctg cac agc gcc agg cac gtg gcg cat ttc agc
ctg gag ctg aag 432Pro Leu His Ser Ala Arg His Val Ala His Phe Ser
Leu Glu Leu Lys 130 135 140aac gtc ctg ctc ggc cag tcg gag cta cgc
cca ggc gat gac gac cga 480Asn Val Leu Leu Gly Gln Ser Glu Leu Arg
Pro Gly Asp Asp Asp Arg145 150 155 160cgc ttt tcc gat ccg gcc tgg
agc cag aat cca ctg tac aag cgc tac 528Arg Phe Ser Asp Pro Ala Trp
Ser Gln Asn Pro Leu Tyr Lys Arg Tyr 165 170 175atg cag acc tac ctg
gcc tgg cgc aag gag ctg cac agc tgg atc agc 576Met Gln Thr Tyr Leu
Ala Trp Arg Lys Glu Leu His Ser Trp Ile Ser 180 185 190cac agc gac
ctg tcg ccg cag gac atc agt cgt ggc cag ttc gtc atc 624His Ser Asp
Leu Ser Pro Gln Asp Ile Ser Arg Gly Gln Phe Val Ile 195 200 205aac
ctg ctg acc gag gcg atg tcg ccg acc aac agc ctg agc aac ccg 672Asn
Leu Leu Thr Glu Ala Met Ser Pro Thr Asn Ser Leu Ser Asn Pro 210 215
220gcg gcg gtc aag cgc ttc ttc gag acc ggc ggc aag agc ctg ctg gac
720Ala Ala Val Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu
Asp225 230 235 240ggc ctc ggc cac ctg gcc aag gac ctg gtg aac aac
ggc ggg atg ccg 768Gly Leu Gly His Leu Ala Lys Asp Leu Val Asn Asn
Gly Gly Met Pro 245 250 255agc cag gtg gac atg gac gcc ttc gag gtg
ggc aag aac ctg gcc acc 816Ser Gln Val Asp Met Asp Ala Phe Glu Val
Gly Lys Asn Leu Ala Thr 260 265 270acc gag ggc gcc gtg gtg ttc cgc
aac gac gtg ctg gaa ctg atc cag 864Thr Glu Gly Ala Val Val Phe Arg
Asn Asp Val Leu Glu Leu Ile Gln 275 280 285tac cgg ccg atc acc gag
tcg gtg cac gaa cgc ccg ctg ctg gtg gtg 912Tyr Arg Pro Ile Thr Glu
Ser Val His Glu Arg Pro Leu Leu Val Val 290 295 300ccg ccg cag atc
aac aag ttc tac gtc ttc gac ctg tcg ccg gac aag 960Pro Pro Gln Ile
Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys305 310 315 320agc
ctg gcg cgc ttc tgc ctg cgc aac ggc gtg cag acc ttc atc gtc 1008Ser
Leu Ala Arg Phe Cys Leu Arg Asn Gly Val Gln Thr Phe Ile Val 325 330
335agt tgg cgc aac ccg acc aag tcg cag cgc gaa tgg ggc ctg acc acc
1056Ser Trp Arg Asn Pro Thr Lys Ser Gln Arg Glu Trp Gly Leu Thr Thr
340 345 350tat atc gag gcg ctc aag gag gcc atc gag gta gtc ctg tcg
atc acc 1104Tyr Ile Glu Ala Leu Lys Glu Ala Ile Glu Val Val Leu Ser
Ile Thr 355 360 365ggc agc aag gac ctc aac ctc ctc ggc gcc tgc tcc
ggc ggg atc acc 1152Gly Ser Lys Asp Leu Asn Leu Leu Gly Ala Cys Ser
Gly Gly Ile Thr 370 375 380acc gcg acc ctg gtc ggc cac tac gtg gcc
agc ggc gag aag aag gtc 1200Thr Ala Thr Leu Val Gly His Tyr Val Ala
Ser Gly Glu Lys Lys Val385 390 395 400aac gcc ttc acc caa ctg gtc
agc gtg ctc gac ttc gaa ctg aat acc 1248Asn Ala Phe Thr Gln Leu Val
Ser Val Leu Asp Phe Glu Leu Asn Thr 405 410 415cag gtc gcg ctg ttc
gcc gac gag aag act ctg gag gcc gcc aag cgt 1296Gln Val Ala Leu Phe
Ala Asp Glu Lys Thr Leu Glu Ala Ala Lys Arg 420 425 430cgt tcc tac
cag tcc ggc gtg ctg gag ggc aag gac atg gcc aag gtg 1344Arg Ser Tyr
Gln Ser Gly Val Leu Glu Gly Lys Asp Met Ala Lys Val 435 440 445ttc
gcc tgg atg cgc ccc aac gac ctg atc tgg aac tac tgg gtc aac 1392Phe
Ala Trp Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn 450 455
460aac tac ctg ctc ggc aac cag ccg ccg gcg ttc gac atc ctc tac tgg
1440Asn Tyr Leu Leu Gly Asn Gln Pro Pro Ala Phe Asp Ile Leu Tyr
Trp465 470 475 480aac aac gac acc acg cgc ctg ccc gcc gcg ctg cac
ggc gag ttc gtc 1488Asn Asn Asp Thr Thr Arg Leu Pro Ala Ala Leu His
Gly Glu Phe Val 485 490 495gaa ctg ttc aag agc aac ccg ctg aac cgc
ccc ggc gcc ctg gag gtc 1536Glu Leu Phe Lys Ser Asn Pro Leu Asn Arg
Pro Gly Ala Leu Glu Val 500 505 510tcc ggc acg ccc atc gac ctg aag
cag gtg act tgc gac ttc tac tgt 1584Ser Gly Thr Pro Ile Asp Leu Lys
Gln Val Thr Cys Asp Phe Tyr Cys 515 520 525gtc gcc ggt ctg aac gac
cac atc acc ccc tgg gag tcg tgc tac aag 1632Val Ala Gly Leu Asn Asp
His Ile Thr Pro Trp Glu Ser Cys Tyr Lys 530 535 540tcg gcc agg ctg
ctg ggt ggc aag tgc gag ttc atc ctc tcc aac agc 1680Ser Ala Arg Leu
Leu Gly Gly Lys Cys Glu Phe Ile Leu Ser Asn Ser545 550 555 560ggt
cac atc cag agc atc ctc aac cca ccg ggc aac ccc aag gca cgc 1728Gly
His Ile Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ala Arg 565 570
575ttc atg acc aat ccg gaa ctg ccc gcc gag ccc aag gcc tgg ctg gaa
1776Phe Met Thr Asn Pro Glu Leu Pro Ala Glu Pro Lys Ala Trp Leu Glu
580 585 590cag gcc ggc aag cac gcc gac tcg tgg tgg ttg cac tgg cag
caa tgg 1824Gln Ala Gly Lys His Ala Asp Ser Trp Trp Leu His Trp Gln
Gln Trp 595 600 605ctg gcc gaa cgc tcc ggc aag acc cgc aag gcg ccc
gcc agc ctg ggc 1872Leu Ala Glu Arg Ser Gly Lys Thr Arg Lys Ala Pro
Ala Ser Leu Gly 610 615 620aac aag acc tat ccg gcc ggc gaa gcc gcg
ccc gga acc tac gtg cat 1920Asn Lys Thr Tyr Pro Ala Gly Glu Ala Ala
Pro Gly Thr Tyr Val His625 630 635 640gaa cga tga 1929Glu
Arg26642PRTP. aeruginosa 26Met Ala Ser Met Ile Ser Ser Ser Ala Val
Thr Thr Val Ser Arg Ala1 5 10 15Ser Arg Gly Gln Ser Ala Ala Met Ala
Pro Phe Gly Gly Leu Lys Ser 20 25 30Met Thr Gly Phe Pro Val Lys Lys
Val Asn Thr Asp Ile Thr Ser Ile 35 40 45Thr Ser Asn Gly Gly Arg Val
Lys Cys Met Gln Val Trp Pro Pro Ile 50 55 60Gly Lys Lys Lys Phe Glu
Thr Leu Ser Tyr Leu Pro Pro Leu Thr Arg65 70 75 80Asp Ser Arg Val
Ser Gln Lys Asn Asn Asn Glu Leu Pro Lys Gln Ala 85 90 95Ala Glu Asn
Thr Leu Asn Leu Asn Pro Val Ile Gly Ile Arg Gly Lys 100 105 110Asp
Leu Leu Thr Ser Ala Arg Met Val Leu Leu Gln Ala Val Arg Gln 115 120
125Pro Leu His Ser Ala Arg His Val Ala His Phe Ser Leu Glu Leu Lys
130 135 140Asn Val Leu Leu Gly Gln Ser Glu Leu Arg Pro Gly Asp Asp
Asp Arg145 150 155 160Arg Phe Ser Asp Pro Ala Trp Ser Gln Asn Pro
Leu Tyr Lys Arg Tyr 165 170 175Met Gln Thr Tyr Leu Ala Trp Arg Lys
Glu Leu His Ser Trp Ile Ser 180 185 190His Ser Asp Leu Ser Pro Gln
Asp Ile Ser Arg Gly Gln Phe Val Ile 195 200 205Asn Leu Leu Thr Glu
Ala Met Ser Pro Thr Asn Ser Leu Ser Asn Pro 210 215 220Ala Ala Val
Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp225 230 235
240Gly Leu Gly His Leu Ala Lys Asp Leu Val Asn Asn Gly Gly Met Pro
245 250 255Ser Gln Val Asp Met Asp Ala Phe Glu Val Gly Lys Asn Leu
Ala Thr 260 265 270Thr Glu Gly Ala Val Val Phe Arg Asn Asp Val Leu
Glu Leu Ile Gln 275 280 285Tyr Arg Pro Ile Thr Glu Ser Val His Glu
Arg Pro Leu Leu Val Val 290 295 300Pro Pro Gln Ile Asn Lys Phe Tyr
Val Phe Asp Leu Ser Pro Asp Lys305 310 315 320Ser Leu Ala Arg Phe
Cys Leu Arg Asn Gly Val Gln Thr Phe Ile Val 325 330 335Ser Trp Arg
Asn Pro Thr Lys Ser Gln Arg Glu Trp Gly Leu Thr Thr 340 345 350Tyr
Ile Glu Ala Leu Lys Glu Ala Ile Glu Val Val Leu Ser Ile Thr 355 360
365Gly Ser Lys Asp Leu Asn Leu Leu Gly Ala Cys Ser Gly Gly Ile Thr
370 375 380Thr Ala Thr Leu Val Gly His Tyr Val Ala Ser Gly Glu Lys
Lys Val385 390 395 400Asn Ala Phe Thr Gln Leu Val Ser Val Leu Asp
Phe Glu Leu Asn Thr 405 410 415Gln Val Ala Leu Phe Ala Asp Glu Lys
Thr Leu Glu Ala Ala Lys Arg 420 425 430Arg Ser Tyr Gln Ser Gly Val
Leu Glu Gly Lys Asp Met Ala Lys Val 435 440 445Phe Ala Trp Met Arg
Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn 450 455 460Asn Tyr Leu
Leu Gly Asn Gln Pro Pro Ala Phe Asp Ile Leu Tyr Trp465 470 475
480Asn Asn Asp Thr Thr Arg Leu Pro Ala Ala Leu His Gly Glu Phe Val
485 490 495Glu Leu Phe Lys Ser Asn Pro Leu Asn Arg Pro Gly Ala Leu
Glu Val 500 505 510Ser Gly Thr Pro Ile Asp Leu Lys Gln Val Thr Cys
Asp Phe Tyr Cys 515 520 525Val Ala Gly Leu Asn Asp His Ile Thr Pro
Trp Glu Ser Cys Tyr Lys 530 535 540Ser Ala Arg Leu Leu Gly Gly Lys
Cys Glu Phe Ile Leu Ser Asn Ser545 550 555 560Gly His Ile Gln Ser
Ile Leu Asn Pro Pro Gly Asn Pro Lys Ala Arg 565 570 575Phe Met Thr
Asn Pro Glu Leu Pro Ala Glu Pro Lys Ala Trp Leu Glu 580 585 590Gln
Ala Gly Lys His Ala Asp Ser Trp Trp Leu His Trp Gln Gln Trp 595 600
605Leu Ala Glu Arg Ser Gly Lys Thr Arg Lys Ala Pro Ala Ser Leu Gly
610 615 620Asn Lys Thr Tyr Pro Ala Gly Glu Ala Ala Pro Gly Thr Tyr
Val His625 630 635 640Glu Arg271941DNAP. aeruginosa 27ggatccatgg
cttctatgat atcctcttcc gctgtgacaa cagtcagccg tgcctctagg 60gggcaatccg
ccgcaatggc tccattcggc ggcctcaaat ccatgactgg attcccagtg
120aagaaggtca acactgacat tacttccatt acaagcaatg gtggaagagt
aaagtgcatg 180caggtgtggc ctccaattgg aaagaagaag tttgagactc
tttcctattt gccaccattg 240accagagatt cccgggtgag tcagaagaac
aataacgagc ttcccaagca agccgcggaa 300aacacgctga acctgaatcc
ggtgatcggc atccggggca aggacctgct cacctccgcg 360cgcatggtcc
tgctccaggc ggtgcgccag ccgctgcaca gcgccaggca cgtggcgcat
420ttcagcctgg agctgaagaa cgtcctgctc ggccagtcgg agctacgccc
aggcgatgac 480gaccgacgct tttccgatcc ggcctggagc cagaatccac
tgtacaagcg ctacatgcag 540acctacctgg cctggcgcaa ggagctgcac
agctggatca gccacagcga cctgtcgccg 600caggacatca gtcgtggcca
gttcgtcatc aacctgctga ccgaggcgat gtcgccgacc 660aacagcctga
gcaacccggc ggcggtcaag cgcttcttcg agaccggcgg caagagcctg
720ctggacggcc tcggccacct ggccaaggac ctggtgaaca acggcgggat
gccgagccag 780gtggacatgg acgccttcga ggtgggcaag aacctggcca
ccaccgaggg cgccgtggtg 840ttccgcaacg acgtgctgga actgatccag
taccggccga tcaccgagtc ggtgcacgaa 900cgcccgctgc tggtggtgcc
gccgcagatc aacaagttct acgtcttcga cctgtcgccg 960gacaagagcc
tggcgcgctt ctgcctgcgc aacggcgtgc agaccttcat cgtcagttgg
1020cgcaacccga ccaagtcgca gcgcgaatgg ggcctgacca cctatatcga
ggcgctcaag 1080gaggccatcg aggtagtcct gtcgatcacc ggcagcaagg
acctcaacct cctcggcgcc 1140tgctccggcg ggatcaccac cgcgaccctg
gtcggccact acgtggccag cggcgagaag 1200aaggtcaacg ccttcaccca
actggtcagc gtgctcgact tcgaactgaa tacccaggtc 1260gcgctgttcg
ccgacgagaa gactctggag gccgccaagc gtcgttccta ccagtccggc
1320gtgctggagg gcaaggacat ggccaaggtg ttcgcctgga tgcgccccaa
cgacctgatc 1380tggaactact gggtcaacaa ctacctgctc ggcaaccagc
cgccggcgtt cgacatcctc 1440tactggaaca acgacaccac gcgcctgccc
gccgcgctgc acggcgagtt cgtcgaactg 1500ttcaagagca acccgctgaa
ccgccccggc gccctggagg tctccggcac gcccatcgac 1560ctgaagcagg
tgacttgcga cttctactgt gtcgccggtc tgaacgacca catcaccccc
1620tgggagtcgt gctacaagtc ggccaggctg ctgggtggca agtgcgagtt
catcctctcc 1680aacagcggtc acatccagag catcctcaac ccaccgggca
accccaaggc acgcttcatg 1740accaatccgg aactgcccgc cgagcccaag
gcctggctgg aacaggccgg caagcacgcc 1800gactcgtggt ggttgcactg
gcagcaatgg ctggccgaac gctccggcaa gacccgcaag 1860gcgcccgcca
gcctgggcaa caagacctat ccggccggcg aagccgcgcc cggaacctac
1920gtgcatgaac gatgaggtac c 1941281137DNAPseudomonas
putidaCDS(1)..(1137) 28atg gct tct atg ata tcc tct tcc gct gtg aca
aca gtc agc cgt gcc 48Met Ala Ser Met Ile Ser Ser Ser Ala Val Thr
Thr Val Ser Arg Ala1 5 10 15tct agg ggg caa tcc gcc gca atg gct cca
ttc ggc ggc ctc aaa tcc 96Ser Arg Gly Gln Ser Ala Ala Met Ala Pro
Phe Gly Gly Leu Lys Ser 20 25 30atg act gga ttc cca gtg aag aag gtc
aac act gac att act tcc att 144Met Thr Gly Phe Pro Val Lys Lys Val
Asn Thr Asp Ile Thr Ser Ile 35 40 45aca agc aat ggt gga aga gta aag
tgc atg cag gtg tgg cct cca att 192Thr Ser Asn Gly Gly Arg Val Lys
Cys Met Gln Val Trp Pro Pro Ile 50 55 60gga aag aag aag ttt gag act
ctt tcc tat ttg cca cca ttg acc aga 240Gly Lys Lys Lys Phe Glu Thr
Leu Ser Tyr Leu Pro Pro Leu Thr Arg65 70 75 80gat tcc cgg gtg agg
cca gaa atc gct gta ctt gat atc caa ggt cag 288Asp Ser Arg Val Arg
Pro Glu Ile Ala Val Leu Asp Ile Gln Gly Gln 85 90 95tat cgg gtt tac
acg gag ttc tat cgc gcg gat gcg gcc gaa aac acg 336Tyr Arg Val Tyr
Thr Glu Phe Tyr Arg Ala Asp Ala Ala Glu Asn Thr 100 105 110atc atc
ctg atc aac ggc tcg ctg gcc acc acg gcc tcg ttc gcc cag 384Ile Ile
Leu Ile Asn Gly Ser Leu Ala Thr Thr Ala Ser Phe Ala Gln 115 120
125acg gta cgt aac ctg cac cca cag ttc aac gtg gtt ctg ttc gac cag
432Thr Val Arg Asn Leu His Pro Gln Phe Asn Val Val Leu Phe Asp Gln
130 135 140ccg tat tca ggc aag tcc aag ccg cac aac cgt cag gaa cgg
ctg atc 480Pro Tyr Ser Gly Lys Ser Lys Pro His Asn Arg Gln Glu Arg
Leu Ile145 150 155 160agc aag gag acc gag gcg cat atc ctc ctt gag
ctg atc gag cac ttc 528Ser Lys Glu Thr Glu Ala His Ile Leu Leu Glu
Leu Ile Glu His Phe 165 170 175cag gca gac cac gtg atg tct ttt tcg
tgg ggt ggc gca agc acg ctg 576Gln Ala Asp His Val Met Ser Phe Ser
Trp Gly Gly Ala Ser Thr Leu 180 185 190ctg gcg ctg gcg cac cag ccg
cgg tac gtg aag aag gca gtg gtg agt 624Leu Ala Leu Ala His Gln Pro
Arg Tyr Val Lys Lys Ala Val Val Ser 195 200 205tcg ttc tcg cca gtg
atc aac gag ccg atg cgc gac tat ctg gac cgt 672Ser Phe Ser Pro Val
Ile Asn Glu Pro Met Arg Asp Tyr Leu Asp Arg 210 215 220ggc tgc cag
tac ctg gcc gcc tgc gac cgt tat cag gtc ggc aac ctg 720Gly Cys Gln
Tyr Leu Ala Ala Cys Asp Arg Tyr Gln Val Gly Asn Leu225 230 235
240gtc aat gac acc atc ggc aag cac ttg ccg tcg ctg ttc aaa cgc ttc
768Val Asn Asp Thr Ile Gly Lys His Leu Pro Ser Leu Phe Lys Arg Phe
245 250 255aac tac cgc cat gtg agc agc ctg gac agc cac gag tac gca
cag atg 816Asn Tyr Arg His Val Ser Ser Leu Asp Ser His Glu Tyr Ala
Gln Met 260 265 270cac ttc cac atc aac cag gtg ctg gag cac gac ctg
gaa cgt gcg ctg 864His Phe His Ile Asn Gln Val Leu Glu His Asp Leu
Glu Arg Ala Leu 275 280 285caa ggc gcg cgc aat atc aac atc ccg gtg
ctg ttc atc aac ggc gag 912Gln Gly Ala Arg Asn Ile Asn Ile Pro Val
Leu Phe Ile Asn Gly Glu 290 295 300cgc gac gag tac acc aca gtc gag
gat gcg cgg cag ttc agc aag cat 960Arg Asp Glu Tyr Thr Thr Val Glu
Asp Ala Arg Gln Phe Ser Lys His305 310 315 320gtg ggc aga agc cag
ttc agc gtg atc cgc gat gcg ggc cac ttc ctg 1008Val Gly Arg Ser Gln
Phe Ser Val Ile Arg Asp Ala Gly His Phe Leu 325 330 335gac atg gag
aac aag acc gcc tgc gag aac acc cgc aat gtc atg ctg 1056Asp Met Glu
Asn Lys Thr Ala Cys Glu Asn Thr Arg Asn Val Met Leu 340 345 350ggc
ttc ctc aag cca acc gtg cgt gaa ccc cgc caa cgt tac caa ccc 1104Gly
Phe Leu Lys Pro Thr Val Arg Glu Pro Arg Gln Arg Tyr Gln Pro 355 360
365gtg cag cag ggg cag cat gca ttt gcc atc tga 1137Val Gln Gln Gly
Gln His Ala Phe Ala Ile 370 37529378PRTPseudomonas putida 29Met Ala
Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala1
5 10 15Ser Arg Gly Gln Ser Ala Ala Met Ala Pro Phe Gly Gly Leu Lys
Ser 20 25 30Met Thr Gly Phe Pro Val Lys Lys Val Asn Thr Asp Ile Thr
Ser Ile 35 40 45Thr Ser Asn Gly Gly Arg Val Lys Cys Met Gln Val Trp
Pro Pro Ile 50 55 60Gly Lys Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro
Pro Leu Thr Arg65 70 75 80Asp Ser Arg Val Arg Pro Glu Ile Ala Val
Leu Asp Ile Gln Gly Gln 85 90 95Tyr Arg Val Tyr Thr Glu Phe Tyr Arg
Ala Asp Ala Ala Glu Asn Thr 100 105 110Ile Ile Leu Ile Asn Gly Ser
Leu Ala Thr Thr Ala Ser Phe Ala Gln 115 120 125Thr Val Arg Asn Leu
His Pro Gln Phe Asn Val Val Leu Phe Asp Gln 130 135 140Pro Tyr Ser
Gly Lys Ser Lys Pro His Asn Arg Gln Glu Arg Leu Ile145 150 155
160Ser Lys Glu Thr Glu Ala His Ile Leu Leu Glu Leu Ile Glu His Phe
165 170 175Gln Ala Asp His Val Met Ser Phe Ser Trp Gly Gly Ala Ser
Thr Leu 180 185 190Leu Ala Leu Ala His Gln Pro Arg Tyr Val Lys Lys
Ala Val Val Ser 195 200 205Ser Phe Ser Pro Val Ile Asn Glu Pro Met
Arg Asp Tyr Leu Asp Arg 210 215 220Gly Cys Gln Tyr Leu Ala Ala Cys
Asp Arg Tyr Gln Val Gly Asn Leu225 230 235 240Val Asn Asp Thr Ile
Gly Lys His Leu Pro Ser Leu Phe Lys Arg Phe 245 250 255Asn Tyr Arg
His Val Ser Ser Leu Asp Ser His Glu Tyr Ala Gln Met 260 265 270His
Phe His Ile Asn Gln Val Leu Glu His Asp Leu Glu Arg Ala Leu 275 280
285Gln Gly Ala Arg Asn Ile Asn Ile Pro Val Leu Phe Ile Asn Gly Glu
290 295 300Arg Asp Glu Tyr Thr Thr Val Glu Asp Ala Arg Gln Phe Ser
Lys His305 310 315 320Val Gly Arg Ser Gln Phe Ser Val Ile Arg Asp
Ala Gly His Phe Leu 325 330 335Asp Met Glu Asn Lys Thr Ala Cys Glu
Asn Thr Arg Asn Val Met Leu 340 345 350Gly Phe Leu Lys Pro Thr Val
Arg Glu Pro Arg Gln Arg Tyr Gln Pro 355 360 365Val Gln Gln Gly Gln
His Ala Phe Ala Ile 370 375301149DNAPseudomonas putida 30ggatccatgg
cttctatgat atcctcttcc gctgtgacaa cagtcagccg tgcctctagg 60gggcaatccg
ccgcaatggc tccattcggc ggcctcaaat ccatgactgg attcccagtg
120aagaaggtca acactgacat tacttccatt acaagcaatg gtggaagagt
aaagtgcatg 180caggtgtggc ctccaattgg aaagaagaag tttgagactc
tttcctattt gccaccattg 240accagagatt cccgggtgag gccagaaatc
gctgtacttg atatccaagg tcagtatcgg 300gtttacacgg agttctatcg
cgcggatgcg gccgaaaaca cgatcatcct gatcaacggc 360tcgctggcca
ccacggcctc gttcgcccag acggtacgta acctgcaccc acagttcaac
420gtggttctgt tcgaccagcc gtattcaggc aagtccaagc cgcacaaccg
tcaggaacgg 480ctgatcagca aggagaccga ggcgcatatc ctccttgagc
tgatcgagca cttccaggca 540gaccacgtga tgtctttttc gtggggtggc
gcaagcacgc tgctggcgct ggcgcaccag 600ccgcggtacg tgaagaaggc
agtggtgagt tcgttctcgc cagtgatcaa cgagccgatg 660cgcgactatc
tggaccgtgg ctgccagtac ctggccgcct gcgaccgtta tcaggtcggc
720aacctggtca atgacaccat cggcaagcac ttgccgtcgc tgttcaaacg
cttcaactac 780cgccatgtga gcagcctgga cagccacgag tacgcacaga
tgcacttcca catcaaccag 840gtgctggagc acgacctgga acgtgcgctg
caaggcgcgc gcaatatcaa catcccggtg 900ctgttcatca acggcgagcg
cgacgagtac accacagtcg aggatgcgcg gcagttcagc 960aagcatgtgg
gcagaagcca gttcagcgtg atccgcgatg cgggccactt cctggacatg
1020gagaacaaga ccgcctgcga gaacacccgc aatgtcatgc tgggcttcct
caagccaacc 1080gtgcgtgaac cccgccaacg ttaccaaccc gtgcagcagg
ggcagcatgc atttgccatc 1140tgaggtacc 114931519DNAAeromonas
caviaeCDS(1)..(519) 31atg agc gca caa tcc ctg gaa gta ggc cag aag
gcc cgt ctc agc aag 48Met Ser Ala Gln Ser Leu Glu Val Gly Gln Lys
Ala Arg Leu Ser Lys1 5 10 15cgg ttc ggg gcg gcg gag gta gcc gcc ttc
gcc gcg ctc tcg gag gac 96Arg Phe Gly Ala Ala Glu Val Ala Ala Phe
Ala Ala Leu Ser Glu Asp 20 25 30ttc aac ccc ctg cac ctg gac ccg gcc
ttc gcc gcc acc acg gcg ttc 144Phe Asn Pro Leu His Leu Asp Pro Ala
Phe Ala Ala Thr Thr Ala Phe 35 40 45gag cgg ccc ata gtc cac ggc atg
ctg ctc gcc agc ctc ttc tcc ggg 192Glu Arg Pro Ile Val His Gly Met
Leu Leu Ala Ser Leu Phe Ser Gly 50 55 60ctg ctg ggc cag cag ttg ccg
ggc aag ggg agc atc tat ctg ggt caa 240Leu Leu Gly Gln Gln Leu Pro
Gly Lys Gly Ser Ile Tyr Leu Gly Gln65 70 75 80agc ctc agc ttc aag
ctg ccg gtc ttt gtc ggg gac gag gtg acg gcc 288Ser Leu Ser Phe Lys
Leu Pro Val Phe Val Gly Asp Glu Val Thr Ala 85 90 95gag gtg gag gtg
acc gcc ctt cgc gag gac aag ccc atc gcc acc ctg 336Glu Val Glu Val
Thr Ala Leu Arg Glu Asp Lys Pro Ile Ala Thr Leu 100 105 110acc acc
cgc atc ttc acc caa ggc ggc gcc ctc gcc gtg acg ggg gaa 384Thr Thr
Arg Ile Phe Thr Gln Gly Gly Ala Leu Ala Val Thr Gly Glu 115 120
125gcc gtg gtc aag ctg cct tca aaa gct ttg ggc aaa ggt gtt acc gag
432Ala Val Val Lys Leu Pro Ser Lys Ala Leu Gly Lys Gly Val Thr Glu
130 135 140gaa caa ttc aaa gag acc tgg acg agg ccg gga gct gct gga
atg ggc 480Glu Gln Phe Lys Glu Thr Trp Thr Arg Pro Gly Ala Ala Gly
Met Gly145 150 155 160gaa ggg act agc ctt gtg gtg gcc aag tcc aga
atg taa 519Glu Gly Thr Ser Leu Val Val Ala Lys Ser Arg Met 165
17032172PRTAeromonas caviae 32Met Ser Ala Gln Ser Leu Glu Val Gly
Gln Lys Ala Arg Leu Ser Lys1 5 10 15Arg Phe Gly Ala Ala Glu Val Ala
Ala Phe Ala Ala Leu Ser Glu Asp 20 25 30Phe Asn Pro Leu His Leu Asp
Pro Ala Phe Ala Ala Thr Thr Ala Phe 35 40 45Glu Arg Pro Ile Val His
Gly Met Leu Leu Ala Ser Leu Phe Ser Gly 50 55 60Leu Leu Gly Gln Gln
Leu Pro Gly Lys Gly Ser Ile Tyr Leu Gly Gln65 70 75 80Ser Leu Ser
Phe Lys Leu Pro Val Phe Val Gly Asp Glu Val Thr Ala 85 90 95Glu Val
Glu Val Thr Ala Leu Arg Glu Asp Lys Pro Ile Ala Thr Leu 100 105
110Thr Thr Arg Ile Phe Thr Gln Gly Gly Ala Leu Ala Val Thr Gly Glu
115 120 125Ala Val Val Lys Leu Pro Ser Lys Ala Leu Gly Lys Gly Val
Thr Glu 130 135 140Glu Gln Phe Lys Glu Thr Trp Thr Arg Pro Gly Ala
Ala Gly Met Gly145 150 155 160Glu Gly Thr Ser Leu Val Val Ala Lys
Ser Arg Met 165 17033598DNAAeromonas caviae 33ggatccatga gcgcacaatc
cctggaagta ggccagaagg cccgtctcag caagcggttc 60ggggcggcgg aggtagccgc
cttcgccgcg ctctcggagg acttcaaccc cctgcacctg 120gacccggcct
tcgccgccac cacggcgttc gagcggccca tagtccacgg catgctgctc
180gccagcctct tctccgggct gctgggccag cagttgccgg gcaaggggag
catctatctg 240ggtcaaagcc tcagcttcaa gctgccggtc tttgtcgggg
acgaggtgac ggccgaggtg 300gaggtgaccg cccttcgcga ggacaagccc
atcgccaccc tgaccacccg catcttcacc 360caaggcggcg ccctcgccgt
gacgggggaa gccgtggtca agctgccttc aaaagctttg 420ggcaaaggtg
ttaccgagga acaattcaaa gagacctgga cgaggccggg agctgctgga
480atgggcgaag ggcgaaggga ctagccttgt ggtggccaag tccagaatgt
aagacagacg 540ttcattgcgg cggagcggcc aaggcggttc ggcatcttcg
cagaaaaaca actagggg 5983431DNAArtificial SequencePCR primer TphaF
34nnnnnnggat ccatggcttc tatgatatcc t 313530DNAArtificial
SequencePCR primer PhaF 35nnnnnnggat ccatgactga cgttgtcatc
303630DNAArtificial SequencePCR primer PhbF 36nnnnnnggat ccatgactca
gcgcattgcg 303730DNAArtificial SequencePCR primer PhcF 37nnnnnnggat
ccatggcgac cggcaaaggc 303818DNAArtificial SequencePCR primer PhaR
38ctgagtcatg tccactcc 183918DNAArtificial SequencePCR primer PhbR
39ctgccgactg gtggaacc 184018DNAArtificial SequencePCR primer PhcR
40gaagcgtcat gccttggc 184127DNAArtificial SequencePCR primer
PhaC1Cf 41nnnnnnggat ccatgagcca gaagaac 274228DNAArtificial
SequencePCR primer PhaC1Cr 42nnnnnnggta cctcatcgtt catgcacg
284332DNAArtificial SequencePCR primer PhaC1Pf 43nnnnnncccg
ggtgagccag aagaacaata ac 324425DNAArtificial SequencePCR primer
PhaJF 44ggatccatga gcgcacaatc cctgg 254527DNAArtificial SequencePCR
primer PhaJR 45aagcttttga aggcagcttg accacgg 274627DNAArtificial
SequencePCR primer PhaGF 46cccgggtgag gccagaaatc gctgtac
274725DNAArtificial SequencePCR primer PhaGR 47ggtacctcag
atggcaaatg catgc 254836DNAArtificial SequencePCR primer SSP-F
48nngagctcga tgggaggtgc tcgaagacat attacc 364926DNAArtificial
SequencePCR primer SSP-R 49nnggatcctg tactagatat ggcagc
265038DNAArtificial SequencePCR primer 3 50ctactcattt actagtcacc
atggcgccca ccgtgatg 385135DNAArtificial SequencePCR primer 4
51catcttactg gtacctttag tacaacggtg acgcc 355239DNAArtificial
SequencePCR primer 5 52ctactcattt actagtcacc atgagcacat acgaaggtc
395336DNAArtificial SequencePCR primer 6 53catcttactg gtaccttcag
cgtttatacg cttgca 365427DNAArtificial SequencePCR primer 1
54ctactcataa ccatggcgcc caccgtg 275543DNAArtificial SequencePCR
primer 2 55catcttactc atatgccgca cctgcatgca ccggatcctt ccg
43565PRTArtificial SequenceFive extra N-terminal amino acid
residues in a CPL variant 56Met Gln Val Arg His1 5
* * * * *